AU2022201608B2 - Heterodimeric antibody FC-containing proteins and methods for production thereof - Google Patents

Abstract

HETERODIMERIC ANTIBODY FC-CONTAINING PROTEINS AND METHODS FOR PRODUCTION THEREOF 5 Novel heterodimeric antibody-Fc-containing proteins, such as bispecific antibodies, and novel methods for producing such proteins HETERODIMERIC ANTIBODY FC-CONTAINING PROTEINS AND METHODS FOR PRODUCTION THEREOF 08 Mar 2022

Description

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2022201608 08 Mar 2022 HETERODIMERIC ANTIBODY HETERODIMERIC ANTIBODY FC-CONTAINING FC-CONTAINING PROTEINS PROTEINS ANDAND METHODS METHODS FOR FOR PRODUCTION THEREOF PRODUCTION THEREOF

FIELDOFTHE FIELD OF THE INVENTION INVENTION 5 TheThe 5 present present invention invention relates relates to novel to novel heterodimeric heterodimeric antibody-Fc-containing antibody-Fc-containing proteins, such proteins, such

as bispecific as bispecific antibodies, antibodies, and novel methods and novel methodsforfor producing producing such such proteins. proteins.

The entire The entire disclosure disclosure in in the the complete specificationofofour completespecification ourAustralian AustralianPatent Application PatentApplication No. 2011244282 No. 2011244282 is by is by this this cross-reference cross-reference incorporated incorporated into into the the present present specification. specification.

0 BACKGROUND 0 BACKGROUND OFOF THEINVENTION THE INVENTION Monoclonal antibodies Monoclonal antibodies have have in in recent recent years years become becomesuccessful successfultherapeutic therapeutic molecules, molecules,inin particular for particular for the treatmentofofcancer. the treatment cancer. Unfortunately, Unfortunately, however, however, monoclonal monoclonal antibodies antibodies are are often unable often unable to to cure curediseases diseaseswhen when usedused as monotherapy. as monotherapy. Bispecific Bispecific antibodies antibodies can can potentially overcome potentially some overcome some of the of the limitations limitations of of monoclonal monoclonal antibody antibody therapy, therapy, e.g. could e.g. they they could 5 be be 5 used used as as mediators mediators to target to target or or a drug a drug toxiccompound toxic compound to target to target cells,asasmediators cells, mediatorstoto retarget effector retarget effector mechanisms mechanisms totodisease-associated disease-associatedsites sites ororasasmediators mediators to to increase increase specificity for specificity fortumor tumor cells, cells,for forexample by binding example by bindingtotoaacombination combinationof of targets targets molecules molecules thatthat

is exclusively is exclusively found on tumor found on tumorcells. cells. Different formats Different formats and anduses uses of bispecific of bispecific antibodies antibodies havehave recently recently been reviewed been reviewed by by 0 Chames 0 Chames and and Baty Baty (2009) (2009) Curr Curr Opin Opin Drug Drug Disc Disc Dev 276. Dev 12: 12: One 276. ofOne theofmajor the major obstacles obstacles in in the development the development of of bispecificantibodies bispecific antibodies hashas beenbeen the difficulty the difficulty of producing of producing the material the material in in sufficient quality sufficient quality and and quantity by traditional quantity by traditional technologies, technologies, such suchasasthe thehybrid hybridhybridoma hybridoma and and chemical conjugation chemical conjugation methods methods (Marvin (Marvin and andZhu Zhu(2005) (2005) Acta Acta Pharmacol Pharmacol Sin Sin 26:649). 26:649). Co- Co expressioninina ahost expression hostcell cellofoftwo two antibodies, antibodies, consisting consisting of different of different heavy heavy and chains, and light light chains, 5 leads 5 leads to to a mixture a mixture of possible of possible antibody antibody products products in addition in addition to desired to the the desired bispecific bispecific antibody. antibody.

Several strategies Several strategies have havebeen been described described to favor to favor the the formation formation of a of a heterodimeric, heterodimeric, i.e. i.e. bispecific, product bispecific, product upon co-expressionofofdifferent upon co-expression differentantibody antibody constructs. constructs.

Lindhofer et Lindhofer et al. al. (1995 (1995 JJ Immunol 155:219)have Immunol 155:219) havedescribed describedthat thatfusion fusionofofrat rat and and mouse 30 mouse 30 hydridomas hydridomas producing producing differentdifferent antibodies antibodies leads to leads to enrichment enrichment of functional of functional bispecificbispecific

antibodies, because antibodies, because of preferential of preferential species-restricted species-restricted heavy/light heavy/light chain pairing. chain pairing. Another Another

strategy to strategy to promote promote formation formationofofheterodimers over heterodimersover homodimers homodimers is a is a "knob-into-hole" "knob-into-hole" strategy in strategy in which which aa protuberance protuberanceisisintroduced introducedatatthe theinterface interfaceofofa afirst first heavy-chain heavy-chain polypeptideand polypeptide anda acorresponding corresponding cavity cavity in the in the interface interface of of a second a second heavy-chain heavy-chain polypeptide, polypeptide,

such 35 such 35 that that thethe protuberance protuberance cancan be positioned be positioned in in thethe cavitysosoasastotopromote cavity promote heterodimer heterodimer formation and formation andhinder hinder

18484534_1 (GHMatter)P36631AU05 18484534_1 (GHMatters) P36631AU05

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homodimerformation. homodimer formation."Protuberances" constructedbybyreplacing areconstructed "Protuberances" are smallamino- replacingsmall amino acid side-chains acid side-chains from fromthethe interface interface of of thethe first first withwith polypeptide polypeptide larger larger side side chains. chains.

Compensatory Compensatory "cavities" "cavities" of of identicalor or identical similarsize similar sizetotothe theprotuberances protuberances are created are created 2022201608 08

in the in interface of the interface of the secondpolypeptide the second polypeptide by by replacing replacing large large amino-acid amino-acid side-chains side-chains

5 with 5 with smallerones smaller ones(US (USpatent patent5,731,168). 5,731,168).EP1870459 EP1870459 (Chugai) (Chugai) andand WO WO 2009089004 2009089004 (Amgen) describeother (Amgen) describe otherstrategies strategies for for favoring favoring heterodimer heterodimerformation formationupon upon co-co expression ofofdifferent expression differentantibody antibody domains domains in a in host cell. a cell. host In these In these methods, methods, one or one or more residues more residues that that make makeup up the the CH3-CH3 CH3-CH3 interface interface in CH3 in both bothdomains CH3 domains are are replaced with replaced with aa charged charged amino aminoacid acid such such thatthat homodimer homodimer formation formation is is 10 electrostatically 10 electrostatically unfavorable unfavorable and heterodimerization and heterodimerization is electrostatically is electrostatically favorable. favorable.

W02007110205 WO2007110205 (Merck) (Merck) describe describe yetyet another another strategy,wherein strategy, whereindifferences differences between between andIgG IgA and IgA IgGCH3 CH3 domains domains are exploited are exploited to promote to promote heterodimerization. heterodimerization.

Dall'acqua et Dall'acqua et al. al. (1998 (1998Biochemistry Biochemistry 37:9266) 37:9266) have identified have identified five five energetically key energetically amino-acid residues key amino-acid residues (366, (366, 368, 368,405, 405,407407 and and 409) 409) that that are are 15 involved 15 involved in inthe theCH3-CH3 CH3-CH3contact contactininthe the interface interface of ofa aCH3 CH3 homodimer. homodimer. WO 2008119353 WO 2008119353(Genmab) (Genmab) describesan an describes in invitro vitromethod methodfor forproducing producing bispecific antibodies bispecific whereina abispecific antibodies wherein bispecificantibody antibodyis is formed formed by "Fab-arm" by "Fab-arm" or "half or "half-

molecule" exchange molecule" (swapping ofof aa heavy exchange (swapping heavychain chainand andattached attachedlight light chain) chain) between between two monospecific two monospecific IgG4- IgG4-ororIgG4-like IgG4-likeantibodies antibodiesupon uponincubation incubationunder under reducing reducing 20 conditions. 20 conditions.This This Fab-arm Fab-arm exchange exchange reaction reaction is theisresult the result of a disulfide-bond of a disulfide-bond isomerization reaction isomerization reactionand anddissociation-association dissociation-association of CH3 of CH3 domains domains whereinwherein heavy- heavy chain disulfide chain disulfide bonds bondsin inthethe hinge hinge regions regions ofparent of the the parent (originally (originally monospecific) monospecific)

antibodies are antibodies arereduced reduced andand the the resulting resulting free free cysteines cysteines form form an an heavy-chain inter inter heavy-chain disulfide bond disulfide with cysteine bond with cysteineresidues residuesofofanother another parent parent antibody antibody molecule molecule (originally (originally

25 with 25 with a different a different specificity),and specificity), andsimultaneously simultaneously CH3 CH3 domains domains of the of the parent parent antibodies release antibodies release and andreform reform by by dissociation-association. dissociation-association. The The resulting resulting product product is a is a bispecific antibody bispecific havingtwotwo antibody having Fab Fab arms arms which which potentially potentially are compased are compased different different sequences. It sequences. It should should be be noted noted that that the the process process isis random randomhowever however andand Fab-arm Fab-arm exchange can exchange canalso alsooccur occurbetween betweentwotwo molecules molecules withwith identical identical sequence sequence or or two two 30 bispecific 30 bispecificmolecules moleculescancan engage engage in Fab-arm in Fab-arm exchange exchange to regenerate to regenerate antibodies antibodies comprisingthe comprising theoriginal original monospecific monospecific parent parent antibody antibody specificity. specificity.

It has It has now nowsurprisingly surprisinglybeen been found found that that by introducing by introducing asymmetrical asymmetrical mutationsininthe mutations theCH3 CH3 regions regions of of thethe twotwo monospecific monospecific starting starting proteins, proteins, the Fab-arm the Fab-arm

exchange reaction exchange reaction can can bebeforced forcedtotobecome become directionaland directional and thereby thereby yieldhighly yield highly 35 stable 35 stable heterodimericproteins. heterodimeric proteins.

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SUMMARYOFOFTHE SUMMARY THEINVENTION INVENTION Accordingly, ininone Accordingly, one aspect, aspect, the the present present invention invention providesprovides an efficient an efficient in vitro in vitro

methodforforthetheproduction method production of highly of highly stable stable heterodimeric heterodimeric Fc-containing Fc-containing proteins proteins on on 5 thethe 5 basis basis of of stablehomodimeric stable homodimeric Fc-containing Fc-containing startingmaterials. starting materials.For Forexample, example, a a highly stable highly stable bispecific bispecific antibody antibodycan can be be formed formed with yield with high high and yieldpurity and on purity the on the basis of basis of two stable monospecific two stable monospecificantibodies antibodiesasasstarting startingmaterial. material. Thus, in Thus, in one one aspect, aspect,the theinvention inventionrelates relatestotoananininvitro vitromethod methodforfor generating generating

heterodimericprotein, a heterodimeric a protein,said saidmethod method comprising comprising the the following following steps: steps:

10 10 a) providing a) providing aa first first homodimeric protein comprising homodimeric protein comprisingananFc Fc region region of of an an immunoglobulin, immunoglobulin, saidFc Fcregion said region comprising comprising a first a first CH3CH3 region, region,

b) providing b) providing aa second homodimericprotein second homodimeric proteincomprising comprisingananFc Fcregion regionof ofanan immunoglobulin, immunoglobulin, saidFc Fcregion said region comprising comprising a second a second CH3 region, CH3 region,

15 15 whereinthe wherein thesequences sequences of said of said firstandand first second second CH3 CH3 regions regions are different are different and and are such are such that that the the heterodimeric heterodimericinteraction interactionbetween between saidsaid first first andand second second CH3 CH3 regions is regions is stronger than each stronger than eachofofthe thehomodimeric homodimeric interactions interactions of said of said first first andand

secondCH3 second CH3regions, regions, c) incubating c) incubating said said first first protein protein together together with said second with said second protein protein under under 20 20 reducing conditions reducing conditionssufficient sufficienttotoallow allowthethe cysteines cysteines in hinge in the the hinge regionregion to to undergodisulfide-bond undergo disulfide-bondisomerization, isomerization, andand

d) obtaining d) said heterodimeric obtaining said heterodimericprotein. protein.

The method The methodcancan for for example example be for be used usedinfor in vitro vitro production production of heterodimeric of heterodimeric

25 proteins, 25 proteins,such such as as bispecificantibodies, bispecific antibodies, for for various various uses, uses, such suchasastherapeutic therapeuticoror diagnostic uses. diagnostic uses. An Anadvantage advantage of this of this in in vitromethod vitro method is that is that heavy-chain/light-chain heavy-chain/light-chain

pairing stays pairing intact during stays intact the reaction, during the reaction, so so no no undesired undesiredcombinations combinations of heavy of heavy chainchain

and light and light chains chainsare areobtained obtained in the in the product. product. This This in contrast in contrast to ofsome to some of the co- the co expression methods expression methods described described inin the the prior prior art art (see (see above) where aa common above) where common light light 30 chain 30 chain which which cancan form form a functionalantibody a functional antibodywith with both both heavy heavychain chain needs needs to to be be found found in order in to avoid order to avoidthe theformation formation of non-functional of non-functional heavy-chain/light-chain heavy-chain/light-chain products, products,

becauseofofrandom because random heavy-chain/light-chain heavy-chain/light-chain pairingpairing in the in the Incell. cell. In addition, addition, the in the in vitro process vitro process can be performed can be performedin inthethelaboratory which laboratorywhich allows allows greater greater control, control, flexibility and flexibility andyield yieldofofthe theheterodimeric heterodimeric protein protein than is allowed than is by co-expression. allowed by co-expression.

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2022201608 08 Mar The in The in vitro vitro method the invention of the method of invention can can also also be used to be used to create create compound compound libraries ofof larger libraries larger size, size,e.g. e.g. inin aa screening screening method methodto toidentify identifyadvantageous advantageous combinations of combinations of specificities. specificities. For For example, for some example, for somecombinations combinationsof of antibody antibody targets, not targets, not any anybispecific bispecificantibody antibodywill willbe be functional, functional, i.e.be be i.e. able able to bind to bind to to both both 5 targets 5 targets at the at the samesame time time and mediate and mediate the desired the desired functional functional effects.effects. In suchIn such acases, cases, a bispecific antibody bispecific havinga desired antibody having a desired property, property, e.g. e.g. optimal optimal target target binding binding or cell or cell killing, may killing, may bebe identified identified by: by:

a) providing a) providing a afirst first set setofofhomodimeric homodimeric antibodies antibodies having having different different variable variable

regions, wherein regions, whereinsaid saidantibodies antibodiesofofsaid saidfirst first set set comprise first CH3 comprise aafirst region, CH3 region,

10 10 b) providing b) providing aa second secondsetsetofofhomodimeric homodimeric antibodies antibodies having having different different variable variable

regions, wherein regions, wherein said said antibodies antibodiesofofsaid saidsecond secondset setcomprise comprisea asecond second CH3 CH3 region, region,

whereinthe wherein thesequences sequences of said of said firstandand first second second CH3 CH3 regions regions are different are different and and are such are such that that the the heterodimeric heterodimericinteraction interactionbetween between saidsaid first first andand second second CH3 CH3 15 15 regions is regions is stronger eachofofthe than each stronger than thehomodimeric homodimeric interactions interactions of said of said first first andand

secondCH3 second CH3regions, regions, c) incubating c) incubating combinations combinationsof of antibodies antibodies of said of said first first setset and and of said of said second second

set under set underreducing reducing conditions conditions sufficient sufficient to allow to allow the the cysteines cysteines in thein hinge the hinge region to region to undergo disulfide-bond isomerization, undergo disulfide-bond isomerization, thus thus generating generating aa set setofof 20 20 bispecific antibodies, bispecific antibodies, d) optionally d) restoring the optionally restoring the conditions conditions to to non-reducing, non-reducing, e) assaying e) assayingthethe resulting resulting set set of bispecific of bispecific antibodies antibodies for a for given given a desired desired property, and property, and f) selecting f) selecting aa bispecific bispecificantibody antibody having the desired having the desired property. property. 25 25 In further In further aspects, aspects,the thepresent present invention invention relates relates to heterodimeric to heterodimeric proteinsproteins

obtained ororobtainable obtained obtainablebybythethemethod method of the of the invention invention andmethods and to to methods for producing for producing

heterodimericproteins heterodimeric proteinsofofthe theinvention inventionbybyco-expression co-expression in ain suitable a suitable host host cell. cell.

30 BRIEF 30 BRIEF DESCRIPTION DESCRIPTION OFOF THE THE DRAWINGS DRAWINGS Figure 1: Figure 1: Generation Generation ofofbispecific bispecific antibodies antibodies bybyinterspecies interspeciesFab-arm Fab-arm exchange. exchange. TheThe generation generation of bispecific of bispecific antibodies antibodies after after GSH-induced GSH-induced in vitro in vitro Fab- Fab arm exchange arm exchangebetween betweenthethe EGFR indicatedEGFR indicated (2F8)andand (2F8) CD20 CD20 (7D8) (7D8) IgG4IgG4 antibodies antibodies was determined was determined by ELISA. by an an ELISA. A concentration A concentration series series (total (total antibody) antibody) of 0-1 of 0-1 µg/mL pg/mL 35 waswas 35 analyzed analyzed in the in the ELISA. ELISA. Bispecificbinding Bispecific bindingwas was higher higher afterFab-arm after Fab-arm exchange exchange

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2022201608 08 Mar between rhesus between rhesus (Rh) (Rh) and and human (Hu)IgG4 human(Hu) IgG4 antibodiesthan antibodies thanbetween between twotwo antibodies antibodies of the of the same species. same species.

Figure 2: Figure 2: Alignment Alignmentofofthe theamino amino acid acid sequences sequences of core of the the core hingehinge (i.e. (i.e. the the CPPC sequence CPPC sequenceininhuman human IgGI IgG1 which which includes includes the the two two cysteine cysteine residues residues 5 that 5 thatpotentially potentially form form the theinterheavy interheavychain chain disulphide disulphide bonds bonds and and correspondingresidues corresponding residuesin in other other human human isotypes isotypes or other or other species) species) and CH3 and CH3- CH3 interface CH3 interface of of the the human humanandand rhesus rhesus antibody antibody isotypes. isotypes. Figure 3: Figure 3: Generation Generation of of bispecific bispecific antibodies antibodies using using mutant humanIgG1 mutant human IgGI engagedforfor engaged Fab-arm Fab-arm exchange. exchange. The generation The generation of bispecific of bispecific antibodies antibodies after after 10 GSH-induced 10 GSH-induced in vitro in vitro Fab-arm Fab-arm exchange exchange between between human human CD20 (7D8) CD20 (7D8) IgG4 antibody IgG4 antibody and the and the indicated indicated human EGFR(2F8) human EGFR (2F8) IgG1 IgG1antibodies antibodies was wasdetermined determinedbybyananELISA. ELISA. The presented The presented graph graph shows shows average average numbers numbersof ofthree threeindependent independentFab-arm Fab-arm exchangeexperiments, exchange experiments, in which in which a total a total antibody antibody concentration concentration of 1 pg/mL of 1 µg/mL was usedwas used for ELISA. for ELISA. Bispecific Bispecific binding binding was washigher higherafter afterFab-arm Fab-arm exchange exchange between between IgGl-2F8 IgG1-2F8-

15 CPSC-ITL 15 CPSC-ITL and and IgG4-7D8 IgG4-7D8 than between than between twoantibodies. two IgG4 IgG4 antibodies. Combining Combining IgG4-7D8IgG4-7D8 with either with either IgG1-2F8-CPSC IgG1-2F8-CPSC or IgGl-2F8-ITL or IgG1-2F8-ITL did not did notinresult result in bispecific bispecific antibodies antibodies

underthe under the conditions conditionsused. used. Figure 4: Figure 4: Generation Generation ofofbispecific bispecific antibodies antibodiesby byininvivo vivoFab-arm Fab-armexchange exchange of of humanIgG4 human IgG4 andand mutant mutant IgG1 IgG antibodies. antibodies. The generation The generation of bispecific of bispecific antibodies antibodies 20 after 20 after inin vivo vivo Fab-arm Fab-arm exchange exchange in in immunodeficient immunodeficient mice mice between between human CD20 human CD20 (7D8) IgG4 and (7D8) IgG4 and the the indicated indicated human humanEGFR EGFR(2F8) (2F8)IgG1 IgG1 andand IgG4 IgG4 mutant mutant antibodies antibodies was determined was determined bybyananELISA. ELISA.The Thepresented presentedgraph graphshows shows average average numbers numbers (n=4). (n=4). Bispecific reactivity Bispecific reactivity isispresented presented as as the concentrationbispecific the concentration bispecific antibodies antibodiesrelative relative toto the total the total IgG concentration(percentage). IgG concentration (percentage).Human Human IgG4 IgG4 with awith a stabilized stabilized (CPPC)(CPPC) hinge hinge

25 or orR409K 25 R409K mutation mutation in in thethe CH3CH3 domain domain is not is not ableable to participate inin Fab-arm to participate Fab-arm exchange. IgG1 exchange. IgG1 with with both both aa CPSC CPSCsequence sequence in inthe thehinge hingeand anda K409R a K409R mutation mutation in in the CH3 the CH3 domain domainis isengaged engaged forfor Fab-arm Fab-arm exchange. exchange. (*) (*) Bispecific Bispecific binding binding forfor the the mixtures containing mixtures containing either eitherIgG1-2F8, IgG1-2F8,IgG4-2F8-CPPC IgG4-2F8-CPPC or or IgG4-2F8-R409K wasbelow IgG4-2F8-R409K was below the detection the detection limit limit andand therefore therefore arbitrarily arbitrarily set to set to zero. zero. 30 Figure 30 Figure 5: 5:Generation Generation of bispecific of bispecific antibodies antibodies using using 2-mercapto 2-mercapto- ethylamineeHCI- ethylamine (2-MEA-) induced HCI- (2-MEA-) induced Fab-arm Fab-arm exchange exchangebetween between human human IgG1IgGI andIgG4 and IgG4 antibodies. antibodies. The generation The generation of bispecific of bispecific antibodies antibodies after 2-MEA-induced after 2-MEA-induced

in in vitro vitro Fab-arm exchangebetween Fab-arm exchange betweenthethe indicatedhuman indicated human (2F8) (2F8) EGFREGFR and CD20 and CD20 (7D8) antibodieswas (7D8) antibodies was determined determined byELISA. by an an ELISA. A concentration A concentration series series of 0-40ofmM0-40 2- mM 2

35 MEAMEA 35 was was tested. tested. TheThe presented presented graph graph shows shows the the result result of of theELISA the ELISA in inwhich whicha atotal total

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concentration of antibody concentration antibody of 20 wasused. pg/mL was 20 µg/mL 2-MEA used.2-MEA efficiently induced efficiently induced Fab-arm Fab-arm exchange,also exchange, alsobetween between antibodies antibodies containing containing a stabilized a stabilized hingehinge (CPPC). (CPPC). Concerning Concerning

the CH3 the CH3 domains, domains,a combination a combination of of human human IgG4 IgG4 x human X human IgG1 IgG1 with thewith the triple triple mutation T350I-K370T-F405L, mutation T3501-K370T-F405L,resulted resulted in higher in higher levels levels of bispecific of bispecific reactivity reactivity 5 compared 5 compared to two to two wild wild type type IgG4 IgG4 antibodies. antibodies. Figure 6: Figure 6: Generation Generationofofbispecific antibodiesusing bispecific antibodies using 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange between exchange betweenhuman human IgGI IgG1 andand IgG4 IgG4 antibodies. antibodies. The generation The generation ofofbispecific bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-inducedin vitro in vitro Fab-arm Fab-arm exchange between exchange betweenthe theindicated indicated human humanEGFR EGFR (2F8)andand (2F8) CD20 CD20 (7D8) (7D8) antibodies antibodies waswas 10 determined 10 determined by mass by mass spectrometry spectrometry for all for all samples samples of the of the concentration concentration seriesofof0-0 series 40 mM 40 mM2-MEA. 2-MEA.(A)(A)Representative Representativeexamples examples of of thethe mass mass spectrometry spectrometry profiles profiles forfor samples of samples of Fab-arm Fab-armexchange exchange reactionsbetween reactions between IgG-2F8-ITL IgG1-2F8-ITL x IgG4-7D8-CPPC X IgG4-7D8-CPPC with 00 mM, with mMand mM, 77 mM and4040mMmM 2-MEA 2-MEA are are shown. shown. (B) (B) After After quantification of quantification of the the mass mass spectrometrydata, spectrometry data, thethe percentage percentage bispecific bispecific antibody antibody was calculated was calculated and and plotted plotted 15 against 15 against thethe concentration concentration 2-MEA 2-MEA in the in the Fab-arm Fab-arm exchange exchange reaction. reaction. IgG4-2F8 IgG4-2F8 X x IgG4-7D8 resulted IgG4-7D8 resulted inin ~50% ~50% bispecificantibody. bispecific antibody.IgG1-2F8-ITL IgGl-2F8-ITL x IgG4-7D8-CPPC X IgG4-7D8-CPPC resulted in resulted in ~95% ~95% bispecificantibody. bispecific antibody. Figure 7: Figure 7: Stability Stability analysis analysis of of heterodimeric heterodimericbispecific bispecific antibodies antibodies obtained obtained by 2-MEA-induced by 2-MEA-inducedFab-arm Fab-arm exchange. exchange. The stability The stability of bispecificsamples of bispecific samples 20 generated 20 generated by by 2-MEA 2-MEA induced induced Fab-arm Fab-arm exchange exchange by combining by combining either either IgGl-2F8-ITL IgG1-2F8-ITL X x IgG4-7D8-CPPC (A), IgG4-7D8-CPPC (A), or or IgG4-2F8 IgG4-2F8 Xx IgG4-7D8 IgG4-7D8(B)(B)waswas tested tested by by measuring measuring EGFR/CD20bispecific EGFR/CD20 bispecific binding binding in in an ELISA after an ELISA after aa GSH-induced GSH-induced Fab-arm Fab-armexchange exchange reaction in reaction in the the presence presenceof of thethe indicated indicated concentrations concentrations irrelevant irrelevant IgG4. IgG4. Bispecific Bispecific

binding isis presented binding presented relative relative to bispecific to the the bispecific binding binding of the starting of the starting material material 25 (control), 25 (control),which whichwaswas set set to 100%. to 100%. (A) Bispecific (A) Bispecific binding binding of 2-MEA-induced of the the 2-MEA-induced bispecific product bispecific product derived derived from IgGl-2F8-ITL X xIgG4-7D8-CPPC from IgG1-2F8-ITL IgG4-7D8-CPPC was was preserved, preserved, indicating aa stable indicating stable product productthat thatdid didnot notparticipate participateininFab-arm Fab-arm exchange exchange under under GSH GSH conditions. (B) conditions. (B) Bispecific Bispecific EGFR/CD20 binding EGFR/CD20 binding of the of the 2-MEA-induced 2-MEA-induced bispecific bispecific product product

derived from derived from IgG4-2F8 IgG4-2F8X xIgG4-7D8 IgG4-7D8 was was diminished, diminished, indicating indicating thatthat the the product product 30 participated 30 participated in Fab-arm in Fab-arm exchange exchange with with the the irrelevant irrelevant IgG4GSH IgG4 under under GSH conditions. conditions.

Figure 8: Figure 8: Plasma Plasma clearance clearance rate rateofofa aheterodimeric heterodimeric bispecificantibody bispecific antibody generatedbyby2-MEA-induced generated 2-MEA-induced Fab-arm Fab-arm exchange. exchange. Three of Three groups groups miceof(3mice mice(3 mice per group) per group) were wereinjected injectedwith withthethe indicatedantibodies: indicated antibodies:(1)(1)100100 pg bispecific µg bispecific antibody, generated antibody, by in generated by in vitro vitro 2-MEA-induced Fab-armexchange 2-MEA-induced Fab-arm exchange between between IgG1 IgG1- 35 2F8-ITL 35 2F8-ITL x IgG4-7D8-CPPC; X IgG4-7D8-CPPC; (2) µg100 (2) 100 pg bispecific bispecific antibody antibody + 1,000 + 1,000 pg irrelevant µg irrelevant

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IgG4; (3) IgG4; (3) 50 50 µg IgGl-2F8-ITL ++5050µg pg pg IgG1-2F8-ITL IgG4-7D8-CPPC. (A) (A) IgG4-7D8-CPPC. Total Total antibody antibody concentrations over concentrations over time, time, determined by ELISA. determined by ELISA. The Thecurves curvesofofthe thetotal total antibody antibody plasma concentrations plasma concentrations were werethe thesame same forfor allallantibodies. antibodies.(B) (B)Bispecific Bispecific antibody antibody concentration asasdetermined concentration determined by ELISA. by an an ELISA. The bispecificity The bispecificity of theofinjected the injected antibody antibody

5 waswas 5 the the samesame with with and without and without the addition the addition of an of an excess excess irrelevant irrelevant IgG4. IgG4. (*) (*) 2022201608 Bispecific binding Bispecific bindingfor forthe IgG1-2F8-ITL++ IgG4-7D8-CPPC theIgGl-2F8-ITL mixturewas IgG4-7D8-CPPC mixture was below below thethe detection limit detection limit and and therefore thereforethe thecorresponding corresponding symbols symbols couldcould not benot be plotted plotted in thisin this graph. Mean graph. Meanvalues values of of two two ELISA ELISA experiments experiments are shown. are shown.

Figure 9: Figure 9: Purity Purity of of bispecific bispecific antibody antibody generated by Fab-arm generated by Fab-armexchange exchange 10 between 10 between human human IgG1-2F8 IgG1-2F8 and IgG4-7D-CPPC. and IgG4-7D8-CPPC. (A) Reducing (A) Reducing SDS-PAGE SDS-PAGE (a) (a) showsbands shows bands of the of the heavy heavy and chains and light light chains forthe for both both the bispecific bispecific sample sample and the and the IgG1 control IgG1 control sample. sample.Non-reducing Non-reducing SDS-PAGE SDS-PAGE (b).The(B)peak (b). (B) Theresults peak results from thefrom HP- the HP SEC analysis SEC analysis shows showsthat >98% that>98% of bispecific of the the bispecific sample sample is homogenous, is homogenous, and and practically nonoantibody practically antibodyaggregates aggregates were were detectable. detectable. (C) (C) Mass Mass spectrometry shows spectrometry shows 15 that 15 that Fab-arm Fab-arm exchange exchange resulted resulted in inapproximately approximately100% 100% bispecific product. bispecific product. Figure 10: Figure 10: Comparison Comparison between between triple triple mutant mutant (ITL), (ITL), double double mutants mutants (IT, (IT, IL, IL, and single TL) and TL) single mutant humanIgG1-2F8 (L) human mutant (L) IgG1-2F8in inthe thegeneration generationofofbispecific bispecific antibodies by antibodies Fab-arm exchange by Fab-arm exchangewith with human human IgG4-7D8. IgG4-7D8. The generation The generation of of bispecific antibodies bispecific antibodiesafter after 2-MEA-inducedininvitro 2-MEA-induced Fab-arm vitro Fab-armexchange between the exchange between the 20 human 20 human IgG1-2F8 IgG1-2F8 triple triple and and double double mutants mutants and type and wild wild IgG4-7D8 type IgG4-7D8 with a with CPSC a CPSC hinge (A) hinge (A) or or mutant mutantIgG4-7D8-CPPC IgG4-7D8-CPPC with awith a stabilized stabilized hinge hinge (B), (B), or theorsingle the single mutantmutant

IgG1-2F8-F405L andIgG4-7D8 IgG1-2F8-F405L and IgG4-7D8 with with a wildtype a wild typeCPSC CPSC or or stabilized CPPC stabilized CPPChinge hinge(C), (C), was determined was determinedby by an ELISA. an ELISA. A concentration A concentration series series (total (total antibody) antibody) of 0-20ofµg/mL 0-20 pg/mL or 0-10 or 0-10 µg/mL pg/mLwaswas analyzed analyzed in ELISA in the the ELISA for thefor the experiments experiments includingincluding the the double double 25 25 andand singlemutants, single mutants,respectively. respectively. Combinations Combinations with with the the double double mutants IgG1-2F8-IL mutants IgG1-2F8-IL and -TL and -TL result resultinin bispecific bispecific EGFR/CD20 EGFR/CD20 binding binding similar similar as the as the triple triple mutant mutant IgG-ITL. IgG1-ITL.

Combinations with Combinations withthethe IgGl-2F8-IT IgG1-2F8-IT doresult do not not in result in a bispecific a bispecific product. product. Combinationswith Combinations with thethe single single mutant mutant IgG1-2F8-F405L IgG1-2F8-F405L result result in bispecific in bispecific EGFR/CD20 EGFR/CD20

binding. binding.

30 Figure11: 30 Figure 11:Generation Generationofofbispecific bispecific antibodies antibodiesusing using2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchangeatatdifferent exchange differenttemperatures. temperatures.The The generation generation of bispecificantibodies of bispecific antibodiesbyby combining the combining the indicated indicated human EGFR human EGFR (2F8)andand (2F8) CD20 CD20 (7D8) (7D8) antibodies antibodies in 2-MEA in 2-MEA- induced inin vitro induced vitro Fab-arm Fab-armexchange exchange reactions reactions at 0°C, at 0°C, 20°C 20°C andwas and 37°C 37°C was followed followed in in time by time byananELISA. ELISA.Bispecific Bispecificbinding bindingwaswas most most efficient efficient at 37°C, at 37°C, and and slower slower at 20°C. at 20°C.

35 At 0°C, 35 At 0°C, no generation no generation of bispecific of bispecific binding binding was measured. was measured.

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2022201608 08 Mar Figure 12: Figure Generationofofbispecific 12: Generation bispecificantibodies antibodiesbyby in in vitroFab-arm vitro Fab-arm exchange exchange induced by induced by different different reducing reducing agents. agents. An An ELISA wasused ELISAwas usedtotomeasure measurethethe generation ofof bispecific generation bispecific antibodies antibodies by by combining combining human human IgGl-2F8-ITL IgG1-2F8-ITL and IgG4-7D8 and IgG4-7D8-

CPPCinina reduction CPPC a reduction reaction reaction with with concentration concentration series series of the indicated of the indicated reducing reducing 5 agents. 5 agents. Bispecificbinding Bispecific binding was wasmeasured measured after after thethe reactionswith reactions DTTDTT with (maximum (maximum obtained at obtained at 2.5 2.5 mM DTT) and mM DTT) and 2-MEA 2-MEA(maximum (maximum obtained obtained at 25 at 25 mM mM 2-MEA), 2-MEA), but but not not with GSH. with GSH. (*) (*) Data DataforforGSH GSH concentration concentration >10 >10 mM excluded mM were were excluded due due to the to the formationofofantibody formation antibodyaggregates. aggregates. Figure 13: 2-MEA-induced Figure 13: 2-MEA-induced Fab-arm Fab-armexchange exchange between between IgG1-2F8-ITL IgG1-2F8-ITL and and 10 IgG1-7D8-K409X 10 IgG1-7D8-K409X mutants. mutants. The generation The generation of bispecific of bispecific antibodies antibodies after 2-MEA after 2-MEA- induced in induced in vitro vitro Fab-arm Fab-arm exchange betweenIgG1-2F8-ITL exchange between IgG1-2F8-ITLandand thethe indicatedIgG1- indicated IgG1 7D8-K409X mutantswaswas 7D8-K409X mutants determined determined by by an an ELISA. ELISA. (A) (A) A concentration A concentration series(total series (total antibody) ofof0-20 antibody) 0-20µg/mL pg/mL was analyzed. was analyzed. The positive The positive control control is a purified is a purified batch of batch of bispecific antibody, bispecific derived from antibody, derived fromIgG1-2F8-ITL IgG1-2F8-ITL x IgG4-7D8-CPPC. X IgG4-7D8-CPPC. (B) The(B) The exchange exchange

15 is presented 15 is presented as bispecific as bispecific binding binding at 20atµg/mL 20 pg/mL relative relative to the to the positive positive controlcontrol (black (black bar). Dark bar). grey bars Dark grey bars represents the bispecific represents the bispecific binding binding between the IgG4 between the IgG4control control (IgG4-7D8 IgG4-2F8),the (IgG4-7D8 X xIgG4-2F8), thenegative negativecontrol control(IgG1-2F8 (IgG1-2F8X IgG1-7D8-K409R) x IgG1-7D8-K409R) and and between IgG1-2F8-ITL between IgG1-2F8-ITLand andIgG4-7D8-CPPC. IgG4-7D8-CPPC. Light Light grey grey bars bars represent represent resultsfrom results from simultaneously performed simultaneously Fab-arm-exchangereactions performed Fab-arm-exchange reactions between betweenthe theindicated indicated IgG1- IgG1 20 7D8-K409X 20 7D8-K409X mutants mutants andIgG1-2F8-ITL. and IgGl-2F8-ITL. Figure 14: Figure 14: Antibody Antibodydeglycosylation deglycosylation does doesnotnot affectthethe affect generation generation of of bispecific antibodies bispecific by 2-MEA-induced antibodies by 2-MEA-induced Fab-arm Fab-arm exchange. exchange. The generation The generation of of bispecific antibodies bispecific antibodiesafter 2-MEA-induced after 2-MEA-inducedininvitro Fab-arm vitro Fab-armexchange between the exchange between the indicated EGFR indicated (2F8) and EGFR (2F8) andCD20 CD20 (7D8) (7D8) antibodies antibodies was was determined determined by anby an ELISA. ELISA. 25 Exchange 25 Exchange with with the the 7D8 7D8 antibodies antibodies was was compared compared with enzymatically with their their enzymatically deglycosylatedvariants. deglycosylated variants.A Aconcentration concentration series series (total (total antibody) antibody) of 0-20 of 0-20 µg/mL pg/mL was was analyzedinin the analyzed the ELISA. ELISA.Fab-arm Fab-arm exchange exchange reactions reactions involving involving deglycosylated deglycosylated (deglyc) (deglyc)

antibodies showed antibodies showed identical identical bispecific bispecific binding binding curves curves as theasglycosylated the glycosylated variantsvariants

from which from whichthey theywere were derived. derived.

30 Figure 30 Figure15:15:The The abilityto ability to engage engage inin Fab-arm Fab-armexchange exchange is is correlatedtotothe correlated the CH3-CH3 interaction CH3-CH3 interaction strength. strength. (A), (A),(B) (B)andand (c) (C) Generation Generation of bispecific of bispecific antibodies by antibodies by GSH-induced Fab-arm exchange GSH-induced Fab-arm exchangebetween between IgG1-2F8 IgG1-2F8 andand IgG1-7D8 IgG1-7D8 (A) (A) or IgG4-2F8 or IgG4-2F8 and andIgG4-7D8 IgG4-7D8 (B and (B and C) constructs C) constructs with with the indicated the indicated mutations, mutations, presentedasasbispecific presented bispecificbinding binding in ELISA in an an ELISA over Bispecificity over time. time. Bispecificity is presented is presented

35 relative 35 relativetotothe theIgG4-2F8 IgG4-2F8 x IgG4-7D8 X IgG4-7D8 control control afterafter 24 h.24(D) h. and (D) (E) andRelation (E) Relation

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2022201608 08 Mar between apparent between (Table2) 2) apparentKDKD(Table andand bispecific bispecific antibody antibody generation generation after24 24 after hrshrs (Figures forIgG1-based 15A/B/C)for (Figures 15A/B/C) IgG1-based(D) (D) or IgG4-based or IgG4-based (E) molecules (E) molecules .

. Figure 16: Figure 16: Sequence Sequence alignment alignment of anti-EGFr of anti-EGFr antibody antibody 2F8 in2F8 an in an IgG4 IgG1, IgGI, IgG4 and (partial) and (partial) IgG3 IgG3backbone. backbone. AminoAmino acid numbering acid numbering according according to and to Kabat Kabat and 5 according 5 according to the to the EU-index EU-index are depicted are depicted (both described (both described in Kabatinet Kabat et al., Sequences al., Sequences of of Proteins of Proteins of Immunological ImmunologicalInterest, Interest,5th5th Ed.Ed. Public Public Health Health Service, Service, National National Institutes Institutes

of Health, of Bethesda,MD. Health, Bethesda, MD.(1991)). (1991)). Figure 17: Figure 17: Generation Generationofofbispecific bispecificantibodies antibodiesbyby ininvitro vitroFab-arm-exchange Fab-arm-exchange induced by induced by different different reducing reducing agents. agents. An An ELISA wasused ELISAwas usedtotomeasure measurethethe 10 generation 10 generation of of bispecific antibodies bispecific antibodies by by combining humanIgG1-2F8-F405L combining human IgG1-2F8-F405L andand IgG1 IgG1- 7D8-K409Rin ina reduction 7D8-K409R a reduction reaction reaction withwith concentration concentration series series of indicated of the the indicated reducing agents.Measured reducing agents. Measured OD values OD values were normalized were normalized to theofsignal to the signal of a bispecific a bispecific

control sample control sample derived derived from 2-MEA-induced Fab-arm-exchange from 2-MEA-induced Fab-arm-exchange between between IgG1-2F8 IgG1-2F8- ITL Xx IgG4-7D8-CPPC, ITL IgG4-7D8-CPPC,which which was was set set to 100%. to 100%. Maximal Maximal bispecific bispecific binding binding was was 15 measured 15 measured after after thethe reactionswith reactions withDTT DTTininthe the concentration concentration range 0.5-50 mM, range 0.5-50 2-MEA mM, 2-MEA in in the the concentration concentration range range 25-50 mMand 25-50 mM andtris(2-carboxyethyl)phosphine tris(2-carboxyethyl)phosphine (TCEP) (TCEP) inin the concentration the concentration range range 0.5-5.0 0.5-5.0 mM, but not mM, but not with with GSH. GSH. (*) (*) Data Data for for GSH GSH concentration 25 concentration > 25 mM mM werewere excluded excluded due todue the to the formation formation of antibody of antibody aggregates. aggregates.

Figure 18: Generation Figure 18: Generation of of bispecific bispecific antibodies antibodies using using 2-MEA-induced 2-MEA-induced Fab- Fab 20 arm-exchange 20 arm-exchangebetween betweenhuman humanIgG1-2F8-F405L IgG1-2F8-F405Land and IgG1-7D8-K409R. IgG1-7D8-K409R. (A) Thegeneration (A) The generation of bispecific of bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-induced in vitroin Fab-arm- vitro Fab-arm exchangewaswas exchange determined determined by anby an ELISA. ELISA. The presented The presented graph graph shows theshows resultthe result of the of the ELISA inin which ELISA whicha atotal totalantibody antibodyconcentration concentrationof of 20 20 pg/mL µg/mL was used. was used. 2-MEA 2-MEA efficiently induced efficiently Fab-arm-exchange. induced Fab-arm-exchange. (B) generation (B) The The generation of bispecific of bispecific antibodies antibodies

25 after2-MEA-induced 25 after 2-MEA-inducedin invitro vitroFab-arm-exchange Fab-arm-exchange was was determined determined by mass by mass spectrometry for spectrometry for all all samples samples of of the the concentration concentration series seriesofof0-40 0-40mM mM 2-MEA. After 2-MEA. After quantification of quantification of the massspectrometry the mass spectrometry data, data, thethe percentage percentage bispecific bispecific antibody antibody was was calculated and calculated andplotted plottedagainst againstthethe concentration concentration of 2-MEA of 2-MEA in Fab-arm-exchange in the the Fab-arm-exchange reaction. IgG1-2F8-F405L reaction. x IgG1-7D8-K409R IgG1-2F8-F405L X IgG1-7D8-K409R resulted resulted in ~100%in bispecific ~100% bispecific antibody, antibody,

30 confirming 30 confirming theELISA the ELISA data. data. Figure 19: Figure 19: Purity Purity of bispecific antibody of bispecific antibody generated generated by Fab-arm-exchange by Fab-arm-exchange between human between humanIgG1-2F8-F405L IgG1-2F8-F405LX IgG1-7D8-K409R. x IgG1-7D8-K409R. MassMass spectrometry spectrometry showsthat shows thatFab-arm-exchange Fab-arm-exchange resulted resulted in approximately in approximately 100% bispecific 100% bispecific product.product.

Figure 20: Figure 20: Plasma Plasmaclearance clearance of aofbispecific a bispecific antibody antibody generated generated by by 2-MEA- 2-MEA 35 induced 35 induced Fab-arm-exchange. Fab-arm-exchange. Two groups Two groups of miceof(3mice mice(3per mice per group) group) were injected were injected

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with the with the indicated antibodies: (1) indicated antibodies: (1) 100 100µgpgbispecific antibody,generated bispecificantibody, generated by vitro by in in vitro 2- 2 MEA-induced Fab-arm-exchange MEA-induced Fab-arm-exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x IgGl-7D8-K409R; X IgG1-7D8-K409R; (2) (2) 100 µgpgbispecific 100 bispecific antibody antibody + + 1,000 1,000 pg irrelevant µg irrelevant IgG4.IgG4. (A) antibody (A) Total Total antibody 2022201608 08

concentrations over concentrations over time, time, determined by ELISA. determined by ELISA. The Thecurves curvesofofthe thetotal total antibody antibody 5 plasma 5 plasma concentrations concentrations were were the the same same for antibodies. for all all antibodies. (B) Bispecific (B) Bispecific antibody antibody concentrationasasdetermined concentration determined by ELISA. by an an ELISA. The bispecificity The bispecificity of theofinjected the injected antibody antibody

was the was thesame same with with andand without without the the addition addition of excess of an an excess irrelevant IgG4.IgG4. irrelevant Figure 21: Figure 21: CDC-mediated CDC-mediatedcellcell killkill of of CD20-expressing CD20-expressing cells cells by a bispecific by a bispecific antibody generated antibody generated by by2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1- IgG1 10 2F8-F405L 10 2F8-F405L x IgG1-7D8-K409R. X IgG1-7D8-K409R. Concentration Concentration series ofseries of the indicated the indicated antibodies antibodies were used were usedto totest test their their capacity capacity to to mediate mediate CDC CDC on on (A) Daudi Daudi (A) and and Raji Raji (B) (B) cells. cells. Both cell Both cell lines lines express CD20 express CD20 butbut not not EGFR. EGFR. Introduction Introduction of theofK409R the K409R in IgG1-7D8 in IgG1-7D8

did not did not influence influence its its capacity to induce capacity to induceCDC. CDC.TheThe bispecific bispecific antibody antibody derived derived fromfrom 2- 2 MEA-induced Fab-arm-exchange MEA-induced Fab-arm-exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R X IgG1-7D8-K409R was was 15 still 15 stillcapable capable to to induce induce CDC. CDC.

Figure 22: Figure 22: ADCC-mediated ADCC-mediatedcellcell killkill of of EGFR-expressing EGFR-expressing cellscells by a by a bispecific bispecific antibody generated antibody generated by by2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1- IgG1 2F8-F405LX IgG1-7D8-K409R. 2F8-F405L x IgGl-7D8-K409R. Concentration Concentration seriesseries of theofindicated the indicated antibodies antibodies were used were used to to test test their theircapacity capacitytoto mediate mediateADCC ADCC on on A431 cells. IgG1-7D8 A431 cells. IgG1-7D8 can not can not 20 bind 20 bind thethe CD20-negative CD20-negative A431 A431 cellsand cells andconsequently consequentlydid didnot notinduce induce ADCC. ADCC. ADCC ADCCwaswas induced by induced by the theEGFR EGFR antibody antibody IgG1-2F8, IgG1-2F8, also also afterafter introduction introduction of the of the F405LF405L mutations in mutations in the the CH3 TheADCC domain. The CH3 domain. ADCC effectorfunction effector functionofofIgG1-2F8-F405L IgG1-2F8-F405Lwaswas retained in retained in the the bispecific format obtained bispecific format obtainedby by Fab-arm-exchange Fab-arm-exchange betweenbetween IgG1-2F8 IgG1-2F8-

F405L Xx IgG1-7D8-K409R. F405L IgG1-7D8-K409R. 25 Figure 25 Figure 23: 23:2-MEA-induced 2-MEA-inducedFab-arm-exchange Fab-arm-exchange between between IgG1-2F8-F405X IgG1-2F8-F405X mutantsand mutants and IgG1-7D8-K409R. IgG1-7D8-K409R. The generation The generation of bispecific of bispecific antibodies antibodies afterafter 2- 2 MEA-induced inin vitro MEA-induced vitro Fab-arm-exchange Fab-arm-exchange between between the indicated the indicated IgG1-2F8-F405X IgG1-2F8-F405X mutants and mutants and IgG1-7D8-K409R IgG1-7D8-K409Rwas was determined determined by anbyELISA. an ELISA. (A) A (A) A concentration concentration series (total series (total antibody) of 0-20 antibody) of pg/mLwaswas 0-20 µg/mL analyzed analyzed in the in the ELISA. ELISA. The positive The positive control control

30 is aispurified 30 a purified batch batch of bispecific of bispecific antibody, antibody, derived derived from from IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8 X IgG1-7D8-

K409R. (B) K409R. (B) The Theexchange exchangeis ispresented presentedasasbispecific bispecific binding binding at at 20 pg/mL antibody 20 µg/mL antibody concentration relative concentration relative toto the thepositive positivecontrol control(black (blackbar). bar).Dark Dark grey grey barsbars represents represents

the bispecific the bispecific binding bindingbetween between the IgG4 control the IgG4 control (IgG4-7D8 (IgG4-7D8 X xIgG4-2F8) IgG4-2F8)andand thethe negative control negative control (IgGl-2F8 (IgG1-2F8 x IgG1-7D8-K409R).Light X IgG1-7D8-K409R). Lightgrey greybars barsrepresent representresults results

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from simultaneously from performed Fab-arm-exchange simultaneously performed Fab-arm-exchangereactions between reactionsbetween the the indicatedIgG1-2F8-F405X mutants indicatedIgG1-2F8-F405X mutantsand andIgG1-7D8-K409R IgGl-7D8-K409Ror or controls. controls. Figure 24: Figure 24: 2-MEA-induced Fab-arm-exchange between 2-MEA-induced Fab-arm-exchange between IgG1-2F8-Y407X IgG1-2F8-Y407X mutantsand mutants and IgG1-7D8-K409R. IgG1-7D8-K409R. The generation The generation of bispecific of bispecific antibodies antibodies afterafter 2- 2 5 MEA-induced 5 MEA-inducedin invitro vitro Fab-arm-exchange Fab-arm-exchange between between the the indicated indicated IgG1-2F8-Y407X IgGl-2F8-Y407X mutants and mutants and IgG1-7D8-K409R IgGl-7D8-K409Rwas was determined determined by anbyELISA. an ELISA. (A) A (A) A concentration concentration series (total series (total antibody) of 0-20 antibody) of pg/mLwaswas 0-20 µg/mL analyzed analyzed in the in the ELISA. ELISA. The positive The positive control control

is aa purified is purified batch of bispecific batch of bispecific antibody, derived from antibody, derived fromIgG1-2F8-F405L IgG1-2F8-F405L x IgGl-7D8 X IgG1-7D8-

K409R. (B) K409R. (B) The Theexchange exchangeis ispresented presentedasasbispecific bispecific binding binding at at 20 20 pg/mL antibody µg/mL antibody 10 concentration 10 concentration relative relative to positive to the the positive control control (black (black bar). bar). Dark bars Dark grey greyrepresents bars represents the bispecific the bispecific binding bindingbetween between the IgG4 control the IgG4 control (IgG4-7D8 (IgG4-7D8 X xIgG4-2F8) IgG4-2F8)andand thethe negative control negative control (IgGl-2F8 (IgG1-2F8 x IgG1-7D8-K409R).Light X IgG1-7D8-K409R). Lightgrey greybars barsrepresent representresults results from simultaneously from simultaneously performed performed Fab-arm-exchange Fab-arm-exchange reactionsbetween reactions between thethe indicated indicated IgGl-2F8-Y407Xmutants IgG1-2F8-Y407X mutantsand andIgG1-7D8-K409R IgGl-7D8-K409R or controls. or controls. 15 Figure 15 Figure 25:25: ofofbispecific Analysis Analysis bispecific antibody antibody generated generated by by 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange by exchange by SDS-PAGE SDS-PAGEunder under non-reducing(Figure non-reducing (Figure 25(A)) 25(A))and andreducing reducing (Figure (Figure 25(B)) conditions. 25(B)) conditions. Figure 26: Figure 26:HP-SEC HP-SEC profilesofofthe profiles thehomodimer homodimer starting starting material material IgG1-2F8-F405L IgG1-2F8-F405L (Figure (Figure 26(B)), 26(B)), the the homodimer starting material homodimer starting material IgG1-7D8-K409R IgG-7D8-K409R (Figure26(A)), (Figure 26(A)), 20 thethe 20 mixture mixture (1:1) (1:1) of both of both homodimers homodimers (Figure (Figure 26(C)), 26(C)), and bispecific and the the bispecific product product generated by generated by 2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x X IgG1- IgG1 7D8-K409R (Figure 26(D)). 7D8-K409R (Figure 26(D)). Figure 27: Figure 27: Mass Massspectrometry spectrometry(ESI-MS) (ESI-MS)ofofthe thehomodimer homodimer startingmaterial starting materialIgG1- IgG1 2F8-F405L(Figure 2F8-F405L (Figure27(B)), 27(B)), thethe homodimer homodimer starting starting material material IgG-7D8-K409R IgG1-7D8-K409R (Figure (Figure 25 27(A)), 25 27(A)), thethe mixture mixture (1:1) (1:1) of of both both homodimers homodimers (Figure (Figure 27(C)), 27(C)), and bispecific and the the bispecific product generated product by 2-MEA-induced generated by 2-MEA-induced Fab-arm Fab-armexchange exchange between between IgG1-2F8-F405L IgG1-2F8-F405L X x IgGl-7D8-K409R (Figure 27(D)). IgG1-7D8-K409R (Figure 27(D)). Figure28: Figure 28: Capillary Capillary isoelectrofocussing isoelectrofocussing (cIEF) (cIEF) profiles profiles of homodimer of the the homodimer startingstarting

material IgG1-2F8-F405L material IgG1-2F8-F405L (Figure (Figure 28(A)), 28(A)), the the homodimer homodimer starting starting material material IgGl-7D8 IgG1-7D8-

30 K409R 30 K409R (Figure (Figure 28(B)), 28(B)), thethemixture mixture(1:1) (1:1)ofof both both homodimers homodimers(Figure (Figure 28(C)), 28(C)), and and the the bispecific product bispecific productgenerated generated by by 2-MEA-induced Fab-armexchange 2-MEA-induced Fab-arm exchange between between IgG1 IgG1- 2F8-F405L Xx IgG1-7D8-K409R 2F8-F405L IgGl-7D8-K409R (Figure28(D)). (Figure 28(D)). Figure 29: Figure 29: HPLC-CIEX profiles of HPLC-CIEXprofiles of the the homodimer starting material homodimer starting material IgG1-2F8-F405L IgG1-2F8-F405L (Figure (Figure 29(A)), 29(A)), the the homodimer starting material homodimer starting IgG-7D8-K409R material IgG1-7D8-K409R (Figure29(B)), (Figure 29(B)), 35 thethe 35 mixture mixture (1:1) (1:1) of both of both homodimers homodimers (Figure (Figure 29(C)), 29(C)), and bispecific and the the bispecific product product

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2022201608 08 Mar generated by generated by 2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x X IgG1- IgG1 7D8-K409R (Figure 29(D)). 7D8-K409R (Figure 29(D)). Figure30: Figure 30:Electrospray Electrospray ionizationmass ionization mass specspec analysis analysis of IgG of IgG obtained obtained by by co- co transfection of transfection of the expressionvectors the expression vectorsencoding encodingthethe heavy heavy and and lightlight chain chain of IgG1 of IgG1-

5 7D8-K409R 5 7D8-K409R or IgGl-2F8-F405. or IgG1-2F8-F405. Heterodimer Heterodimer peakspeaks are indicated are indicated with with an an*. *. Homodimer Homodimer peaks peaks are are indicated indicated withwith an an t. t. Figure 31: Figure 31: Exchange Exchangereaction reactionofof the the homodimers homodimers IgG1-2F8-F405L IgG1-2F8-F405L and and IgGl-7D8 IgG1-7D8- K409Rasasmonitored K409R monitoredby by HighHigh Pressure Pressure Liquid Liquid Chromatography Chromatography CationCation Exchange Exchange (HPLC-CIEX) afterinjection (HPLC-CIEX) after injectionatatdifferent different intervals. intervals. 10 Figure 10 Figure 32:32: Residual Residual homodimers homodimers of the of the exchange exchange reaction reaction as as shown shown in Figure in Figure 32 32 as as detected with detected with the theCIEX CIEXmethod method (indicated (indicated by arrows). by arrows).

Figure33: Figure 33:Generation Generation of of bispecificantibodies bispecific antibodies at at various various IgGIgG concentrations, concentrations, 2-MEA 2-MEA

concentrations, incubation concentrations, incubationtemperatures temperaturesand and times times as determined as determined by an by an ELISA. ELISA. Figure34: Figure 34:Generation Generation of of bispecificantibodies bispecific antibodies at at various various IgGIgG concentrations, concentrations, 2-MEA 2-MEA

15 concentrations, 15 concentrations,incubation incubation temperatures temperaturesand andtimes times as as determined determined by by an an ELISA and ELISA and comparedtotocontrol compared controlwhich which waswas arbitrarily arbitrarily setset to to 100%. 100%.

Figure35: Figure 35:Generation Generation of of bispecificantibodies bispecific antibodies at at various various IgGIgG concentrations, concentrations, 2-MEA 2-MEA

concentrations, incubation concentrations, incubationtemperatures temperaturesand and times times as analysed as analysed by HPLC-CIEX. by HPLC-CIEX.

Figure36: Figure 36:Generation Generation of bispecific of bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-induced in vitro in vitro Fab-arm Fab-arm

20 exchange 20 exchange between between the the indicated indicated IgGl-2F8-L368X IgG1-2F8-L368X mutants mutants and IgGl-7D8-K409R and IgG1-7D8-K409R was was determinedbyby determined an an ELISA ELISA using using a concentration a concentration seriesseries (total(total antibody) antibody) of µg/mL of 0-20 0-20 pg/mL (Figure 37(A)). The (Figure 37(A)). Thepositive positivecontrol controlisis aa purified purified batch batchofofbispecific bispecific antibody, antibody,derived derived from IgG1-2F8-F405L from IgG1-2F8-F405LX xIgG1-7D8-K409R. IgG1-7D8-K409R. Figure Figure 37(B) 37(B) shows shows the the bispecificbinding bispecific binding at 20 at 20 µg/mL pg/mL relative relative to to thethe positive positive control control (black (black bar). bar). DarkDark grey grey bars represents bars represents

25 thethe 25 bispecificbinding bispecific bindingbetween betweenthethe IgG4 IgG4 control control (IgG4-7D8 (IgG4-7D8 x IgG4-2F8) X IgG4-2F8) and and the the negative control negative control (IgG1-2F8 (IgG1-2F8 x IgG1-7D8-K409R).Light X IgG1-7D8-K409R). Lightgrey greybars barsrepresent representresults results from simultaneously from simultaneously performed performed Fab-arm-exchange Fab-arm-exchange reactionsbetween reactions between thethe indicated indicated IgGl-2F8-L368Xmutants IgG1-2F8-L368X mutantsand andIgG1-7D8-K409R. IgG1-7D8-K409R. Figure37: Figure 37:Generation Generation of bispecific of bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-induced in vitro in vitro Fab-arm Fab-arm

30 exchange 30 exchange between between the the indicated indicated IgG1-2F8-K370X IgG1-2F8-K370X mutants mutants and IgG-7D8-K409R and IgG1-7D8-K409R was was determinedbyby determined an an ELISA ELISA using using a concentration a concentration seriesseries (total(total antibody) antibody) of µg/mL of 0-20 0-20 pg/mL (Figure 37(A)). The (Figure 37(A)). Thepositive positivecontrol controlisis aa purified purified batch batchofofbispecific bispecific antibody, antibody,derived derived from IgG1-2F8-F405L from IgG1-2F8-F405LX xIgG1-7D8-K409R. IgG1-7D8-K409R. Figure Figure 37(B) 37(B) shows shows the the bispecificbinding bispecific binding at 20 at 20 µg/mL pg/mL relativeto to relative thethe positive positive control control (black (black bar). bar). DarkDark grey grey bars represents bars represents

35 thethe 35 bispecificbinding bispecific bindingbetween betweenthethe IgG4 IgG4 control control (IgG4-7D8 (IgG4-7D8 x IgG4-2F8) X IgG4-2F8) and and the the

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negative control negative control (IgG1-2F8 (IgG1-2F8 x IgG1-7D8-K409R).Light X IgG1-7D8-K409R). Lightgrey barsrepresent greybars representresults results from simultaneously from simultaneously performed performed Fab-arm-exchange Fab-arm-exchange reactionsbetween reactions between thethe indicated indicated IgGl-2F8-D370Xmutants IgG1-2F8-D370X mutantsand andIgG1-7D8-K409R. IgG1-7D8-K409R. Figure38: Figure 38:Generation Generation of bispecific of bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-induced in vitro in vitro Fab-arm Fab-arm

5 exchange 5 exchangebetween betweenthethe IgG-2F8-D399X mutants indicated IgG1-2F8-D399X indicated mutantsand andIgG1-7D8-K409R IgG-7D8-K409R was determined was determined by ELISA by an an ELISA using using a concentration a concentration series antibody) series (total (total antibody) of 0-20 of 0-20 pg/mL (Figure µg/mL (Figure 38(A)). 38(A)). Figure Figure38(B) 38(B)shows shows the the bispecific bispecific binding binding at µg/mL at 20 20 pg/mL antibody concentration antibody concentrationrelative relativeto to thethe positive positive control control (black (black bar).bar). Dark Dark grey grey bars bars represents the represents thebispecific bispecificbinding bindingbetween between the the IgG4 IgG4 control control (IgG4-7D8 (IgG4-7D8 x IgG4-2F8) X IgG4-2F8)

10 andand 10 thethe negative negative control(IgG1-2F8 control (IgG1-2F8 x IgG1-7D8-K409R). X IgG1-7D8-K409R). LightLight greygrey barsbars represent represent results from results simultaneously performed from simultaneously performed Fab-arm-exchange Fab-arm-exchange reactions reactions between between the the indicated IgGl-2F8-D399X indicated mutantsand IgG1-2F8-D399X mutants andIgG1-7D8-K409R. IgG1-7D8-K409R. Figure 39: Figure 39: 2-MEA-induced 2-MEA-induced Fab-arm Fab-armexchange exchange between between four four different IgG1 different IgG1 mutant mutant combinationsatat15°C combinations 15°C after0, 0,30,30, after 60, 60, 105105 andand 200 200 min incubations min incubations as determined as determined by by 15 sandwich 15 sandwichELISA. ELISA. Figure 40: Figure 40: 2-MEA-induced 2-MEA-induced Fab-arm Fab-armexchange exchange between between differentIgG1 different IgG1mutant mutant combinationsafter combinations afterantibody antibodyincubation incubation at at 15°C 15°C for for 90 min 90 min as determined as determined by by sandwich ELISA. sandwich ELISA. Figure41: Figure 41:Phosphorylation Phosphorylation of of c-Met c-Met by c-Met by c-Met specific specific antibodies. antibodies. A549A549 cellscells are are 20 incubated 20 incubated formin for 15 15 with min with HGF HGF or or a of a panel panel of different different antibodies. antibodies. Proteins Proteins are are separatedbybySDS-page separated SDS-pagegel gel electrophoresis electrophoresis and transferred and and and transferred to membranes to membranes by by westernblotting. western blotting. Phosphorylated Phosphorylatedc-Met, c-Met, totalc-Met total c-Met andand are are p-actin ß-actin detected detected by by antibodies against antibodies againstphosphorylated phosphorylated c-Met, c-Met, total total c-Met c-Met or p-actin. or ß-actin.

Figure42: Figure 42:Proliferation assaywith Proliferation assay withNCI-H441 NCI-H441 cells. cells. NCI-H441 NCI-H441 cellscells were were incubated incubated

25 for for 25 seven seven daysdays with with monovalent monovalent bispecific bispecific IgG1 069/b12, IgG1 069/b12, control antibodies control antibodies (IgG1- (IgG1 069, UniBody-069, 069, UniBody-069, IgG1-b12) IgG1-b12) left left untreated. untreated. CellCell massmass was determined was determined and plotted and plotted

as percentage as percentageofofnon-treated non-treated samples samples (set(set as 100%) as 100%)

Figure43: Figure 43:CDC-mediated CDC-mediated cell kill cell kill of CD20 of CD20 expressing expressing cells cells by by bispecific bispecific antibodies antibodies

generated by generated by 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1-7D8-F405L IgG1-7D8-F405L or IgG1 or IgG1- 30 2F8-F405L 30 2F8-F405L and and IgG1-7D8-K409R. IgG1-7D8-K409R. Concentration Concentration series series of theofindicated the indicated antibodies antibodies were used were usedtototest testtheir theircapacity capacitytoto mediate mediateCDCCDC on Daudi on Daudi (A) Raji (A) and and (B) Rajicells. (B) cells. Both Both

cell lines cell lines express CD20butbut express CD20 notnot The The EGFR. EGFR. bispecific bispecific antibody antibody generated generated by by 2-MEA- 2-MEA induced Fab-arm-exchange induced Fab-arm-exchange between between IgG1-7D8-F405L IgG1-7D8-K409R was IgG1-7D8-F405L xX IgG1-7D8-K409R was asas effective as effective IgG1-7D8in ininduction as IgG1-7D8 induction of of CDCCDC mediated mediated cell kill. cell kill. The The bispecific bispecific antibody antibody

35 derived 35 derived from from 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG2-2F8-F405L IgG2-2F8-F405L X IgG1- x IgG1-

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2022201608 08 Mar 7D8-K409R results 7D8-K409R results in monovalent in a a monovalent CD20 binding CD20 binding bispecific bispecific antibody, antibody, which slightly which slightly

affectedthe affected theinduction induction of mediated of CDC CDC mediated cell killcell withkill with slightly. slightly.

Figure44: Figure 44:Killing KillingofofA431 A431 cells cells induced induced by anti-kappa-ETA' by anti-kappa-ETA' pre-incubated pre-incubated HER2 X HER2 x HER2bispecific HER2 bispecificantibodies. antibodies.The The viabilityofofA431 viability A431 cells cells after after 3 days 3 days incubation incubation with with 5 HER2 5 HER2 antibodies, antibodies, pre-incubated pre-incubated with with anti-kappa-ETA'. anti-kappa-ETA'. Cellviability Cell viability was was quantified quantified using Alamarblue. using Alamarblue.Data Data shown shown are fluorescence are fluorescence intensities intensities (FI) of(FI) one of one experiment experiment

with A431 with A431cells cellstreated treatedwith withanti-kappa-ETA'-conjugated anti-kappa-ETA'-conjugated HER2 antibodies HER2 antibodies and HER2and HER2 HER2bispecific x HER2 X bispecificantibodies. antibodies.Staurosporin Staurosporin waswas usedused as positive as positive control, control, whereas whereas an an isotype control isotype control antibody antibodywas wasused used as as negative negative control. control.

10 Figure 10 Figure 45: 45: HER2HER2 HER2 bispecific x bispecific X HER2 molecules molecules induced induced downmodulation downmodulation of HER2 of HER2 receptor. Relative receptor. Relative percentage percentageofofHER2 HER2 expression expression levels levels in AU565 in AU565 cell lysates cell lysates afterafter 3 3 days incubation days incubation with with 10 pg/mL mAb. 10 µg/mL mAb.The The amount amount of HER2 of HER2 was quantified was quantified using using a a HER2-specific capture HER2-specific ELISA and capture ELISA anddepicted depictedasaspercentage percentage inhibitioncompared inhibition compared to to untreated cells. untreated cells. Data Data shown shownis isthe themean mean of two of two experiments experiments plus standard plus standard deviation. deviation.

15 Figure 15 Figure 46: Colocalization 46: Colocalization analysis analysis of XHER2 of HER2 x HER2 bispecific HER2 bispecific antibodies antibodies (FITC) (FITC) with with lysosomalmarker lysosomal marker LAMP1 LAMP1 (Cy5). (Cy5). FITC FITC pixel pixel intensity intensity overlapping overlapping withforCy5 with Cy5 for various various

monospecificHER2 monospecific HER2 antibodies antibodies and and HER2 HER2 HER2 bispecific X HER2x bispecific antibodies antibodies (Figure (Figure 46(B)) 46(B)) FITC pixel FITC pixel intensity intensity in in LAMP1/Cy5 LAMP1/Cy5 positive positive pixels pixels of of three three different different images images is plotted is plotted

for each for each antibody antibody tested. tested. Monospecifics Monospecifics show FITC show lower lower FITC pixel pixel intensities intensities in the in the 20 LAMP1/Cy5 20 LAMP1/Cy5 positive positive pixelspixels compared compared to bispecifics. to bispecifics. Figure Figure 46(B) represents 46(B) represents the mean the mean value of value of FITC FITCpixel pixelintensity intensityper perLAMP1/Cy5 LAMP1/Cy5 positive positive pixel pixel calculated calculated from from the three the three

different images. different images.Together Together these these results results indicate indicate that after that after internalization internalization higher higher

levels of levels of bispecific bispecific antibodies, antibodies,compared compared to monospecifics to monospecifics antibodies, antibodies, localizelocalize to to Lamp1/Cy5 Lamp1/Cy5 positive positive vesicles. vesicles.

25 Figure 25 Figure 47: 47: Inhibition Inhibition of proliferationbybyHER-2 of proliferation HER-2 monomono and bispecific and bispecific antibodies. antibodies. AU565cells AU565 cells were were seeded seededininthe thepresence presenceofof1010µg/mL pg/mL HER2 HER2 antibody antibody or HER2 or HER2 X x HER2bispecific HER2 bispecificantibody antibodyin in serum-free serum-free cell cell culture culture medium. medium. After days, After three threethe days, the amount ofofviable amount viablecells cellswas was quantifiedwith quantified with Alamarblue Alamarblue and and cell cell viability viability was was presentedasasa apercentage presented percentage relative relative to untreated to untreated cells.cells. An isotype An isotype controlcontrol antibody antibody

30 waswas 30 usedused as negative as negative control. control. Data Data shown shown are percentage are percentage viablecells viable AU565 AU565 cells compared totountreated compared untreatedcells cells measured measured in five-fold± in five-fold ± thethe standard standard deviation. deviation. * *

indicates only indicates one data only one data point pointwas wasdepicted. depicted. Figure48: Figure 48:Binding Binding of of mono mono and bispecific and bispecific IgG1 IgG1 and hinge-deleted and hinge-deleted IgG1 antibodies IgG1 antibodies

to human to andmouse human and mouseFcRn FcRn atatdifferent different pH. pH. Plates Plates with with human and mouse human and mouseFcRn were FcRnwere 35 incubated 35 incubated with with different different mono-mono- and bispecific and bispecific IgG1 antibodies IgG1 antibodies or hinge-deleted or hinge-deleted IgG1 IgG1

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2022201608 08 Mar molecules. Binding molecules. Binding to FcRn was to FcRn was analyzed analyzed by by ELISA ELISA at at 405 405 nm. (A) Binding nm. (A) Binding of of mono mono and bispecific and bispecific IgG1 IgG1antibodies antibodiesand and hinge-deleted hinge-deleted IgG1IgG1 (Uni-G1) (Uni-G1) molecules molecules to to human human FcRn at FcRn at pH pH7.4 7.4and and 6.0.Binding 6.0. Binding to to human human FcRn FcRn is low is very veryatlow at neutral neutral pH. At pH. At pH 6.0 pH 6.0 (bispecific) (bispecific) antibodies antibodies bind bind efficiently efficientlyto tohuman FcRn,unless human FcRn, unlessthey they contain contain thethe H435A H435A

5 mutation. 5 mutation. Hinge-deleted Hinge-deleted IgG1 IgG1 (Uni-G1) (Uni-G1) molecules molecules bind bind human humanFcRn FcRnwith withlowlow efficiency. (B) efficiency. Bindingofofmono (B) Binding mono and bispecific and bispecific IgG1 antibodies IgG1 antibodies and hinge-deleted and hinge-deleted

IgG1 (Uni-G1) IgG1 (Uni-G1)molecules molecules to to mouse mouse FcRn FcRn at pH at 7.4pHand 7.46.0. andBinding 6.0. Binding to FcRn to mouse mouse is FcRn is very low very lowatatneutral neutral pH.pH. At 6.0 At pH pH (bispecific) 6.0 (bispecific) antibodies antibodies bind bind very very efficiently efficiently to to mouse FcRn, mouse FcRn,unless unlessthey they contain contain the the H435A H435A mutation mutation in Fab-arms. in both both Fab-arms. The The 10 bispecific 10 bispecific molecule molecule harboring harboring the H435A the H435A mutation mutation in only in only one one is Fab-arm Fab-arm is still still able to able to bind mouse bind mouseFcRn. FcRn.Hinge-deleted Hinge-deletedIgG1 IgG1(Uni-G1) (Uni-G1)molecules molecules bind bind mouse mouse FcRn FcRn with with intermediate efficiency intermediate efficiency and and the hinge-deleted IgG1 the hinge-deleted IgG1 (Uni-G1) (Uni-G1) bispecific bispecific molecule molecule harboring the harboring theH435A H435A mutation mutation in only in only one one Fab-arm Fab-arm is slightly is slightly less less efficient. efficient.

Figure49: Figure 49:T Tcell cell mediated cytotoxicity mediatedcytotoxicity ofof AU565 AU565 cells cells by by Her2 Her2 x CD3 X CD3 bispecific bispecific

15 antibodies 15 antibodies as well as well as N297Q as by by N297Q mutants mutants of Her2 of X Her2xCD3 bispecific CD3 bispecific antibodies. antibodies.

DETAILED DESCRIPTION DETAILED OFTHE DESCRIPTION OF INVENTION THE INVENTION Definitions Definitions

The term The term"immunoglobulin" "immunoglobulin" refersrefers to a of to a class class of structurally structurally related related glycoproteins glycoproteins

20 consisting 20 consistingof oftwotwopairs pairsof ofpolypeptide polypeptidechains, chains,one onepair pairofoflight (L) low light (L) low molecular molecular weight chains weight chainsand andoneone pair pair of of heavy heavy (H) (H) chains, chains, all four all four inter-connected inter-connected by disulfide by disulfide

bonds. The bonds. The structure structure ofof immunoglobulins immunoglobulinshashas beenbeen well well characterized. characterized. See See for for instance Fundamental instance ImmunologyCh.Ch.7 (Paul, Fundamental Immunology 7 (Paul,W., W.,ed., ed., 2nd 2nded. ed. Raven RavenPress, Press,N.Y. N.Y. (1989)). Briefly, each (1989)). Briefly, each heavy heavy chain chain typically typically is is comprised comprised of a of a heavy heavy chain chain variable variable

25 region 25 region (abbreviatedherein (abbreviated hereinas as VH)VH) andand a heavy a heavy chain chain constant constant region. region. The The heavyheavy chain constant chain constantregion regiontypically typicallyisiscomprised comprisedof of three three domains, domains, CH1, CH1, CH2, CH2, and and CH3. CH3. The heavy The heavychains chainsare areinter-connected inter-connected via via disulfide disulfide bonds bonds in in the the so-called so-called "hinge "hinge region". Each region". Eachlight lightchain chain typically typically is comprised is comprised of a chain of a light chain variable light variable region region (abbreviated herein (abbreviated hereinasasVL) VL)and and a lightchain a light chainconstant constant region. region. TheThe light light chain chain constant constant

30 region 30 region typicallyisis comprised typically comprised of of one one domain, domain,CL. CL. Typically, Typically, the the numbering of amino numbering of amino acid residues acid residues in in the constant region the constant region is is performed performed according according to to the theEU-index EU-indexasas described inin Kabat described Kabatetetal., al.,Sequences Sequences of Proteins of Proteins of Immunological of Immunological Interest, Interest, 5th Ed.5th Ed. Public Health Public Health Service, Service, National NationalInstitutes Institutes ofof Health, Health,Bethesda, Bethesda,MD.MD. (1991). (1991). Figure Figure 16 16 gives an gives overview of an overview of the the EU EU and andKabat numbering Kabatnumbering for for differentisotype different isotypeforms formsof of 35 antibody 35 antibody 2F82F8 (WO (WO 02/100348). 02/100348). The The VH VHVLand and VL regions regions may be may be further further subdivided subdivided

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into regions into hypervariability (or of hypervariability regions of (or hypervariable hypervariable regions whichmaymay regionswhich be hypervariable be hypervariable

in sequence in and/orform sequence and/or form of of structurally structurally defined defined loops), loops), also also termed termed complementarity complementarity

determining regions determining regions (CDRs), (CDRs), interspersed interspersed with with regions regions that that are are more moreconserved, conserved, termed framework termed frameworkregions regions(FRs). EachVHVH (FRs). Each andand VL typically VL is is typicallycomposed composed of three of three 5 CDRs 5 CDRs and and four four FRs, FRs, arranged arranged from amino-terminus from amino-terminus to carboxy-terminus to carboxy-terminus in the in the following order: following order:FR1, FR1,CDR1, CDR1, FR2, FR2, CDR2, CDR2, FR3, FR3, CDR3, FR4 (see CDR3, FR4 (see also also Chothia Chothia and Lesk and Lesk J. Mol. J. Mol. Biol. Biol. 196, 196, 901 917 (1987)). 901 917 (1987)). Whenused When used herein, herein, the the termterm "Fab-arm" "Fab-arm" refersrefers to one to one chain-light heavy heavy chain-light chain chain pair. pair.

10 10 Whenused When usedherein, herein, thethe termterm "Fc region" "Fc region" refers refers to antoantibody an antibody region region comprisingatatleast comprising least the the hinge hingeregion, region,a aCH2 CH2 domain domain and and CH3 domain. a CH3a domain.

The term The term"antibody" "antibody" (Ab) (Ab) in the in the context context of the of the present present invention invention refers refers to anto an immunoglobulin molecule, immunoglobulin molecule, aa fragment fragment of of an an immunoglobulin immunoglobulin molecule, molecule, or or aa derivative of derivative of either either thereof, thereof, which whichhashas thethe ability ability to to specificallybind specifically bind to to an an antigen antigen

15 under 15 under typical typical physiological physiological conditions conditions with awith halfoflifesignificant halfalife of significant periods periods of of time, time, such asasatatleast such leastabout about30 30 min,min, at least at least about about 45 at 45 min, min, at about least least one about one hour, at hour, at

least about least about two twohours, hours, at at least least about about four four hours, hours, at least at least about about 8 hours, hours, 8 at leastat least about 1212hours about hours(h), (h),about about 24 24 hours hours or more, or more, aboutabout 48 hours 48 hours or about or more, more,3,about 4, 5, 3, 4, 5, 6, 77 or 6, or more moredays, etc.,ororany days,etc., anyother other relevant relevant functionally-defined functionally-defined period period (such (such as a as a

20 time 20 time sufficientto toinduce, sufficient induce, promote, promote, enhance, enhance, and/or and/or modulate modulate a physiological a physiological responseassociated response associatedwith with antibody antibody binding binding to antigen to the the antigen and/orand/or time sufficient time sufficient for for the antibody the antibodytotorecruit recruitananeffector effector activity).The activity). The variable variable regions regions of the of the heavy heavy and and light chains light chains of of the immunoglobulin the immunoglobulin molecule molecule contain contain a binding a binding domain domain that interacts that interacts

with an with an antigen. antigen. The The constant constant regions regions ofof the the antibodies antibodies (Abs) (Abs) may maymediate mediate thethe 25 binding 25 binding of the of the immunoglobulin immunoglobulin totissues to host host tissues or factors, or factors, including including various various cells cells of theof the

immunesystem immune system(such (suchasaseffector effector cells) cells) and and components of the components of the complement complementsystem system such as such as C1q, C1q,the thefirst first component component in the in the classical classical pathway pathway of complement of complement activation. activation.

An antibody An antibody may mayalso alsobebea abispecific bispecific antibody, antibody, diabody, diabody, or or similar similar molecule. molecule. The The term "bispecific term "bispecific antibody" antibody"refers refers to to antibody antibody having having specificities specificities for atfor at two least least two 30 different 30 different epitopes, epitopes, typically typically non-overlapping non-overlapping epitopes. epitopes. As indicated As indicated above, above, the term the term

antibodyherein, antibody herein,unless unless otherwise otherwise stated stated or clearly or clearly contradicted contradicted by the by the context, context, includes fragmentsof of includes fragments an an antibody antibody thatthat retain retain the ability the ability to specifically to specifically bindbind to the to the

antigen. Such antigen. Such fragments fragments may be provided may be provided by by any any known knowntechnique, technique, such such asas enzymatic cleavage, enzymatic cleavage, peptide peptide synthesis synthesis and and recombinant recombinantexpression expressiontechniques. techniques.ItIt 35 35 hashas been been shown shown thatthat the the antigen-binding antigen-binding functionofofananantibody function antibodymay maybe be performed performed

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by fragments by fragmentsof of a full-length a full-length antibody, antibody, e.g.e.g. a F(ab')2 a F(ab')2 fragment. It alsoIt should fragment. also should be be understood that understood that the theterm term antibody, antibody, unless unless specified specified otherwise, otherwise, alsoalso includes includes polyclonal antibodies, polyclonal antibodies,monoclonal monoclonal antibodies antibodies (mAbs), (mAbs), antibody-like antibody-like polypeptides, polypeptides,

such as such as chimeric chimericantibodies antibodiesand and humanized humanized antibodies. antibodies. An antibody An antibody as generated as generated can can 5 possess 5 possess anyany isotype. isotype. The term The term"full-length "full-length antibody" antibody"when when used used herein, herein, refers refers to antibody to an an antibody whichwhich

contains all contains all heavy heavyand andlight lightchain chainconstant constant and and variable variable domains domains that that are are normally normally

found in found in an an antibody antibodyofofthat thatisotype. isotype. As used As usedherein, herein,"isotype" "isotype" refers refers to the to the immunoglobulin immunoglobulin class class (for (for instance instance

10 IgG1, 10 IgG1, IgG2, IgG2, IgG3, IgG3, IgG4, IgG4, IgD,IgD, IgA, IgA, IgE,IgE, or IgM) or IgM) that that is encoded is encoded by heavy by heavy chain chain constant region constant regiongenes. genes. The term The term"human "human antibody", antibody", as used as used herein, herein, is intended is intended to include to include antibodies antibodies

having variable having variable and and constant constant regions regionsderived fromhuman derivedfrom germline immunoglobulin human germline immunoglobulin sequences.The sequences. The human human antibodies antibodies ofinvention of the the invention may include may include amino amino acid acid residues residues 15 notnotencoded 15 encodedby by human human germline germline immunoglobulin immunoglobulin sequences sequences (e.g., (e.g., mutations mutations introduced bybyrandom introduced random or site-specific or site-specific mutagenesis mutagenesis in vitro in vitro or somatic or by by somatic mutation mutation in in vivo). However, vivo). the term However, the term "human "humanantibody", antibody", as as used used herein, herein, is not is not intended intended to to include antibodies include antibodies ininwhich which CDR sequences derived CDR sequences derived from fromthe thegermline germlineofofanother another mammalianspecies, mammalian species,such suchasasa amouse, mouse, have have beenbeen grafted grafted ontoonto human human framework framework 20 sequences. 20 sequences. When used When used herein, herein, the the term term "heavy "heavy chain chain antibody" antibody" oror"heavy-chain "heavy-chain antibody" refers antibody" refers to to an an antibody antibodywhich which consists consists only only of of twotwo heavy heavy chains chains and lacks and lacks the the two light two light chains chains usually usually found foundininantibodies. antibodies.Heavy Heavy chain chain antibodies, antibodies, which which naturally naturally

occur in occur in e.g. e.g. camelids, can bind camelids, can bindantigens antigensdespite despitehaving having only only VH VH domains. domains.

25 25 The term The term"epitope" "epitope"means means a protein a protein determinant determinant capable capable of specific of specific binding binding to to an antibody. an antibody. Epitopes Epitopes usually usually consist consist of of surface surface groupings of molecules groupings of molecules such such asas amino acids amino acids ororsugar sugarside sidechains chains andand usually usually havehave specific specific three three dimensional dimensional structural characteristics, structural characteristics, as as well well as as specific specific charge chargecharacteristics. characteristics.Conformational Conformational and nonconformational and nonconformational epitopes epitopes are distinguished are distinguished in the in that thatbinding the binding to the to the former former 30 butbut 30 notnot thethe latterisislost latter lost in in the of denaturing presence of the presence denaturing solvents. solvents. The The epitope epitope may may comprise amino comprise aminoacid acid residues residues directly directly involved involved in binding in the the binding (also (also called called immunodominant immunodominant component component of the of the epitope) epitope) andand other other amino amino acidacid residues, residues, which which are not are not directly directly involved involved inin the the binding, binding, such such as as amino acid residues amino acid which are residues which are effectively blocked effectively bythe blocked by thespecifically specificallyantigen antigenbinding binding peptide peptide (in (in other other words, words, the the 35 amino 35 amino acid acid residue residue is within is within the footprint the footprint of the of the specifically specifically antigen antigen binding binding peptide). peptide).

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As used As used herein, herein, the theterm "binding"in inthethecontext term"binding" of of context thethe binding binding of of an an antibody to antibody to a apredetermined predetermined antigen antigen typically typically is ais binding a binding with with an affinity an affinity correspondingtotoa aKDKDof ofabout corresponding about M or M 10 10-6 or less, less, e.g. e.g. 10 M 10- M or such or less, less,as such as 10about about 10-8 2022201608 08

or less, M or M less, such such as as about 10 M Mororless, about 10-9 about 10¹ less, about M or 10-10M or less,ororabout less, about10¹¹ 10-11M Mor or 5 even 5 evenless lesswhen when determinedby by determined forfor instancesurface instance surface plasmon plasmon resonance resonance (SPR) (SPR) technology in technology in aa BIAcore 3000 instrument BIAcore 3000 instrument using using the the antigen antigen as as the the ligand ligand and and the the antibody asasthe antibody theanalyte, analyte, andand bindsbinds to predetermined to the the predetermined antigen antigen with with an an affinity affinity correspondingtotoa aKDKDthat corresponding thatis isatatleast leastten-fold ten-foldlower, suchas as lower,such at at least100100 least fold fold lower, lower,

for instance for at least instance at least 1,000 fold lower, 1,000 fold lower, such suchasasatatleast least10,000 10,000fold foldlower, lower,for forinstance instance 10 at least 10 at least 100,000 100,000 fold fold lower lower than than its affinity its affinity forfor binding binding to to a non-specific a non-specific antigen antigen (e.g., (e.g.,

BSA, casein) BSA, casein)other otherthan thanthethe predetermined predetermined antigen antigen or a closely-related or a closely-related antigen. antigen. The The amountwith amount withwhich which the the affinity affinity is lower is lower is dependent is dependent onKDthe on the KD of of the the antibody, antibody, so so that when that whenthe theKDKDofofthe theantibody antibody is is very very low low (that (that is,is, the the antibody antibody is ishighly highlyspeific), specific),

then the then the amount amount with with which which the the affinity affinity forfor theantigen the antigen is is lower lower than than thethe affinity affinity fora for a 15 non-specific 15 non-specific antigen antigen may may be at be at least least 10,00010,000 fold.term fold. The The"KD" term (M), as (M), "KD" as used used herein,herein, refers to refers to the thedissociation dissociation equilibrium equilibrium constant constant of a particular of a particular antibody-antigen antibody-antigen

interaction. interaction.

Whenused When used herein herein the the termterm "heterodimeric "heterodimeric interaction interaction betweenbetween the firstthe andfirst and secondCH3 second CH3 regions" regions" refers refers to the to the interaction interaction between between the first the first CH3 region CH3 region and theand the 20 second 20 second CH3CH3 region region in in a first-CH3/second-CH3heterodimeric a first-CH3/second-CH3 heterodimericprotein. protein. Whenused When used herein herein the the termterm "homodimeric "homodimeric interactions interactions of the of the and first firstsecond and second CH3regions" CH3 regions"refers refersto tothethe interaction interaction between between a first a first CH3 region CH3 region and another and another first first CH3region CH3 regioninina afirst-CH3/first-CH3 first-CH3/first-CH3homodimeric homodimeric protein protein andinteraction and the the interaction between between

second CH3 a second a CH3region region and andanother anothersecond secondCH3 CH3 region region in ina asecond-CH3/second-CH3 second-CH3/second-CH3 25 homodimeric 25 homodimeric protein. protein. An "isolated An "isolated antibody," antibody,"asasused used herein, herein, denotes denotes thatmaterial that the the material has has been been removedfrom removed from itsitsoriginal originalenvironment environment (e.g., (e.g., thethe natural natural environment environment if it ifisit naturally is naturally occurring or occurring or the thehost hostcell cellifif it it is is recombinantly expressed). recombinantly expressed). It isalso It is also advantageous advantageous

that the that the antibodies antibodies bebeinin purified purified form. form. The Theterm term does "purified"does "purified" notnot require require absolute absolute

30 purity; 30 purity; rather, rather, it intended it is is intended as a as a relative relative definition, definition, indicating indicating an increase an increase of the of the antibody concentration antibody concentrationrelative relativetotothe theconcentration concentrationof of contaminants contaminants in aincomposition a composition as compared as comparedto tothethestarting startingmaterial. material. The term The term"host "hostcell", cell",asasused usedherein, herein, is is intended intended to refer to refer to to a cell a cell into into which which

an expression an expression vector vector has has been been introduced, introduced, e.g. e.g. an an expression expression vector vector encoding encoding an an

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19

antibody of the antibody of theinvention. invention.Recombinant Recombinant host host cells cells include, include, for example, for example, transfectomas, suchasasCHO transfectomas, such CHO cells,HEK293 cells, HEK293 cells, cells, NS/0 NS/0 cells, cells, and and lymphocytic lymphocytic cells. cells.

When usedherein, When used herein,the theterm term"co-expression" “co-expression”ofof two twoorormore more nucleicacid nucleic acid constructs, refers constructs, refers to to expression expression of two of the theconstructs two constructs in ahost in a single single host cell. cell.

5 5 The term The term"tumor “tumor cellprotein" cell protein”refers refersto to aa protein protein located locatedon onthe thecell cell surface surface of of a tumor cell. 2022201608

a tumor cell.

As used As usedherein, herein,the the term term "effector "effector cell" cell" refers refers to to an immune an immune cell which cell which is is involved in the involved in the effector effector phase phaseofofananimmune immune response, response, as opposed as opposed to the to the cognitive cognitive

and activation phases and activation phasesof of an animmune immune response. response. Exemplary Exemplary immune immune cells include cells include a cell a cell

10 ofmyeloid 10 of a a myeloid or lymphoid or lymphoid origin, origin, for for instance instance lymphocytes lymphocytes (such(such as B cells as B cells and Tand T cells cells

including cytolytic TT cells including cytolytic cells (CTLs)), (CTLs)),killer killer cells, cells, natural natural killer killer cells, cells, macrophages, macrophages, monocytes, eosinophils,polymorphonuclear monocytes, eosinophils, polymorphonuclear cells, cells, suchsuch as neutrophils, as neutrophils, granulocytes, granulocytes,

mast cells, and mast cells, basophils. Some and basophils. Someeffector effectorcells cells express expressspecific specific Fc Fc receptors receptors and andcarry carry out specific immune out specific functions.InInsome immune functions. some embodiments, embodiments, an effector an effector cell cell is is capable capable of of 15 inducing 15 inducing antibody-dependent antibody-dependent cellular cellular cytotoxicity cytotoxicity (ADCC), (ADCC), such as such as akiller a natural natural killer cell, cell, capable capable of of inducing inducing ADCC. Insome ADCC. In some embodiments, embodiments, an effector an effector cell cell may may phagocytose phagocytose a atarget targetantigen antigenorortarget targetcell. cell. The term The term"reducing "reducingconditions" conditions"oror"reducing "reducingenvironment" environment" refers refers to to a a condition or an condition or anenvironment environment in which in which a substrate, a substrate, here here a a cysteine cysteine residueresidue in the in the

20 hinge 20 hinge region region of antibody, of an an antibody, is more is more likely likely to become to become reduced reduced than oxidized. than oxidized.

The term The term"disulfide "disulfidebond bond isomerization" isomerization" refers refers to exchange to an an exchange of disulfide of disulfide

bonds between bonds between different different cysteines, cysteines, i.e.,shuffling i.e., the the shuffling of disulfide of disulfide bonds. bonds.

In In the the claims claims which follow and which follow and in in the the preceding precedingdescription descriptionof of the the invention, invention, except wherethe except where thecontext contextrequires requiresotherwise otherwise duedue to to express express language language or necessary or necessary

25 implication, 25 implication, thethe word word “comprise” "comprise" or variations or variations suchsuch as “comprises” as "comprises" or “comprising” or "comprising" is is used used ininan aninclusive inclusive sense, sense, i.e.i.e. to specify to specify the presence the presence of thefeatures of the stated stated features but not but not to preclude to the presence preclude the presenceororaddition additionof of further further features features in in various various embodiments embodiments of of

the invention. the invention.

30 Further 30 Further aspectsand aspects andembodiments embodimentsofofthe theinvention invention As described As describedabove, above, in in a first a first aspect, aspect, the the invention invention relates relates to antoinan in vitro vitro method forgenerating method for generatinga aheterodimeric heterodimeric protein, protein, said said method method comprising comprising the following the following

steps: steps:

a) providing a a) providing first homodimeric a first protein comprising homodimeric protein comprising an an Fc Fc region region ofof anan 35 immunoglobulin, said 35 immunoglobulin, said FcFc region regioncomprising comprisinga first a first CH3 region, CH3 region,

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19a

May b) b) providing providing aa second second homodimeric protein comprising homodimeric protein comprising an an Fc Fc region region of of an an immunoglobulin, saidFcFcregion immunoglobulin, said regioncomprising comprising a second a second CH3CH3 region, region,

5 5 wherein thesequences wherein the sequencesof of said said firstand first andsecond second CH3CH3 regions regions are are different different and and

are such that that the the heterodimeric heterodimeric interaction interaction between said first first and and second CH3 2022201608

are such between said second CH3

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regions is regions is stronger eachofofthe than each stronger than thehomodimeric homodimeric interactions interactions of said of said firstandand first second second

CH3regions, CH3 regions,

2022201608 08

c) incubating c) incubating said said first first protein protein together together with said second with said second protein protein under under 5 reducing 5 reducing conditions conditions sufficient sufficient to allow to allow the cysteines the cysteines in theinhinge the region hinge region to to undergo undergo disulfide-bond isomerization, disulfide-bond isomerization,and and

d) obtaining d) said heterodimeric obtaining said heterodimericprotein. protein.

10 10 The bispecific The bispecific format maybe be format may used used in many in many ways ways to to generate generate desired desired combinations of combinations of bispecific bispecific antibodies. antibodies. In In addition addition to to being being able ableof ofcombining combining antibodies targeting antibodies targetingdifferent differentantigens antigens in very in a a very selective selective way way it can itbecan usedbe to used to change a adesired change desiredproperty, property,e.g. e.g.totoincrease increaseCDC, CDC, by combining by combining two different two different antibodies targeting antibodies targeting the the same same antigen. antigen. Furthermore, Furthermore, it can it can be used be used to remove to remove partialpartial

15 agonistic 15 agonistic activity activity of of an an antagonistic antagonistic antibody antibody or convert or convert an agonistic an agonistic antibody antibody into into an an antagonistic antbody antagonistic by making antbody by makinga bispecific a bispecific antibody antibody thereof thereofwith with ananirrelevant irrelevant (inactive) (inactive) antibody. antibody.

In one In embodiment,the one embodiment, thehomodimeric homodimeric proteinsareare proteins selectedfrom selected fromthe thegroup group consisting of consisting of (i) (i) an Fc region, an Fc region, (ii) (ii) an an antibody, (iii) a afusion antibody, (iii) protein comprising fusionprotein an Fc comprising an Fc 20 region, 20 region, suchsuch as anFc as anFc regionregion fused fused to a receptor, to a receptor, cytokine cytokine or hormone, or hormone, and (iv) aand Fc (iv) a Fc region conjugated region conjugatedtotoa aprodrug, prodrug, peptide, peptide, drug drug or or a toxin. a toxin.

In some In some embodiments, embodiments,said saidfirst first and/or and/or second second homodimeric homodimericprotein protein comprise,inin addition comprise, additiontotothe theFcFcregion, region,oneone or or more more or of or all all the of the other other regions regions of anof an antibody, i.e. antibody, i.e. aa CH1 CH1region, region,a VH a VH region, region, CL region a CLa region and/orand/or a VL region. a VL region. Thus, inThus, in 25 one one 25 embodiment, embodiment, said homodimeric said first first homodimeric protein protein is is a full-length a full-length antibody. antibody. In In another another embodiment, embodiment, said said second second homodimeric homodimeric protein protein is a full-length is a full-length antibody. antibody.

In an In an important importantembodiment, embodiment, said said firstfirst and and second second homodimeric proteinsproteins homodimeric are are both antibodies, both antibodies,preferably preferablyfull-length full-lengthantibodies, antibodies, and and bind different bind different epitopes. epitopes. In In such an such an embodiment, embodiment,thethe heterodimeric heterodimeric proteinthat protein that is isgenerated generatedis isa bispecific a bispecific 30 antibody. 30 antibody. SaidSaid epitopes epitopes may may be be located located on different on different antigens antigens or on or on the samethe same antigen. antigen. In other In other embodiments, however,only embodiments, however, onlyone oneofofthe thehomodimeric homodimericproteins proteinsisisa a full-length antibody full-length andthe antibody and theother otherhomodimeric homodimeric protein protein is anot is not a full-length full-length antibody, antibody,

e.g. an e.g. an FcFcregion regionwithout without a variable a variable region, region, expressed expressed in conjunction in conjunction to to another another protein or protein or peptide peptide sequence sequence likea areceptor, like receptor,cytokine cytokine or or hormone, hormone, or conjugated or conjugated to a to a 35 prodrug, 35 prodrug, peptide, peptide, a drug a drug or aortoxin. a toxin. In a In a further further embodiment, embodiment, neitherneither of the of the

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2022201608 08 Mar proteins isis aa full-length homodimeric proteins homodimeric full-length antibody. For example, antibody. For bothhomodimeric example, both homodimeric proteins may proteins maybebeFcFcregions regionsthat that are are fused fused to to another another protein protein or peptide or peptide sequence sequence like like receptor, cytokine a receptor, a cytokineororhormone, hormone, or conjugated or conjugated to a prodrug, to a prodrug, peptide, peptide, a drug a drug or a or a toxin. toxin.

5 5 In one In one embodiment, embodiment,the the Fc region Fc region of theoffirst the first protein protein homodimeric homodimeric is of an is of an isotype selected isotype selected from fromthe thegroup group consisting consisting of of IgG1, IgG1, IgG2, IgG2, IgG3 IgG3 and and and IgG4 IgG4 theand Fc the Fc region of region of the the second secondhomodimeric homodimeric protein protein is of isanof an isotype isotype selected selected from from the the group group consisting of consisting of IgG1, IgG1, IgG2, IgG2,IgG3 IgG3 andand IgG4. IgG4. In aInpreferred a preferred embodiment, embodiment, the Fc the Fc regions regions of both of both said said first first and and said said second second homodimeric homodimeric protein protein are ofare the of theisotype. IgG1 IgG1 isotype. In In 10 another 10 another preferred preferred embodiment, embodiment, one Fcof regions one of the the Fc of regions of said homodimeric said homodimeric proteins isproteins is of the of IgG1 isotype the IgG1 isotypeand andthethe other other of of thethe IgG4 IgG4 isotype. isotype. In the In the latter latter embodiment, embodiment, the the resulting heterodimeric resulting comprises heterodimeric comprises an an Fc region Fc region of IgG1 of an an IgG1 and and an Fc an Fc region region of IgG4of IgG4 and may and may thus thus have have interesting interesting intermediate intermediate properties properties with respect with respect to activation to activation of of effector functions. effector functions. AA similar similar product productcan canbebe obtained obtained if said if said firstand/or first and/or said said second second

15 homodimeric 15 homodimeric protein protein comprises comprises a mutation a mutation removing removing thethe acceptor acceptor sitefor site for Asn-linked Asn-linked glycosylation or glycosylation or is is otherwise manipulatedto tochange otherwise manipulated change the the glycosylation glycosylation properties. properties.

In aa further In further embodiment, embodiment,one one or both or both ofhomodimeric of the the homodimeric proteins proteins is glyco-is glyco engineeredtotoreduce engineered reduce fucose fucose andand thusthus enhance enhance ADCC, ADCC, e.g. bye.g. by addition addition of compounds of compounds

to the to the culture mediaduring culture media duringantibody antibody production production as described as described in US2009317869 in US2009317869 or as or as 20 described 20 described in in van van Berkeletetal. Berkel al. (2010) (2010) Biotechnol. Biotechnol. Bioeng. Bioeng. 105:350 or by 105:350 or by using using FUT8 FUT8 knockoutcells, knockout cells, e.g. e.g. as as described describedininYamane-Ohnuki et al et Yamane-Ohnuki al (2004) (2004) Biotechnol. Biotechnol. Bioeng Bioeng 87:614. ADCC 87:614. ADCCmay mayalternatively alternatively be be optimized optimized using using the method described the method described by by Umana Umaña et al. et al. (1999) Nature Biotech (1999) Nature Biotech17:176. 17:176. In aa further In furtherembodiment, one or embodiment, one or both both of of the the homodimeric proteins has homodimeric proteins has been been 25 engineered 25 engineered to to enhance enhance complement complement activation, activation, e.g.e.g. as described as described in in Natsume Natsume et al. et al. (2009) CancerSci. (2009) Cancer Sci.100:2411. 100:2411. In aa further In furtherembodiment, one or embodiment, one or both both of of the the homodimeric proteins has homodimeric proteins has been been engineeredtotoreduce engineered reduce or or increase increase the the binding binding to neonatal to the the neonatal Fc receptor Fc receptor (FcRn) (FcRn) in in order to order to manipulate manipulatethe theserum serum half-lifeofofthe half-life theheterodimeric heterodimeric protein. protein.

30 30 In aa further In further embodiment, embodiment, one one of the of the homodimeric homodimeric starting starting proteins proteins has has been been engineeredtotonot engineered notbind bindProtein ProteinA,A,thus thus allowing allowing to to separate separate the the heterodimeric heterodimeric protein protein

from said from said homodimeric homodimericstarting starting protein protein by by passing passing the theproduct productover overa aprotein proteinA A column. This column. Thismay mayin in particular particular be be useful useful forfor embodiments embodiments wherein wherein an of an excess excess one of one homodimeric homodimeric protein protein is used is used relative relative to other to the the other homodimeric homodimeric protein protein as as starting starting 35 material. 35 material.In Insuch such embodiments, embodiments, it be it may mayuseful be useful to engineer to engineer the homodimeric the homodimeric

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protein that protein that is is in in excess sothat excess so thatisis looses loosesits its ability ability to protein A. bind protein to bind A. Following Followingthe the heterodimerizationreaction, heterodimerization reaction,the theheterodimeric heterodimeric protein protein maymay then then be separated be separated from afrom a surplus of surplus of unexchanged unexchanged homodimeric homodimeric protein protein by passage by passage over a over a protein protein A column. A column.

In aa further In further embodiment, embodiment, oneone of of thethe homodimeric homodimeric proteins proteins is an is Fcan Fc region region or a or a 5 full-length 5 full-length antibody antibodyrecognizing recognizing a anon-relevant non-relevantepitope epitopeorora full-length a full-length antibody antibody containing germline-derived containing germline-derived sequences that have sequences that have not notundergone undergone somatic somatic hypermutation and hypermutation do not and do not bind bindself-antigens. self-antigens. In such an In such an embodiment embodimentthethe heterodimericprotein heterodimeric proteinfunctions functionsas as a monovalent a monovalent antibody. antibody. In another In another embodiment, embodiment,

both homodimericproteins both homodimeric proteins comprises comprisesthe thesame same heavy heavy chain, chain, butbut only only oneone of the of the 10 homodimeric 10 homodimeric proteins proteins contains contains light chain a lighta chain which aforms which forms a functional functional antigen-binding antigen-binding

site with site said heavy with said heavychain, chain,whereas whereas the the other other homodimeric homodimeric protein protein containscontains a non- a non functional light functional light chain, chain, which whichdoes does not not bind bind any antigen any antigen in combination in combination with said with said heavy chain. In heavy chain. In such suchananembodiment, embodiment, the the heterodimeric heterodimeric protein protein functions functions as aas a monovalent antibody. Such monovalent antibody. Sucha anon-functional non-functionallight light chain chain can can e.g. e.g. bebea agermline- germline 15 derived 15 derived sequence sequence that that hashas notnot undergone undergone somatic somatic hypermutation hypermutation and and doesdoes not not bindbind self-antigens. self-antigens.

Antibodies to Antibodies to be be used usedasashomodimeric homodimeric starting starting material material of the of the present present invention may invention maye.g. e.g.bebe produced produced by hybridoma by the the hybridoma method method first described first described by Kohlerby Kohler et al., et al.,Nature Nature256, 256,495 495(1975), (1975),orormay may be produced by be produced DNAmethods. recombinant DNA by recombinant methods. 20 Monoclonal 20 Monoclonal antibodies antibodies maymay alsoalso be be isolatedfrom isolated fromphage phage antibody antibody libraries using libraries using the the techniquesdescribed techniques describedin,in,forforexample, example, Clackson Clackson et al., et al., Nature Nature 352,628624 352, 624 628 (1991) (1991) and Marks and Markset etal., al.,J. J. Mol. Biol. 222, Mol.Biol. 222,581581 597597 (1991). (1991). Monoclonal Monoclonal antibodies antibodies may be may be obtained from obtained fromany anysuitable suitablesource. source. Thus, Thus, forfor example, example, monoclonal monoclonal antibodies antibodies may be may be obtained from obtained fromhybridomas hybridomas prepared prepared from murine from murine splenic splenic cells obtained B cells Bobtained from micefrom mice 25 immunized 25 immunized with with an an antigen antigen of interest, of interest, for instance for instance in cells in form of form expressing of cells expressing the the antigen on antigen onthe thesurface, surface,orora anucleic nucleicacid acidencoding encoding an an antigen antigen of interest. of interest. Monoclonal Monoclonal

antibodies may antibodies mayalso alsobe be obtained obtained fromfrom hybridomas hybridomas derivedderived from antibody-expressing from antibody-expressing

cells ofofimmunized cells immunized humans or non-human humans or non-human mammals mammals such such as rats, as rats, dogs,dogs, primates, primates, etc. etc.

30 30 Antibodies to Antibodies to be be used homodimeric usedasashomodimeric starting starting material material of the of the present present invention may invention e.g. chimeric may e.g. chimeric or or humanized antibodies. In humanized antibodies. In another another embodiment, one embodiment, one or both or of the both of the homodimeric homodimeric starting starting proteins, proteins, except except for for anyany specified specified mutations, mutations, is a is a humanantibody. human antibody. Human Human monoclonal monoclonal antibodies antibodies maymay be generated be generated using using transgenic transgenic or transchromosomal or mice, e.g. transchromosomal mice, e.g. HuMAb HuMAbmice, mice,carrying carrying parts parts of of the the human humanimmune immune 35 system 35 systemrather ratherthan thanthe themouse mouse system.TheThe system. HuMAb HuMAb mouse mouse contains contains a human a human

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2022201608 08 Mar geneminiloci immunoglobulin gene immunoglobulin that encodes miniloci that encodes unrearranged human unrearrangedhuman heavy heavy (p and (µ and ) y) and KKlight and light chain chain immunoglobulin immunoglobulin sequences, sequences, together together with targeted with targeted mutations mutations that that inactivate the inactivate the endogenous endogenous p and µ and K chain K chain loci (Lonberg, loci (Lonberg, N. etNature N. et al., al., Nature 368, 856368, 856 859 (1994)). Accordingly, 859 (1994)). Accordingly, the the mice mice exhibit exhibit reduced reduced expression expression of of mouse mouse IgM or KK IgM or 5 andand 5 in response in response to immunization, to immunization, the introduced the introduced humanhuman heavy heavy and andchain light light chain transgenes,undergo transgenes, undergo class class switching switching and and somatic somatic mutation mutation to generate to generate high high affinity affinity humanIgG,k human IgG,K monoclonal monoclonal antibodies antibodies (Lonberg, (Lonberg, N. (1994), N. et al. et al. (1994), supra; reviewed supra; reviewed in in Lonberg, N. Lonberg, N. Handbook of Experimental Handbook of Experimental Pharmacology Pharmacology 113, 113,49 49101 101(1994), (1994) Lonberg, , Lonberg, N. and N. and Huszar, Huszar,D., D.,Intern. Intern.Rev. Rev. Immunol. Immunol. Vol. Vol. 13 65139365 93 (1995) (1995) and Harding, and Harding, F. and F. and 10 Lonberg, 10 Lonberg, N. N. Ann. Ann. N.Y.N.Y. Acad. Acad. Sci Sci 764764 536 536 546 546 (1995)). (1995)). The preparation The preparation of HuMAb of HuMAb mice is mice is described describedinin detail detail in in Taylor, L. et Taylor, L. et al., al., Nucleic Nucleic Acids Acids Research 20,6287 Research 20, 6287 6295 6295

(1992), Chen,J.J.etetal., (1992), Chen, al., International Immunology International Immunology 5, 647 5, 647 656 (1993), 656 (1993), Tuaillon Tuaillon et al., et al.,

J. Immunol. J. 152, Immunol. 152, 2912 2912 29202920 (1994), (1994), Taylor, Taylor, L. et International L. et al., al., International Immunology Immunology 6, 6, 579 591 579 591(1994), (1994), Fishwild, Fishwild, D. D. et al., et al., Nature Nature Biotechnology Biotechnology 14,851 14, 845 845(1996). 851 (1996). See See 15 also 15 also US US 5,545,806, 5,545,806, US US 5,569,825, 5,569,825, US 5,625,126, US 5,625,126, US 5,633,425, US 5,633,425, US 5,789,650, US 5,789,650, US US 5,877,397, US 5,877,397, 5,661,016, US US 5,661,016, 5,814,318, USUS5,874,299, US 5,814,318, 5,874,299,USUS 5,770,429, 5,770,429, US US 5,545,807, WO 5,545,807, WO 98/24884, 98/24884, WO 94/25585, WO WO 94/25585, 93/1227, WO WO 93/1227, WO 92/22645, 92/22645, WO WO 92/03918 and 92/03918 andWOWO 01/09187. 01/09187. Splenocytes Splenocytes from from these these transgenic transgenic mice mice maymay be used be used to generate to generatehybridomas hybridomas that that secrete secrete human human monoclonal monoclonal antibodies antibodies accordingaccording to well to well 20 known 20 known techniques. techniques. Further, human Further, antibodies ofofthe human antibodies thepresent presentinvention inventionor orantibodies antibodiesof ofthethe present invention present invention from from other otherspecies species maymay be identified be identified through through display-type display-type technologies, including, technologies, including, without without limitation, limitation, phage phagedisplay, display,retroviral retroviral display, display, ribosomaldisplay, ribosomal display,mammalian mammalian display, display, and other and other techniques, techniques, using techniques using techniques well well 25 known 25 known in art in the the and art the and resulting the resulting molecules molecules may bemay be subjected subjected to additional to additional maturation,such maturation, suchas as affinitymaturation, affinity maturation, as such as such techniques techniques areknown are well well in known the in the art. art.

In aa further In further embodiment embodiment of of thethe invention, invention, the the antibody antibody or a or a part part thereof, thereof, e.g. e.g.

one or one or more moreCDRs, CDRs, is of is of a species a species in in thethe family family Camelidae, Camelidae, see see W02010001251, WO2010001251, or a or a 30 species 30 species of of cartilaginousfish, cartilaginous fish, such such asasthe thenurse nurseshark sharkor orheavy-chain heavy-chain or or domain domain antibodies. antibodies.

In one In one embodiment embodimentof ofthethe method method of the of the invention, invention, said said firstand first andsecond second homodimeric homodimeric proteins proteins provided provided in step in step a) and a) and b) are b) are purified. purified.

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In one In one embodiment, thefirst embodiment, the first and/or second homodimeric and/or second protein isis homodimericprotein conjugatedtotoa adrug, conjugated drug, a prodrug a prodrug or a or a toxin toxin or contains or contains an acceptor an acceptor group forgroup the for the same.Such same. Suchacceptor acceptor group group may may e.g. e.g. be anbe an unnatural unnatural amino amino acid. acid.

5 5 As described As describedabove, above, the the sequences sequences of the of the first first andand second second CH3 regions CH3 regions of the of the homodimeric starting homodimeric starting proteins proteins are are different different and are such and are such that that the the heterodimeric heterodimeric interaction between interaction betweensaid saidfirst firstand andsecond second CH3 CH3 regions regions is stronger is stronger thanofeach than each the of the homodimeric homodimeric interactions interactions of of saidfirst said first and andsecond secondCH3CH3 regions. regions.

In one In one embodiment, embodiment,the the increased increased strength strength of the of the heterodimeric heterodimeric interaction interaction

10 as as 10 compared compared to each to each of the of the homodimeric homodimeric interactions interactions is due is due to CH3 to CH3 modifications modifications other than other than the the introduction introductionofofcovalent covalentbonds, bonds, cysteine cysteine residues residues or or charged charged residues. residues.

In some In someembodiments, embodiments, the product the product of theofinvention the invention is highly is highly stablestable and and does does not undergo not undergo Fab-arm Fab-arm exchange exchange underunder mildlymildly reducing reducing conditions conditions in or, in vitro vitro or, importantly, ininvivo importantly, vivo upon upon administration administrationto toa a human being. Thus, human being. Thus, in in one one 15 embodiment, 15 embodiment, the heterodimeric the heterodimeric interaction interaction between between said firstsaid and first andproteins second secondinproteins in the resulting the resulting heterodimeric heterodimericprotein proteinis issuch such that that no Fab-arm no Fab-arm exchange exchange can can occur at occur at 0.5 mM 0.5 mMGSH GSH under under the the conditions conditions described described in Example in Example 13. 13. In another In anotherembodiment, embodiment,the the heterodimeric heterodimeric interaction interaction between between saidand said first first and second proteins second proteins in in the the resulting resulting heterodimeric heterodimeric protein protein is is such such that that no no Fab-arm Fab-arm 20 exchange 20 exchange occurs occurs in in in vivo vivo in under mice mice under the conditions the conditions described described in Example in Example 14. 14. In another In anotherembodiment, embodiment,the the heterodimeric heterodimeric interaction interaction between between saidand said first first and second proteins second proteins in in the the resulting heterodimeric protein resulting heterodimeric protein is is more morethan thantwotwo times times stronger, such stronger, suchasasmore more than than three three times times stronger, stronger, e.g.e.g. moremore than than five times five times stronger stronger

than the than the strongest strongest of of the the two homodimericinteractions, two homodimeric interactions, e.g. e.g. when determined asas when determined 25 described 25 described in inExample Example 30.30. In aa further In further embodiment, embodiment, thethe sequences sequences of said of said firstfirst and and second second CH3 regions CH3 regions

are such are suchthat thatthethe dissociation dissociation constants constants of heterodimeric of the the heterodimeric interaction interaction between between

said first said first and secondproteins and second proteinsin in thethe resulting resulting heterodimeric heterodimeric protein protein is below is below 0.05 0.05 micromolar when micromolar when assayed assayed as described as described in Example in Example 30. 30. 30 30 In aa further In further embodiment, embodiment, thethe sequences sequences of said of said firstfirst and and second second CH3 regions CH3 regions

are such are such that thatthe thedissociation dissociationconstants constants of of both both homodimeric homodimeric interactions interactions are are above above 0.01 micromolar, 0.01 micromolar, such such as as above above0.05 0.05micromolar micromolarpreferably preferablybetween between0.01 0.01 andand 10 10 micromolar, such as micromolar, such as between between0.05 0.05andand 10 10 micromolar, micromolar, moremore preferably preferably between between 0.01 and 0.01 and 5, 5, such such as as between 0.05 and between 0.05 and 55 micromolar, micromolar, even even more more preferably preferably between between 35 0.01 35 0.01 andand 1 micromolar, 1 micromolar, such such as as between between 0.050.05 and and 1 micromolar, 1 micromolar, between between 0.01 0.01 and and

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0.5 or between 0.5 or 0.01 and between 0.01 and0.10.1when when assayed assayed as described as described in Example in Example 21. 21. Embodimentswherein Embodiments whereinthethe homodimeric homodimeric starting starting proteins proteins areare relatively stable relatively stable can can have the have theadvantage advantagethatthat it is it is easier easier to to produce produce a large a large quantity quantity of starting of starting protein protein

and e.g. and e.g. avoid avoid aggregation aggregationorormisfolding. misfolding. 5 5 In some In embodiments, some embodiments, a stable a stable heterodimeric heterodimeric protein protein can becan be obtained obtained at high at high yield using yield using the themethod method of the of the invention invention onbasis on the the basis of two of two homodimeric homodimeric starting starting proteins containing proteins containingonly onlya few, a few, fairlyconservative, fairly conservative, asymmetrical asymmetrical mutations mutations in the in the CH3regions. CH3 regions. Thus, in Thus, in one one embodiment, embodiment,the the sequences sequences of first of said said first and second and second CH3 regions CH3 regions

10 contain 10 contain amino amino acid acid substitutions substitutions at non-identical at non-identical positions. positions.

The amino The amino acid acid substituents substituents may be natural may be natural amino amino acids acids or or unnatural unnatural amino amino acids. Examples acids. Examplesofofunnatural unnatural amino amino acidsacids are disclosed are e.g. e.g. disclosed in XieinJ Xie and JSchultz and Schultz P. P. G., Current G., Current Opinion in Chemical Opinion in Biology (2005), Chemical Biology (2005), 9:548-554, 9:548-554, and andWang Wang Q. Q. et al., et al., Chemistry& & Chemistry Biology(2009), Biology (2009), 16:323-336. 16:323-336.

15 15 In one In embodiment, one embodiment, thethe amino amino acidsacids are natural aminoamino are natural acids.acids.

In one In embodiment,said one embodiment, saidfirst first homodimeric homodimericprotein protein has hasnonomore more than than oneone aminoacid amino acidsubstitution substitutionininthe theCH3 CH3 region, region, and and the second the second homodimeric homodimeric protein protein has has no more no morethan thanoneone amino amino acidacid substitution substitution in the in the CH3 region CH3 region relative relative towild-type to the the wild-type CH3regions. CH3 regions. 20 20 In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 370,370, 399, 399,

405, 407 405, 407 and and 409, 409, and and said said second second homodimeric homodimeric protein protein has has an an amino aminoacid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 370,370, 399, 399,

405, 407 405, 407 and and409, 409,andand wherein wherein saidsaid firsthomodimeric first homodimeric protein protein andand saidsaid second second 25 homodimeric 25 homodimeric protein protein is notissubstituted not substituted in theinsame the positions. same positions. In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at position position 366, 366, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 368, 368, 370,370, 399,399, 405, 405,

407 and 407 and 409. 409. InIn one oneembodiment embodimentthethe amino amino acidacid at position366366 at position is is selectedfrom selected from 30 Arg,Arg, 30 Lys,Lys, Asn,Asn, Tyr,Tyr, Gln,Gln, Glu Glu and and Gly. Gly. In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at position position 368, 368, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 370,370, 399,399, 405, 405,

407 and 407 and 409. 409.

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2022201608 08 Mar In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at position position 370, 370, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 399,399, 405, 405,

407 and 407 and 409. 409. 5 5 In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein has an an acid hasamino amino acid substitution at substitution at position position 399, 399, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 370,370, 405, 405,

407 and 407 and 409. 409. In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid 10 substitution 10 substitution at position at position 405,405, and and said said second second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 370,370, 399, 399,

407 and 407 and 409. 409. In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at position position 407, 407, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid 15 substitution 15 substitution at aatposition a position selected selected fromfrom the group the group consisting consisting of: 368, of: 366, 366,370, 368,399, 370, 399, 405, and 405, and 409. 409. In one In one embodiment, embodiment,the the first first homodimeric homodimeric protein protein hasamino has an an acid amino acid substitution at substitution at position position 409, 409, and andsaid saidsecond second homodimeric homodimeric protein protein has has an an amino amino acid acid substitution at substitution at aa position position selected selected from fromthe thegroup group consisting consisting of:of: 366, 366, 368,368, 370,370, 399, 399,

20 405, 20 405, and407. and 407. Accordingly, inin one Accordingly, oneembodiment, embodiment,the the sequences sequences of saidoffirst said and firstsecond and second CH3 CH3 regions containasymmetrical regions contain asymmetrical mutations, mutations, i.e. i.e. mutations mutations at different at different positions positions in thein the

two CH3 two CH3regions, regions, e.g. e.g. a mutation a mutation at position at position 405 405 in oneinofone the of CH3the CH3 and regions regions a and a mutation mutation atatposition position 409 409ininthe theother otherCH3 CH3 region. region.

25 25 In one In one embodiment, embodiment,the thefirst first homodimeric homodimericprotein protein has has an an amino aminoacid acidother other than Lys, than Lys, Leu or Met Leu or Met at at position position 409 409 and said second and said homodimeric protein second homodimeric protein has has an an amino-acidsubstitution amino-acid substitutionatata aposition positionselected selectedfrom fromthethe group group consisting consisting of: of: 366, 366, 368,368,

370,399,405 370, and 399, 405 and 407. 407.

In one In one such such embodiment, embodiment,said saidfirst first homodimeric homodimeric protein protein has has an an amino aminoacid acid 30 other 30 other than than Lys, Lys, LeuororMet Leu Metatatposition position 409 409 and and said said second second homodimeric homodimericprotein protein has has an amino an aminoacid acidother other than than PhePhe at position at position 405.405. In a In a further further embodiment embodiment hereof, hereof, said said first homodimeric first proteinhashas homodimeric protein an an amino amino acid acid otherother than Leu than Lys, Lys, or Leu Met or Met at position at position

409 and 409 and said said second second homodimeric homodimericprotein protein has has an an amino aminoacid acid other other than than Phe, Phe, Arg Arg or or Gly at Gly at position position 405. 405.

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In another In embodiment,said another embodiment, first homodimeric said first protein comprises homodimeric protein Pheatat comprises aa Phe position 405 position 405 and andananamino amino acidacid other other thanthan Lys, Lys, Leu Leu or MetoratMet at position position 409 409 and and said said secondhomodimeric second homodimeric protein protein comprises comprises an amino an amino acid than acid other otherPhethan Phe at position at position 405 405 and aa Lys and Lysatatposition position409. 409. In In a further a further embodiment embodiment hereof,hereof, saidhomodimeric said first first homodimeric 5 protein 5 proteincomprises comprisesa Phe a Phe at at position405 position 405and and an an amino amino acidacid other other than than Lys, Lys, LeuLeu or or Met at Met at position position 409 409 and and said said second second homodimeric protein comprises homodimeric protein comprises an an amino aminoacid acid other than other than Phe, Phe,Arg ArgororGly Glyatatposition position405 405andand a Lys a Lys at at position position 409. 409.

In In another embodiment,said another embodiment, said first first homodimeric homodimeric protein protein comprises comprises aa Phe Pheatat position 405 position 405 and andananamino amino acidacid other other thanthan Lys, Lys, Leu Leu or MetoratMet at position position 409 409 and and said said 10 second 10 second homodimeric homodimeric protein protein comprises comprises a Leu a Leu at position at position 405405 and and a Lys a Lys at position at position 409. In 409. In aa further further embodiment embodiment hereof, hereof, saidsaid first first homodimeric homodimeric protein protein comprises comprises a Phe a Phe at position at position 405 405 and an Arg and an Arg at at position position 409 409 and and said said second second homodimeric protein homodimericprotein comprises an comprises an amino aminoacid acid other other than than Phe, Phe,Arg Arg ororGly Glyatat position position 405 405 and and aa Lys Lysatat position 409. position 409.

15 15 In another In another embodiment, saidfirst embodiment,said first homodimeric homodimericprotein comprises proteincomprises PhePhe at at position 405 position and ananArg 405 and Argat atposition position409 409 andand saidsaid second second homodimeric homodimeric protein protein comprisesa aLeu comprises Leuatatposition position405 405andand a Lys a Lys at position at position 409. 409.

In aa further In further embodiment, embodiment, said said first first homodimeric homodimeric protein protein comprises comprises an an amino amino acid other acid other than than Lys, Lys,Leu LeuororMet Metatatposition 409 position409 andand saidsaid second second homodimeric homodimeric proteinprotein

20 comprises 20 comprises Lysposition a Lysa at at position 409, 409, Thr a Thraat at position position 370a and 370 and a Leu Leu at at position position 405. 405. In aa further In further embodiment, embodiment, said said first first homodimeric homodimeric protein protein comprises comprises an Arg an at Arg at position 409 position andsaid 409 and saidsecond second homodimeric homodimeric protein protein comprises comprises a Lys a Lys at at position position 409, a 409, a Thr at Thr at position position 370 370 and anda aLeu Leuatatposition position405. 405. In an In even further an even further embodiment, said first embodiment, said first homodimeric protein comprises homodimeric protein comprises aa 25 Lys Lys 25 at position at position 370,370, a Phe a Phe at position at position 405 an 405 and and ArganatArg at position position 409 409 and andsecond said said second homodimeric homodimeric protein protein comprises comprises a Lys a Lys at position at position 409,409, a Thr a Thr at position at position 370 aand 370 and Leu a Leu at position at position 405. 405.

In another In embodiment,said another embodiment, said first first homodimeric protein comprises homodimeric protein comprises an an amino amino acid other acid other than than Lys, Lys,Leu LeuororMet Metatatposition 409 position409 andand saidsaid second second homodimeric homodimeric proteinprotein

30 comprises 30 comprises Lys a Lysa at at position position 409 a)and: 409 and: a) an an Ile Ile at position at position 350 and350 and a Leu Leu at at aposition position 405, or 405, or b) b) aa Thr Thr at at position 370 and position 370 anda aLeu Leuatatposition position405. 405. In another In another embodiment, said first embodiment, said first homodimeric protein comprises homodimeric protein an Arg comprises an Arg at at position 409 position 409 and andsaid saidsecond second homodimeric homodimeric protein protein comprises comprises Lys at position a Lys ata position 409 409 and: a) and: a) an anIle at position Ile at 350and position 350 anda Leu a Leu at at position position 405, 405, or ab)Thr or b) a Thr at position at position 370 370 35 andand 35 a Leu a Leu at at position405. position 405.

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In another In embodiment,said another embodiment, first homodimeric saidfirst protein comprises homodimeric protein Thratat comprises a aThr position 350, position 350, aa Lys Lys at at position position 370, 370,a aPhe Pheatatposition position405 405 andand an Arg an Arg at position at position 409 409 and said and said second secondhomodimeric homodimeric protein protein comprises comprises at Lys a Lys a at position position 409a) and: 409 and: a) an Ile an Ile at position at position 350 350 and anda aLeuLeu at at position position 405, 405, or ab)Thr or b) a Thr at position at position 370a and 370 and a Leu Leu at at 5 position 5 position405. 405. In another In anotherembodiment, embodiment,said said firstfirst homodimeric homodimeric protein protein comprises comprises Thr at a Thr ata position position 350, aa Lys 350, Lysat at position position 370, 370,a aPhe Pheatatposition position405 405 andand an an Arg Arg at position at position 409 409 and and said said secondhomodimeric second homodimeric protein protein comprises comprises an IleanatIle at position position 350, a350, Thr aatThr at position position 370, 370, Leu at a Leu a at position position 405 405 and anda aLys Lysatatposition position409. 409. 10 10 In another In another embodiment, embodiment,said saidfirst first homodimeric homodimericprotein protein has hasananamino amino acid acid other than other than Lys, Lys,Leu LeuororMet Met at at position409409 position andand saidsaid second second homodimeric homodimeric protein protein has has an amino an aminoacid acidother other than than Tyr,Tyr, Asp,Asp, Phe, Phe, Glu, Glu, Lys, Arg, Lys, Gln, Arg, Gln, Ser or Ser Thr or Thr at position at position

407. 407. In another In another embodiment, embodiment,said saidfirst first homodimeric homodimericprotein protein has hasananamino amino acid acid 15 other 15 other than than Lys,Leu Lys, LeuororMet Metatatposition position 409 409 and and said said second second homodimeric homodimeric protein protein has has an Ala, an Ala, Gly, Gly, His, His, Ile, Ile,Leu, Leu, Met, Met, Asn, Asn, Val Val or or Trp Trp at at position position 407. 407.

In another In embodiment,said another embodiment, saidfirst first homodimeric homodimericprotein protein has hasananamino amino acid acid other than other than Lys, Lys,Leu LeuororMet Met at at position409409 position andand saidsaid second second homodimeric homodimeric protein protein has has a Gly, a Leu, Met, Gly, Leu, Met, Asn Asn or orTrp Trpatat position position 407. 407. 20 20 In another In anotherembodiment, embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

407 and 407 andanan amino amino acidacid other other than than Lys, Lys, Leu orLeu Metorat Met at position position 409 and409 saidand said second second homodimeric homodimeric protein protein hashas an amino an amino acid acid otherother than Asp, than Tyr, Tyr, Glu, Asp,Phe, Phe,Gln, Glu, Lys, Lys,Arg, Gln, Arg, Ser or Ser or Thr Thr at at position position 407 407 and anda aLys Lysatatposition 409. position409. In another In anotherembodiment, embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

25 407407 25 and and an amino an amino acidacid other other thanthan Lys,Lys, LeuLeu or Met or Met at position409 at position 409 andand said said second second homodimeric homodimeric protein protein hashas an Ala, an Ala, Gly, Gly, His, His, Leu,Leu, Ile, Ile, Met, Met, Asn, Asn, ValTrporatTrp Val or at position position

407 and 407 anda aLys Lysatatposition position409. 409. In another In anotherembodiment, embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

407 and 407 andananamino amino acidacid other other than than Lys, Lys, Leu orLeu Metorat Met at position position 409 and409 saidand said second second 30 homodimeric 30 homodimeric protein protein has has a Gly, a Gly, Leu, Leu, Met, Met, AsnAsn or Trp or Trp at position407 at position 407 andand a Lys a Lys at at position 409. position 409.

In another In anotherembodiment, embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

407 and 407 and an an Arg Arg at at position position 409 409 and said second and said homodimeric protein second homodimeric protein has has an an amino amino acid other acid other than thanTyr, Tyr,Asp, Asp,Glu, Phe, Glu,Phe, Lys, Lys, Arg,Arg, Gln, Gln, Ser Ser or at or Thr Thrposition at position 407a 407 and and a 35 LysLys 35 at at position 409. position 409.

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In another In anotherembodiment, embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

407 and 407 andananArg Argatatposition position409 409 andand said said second second homodimeric homodimeric proteinprotein has an has Ala,an Ala, Gly, Gly, His, Ile, His, Ile,Leu, Leu, Met, Met, Asn, Asn, Val Val or or Trp Trp at at position position 407 and aa Lys 407 and Lysatat position position 409. 409. 2022201608 08

In anotherembodiment, In another embodiment,said said first first homodimeric homodimeric protein protein has at has a Tyr a Tyr at position position

5 407407 5 andand an an ArgArg at at position409 position 409and andsaid saidsecond secondhomodimeric proteinhas homodimericprotein hasa aGly, Gly, Leu, Leu, Met, Asn Met, Asn or or Trp Trpat at position position 407 407and anda aLysLys at at position409. position 409. In In one embodiment,the one embodiment, thefirst first homodimeric homodimeric protein protein has has an an amino aminoacid acidother other than Lys, than Lys, Leu Leu oror Met Metatatposition position409, 409,and andthethe second second homodimeric homodimeric protein protein has has

10 10 (i) (i) an amino an aminoacid acidother otherthan than Phe, Phe, LeuLeu andand Met Met at position at position 368,368, or or

(ii) (ii) a Trp a Trp at at position position 370, 370, or or

(iii) ananamino (iii) amino acid acid other other thanthan Asp,Asp, Cys,Cys, Pro, Pro, Glu or or at GluGln at position Glnposition 399. 399.

In one In embodiment, one embodiment, the the first first homodimeric homodimeric protein protein has anhas an Ala, Arg, Arg,His Ala,or His Glyor Gly at position at position 409, and the 409, and the second secondhomodimeric homodimeric protein protein has has 15 15 (i) (i) a Lys, a Lys, Gln,Gln, Asp,Asp, Ala,Ala, Gly, Gly, Glu, Glu, His, Ile, Arg, Arg, Ile, Asn, His, Asn, Ser, Val, Ser, Thr, TrporatTrp Val, Thr, or at position 368, position or 368, or

(ii) (ii) a Trp a Trp at at position position 370, 370, or or

(iii) ananAla, (iii) Ala,Gly, Gly,Ile, Leu,Met, Ile, Leu, Met,Asn, Asn, Ser, Ser, Thr, Thr, Trp, Trp, Phe, Phe, His,His, Lys,Lys, Arg Arg or Tyr or Tyr at at 20 20 position 399. position 399.

In one In embodiment, one embodiment, the the first first homodimeric homodimeric protein protein has has an anatArg Arg at position position 409, 409, and the and the second secondhomodimeric homodimeric protein protein has has

an Asp, (i) an Asp, (i) Glu,Glu, Asn,Asn, Gly,Gly, Ser, Ser, Arg, Arg, Val, Val, Thr, Thr, or Trp or Trp at position 368,368, at position or or

25 25 (ii) (ii) a Trp a Trp at at position position 370, 370, or or

(iii) (iii) a aPhe, Phe,His, His,Lys, Lys,Arg ArgororTyr Tyratatposition position399. 399.

In addition In addition totothe theabove-specified above-specified amino-acid amino-acid substitutions, substitutions, said and said first first and secondhomodimeric second homodimeric protein protein may contain may contain furtherfurther amino-acid amino-acid substitutions, substitutions, deletion deletion or insertions or relative to insertions relative to wild-type wild-type Fc Fc sequences. sequences.

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In further embodiment, a further In a embodiment,saidsaid first first and and second second CH3 regions, CH3 regions, except except for the for the

specified mutations, specified mutations,comprise comprisethethesequence sequence set set forth forth in SEQ in SEQ ID NO:1 ID NO:1 (IgG1m(a)): (IgG1m(a)):

SEQ ID SEQ ID NO:1: NO:1: 5 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF 5 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In further embodiment, a further In a embodiment,saidsaid first first and and second second CH3 regions, CH3 regions, except except for the for the

specified mutations, specified mutations,comprise comprisethethesequence sequence set set forth forth in SEQ in SEQ ID NO:2 ID NO:2 (IgG1m(f)): (IgG1m(f)):

10 10 SEQ ID SEQ ID NO:2: NO:2: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

15 15 In aa further In further embodiment, embodiment,saidsaid first first and and second second CH3 regions, CH3 regions, except except for the for the specified mutations, specified mutations, comprise comprisethethesequence sequence set set forth forth in SEQ in SEQ ID NO:3 ID NO:3 (IgG1m(ax)): (IgG1m(ax)):

SEQ ID SEQ ID NO:3: NO:3: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF 20 FLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK 20 FLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK

In aa further In further embodiments, the homodimeric embodiments, the homodimericproteins proteins provided provided may maybebea arat rat antibody and antibody and aa mouse mouse antibody, antibody, whowho showshow preferential preferential pairing, pairing, as as described described by by Lindhofer et Lindhofer et al. al. (1995) (1995)J JImmunol Immunol 155:219 155:219 (see above), (see above), or so-called or so-called knob-in-hole knob-in-hole

25 variant 25 variant antibodies, antibodies, as described as described in USinpatent US patent 5,731,168 5,731,168 (see above). (see above). In some In some cases, cases, however,the however, thelatter latterhomodimeric homodimeric starting starting proteins proteins may may be be difficult more more difficult to produce, to produce,

becauseofoftoo because tooweak weak homodimeric homodimeric CH3-CH3 CH3-CH3 interactions. interactions. Thus, Thus, the thedescribed herein herein described variants having variants having mutations mutationsat atpositions positions350, 350, 370, 370, 405405 and and 409, 409, may may be be preferred. preferred.

30 30 The sequence The sequence ofof the the hinge hinge region of the region of the homodimeric homodimeric starting proteins may starting proteins may vary. However, vary. However,thethe resultingheterodimeric resulting heterodimeric protein protein may may be be stable more more under stablesome under some circumstancesifif the circumstances the hinge hingeregion regionisisnot notIgG4-like, IgG4-like,and, and,preferably preferablyisisIgG1-like. IgG1-like. Thus, in Thus, in one one embodiment, embodiment,neither neithersaid saidfirst first nor nor said said second secondhomodimeric homodimeric protein comprises protein comprisesa aCys-Pro-Ser-Cys Cys-Pro-Ser-Cys sequence sequence in (core) in the the (core) hingehinge region. region.

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In aa further In further embodiment, both embodiment, both said said first first and and said said second second homodimeric homodimeric proteinprotein

comprisea aCys-Pro-Pro-Cys comprise Cys-Pro-Pro-Cys sequence sequence in (core) in the the (core) hingehinge region. region.

In many In manyembodiments embodiments wherein wherein firstand first and saidsecond said second homodimeric homodimeric proteins proteins are antibodies, are antibodies, said saidantibodies antibodiesfurther furthercomprise comprise a light a light chain. chain. As explained As explained above,above,

5 saidsaid 5 light light chains chains may may be different, be different, i.e. i.e. differ differ in in sequence sequence and form and each each aform a functional functional

antigen-bindingdomain antigen-binding domain with with onlyonly one one of heavy of the the heavy chains. chains. In another In another embodiment, embodiment,

however, said however, said first first and and second homodimericproteins second homodimeric proteins are areheavy-chain heavy-chainantibodies, antibodies, which do which do not not need needa alight light chain chain for for antigen antigen binding, binding, see see e.g. e.g. Hamers-Casterman Hamers-Casterman (1993) Nature (1993) Nature 363:446. 363:446. 10 10 As described As described above, above,step stepc) c)of of thethe method method of invention of the the invention comprises comprises incubating said first incubating said first protein protein together together with said second with said second protein protein under underreducing reducing conditions sufficient conditions sufficient to allow the to allow the cysteines cysteinesininthe thehinge hingeregion region to to undergo undergo disulfide disulfide-

bond isomerisation. bond isomerisation.Examples Examples of suitable of suitable conditions conditions are given are given herein.herein. The The minimal minimal requirementsforforthethe requirements cysteines cysteines in hinge in the the hinge regionregion for undergoing for undergoing disulfide-bond disulfide-bond

15 isomerisation 15 isomerisationmaymay differ differ depending depending onhomodimeric on the the homodimeric starting starting proteins, proteins, in in particular depending particular depending onon the the exact exact sequence sequence in hinge in the the hinge region. region. It is It is important important that that the respective the respective homodimeric interactions of homodimeric interactions of said said first firstand andsecond second CH3 regions are CH3 regions are sufficiently weak sufficiently toallow weak to allowcysteines cysteines in in thethe hinge hinge region region to undergo to undergo disulfide-bond disulfide-bond

isomerisation under isomerisation underthe thegiven givenconditions. conditions. 20 20 In one In one embodiment, embodiment,the the reducing reducing conditions conditions in c) in step step c) comprise comprise the addition the addition

of aa reducing of reducing agent, agent,e.g. e.g.a areducing agent reducingagent selected selected fromfrom the group the group consisting consisting of: 2-of: 2 mercaptoethylamine (2-MEA), mercaptoethylamine (2-MEA), dithiothreitol dithiothreitol (DTT), (DTT), dithioerythritol dithioerythritol (DTE), (DTE), glutathione, tris(2-carboxyethyl)phosphine glutathione, tris(2-carboxyethyl)phosphine (TCEP), (TCEP), L-cysteine L-cysteine and beta-mercapto and beta-mercapto-

ethanol, preferably ethanol, preferably aa reducing reducing agent agent selected selected from from the thegroup consistingof:of:2-2 groupconsisting 25 mercaptoethylamine, 25 mercaptoethylamine, dithiothreitol and dithiothreitol and tris(2-carboxyethyl)phosphine tris(2-carboxyethyl)phosphine. In one In one embodiment, embodiment,thethe reducing reducing conditions conditions enabling enabling controlledFab-arm controlled Fab-arm exchange are exchange are described described inin terms termsofofthe therequired requiredredox redoxpotential. potential. The Thetripeptide tripeptide glutathione (GSH) glutathione (GSH)is isthe themajor major low-molecular low-molecular weight weight thiol thiol in cells in cells and and controls controls thiol thiol-

disulphide redox disulphide redoxstate statewhich which is essential is essential for normal for normal redox signaling redox signaling in vivo.inThe vivo. The 30 dynamics 30 dynamics of cellular of cellular redox redox balance balance areare achieved achieved by maintenance by maintenance of thiol-to- of the the thiol-to disulphide status disulphide status ofof reduced reduced GSH and its GSH and its oxidized oxidized form GSSG. The form GSSG. Thevalues valuesfor for the the reduction potential reduction potential can can be measured asasinin Rost be measured Rostand andRapoport, Nature Rapoport,Nature 201: 201: 185 185 (1964) and Aslund (1964) and Aslundet etal., al.,J.J. Biol. Biol. Chem. Chem.272:30780-30786 272:30780-30786 (1997). (1997). The redox The redox potential Eh, potential which takes Eh, which takesinto intoconsideration considerationthethe stoichiometry stoichiometry of GSH of two twooxidized GSH oxidized 35 perper 35 GSSG GSSG is a isquantitative a quantitative measure measure for redox for the the redox state. state. E is Eh is calculated calculated by by the the

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2 2022201608 08 Mar Nernst equation: Nernst equation:EnEh= =E Eo + (RT/nF)ln + (RT/nF)In ([GSSG ([GSSG (ox)]/[GSH (ox)]/[GSH (red)] (red)]²). Eo is ). the Eo standard is the standard potential for potential for the redox couple the redox coupleatatdefined definedpH,pH, R isthethegasgas R is constant, constant, is the T the T is absolute absolute

temperature,F FisisFaraday's temperature, Faraday'sconstant constant andand is the n isn the number number of electrons of electrons transferred. transferred. In In vivo estimates vivo estimatesfor forEhEhfor forthe theGSH/GSSG GSH/GSSG couple couple are inare theinrange the range of -260ofto-260 -200tomV-200 mV 5 (Aw, 5 (Aw, T.,T., News News Physiol. Physiol. Sci.Sci. 18:201-204 18:201-204 (2003)). (2003)). Terminally Terminally differentiated differentiated cellscells therebymaintain thereby maintainan an Eh Eh in the in the order order of -200 of -200 mV, whereas mV, whereas actively actively proliferating proliferating cells cells maintain morereduced maintain aa more reduced Eh approximately Eh of of approximately -260 -260 mV. mV.

The standardredox The standard redox potential potential forfor DTTDTT is -330 is -330 mV (Cleland mV (Cleland et al.etBiochemistry al. Biochemistry 3: 480-482 3: (1964)). TCEP 480-482 (1964)). TCEP has hasbeen beenshown shownto toreduce reduceDTTDTT in in solutionand solution andtherefore therefore 10 hashas 10 a more a more negative negative redox redox potential potential than than DTT. DTT. TheThe precise precise value value however however has has not not been reported. been reported.Reducing Reducing conditions conditions allowing allowing controlled controlled Fab-arm Fab-arm exchange exchange conditions conditions

can therefore can therefore be be described described inin terms termsofofa arequired requiredredox redoxpotential Eh,which potentialEh, whichis is optimally below optimally belowthe thevalue value that that is achieved is achieved underunder normalnormal plasma plasma conditions conditions in vivo in vivo and that and that is is above above the theredox redoxpotential whichreduces potential which reduces antibody antibody disulphidebonds disulphide bonds 15 outside 15 outside those those located located in the in the hingehinge region region and involved and involved in inter-heavy in inter-heavy chain disulphide chain disulphide

bond formation. bond formation.

Thus, in a afurther Thus, in furtherembodiment, embodiment, stepstep c) isc)performed is performed under reducing under reducing conditions with conditions with aa redox redox potential potentialranging rangingbelow below -50 -50 mV, such as mV, such as below below -150 -150 mV, mV, preferably preferably between -150 and between -150 and -600 -600 mV, mV,such suchasasbetween between -200 -200 andand -500 -500 mV, mV, more more 20 preferably 20 preferablybetween between -250 -250 and and -450-450 mV, such mV, such as between as between -250-400 -250 and andmV, -400 mV, even even more preferably between more preferably -260 and between -260 and -300 -300 mV. mV. In aa further In embodiment, further embodiment, step step c) c) comprises comprises incubation incubation forleast for at at least 90 at 90 min mina at a temperature ofof at temperature at least least20°C20°C inin the the presence presence ofof atat least least2525 mM mM 2-2 mercaptoethylamine mercaptoethylamine ororininthe thepresence presence of least of at at least 0.5 0.5 mM dithiothreitol. mM dithiothreitol. The The 25 incubation 25 incubation may may be be performed performed at a pH at pH of ofa from from 5 to 8, 5 to 8, such as such at pH as 7.0ator pHat7.0 pH or at pH 7.4. 7.4.

In aa further In further embodiment, embodiment,step step d) d) comprises comprises the the restoring restoring conditions conditions to to becomenon-reducing become non-reducing or less or less reducing, reducing, for example for example by removal by removal of a reducing of a reducing agent, agent, e.g. by e.g. desalting. by desalting.

In some In someembodiments, embodiments, the the method method of theofinvention the invention yieldsyields an antibody an antibody 30 product 30 product wherein wherein more more than than 80%, 80%, suchsuch as more as more thanthan 90%,90%, e.g. e.g. moremore thanthan 95%,95%, such such as more as morethan than99% 99% of the of the antibody antibody molecules molecules aredesired are the the desired bispecific bispecific antibodies. antibodies.

The post-production The post-productionis is more more flexible flexible and and easier easier to control to control compared compared to the to the prior art prior art methods basedonon methods based co-expression. co-expression.

The post-productionnature The post-production nature of making of making bispecific bispecific antibodies antibodies by Fab-exchange by Fab-exchange

35 under 35 under reducing reducing conditions(such conditions (suchasasbybyaddition addition of of 2-MEA) 2-MEA) as as disclosed disclosed herein herein makes makes

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2022201608 08 Mar it aa highly it suitable strategy highly suitable for (high-throughput) strategy for screeningof ofmultiple (high-throughput) screening multiplecombinations combinations of specificities of specificitiesfor forbispecific bispecificantibody antibodydiscovery. discovery. In In addition, addition, the in vitro the in vitro process can process can

be performed be performedin inthethe laboratory laboratory which which allows allows greater greater control, control, flexibility flexibility and yield and yield of of the heterodimeric the heterodimericprotein proteinthan than is is allowed allowed by by co-expression. co-expression. An additional An additional advantage advantage

5 of this 5 of this strategy strategy is that is that the the screening screening can becan donebein done in the the final final therapeutic therapeutic format, format, precluding the precluding the need needfor forengineering engineeringupon upon lead lead selection. selection.

As explained As explainedabove, above,in in a further a further aspect, aspect, thethe method method of invention of the the invention may bemay be used for used for "matrix" "matrix" screening, screening, i.e. i.e. for for generating generatinga alarge largenumber number of different of different combinationsofofbinding combinations binding specificitiesononthethe specificities basis basis of of twotwo setssets of antibodies, of antibodies, one one set set 10 having 10 having identicalfirst identical first CH3 CH3regions regionsand andthe theother othersetsethaving havingidentical identical second secondCH3 CH3 regions, wherein regions, whereinthethesequences sequences of said of said firstfirst and and second second CH3 regions CH3 regions are different are different

and are and aresuch suchthat thatthethe heterodimeric heterodimeric interaction interaction between between said and said first firstsecond and second CH3 CH3 regions is regions is stronger than each stronger than eachofofthe thehomodimeric homodimeric interactions interactions of said of said firstandand first second second

CH3regions. CH3 regions. 15 15 Thus, in Thus, in one oneembodiment embodiment the invention the invention relates relates to a method for the for to a method the selection selection

of aa heterodimeric of heterodimericprotein protein having having a desired a desired property, property, said method said method comprising comprising the the stepsof: steps of:

a) providing a) providing aa first first set set of of homodimeric proteins comprising homodimeric proteins comprising an an FcFcregion region whereinthe wherein thehomodimeric homodimeric proteins proteins havehave identical identical first first CH3CH3 regions, regions,

20 20 b) providing b) providing aa second set of second set of homodimeric proteins comprising homodimeric proteins comprising an an Fc Fc region region whereinthe wherein thehomodimeric homodimeric proteins proteins havehave identical identical second second CH3 regions, CH3 regions,

whereinthe wherein thesequences sequences of said of said firstandand first second second CH3 CH3 regions regions are different are different and and 25 25 are such are such that that the the heterodimeric interactionbetween heterodimericinteraction between saidsaid first first andand second second CH3 CH3 regions is regions is stronger than each stronger than eachofofthe thehomodimeric homodimeric interactions interactions of said of said first first andand

secondCH3 second CH3regions, regions,

c) incubating c) combinationsofofthe incubating combinations thehomodimeric homodimeric proteins proteins of said of said first first setset andand of of 30 30 said second said secondset setunder under reducing reducing conditions conditions sufficient sufficient to allow to allow the the cysteines cysteines in in the hinge the hingeregion regiontotoundergo undergo disulfide-bond disulfide-bond isomerization, isomerization, thus generating thus generating a a set of set of bispecific bispecificantibodies, antibodies,

d) optionally d) restoring the optionally restoring the conditions conditions to to non-reducing, non-reducing, 35

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e) assaying e) assayingthe resultingsetset theresulting of of heterodimeric heterodimeric proteins for a for proteins a given given desireddesired

property, and property, and

f) selecting f) selecting aa heterodimeric protein having heterodimeric protein havingthe thedesired desiredproperty. property. 5 5

In one In one embodiment, embodiment,the the invention invention relates relates to a to a method method for thefor the selection selection of a of a bispecific bispecific antibody having aa desired antibody having desiredproperty, property,said saidmethod method comprising comprising the steps the steps of: of:

a) a) a) a) providing providinga afirst first set setofofhomodimeric homodimeric antibodies antibodies comprising comprising antibodies antibodies

10 10 with different with different variable variableregions, regions, wherein wherein said antibodies said antibodies of said of saidsetfirst first set compriseidentical comprise identical first first CH3 regions, CH3 regions,

b) providing b) providing aa second set of second set of homodimeric homodimeric antibodies antibodies comprising comprising antibodies antibodies with different with different variable variableregions regions or identical or identical variable variable regions, regions, wherein wherein said said 15 15 antibodies of antibodies of said said second secondset setcomprise comprise identicalsecond identical second CH3CH3 regions, regions,

whereinthe wherein thesequences sequences of said of said firstandand first second second CH3 CH3 regions regions are different are different and and are such are such that that the the heterodimeric heterodimeric interaction interaction between betweensaid saidfirst first and and second secondCH3CH3 regions is stronger regions is than each stronger than eachofofthe thehomodimeric homodimeric interactions interactions of said of said firstandand first second second

20 CH3 20 CH3 regions, regions,

c) incubating c) incubating combinations combinationsof of antibodies antibodies of said of said first first setset and and of said of said second second

set under set underreducing reducingconditions conditions sufficienttotoallow sufficient allowthethe cysteines cysteines in in thethe hinge hinge region region to to undergo disulfide-bondisomerization, undergo disulfide-bond isomerization, thus thus generating generating a set a set of bispecific of bispecific antibodies, antibodies,

25 25 d) optionally d) optionally restoring restoring the the conditions conditions to to non-reducing, non-reducing,

e) assaying e) assayingthethe resulting resulting set set of bispecific of bispecific antibodies antibodies for a for given given a desired desired property, and property, and 30 30 f) selecting f) selecting aa bispecific bispecificantibody antibody having the desired having the desired property. property.

In one In one embodiment, embodiment,thethe homodimeric homodimeric antibodies antibodies of the of the second second set set have have differentvariable different variable regions. regions.

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In one In one embodiment, embodiment,thethe homodimeric homodimeric antibodies antibodies of the of the second second set set have have identical variable identical variable regions, regions,butbut havehave different different different different amino amino acid or acid or structural structural

variations outside variations outside of of the the antigen antigen binding bindingregion. region. The two The two sets sets can can be be composed composedininmany manydifferent different ways waysasasdesired. desired. Thus, Thus, the the 5 twotwo 5 setssets may may target target the epitope the same same epitope or different or different epitopes epitopes on the on samethe same antigen. antigen. The The two sets two sets may mayalso also target target different different antigens, antigens,and and each set may each set contain antibodies may contain antibodies binding to binding to the the same sameepitope epitopeor or epitopesonon differentepitopes different thethe antigen antigen in question. in question. Furthermore, one Furthermore, one of of the the sets sets or or both both sets sets may mayeach eachcontain containantibodies antibodiestargeting targeting different antigens. different antigens.

10 10 In another In another embodiment, embodiment, said said desired desired property property is cell is cell killing,cell killing, celllysis, lysis, inhibition ofofcell inhibition cell proliferation, proliferation,ororbinding binding to cells to cells expressing expressing both antigen both antigen targets. targets. The screening The screeningstrategy strategy includes includes two two panels panels of antibody of antibody vectors vectors containing containing a a range ofofspecificities, range specificities, where whereoneone panel panel is cloned is cloned into ainto a backbone backbone that is that is able to able to engage in engage in Fab-arm Fab-arm exchange exchange under under reducing reducing conditions(such conditions (suchasasbybyaddition additionofof2-2 15 MEA) 15 MEA) withwith thethe backbone backbone of the of the second second panel. panel. For For example, example, the the firstpanel first panelisis cloned cloned into an into an IgGl-F405L backboneandand IgG1-F405L backbone thethe second second panel panel is cloned is cloned into into a IgG-K409R a IgG1-K409R backbone (for backbone (for other other possible possible backbone combination see backbone combination see also also Examples Examples 19, 19,28, 28, 29, 29, 30,35,36,37,38, 30, and39). 35, 36, 37, 38, and 39). Each member Each memberof of the the two two panels panels of antibody of antibody vectors vectors is expressed is then then expressed 20 individually 20 individuallyatatsmall smallscale. scale.ForFor example, example, all antibody all antibody vectors vectors are transfected are transfected transiently inin HEK293 transiently cells and HEK293 cells expressed inin 2.3 and expressed 2.3 mLmLcultures culturesinin24-well 24-wellplates. plates. Alternatively, other Alternatively, other suitable suitable (small-scale) (small-scale) production productionsystems systems known known in theinart themay art may be used. be used. The expressed The expressedantibodies antibodies of of thethe twotwo panels panels of antibodies of antibodies are are thenthen mixedmixed pair- pair 25 wise 25 wise in ain matrix-like a matrix-like fashion fashion at equimolar at equimolar ratios. ratios. For example, For example, all individual all individual antibodies are antibodies purified by are purified by small-scale small-scale protein protein AA chromatography chromatographyand and antibody antibody concentration are concentration measuredbybyabsorbance are measured absorbance at aatwavelength a wavelength of nm. of 280 280Other nm. Other suitable (small-scale) suitable (small-scale) purification purificationmethods methods or methodsfor or methods fordetermining determiningprotein protein concentrationknown concentration known in the in the art art may may alternatively alternatively be used. be used. In another In another embodiment, embodiment,

30 the the 30 purification purification stepstep may may be out be left left if outdown-stream if down-stream applications applications are notare not affected affected by by the expression the expressionmedium. medium. Thereafter, Thereafter, the antibody the antibody concentrations concentrations are normalized are normalized so so that aa suitable that suitable volume volumecontains contains equimolar equimolar amounts amounts of antibodies. of both both antibodies. For example, For example,

panel of a panel a of 88 antibodies antibodiesinin the the F405L F405Lbackbone backbone is individually is individually mixed mixed withwith 8 antibodies 8 antibodies

in the in the K409R backbone K409R backbone so that so that 64 mixtures 64 mixtures of µl of 100 100contain pl contain 80 pg/mL 80 µg/mL of antibody of antibody A A 35 35 (F405L) (F405L) and and 80 80 pg/mL µg/mL of antibody of antibody B (K409R). B (K409R). Alternatively, Alternatively, if the contains if the strategy strategy contains

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a bispecific antibody-specific a bispecific purification step antibody-specific purification stepdown-stream, down-stream, the step the step to normalize to normalize

antibody amounts antibody amountsmaymay be left be left out.out. To the To the mixtures mixturesofofantibodies, antibodies, a suitable a suitable amount amount of reducing of reducing agent agent is is added added 2022201608 08

and incubated and incubated for for a asuitable suitable period period ofoftime timeat at a permissive a permissive temperature. temperature. For For 5 example, 5 example, to to 100 100 pl containing µl containing 80 80 pg/mL µg/mL of antibody of antibody A (F405L) A (F405L) andµg/mL and 80 80 pg/mL of of antibody BB(K409R), antibody (K409R),25 25 pl of µl of 125125 mM 2-MEA mM 2-MEA is (final is added added concentration (final concentration 25 mM 2-25 mM 2 MEA)and MEA) andincubated incubated overnight overnight at 25°C. at 25°C.

The reducing The reducing agent agent is is thereupon thereupon removed from the removed from the mixtures (now containing mixtures (now containing bispecific antibodies) bispecific antibodies)totopromote promote oxidation oxidation of of the disulfide bonds the disulfide and to bonds and to avoid avoid 10 interference 10 interferenceofofthe thereducing reducingagent agentininthe the screening screening assays. assays. For For example, example,2-MEA 2-MEAis is removed bybyperforming removed performinga abuffer buffer exchange exchangeofofthe the 6464mixtures mixturesusing using Zeba ZebaSpin Spin96- 96 well desalting well desalting plates plates(Pierce (PierceBiotechnology, Biotechnology, #89807). #89807). Alternatively, Alternatively, other suitable other suitable

methodstotoremove methods remove the the reducing reducing agent agent knownknown in the inart themayartbemay usedbe used The bispecific The bispecific antibodies antibodiesare arethen thencharacterized characterized biochemically biochemically or functionally or functionally

15 to to 15 identifythethe identify lead lead candidates. candidates. For For example, example, thebispecific the 64 64 bispecific antibodies antibodies are are assessedfor assessed forproliferation proliferation inhibition inhibition of of suitable suitable cell-lines cell-lines or or binding binding totosuitable suitablecell- cell lines. Identified lines. Identified lead lead candidates will then candidates will then bebeproduced produced at larger at larger scale scale and and characterized in characterized in more moredetail. detail.

20 Production 20 Production by by co-expression co-expression The heterodimeric The heterodimericproteins proteinsof ofthe theinvention invention maymay alsoalso be obtained be obtained by co-expression by co-expression of of constructs encoding constructs encodingthe thefirst first and andsecond second polypeptides polypeptides in single in a a single cell. cell.

Thus, inin aa further Thus, furtheraspect, aspect,the theinvention invention relates relates to to a method a method for producing for producing a a heterodimericprotein, heterodimeric protein,said saidmethod method comprising comprising the the following following steps: steps:

25 25 a) providing a) providing aafirst first nucleic-acid nucleic-acid construct constructencoding encoding a firstpolypeptide a first polypeptide comprising comprising a a first Fc first region ofof ananimmunoglobulin, Fc region immunoglobulin, said said first first Fc region Fc region comprising comprising a first aCH3first CH3 region, region,

30 b) b)providing 30 providing a asecond secondnucleic-acid nucleic-acid construct construct encoding encoding aa second second polypeptide polypeptide comprising aa second comprising secondFc Fc region region of immunoglobulin, of an an immunoglobulin, said second said second Fc Fc region region comprisinga afirst comprising first CH3 region, CH3 region,

wherein the wherein the sequences sequences ofof said said first first and and second CH3 regions second CH3 regions are are different different and are and are 35 suchsuch 35 thatthat the the heterodimeric heterodimeric interaction interaction between between saidand said first first and second second CH3 isregions CH3 regions is

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stronger than stronger each of than each of the homodimericinteractions the homodimeric interactions of of said first and said first and second CH3 second CH3 regions, and regions, and

2022201608 08

whereinsaid wherein saidfirst first homodimeric homodimeric protein protein hashas an amino an amino acid acid otherother than Leu than Lys, Lys, Leu 5 5 or Met or at position Met at position 409 409 and and said said second homodimeric protein second homodimeric protein has has an an amino- amino acid substitution acid substitution atat a aposition positionselected selected from from the group the group consisting consisting of: of: 366, 366, 368,370,399,405 and 368, 370, 399, 405 and 407. 407.

and/or and/or 10 10 whereinthe wherein thesequences sequences of said of said firstand first and second second CH3 CH3 regions regions are that are such suchthe that the dissociation constants dissociation of homodimeric constants of homodimeric interactions interactions of each of each of the of the CH3 CH3 regions regions

are between are between 0.01 0.01 and and 1010micromolar, micromolar, such such asasbetween between0.05 0.05andand 10 10 micromolar, more micromolar, more preferably preferably between 0.01 and between 0.01 and 5, 5, such such as as between between 0.05 0.05 and and 15 15 5 micromolar, 5 micromolar, even preferably between more preferably even more 0.01 and between 0.01 and 11 micromolar, such as micromolar, such as between 0.05 between 0.05 and and 11 micromolar, micromolar, between between0.01 0.01and and0.5 0.5ororbetween between0.01 0.01and and 0.1 whenassayed 0.1 when assayedas as described described in Example in Example 21. 21.

c) co-expressing c) saidfirst co-expressing said first and secondnucleic-acid and second nucleic-acidconstructs constructs in ina ahost hostcell, cell,and and 20 20 d) obtaining d) said heterodimeric obtaining said heterodimericprotein proteinfrom from thethe cellculture. cell culture.

Suitable expression vectors, Suitable expression vectors, including including promoters, promoters,enhancers, enhancers, etc., etc., andand suitable host suitable host cells cells for for the the production production of antibodies are of antibodies are well-known well-known inin the theart. art. 25 Examples 25 Examples of cells of host host cells include include yeast, yeast, bacterial bacterial and mammalian and mammalian cells, cells, such as such CHO oras CHO or HEKcells. HEK cells. In one In embodimentofofthis one embodiment this method, method,said said first first CH3 region has CH3 region has an an amino acid amino acid other than other than Lys, Lys,Leu LeuororMet Metatatposition position409 409 andand saidsaid second second CH3 region CH3 region has anhas an amino amino acid other acid other than than Phe Pheatatposition position405. 405. 30 30 and/or and/or

the sequences the sequencesofofsaid saidfirst first and andsecond secondCH3CH3 regions regions are such are such that that the the dissociation constants dissociation constantsofofhomodimeric homodimeric interactions interactions of each of each of theofCH3 theregions CH3 regions are are between 0.01 between 0.01 and and1010micromolar, micromolar,such suchasasbetween between 0.05 0.05 andand 10 10 micromolar, micromolar, more more 35 preferably 35 preferablybetween between 0.01 0.01 andand 5, 5, such such as as between between 0.05 0.05 andand 5 micromolar, 5 micromolar, even even more more

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preferably between preferably between0.01 0.01 andand 1 micromolar, 1 micromolar, such such as between as between 0.05 0.05 and and 1 micromolar, 1 micromolar,

between 0.01 between 0.01 and and0.50.5or or between between 0.010.01 and when and 0.1 0.1 when assayed assayed as described as described in in Example 21. Example 21. 2022201608 08

5 In In 5 another another embodiment embodiment of thismethod: of this method: said first said first CH3 region has CH3 region hasananamino amino acid acid other other than than Lys,Lys, Leu Leu or Met or Met at position at position

409 and 409 andsaid saidsecond secondCH3CH3 region region hasamino has an an amino acid than acid other otherPhe than at Phe at position position

405, such 405, suchasasother otherthan thanPhe, Phe, Arg Arg or or GlyGly at at 405405 position position

or or 10 10 said first said first CH3 region has CH3 region hasananamino amino acid acid other other than than Lys,Lys, Leu Leu or Met or Met at position at position

409 and 409 andsaid saidsecond second CH3CH3 region region hasamino has an an amino acid than acid other other than Tyr, Tyr, Asp, Asp, Glu, Glu, Phe, Lys, Phe, Lys, Gln, Arg, Ser Gln, Arg, Ser or or Thr Thr at at position position 407. 407.

In some In some embodiments, embodiments, said said firstandand first second second polypeptides polypeptides are are full-length full-length 15 heavy 15 heavy chains chains of antibodies of two two antibodies that different that bind bind different epitopes epitopes (i.e. first (i.e. said said first and second and second

nucleic-acid constructs nucleic-acid constructsencode encode full-length full-length heavy heavy chains chains of antibodies of two two antibodies that that bind bind different epitopes), different and thus epitopes), and thusthe theheterodimeric heterodimeric protein protein is aisbispecific a bispecific antibody. antibody. ThisThis

bispecific antibody bispecific canbe be antibody can a heavy-chain a heavy-chain antibody, antibody, or saidorhost saidcell host may cell may further further oneorormore express one express more nucleic-acid nucleic-acid constructs constructs encoding encoding a light-chain. a light-chain. If only one one If only light light-

20 chain 20 chain construct construct is isco-expressed co-expressedwith withthe theheavy heavy chainconstructs, chain constructs,then thena afunctional functional bispecific antibody bispecific is only antibody is only formed formed ifif the the light light chain chain sequence sequenceis issuch such that that it itcan canform form functional antigen-binding a functional a antigen-bindingdomain domain withwith eacheach ofheavy of the the heavy chains.chains. If two If two or more or more different light-chain different light-chain constructs constructsareareco-expressed co-expressed withheavy with the the chain, heavymultiple chain, multiple products will products will be formed. be formed.

25 25 In further In embodiments, further embodiments, thethe co-expression co-expression method method according according to the to the invention invention

comprisesany comprises anyofofthe thefurther furtherfeatures featuresdescribed described under under the the in vitro in vitro method method above. above.

In aa further In further aspect, aspect,the theinvention inventionrelates relatestotoanan expression expression vector vector comprising comprising

the first the first and and second nucleic-acid constructs second nucleic-acid constructs specified specified herein herein above. above. In In an even an even further aspect, further aspect, the theinvention inventionrelates relatesto to a host a host cellcell comprising comprising the first the first and and second second

30 nucleic-acid 30 nucleic-acid constructs constructs specified specified herein herein above. above.

Heterodimericproteins Heterodimeric proteins In aa further In furtheraspect, aspect,thethe invention invention relates relates to a to a heterodimeric heterodimeric obtainedobtained proteinprotein or or obtainable by obtainable bythe themethod methodof of thethe invention. invention.

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Furthermore,the Furthermore, themethod method of the of the invention invention enables enables the formation the formation of of molecules, asymmetricalmolecules, asymmetrical molecules molecules withwith different different characteristics characteristics on each on each of the of the Fab- Fab

armsororononeach arms eachofofthe theCH3 CH3 domains domains or molecules or molecules with distinct with distinct modifications modifications

thoroughoutthe thoroughout themolecules, molecules, e.g. e.g. molecules molecules withwith unnatural unnatural aminoamino acid substitution(s) acid substitution(s)

5 forfor 5 conjugation. conjugation. SuchSuch asymmetrical asymmetrical molecules molecules can be generated can be generated in any in any suitable suitable combinatons.This combinatons. Thisisisillustrated illustrated further further below belowbybysome some non-limiting non-limiting examples. examples.

Bispecificantibodies Bispecific antibodiescancan be used be used to pretarget to pretarget target a target acell cell of interest, of interest,

including but including but not not limited limited to, to, aa tumor cell. Pretargeting tumor cell. of aa target Pretargeting of target cell cell could could be be used used

for imaging for studiesororfor imaging studies for immunotherapeutic immunotherapeutic purposes. purposes.

10 10 In an In embodiment an embodiment of the of the method method of invention, of the the invention, the first the first Fab-arm Fab-arm of of the the bispecific molecule bispecific binds to molecule binds to aa tumor tumorcell, cell, such such asasaatumor tumorcell cellsurface surfaceprotein proteinoror tumorcell tumor cell surface surface carbohydrate, carbohydrate,such such as as oneone of the of the tumor tumor cellcell surface surface proteins proteins listed listed

herein and herein and the thesecond secondFab-arm Fab-arm recognizes recognizes a radioactive a radioactive effector effector molecule molecule including including

but not but not limited limited to, to, aa radiolabel radiolabel coupled or linked coupled or linked (via (via aa chelator) chelator) to to aa peptide or peptide or

15 hapten. 15 hapten. An example An example ofradiolabelled of such such radiolabelled peptidepeptide is indium-labelled is indium-labelled

diethylenetriaminepentaacetic diethylenetriaminepentaacetic acid(anti-DTPA(In) acid (anti-DTPA(In) van van Schaijk Schaijk et Clin. et al. al. Clin. Cancer Cancer

Res. 2005; Res. 2005;11: 11:7230s-7126s). 7230s-7126s). Another Another example example is using is using hapten-labelled hapten-labelled colloidal colloidal

particles such particles such as liposomes,nanoparticles as liposomes, nanoparticlesofofpolymeric polymeric micelles micelles carrying carrying

radionuclides such radionuclides suchasasfor for example example technetium-99 technetium-99 (Jestin (Jestin et al. et al. Q JQNucl Med Med JNuc/ Mol Mol 20 Imaging 20 Imaging 2007;51:51-60). 2007; 51:51-60). In another In embodiment, another embodiment, a hapten-coupled a hapten-coupled alternative alternative cytostatic cytostatic molecule molecule

such as such as aa toxin toxin is is used. used. In aa further In further embodiment embodiment of of thethe method method of the of the invention, invention, the first the first Fab-arm Fab-arm of of the bispecific the bispecific molecule is glycosylated molecule is at position glycosylated at position N297 N297(EU (EUnumbering) numbering) and and the the 25 second 25 second Fab-arm Fab-arm of the of the bispecific bispecific molecules molecules is aglycosylated is aglycosylated (nonglycosylated (nonglycosylated for for instance by instance by mutating mutatingN297 N297 to to Q ororA Eormutation Q or A E mutation (Bolt(Bolt et al., S et Sal., Eur Eur JImmunol J Immunol

1993, 23:403-411)). 1993, 23:403-411)). Asymmetrical Asymmetrical glycosylation glycosylation in Fc-region in the the Fc-region impacts impacts the the interaction to interaction to Fcy-receptors and has Fcy-receptors and hasimpact impactonon antibody-dependent antibody-dependent cell cytotoxicity cell cytotoxicity

effect of effect of the the antibody (Ha et antibody (Ha et al., al., Glycobiology 2011,April Glycobiology 2011, April 5)5) as as well well as as interaction interaction 30 withwith 30 other other effector effector function function molecules molecules such such as as C1q. C1q. In another In embodiment another embodiment of the of the method method ofinvention, of the the invention, the first the first Fab-arm Fab-arm of of the bispecific the bispecific molecule interacts with molecule interacts with FcRn, FcRn,the theneonatal neonatalFcFcreceptor receptor (Roopenian (Roopenian DC, DC, et al. et al. Nat. Nat. Rev. Rev. Immunol. 2007, Immunol. 2007, 7:715-725) 7:715-725) and second and the the second Fab-arm Fab-arm is impaired is impaired in in binding to binding to FcRn FcRn bybymutation mutationof of thetheFcRn FcRn interaction interaction site site on on thethe molecules molecules for for

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2022201608 08 Mar instance by instance by making makinga aH435A H435A mutation mutation (Shields, (Shields, R.L.,R.L., et Jal,Biol et al, J Biol Chem, Chem, 2001,2001, Firan, Firan,

M., et M., et al, IntImmunol, al, Int 2001). Immunol, 2001).

In another In embodiment another embodiment of the of the method method ofinvention, of the the invention, the first the first Fab-arm Fab-arm of of the bispecific the bispecific molecule interacts with molecule interacts with staphylococcal staphylococcalprotein proteinA A(protein (proteinA,A, 5 Deisenhofer 5 Deisenhofer et et al, Biochemistry al, Biochemistry 20, 20, 2361-2370 2361-2370(1981) (1981)and andstreptococcal streptococcal protein protein G G (protein G, (protein G, Derrick Derrick et et al., al., Nature 359,752-754 Nature 359, 752-754 (1992), (1992), often often usedused for purification for purification of of antibodies, and antibodies, and the thesecond secondFab-arm Fab-arm of bispecific of bispecific molecules molecules is impaired is impaired in in the the interaction with interaction protein AA of with protein of G. G. As As aa result, result, removal ofresidual removal of residual amounts amountsof of

homodimer homodimer with with impaired impaired protein protein A orA Gor G binding binding afterafter the exchange the exchange into heterodimer into heterodimer

10 is easily 10 is easily obtained obtained by purification by purification of of thethe bispecificmolecule bispecific molecule with with protein protein A or or A G. G. In another In another embodiment, embodiment,the the binding binding to either to either Fcy-receptors Fcy-receptors or FcRn or FcRn is is improved improved orordecreased decreased on on one one of the of the two two Fab-arms of theofbispecific Fab-arms the bispecific molecule. molecule.

In another In embodiment, another embodiment, the the binding binding to C1q to C1q is improved is improved or decreased or decreased on one on one of the of the two Fab-armsof ofthe two Fab-arms thebispecific bispecificmolecule. molecule. 15 15 In another In embodiment, another embodiment, the the has has protein protein been been engineered engineered to enhance to enhance

complement complement activation activation on on oneone or both or both of the of the two two Fab-arms Fab-arms of theofmolecule. the molecule. In another In embodiment, another embodiment, eacheach of the of the Fab-arms Fab-arms present present in the inbispecific the bispecific molecule molecule isis derived derived from froma adifferent differentIgG IgGsubclass. subclass. In another In embodiment, another embodiment, eacheach of the of the Fab-arms Fab-arms present present in the inbispecific the bispecific 20 molecule 20 molecule carrycarry different different allotypic allotypic mutations mutations (Jefferis (Jefferis & Lefranc, & Lefranc, 2009,2009, MABs MABs 1 :332-1 :332 8). 8).

In another In another embodiment, another category embodiment, another category of of asymmetric asymmetric immunotherapeutic immunotherapeutic molecules molecules isis generated generatedbyby replacement replacement of the of the Fab Fab of one of one of Fab-arms of the the Fab-arms of theof the

bispecific molecule bispecific by an molecule by an immuno immuno active, active, stimulating stimulating or inhibiting or inhibiting cytokine. cytokine. Non Non-

25 limitingexamples 25 limiting examplesofofsuch suchcytokines cytokines are are IL-2, IL-2,FN-, I FN-p, IFN-, IFN-ß, TNF-a, G-CSF, IFN-y, IFN-, TNF-,GM G-CSF, GM- CSF, IL-10, CSF, IL-10,IL-4, IL-4, IL-6, IL-6, IL-13. IL-13. Alternatively, Alternatively, aa (growth) (growth) factor factoror or hormone hormone stimulating stimulating

or inhibition or inhibition agent is included agent is included in in the the molecules. molecules.

In another In embodiment, another embodiment, a Fab a Fab of one of one of the of the Fab-arms Fab-arms is replaced is replaced by a by a lytic lytic peptide,i.i. e.e. peptides peptide, peptides that that are are able able to tumor to lyse lyse tumor cells, bacteria, cells, bacteria, fungi fungi etc, etc, including including 30 30 but but not not limited limited to antimicrobial to antimicrobial peptides peptides like like magainin, magainin, mellitin, mellitin, cecropin, cecropin, KLAKKLAK KLAKKLAK

and variants and variants thereof thereof(Schweizer (Schweizeret et al.Eur. al. Eur.J.J. Pharmacology Pharmacology 2009; 2009; 625: 625: 190-194, 190-194,

Javadpour,J.J. Med. Javadpour, Med.Chem., Chem., 1996, 1996, 39: 39: 3107-3113, 3107-3113, Marks Marks et et al, Cancer al, Cancer Res Res 2005; 2005; 65:2373-2377, 65:2373-2377, Rege Rege et al, et al, Cancer Cancer Res.Res. 2007; 2007; 67:6368-6375) 67:6368-6375) or cationic or cationic lytic lytic peptides (CLYP peptides (CLYP technology, technology, US2009/0269341). US2009/0269341).

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In another In embodiment, another embodiment, one one or both or both of the of the Fabs Fabs onFab on the thearms Fab is arms is replaced replaced

by receptors by receptors for for cytokines cytokinesand/or and/orgrowth growth factors, factors, creating creating socalled socalled decoy decoy receptors, receptors,

of which of Enbrel®(etanercept) which Enbrel® (etanercept) targeting targeting TNF-a TNF- and VEGF-trap, and VEGF-trap, targeting targeting VEGF, VEGF, are are 2022201608 08

well-knownexamples. well-known examples. Combining Combining thesethese two decoy two decoy receptors receptors into oneinto one molecule molecule

5 showed 5 showed superior superior activity activity over over the single the single decoydecoy receptors receptors (Jung,(Jung, J.Biol. J.Biol. Chem. Chem. 2011; 2011; 286:14410-14418). 286:14410-14418). In another In another embodiment, another category embodiment, another category of of asymmetric asymmetric immunotherapeutic immunotherapeutic molecules molecules isis generated generatedbyby fusionof ofimmuno-active, fusion immuno-active, -stimulating -stimulating or inhibiting or inhibiting

cytokines to cytokines to the the N-terminus N-terminusororC-terminus C-terminus of one, of one, or both, or both, of the of the Fab-arms Fab-arms present present

10 in the 10 in the bispecific bispecific molecules. molecules. This This may may positively positively impact impact anti-tumor anti-tumor activity activity of theof the bispecific molecule. bispecific Examplesofofsuch molecule. Examples such molecules, molecules, however however not limited not limited to list to the the list below, are below, are IL-2 IL-2 (Fournier (Fournier etetal., al., 2011, Int.J. 2011, Int. J. Oncology, doi: 10.3892/ijo.2011.976), Oncology, doi: 10.3892/ijo.2011.976), IFN-, IFN-p IFN-a,IFN- or IFN-y or IFN- (Huan (Huan et al., et al., 2007;2007; J./mmunol. J.Immunol. 179:6881-6888, 179:6881-6888, Rossie et Rossie al., et al., 2009;Blood 2009; Blood114: 114: 3864-3871), 3864-3871), TNF-a. TNF-. Alternatively, Alternatively, N-terminal N-terminal or C-terminal or C-terminal fusion fusion 15 of cytokines, 15 of cytokines, suchsuch as example as for for example G-CSF,G-CSF, GM-CSF,GM-CSF, IL-10, IL-10, IL-4, IL-4, IL-6, or IL-6, IL-13or IL-13 may may positively impact positively the bispecific impact the bispecific antibody moleculeeffector antibody molecule effectorfunction. function.Alternatively Alternativelya a (growth) factor (growth) factor or or hormone hormone stimulating stimulating or or inhibition inhibition agent agent is is included included in in thethe

moleculesononthe molecules theN-terminus N-terminus or C-terminus. or C-terminus.

In another In embodiment, another embodiment, N-terminal N-terminal or C-terminal or C-terminal fusion fusion of a lytic of a lytic peptide, peptide,

20 20 suchsuch as for as for example example antimicrobial antimicrobial peptides peptides like magainin, like magainin, mellitin, mellitin, cecropin, cecropin,

KLAKKLAK KLAKKLAK andand variants variants thereof thereof (Schweizer (Schweizer et Eur. et al. al. Eur. J. Pharmacology J. Pharmacology 2009; 2009; 625: 625: 190-194,Javadpour, 190-194, Javadpour,J. J. Med. Med. Chem., Chem., 1996, 1996, 39: 3107-3113, 39: 3107-3113, Marks etMarks et al, Res al, Cancer Cancer Res 2005;65:2373-2377, 2005; 65:2373-2377,RegeRege et Cancer et al, al, Cancer Res. Res. 2007;2007; 67:6368-6375) 67:6368-6375) or cationic or cationic lytic lytic peptides peptides (CLYP (CLYP technology, technology, US2009/0269341) on one US2009/0269341) on oneor or both both of of the the Fab-ams may Fab-ams may 25 enhance 25 enhance the activity the activity of molecule. of the the molecule. In another In another embodiment, another category embodiment, another category of of asymmetric asymmetric immunotherapeutic immunotherapeutic molecules molecules isis monovalent monovalent antibodies, antibodies, molecules molecules which which interact interact with with one Fab-arm one Fab-arm to to the target the target of of choice. choice. In In such moleculeone such molecule one of of theFab-arms the Fab-arms present present in the in the bispecific bispecific

molecule molecule isis directed directed against againstthe thetarget targetmolecule moleculeofof choice,the choice, thesecond second Fab-arm Fab-arm of the of the

30 molecule 30 molecule does does not carry not carry a Fab aorFab hasora has a non-binding/non-functional non-binding/non-functional Fab such Fab as such as

decribed for decribed for MetMab MetMab(Genentech; (Genentech; WO 96/38557). WO 96/38557). Alternatively, Alternatively, monomeric monomeric Fc-fusion Fc-fusion

proteins such as proteins such as those thosedescribed describedforforFactor FactorVIII VIIIand and IX IX (Peters (Peters et et al.,Blood al., Blood2010; 2010; 115: 2057-2064) 115: maybebegenerated. 2057-2064) may generated. Alternatively, combinations Alternatively, combinations ofofany anyofofthe theabove above mentioned mentioned asymmetrical asymmetrical

35 molecules 35 molecules maymay be generated be generated by the by the method method of the of the invention. invention.

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In an In evenfurther an even furtheraspect, aspect, the the invention invention relates relates to a to a heterodimeric heterodimeric protein protein

comprisinga afirst comprising first polypeptide polypeptide comprising comprisinga first a firstFcFcregion regionofofananimmunoglobulin, immunoglobulin, saidsaid

first FcFc region first region comprising first CH3 comprising aafirst CH3region, region,and anda second a second polypeptide polypeptide comprising comprising a a 5 second 5 second Fc Fc region region of of an an immunoglobulin, immunoglobulin, said said second second Fc Fc regioncomprising region comprisinga asecond second CH3region, CH3 region,wherein whereinthethe sequences sequences of said of said first first andand second second CH3 regions CH3 regions are different are different

and are and aresuch suchthat that thethe heterodimeric heterodimeric interaction interaction between between said and said first firstsecond and second CH3 CH3 regions is regions is stronger than each stronger than eachofofthe thehomodimeric homodimeric interactions interactions of said of said first first and and second second

CH3 regions, CH3 regions, and and 10 10 whereinsaid wherein saidfirst first homodimeric homodimeric protein protein hashas an amino an amino acid acid otherother than Leu than Lys, Lys, Leu or Met or Met at at position position 409 409 and and said said second homodimeric protein second homodimeric protein has has an an amino- amino acid substitution acid substitution atata aposition positionselected selected from from the group the group consisting consisting of: of: 366, 366, 368,370,399,405 368, 370, 399, 405 and and 407 407

15 15 and/or and/or

whereinthe wherein thesequences sequences of said of said firstandand first second second CH3 CH3 regions regions are that are such suchthe that the dissociation constants dissociation constants ofofhomodimeric homodimeric interactions interactions of each of each of the of the CH3 CH3 regions regions

are between are between 0.01 0.01 and and 1010micromolar, micromolar, such such asasbetween between0.05 0.05andand 10 10 micromolar, more micromolar, more preferably preferably between 0.01 and between 0.01 and 5, 5, such such as as between between 0.05 0.05 and and 20 20 55 micromolar, micromolar, even more preferably even more preferably between 0.01 and between 0.01 and 11 micromolar, micromolar, such such as as between 0.05 between 0.05 and and 11 micromolar, micromolar, between between0.01 0.01and and0.5 0.5ororbetween between0.01 0.01and and 0.1 when 0.1 whenassayed assayedas as described described in Example in Example 21. 21.

In one In one embodiment, embodiment,saidsaid first first CH3CH3 region region hasamino has an an amino acid than acid other other than Lys, Leu Lys, or Leu or 25 25 MetMet at at position409 position 409and andsaid saidsecond secondCH3 CH3region regionhas hasananamino aminoacid acidother otherthan than Phe Phe at at position 405 position 405

and/or and/or the sequences the sequencesofofsaid saidfirst first and andsecond second CH3CH3 regions regions are such are such that that the the dissociation constants dissociation constantsofofhomodimeric homodimeric interactions interactions of each of each of theofCH3 theregions CH3 are regions are 30 between 30 between 0.010.01 and and 10 micromolar, 10 micromolar, such such as between as between 0.0510and 0.05 and 10 micromolar, micromolar, more more preferably preferably between between 0.01 0.01 and and 5, 5, such such as as between 0.05 and between 0.05 micromolar, even 5 micromolar, and 5 more even more preferably between0.01 preferably between 0.01 andand 1 micromolar, 1 micromolar, such such as between as between 0.05 and0.05 and 1 micromolar, 1 micromolar,

between 0.01 and between 0.01 and0.50.5or or between between 0.010.01 and when and 0.1 0.1 when assayed assayed as described as described in in Example 21. Example 21.

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In aa further In embodiment of of further embodiment thethe heterodimeric heterodimeric protein protein

said first said first CH3 region has CH3 region hasananamino amino acid acid other other than than Lys,Lys, Leu Leu or Met or Met at position at position

409 and 409 andsaid saidsecond secondCH3CH3 region region hasamino has an an amino acid than acid other otherPhe than at Phe at position position

5 5 405, such 405, suchasasother otherthan thanPhe, Phe, Arg Arg or or Gly, Gly, at at position405405 position

or or said first said first CH3 region has CH3 region hasananamino amino acid acid other other than than Lys,Lys, Leu Leu or Met or Met at position at position

409 and 409 andsaid saidsecond second CH3CH3 region region hasamino has an an amino acid than acid other other than Tyr, Tyr, Asp, Asp, Glu, Glu, Phe, Lys, Phe, Lys, Gln, Arg, Ser Gln, Arg, Ser or or Thr Thr at at position position 407. 407. 10 10 In further In embodiments, further embodiments, thethe heterodimeric heterodimeric protein protein according according to thetoinvention the invention comprisesany comprises anyofofthe thefurther furtherfeatures featuresdescribed described above above for for the the methods methods of production. of production.

Thus, inin aa further Thus, further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of theof the invention, invention,

said first said first polypeptide polypeptide is is aa full-length full-lengthheavy chain of heavy chain of an an antibody, antibody,preferably preferablya ahuman human 15 antibody. 15 antibody. In another In embodimentofofthe another embodiment theheterodimeric heterodimericprotein proteinofofthe theinvention, said invention, said secondpolypeptide second polypeptideis isa full-length a full-lengthheavy heavy chain chain of antibody, of an an antibody, preferably preferably a a human human antibody. antibody.

In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said 20 first 20 firstand andsecond secondpolypeptides polypeptidesare areboth both full-length heavy full-length heavychains chainsofoftwo twoantibodies, antibodies, preferably both preferably both human human antibodies antibodies thatthat bindbind different different epitopes, epitopes, and and thus thus the resulting the resulting

heterodimericprotein heterodimeric proteinis is a bispecific a bispecific antibody. antibody. This This bispecific bispecific antibody antibody can be can a be a heavy-chain antibody, heavy-chain antibody, ororananantibody antibody which which in addition in addition to heavy to the the heavy chains chains comprisestwo comprises twofull-length full-lengthlight light chains, chains, which whichmay may be be identical identical or or different. different.

25 25 In a further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of invention, of the the invention, the the Fc Fc region of region of the the first first polypeptide is of polypeptide is of an an isotype isotype selected selectedfrom from thethe group group consisting consisting of of IgG1, IgG2, IgG1, IgG2,IgG3 IgG3andand IgG4IgG4 (except (except for specified for the the specified mutations) mutations) and and the Fc the Fc region region of of the second the secondpolypeptide polypeptide is is of of an an isotype isotype selected selected fromfrom the group the group consisting consisting of of IgG1, IgG1, IgG2, IgG3 IgG2, IgG3and andIgG4 IgG4 (except (except for for the the specified specified mutations). mutations).

30 30 In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of invention, of the the invention, the the Fc Fc regions of regions of both both said said first first and and said said second polypeptidesareareofofthe second polypeptides theIgG1 IgG1 isotype. isotype.

In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of invention, of the the invention, one one of of the Fc the Fc regions regionsofofsaid saidpolypeptides polypeptidesis isofofthe theIgG1 IgG1 isotype isotype and and the other the other of IgG4 of the the IgG4 isotype. isotype.

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In aa further In further embodiment embodiment of heterodimeric of the the heterodimeric proteinprotein of the invention, of the invention, the the increased strength increased strength of of the the heterodimeric heterodimeric interaction interaction as as compared to each compared to eachofofthethe homodimeric homodimeric interactions interactions is is duedue to CH3 to CH3 modifications modifications other other thanintroduction than the the introduction of of covalent bonds, covalent bonds,cysteine cysteineresidues residuesor orcharged charged residues. residues.

5 5 In aa further In further embodiment embodiment of heterodimeric of the the heterodimeric proteinprotein of the invention, of the invention, the the heterodimeric interaction heterodimeric interaction between between said saidfirst first and andsecond second polypeptides polypeptides in in the the heterodimeric protein heterodimeric protein isissuch suchthat thatnonoFab-arm Fab-arm exchange can occur exchange can occur at at 0.5 0.5 mM GSH mM GSH under the under the conditions conditionsdescribed describedininExample Example 13. 13.

In aa further In further embodiment embodiment of heterodimeric of the the heterodimeric proteinprotein of the invention, of the invention, the the 10 heterodimeric 10 heterodimeric interaction interaction between between said and said first firstsecond and second polypeptides polypeptides in the resulting in the resulting

heterodimericprotein heterodimeric proteinisis such suchthat thatnonoFab-arm Fab-arm exchange exchange occursoccurs in in in vivo vivo in under mice mice under the conditions the conditions described describedininExample Example14.14.

In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said first CH3 first region comprises CH3 region comprisesa Phe a Phe at position at position 405 405 andamino and an an amino acid than acid other other Lys, than Lys,

15 LeuLeu 15 or or MetMet at position at position 409409 andand saidsaid second second CH3 CH3 region region comprises comprises an amino an amino acid acid other than other than Phe Pheatatposition position405 405and and a Lys a Lys at at position position 409. 409.

In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said first CH3 first region comprises CH3 region comprisesa Phe a Phe at position at position 405 405 and and an amino an amino acid than acid other other than Lys, Lys, Leu or Leu or Met Metatatposition position409 409 andand saidsaid second second CH3 region CH3 region comprises comprises Leu at a Leu ata position position 20 405405 20 andand a Lys a Lys at at position409. position 409. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said first CH3 first region comprises CH3 region comprisesPhePhe at position at position 405 405 andArganatArg and an at position position 409 409 and and said said secondCH3 second CH3region region comprises comprises a Leu a Leu at position at position 405 405 and aand Lys aatLys at position position 409. 409. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said 25 first 25 first CH3CH3 region region comprises comprises an amino an amino acidthan acid other other than Lys, Leu Lys, Leuatorposition or Met Met at 409 position 409 and said and said second second CH3 CH3region regioncomprises comprises a Lys a Lys at position at position 409409 and:and: a) Ile a) an an at Ile at position 350 position 350and anda Leu a Leu at position at position 405,405, or b)ora Thr b) aatThr at position position 370 and370 and a Leu at a Leu at position 405. position 405.

In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said 30 first 30 firstCH3 CH3region region comprises comprises an Arg an Arg at position at position 409 409 and second and said said second CH3 CH3 region region comprisesa aLys comprises Lysatatposition position409 409 and: and: a) Ile a) an an at at position Ile position 350 350 and aand Leu aatLeu at position position

405, or 405, or b) b) aa Thr Thr at at position 370 and position 370 anda aLeu Leuatatposition position405. 405. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said first CH3 first regioncomprises CH3 region comprises a Thr a Thr at position at position 350, 350, a Lys aatLys at position position 370, a 370, Phe ata Phe at 35 position 35 position405 405and and an an Argatatposition Arg position 409 409 and andsaid said second second CH3 CH3region region comprises comprises aa Lys Lys

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2022201608 08 Mar at position at position 409 and:a)a)ananIleIleatatposition 409 and: position350 350 andand a Leu a Leu at position 405,405, at position ora b) or b) Thra Thr at position at position 370 anda aLeu 370 and Leuatatposition position405. 405. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said first CH3 first regioncomprises CH3 region comprises a Thr a Thr at position at position 350, 350, a Lys aatLys at position position 370, a 370, Phe ata Phe at 5 position 5 position 405405 and and an at an Arg Argposition at position 409said 409 and andsecond said second CH3comprises CH3 region region comprises an Ile an Ile at position at position 350, 350, a aThr Thratatposition position370, 370, a Leu a Leu at position at position 405 a and 405 and at Lys Lys a at position position

409. 409. In aa further In further embodiment embodiment of of thethe heterodimeric heterodimeric protein protein of the of the invention, invention, neither neither

said first said first nor nor said said second comprises polypeptidecomprises second polypeptide a Cys-Pro-Ser-Cys a Cys-Pro-Ser-Cys sequence sequence in the in the 10 hinge 10 hinge region. region. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of theof the invention, invention, both both said first said first and said second and said secondpolypeptide polypeptide comprise comprise a Cys-Pro-Pro-Cys a Cys-Pro-Pro-Cys sequence sequence in the in the hinge region. hinge region. In aa further In further embodiment embodiment of the of the heterodimeric heterodimeric protein protein of the of the invention, invention, said said 15 first 15 first and/or and/or said said second second polypeptide polypeptide comprises comprises a mutation a mutation removingremoving the site the acceptor acceptor site for Asn-linked for glycosylation. Asn-linked glycosylation.

Target antigens Target antigens As explained As explainedabove, above,in inanan important important embodiment embodiment of the of the invention, invention, the heterodimeric the heterodimeric

20 protein 20 protein is aisbispecific a bispecific antibody antibody comprising comprising two variable two variable regions regions that differ that differ in binding in binding

specificity, i.e. specificity, i.e. bind binddifferent differentepitopes. epitopes. In principle, In principle, any combinationofofspecificities any combination specificities is is possible. possible. As As mentioned mentioned above, above,

bispecific antibodies bispecific can potentially antibodies can potentially be beused usedtotoovercome overcome some some of theof the limitations limitations of of monospecific antibodies.One monospecific antibodies. One possible possible limitation limitation of of a monospecific a monospecific antibody antibody is a lack is a lack

25 of specificity 25 of specificity forfor thethe desired desired target target cells cells due due to expression to expression of theoftarget the target antigen antigen on on other cell other cell types towardswhich types towards which no no antibody antibody binding binding is desired. is desired. For For example, example, a target a target

antigen overexpressed antigen overexpressedon on tumor tumor cells cells may may also also be expressed be expressed in healthy in healthy tissues tissues which which could result could result in in undesired side-effects upon undesired side-effects upon treatment with an treatment with an antibody antibodydirected directed against that against thatantigen. antigen.A bispecific A bispecific antibody antibody having having a further a further specificity specificity againstagainst a a 30 proteinwhich 30 protein which is exclusivelyexpressed is exclusively expressed on the on the target target typetype cellcell could could potentially potentially improvespecific improve specific binding bindingtototumor tumorcells. cells. Thus, in one Thus, in oneembodiment embodiment of invention, of the the invention, said said firstfirst and and second second epitopes epitopes are are located on located on the thesame same cell,e.g. cell, e.g.a tumor a tumor cell. cell. Suitable Suitable targets targets on tumor on tumor cells cells include, include,

but are but are not notlimited limitedto, to,the thefollowing: following:erbB1 erbB1 (EGFR), (EGFR), erbB2 erbB2 (HER2),(HER2), erbB3, erbB3, erbB4, erbB4, 35 MUC-1, 35 MUC-1, CD19, CD19, CD20, CD20, CD4, CD4, CD38, CD38, CD138, CD138, CXCR5, CXCR5, c-Met, c-Met, HERV-envelop HERV-envelop protein, protein,

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2022201608 08 Mar Bigh3,SPARC, periostin, Bigh3, periostin, SPARC, BCR, CD79, CD37, BCR, CD79, CD37,EGFrvIII, EGFrvIII, L1-CAM, AXL, L-CAM,AXL, Tissue Tissue Factor Factor (TF), CD74, (TF), CD74,EpCAM EpCAM and and MRP3.MRP3. Possible Possible combinations combinations of tumor of tumor cell cellinclude, targets targets include, but are but are not not limited limited to: to: erbB1 + erbB2, erbB1 + erbB2, erbB2 erbB2 + +erbB3, erbB3,erbB1 erbB1 + erbB3, + erbB3, CD19CD19 +

+ CD20, CD38 CD20, CD38 ++ CD34, CD34,CD4 CD4+ CXCR5, + CXCR5, CD38 CD38 + RANKL, + RANKL, CD38CD38 + CXCR4, + CXCR4, CD20 CD20 +

+ 5 CXCR4, 5 CXCR4, CD20 CD20 + CCR7, + CCR7, CD20 CD20 + CXCR5, + CXCR5, CD20 + CD20 RANKL, RANKL,+ erbB2 + erbB2 + AXL, AXL, erbB1 + erbB1 cMet, + cMet, erbB2 ++ c-Met, erbB2 c-Met, erbB2 erbB2 ++EpCAM, EpCAM, c-Met c-Met + AXL, + AXL, c-Met c-Met + TF, + TF, CD38 CD38 + CD20, + CD20, CD38 CD38 +

+ CD138. CD138. In aa further In further embodiment, said first embodiment, said first and and second second epitopes epitopes may be located may be located on on the same the same target target antigen, antigen, wherein wherein the thelocation location of of the the two two epitopes epitopes on onthe thetarget target 10 antigen 10 antigen is is such such thatbinding that bindingofofananantibody antibodytotoone oneepitope doesnot epitopedoes notinterfere interfere with with antibody binding antibody bindingtotothe theother otherepitope. epitope.InIna afurther further embodiment embodiment hereof, hereof, said first said first and and second homodimeric second homodimericproteins proteinsare areantibodies antibodiesthat thatbind bindto totwotwo differentepitopes different epitopes located on located on the thesame same target target antigen, antigen, but have but have a different a different mode-of-action mode-of-action for for killing killing the target the target cell, cell, e.g. e.g.a a tumor cell. For tumor cell. For example, in one example, in one embodiment, embodiment,thethetarget target 15 antigen 15 antigen is is erbB2 erbB2 (HER2) (HER2) andand the the bispecificantibody bispecific antibodycombines combines thepertuzumab the pertuzumab and and trastuzumab antigen-binding trastuzumab antigen-binding sites. sites. In In another embodiment,the another embodiment, thetarget targetantigen antigenisis erbB1 (EGFr) erbB1 (EGFr) and and the thebispecific bispecific antibody antibody combines combines the the zalutumumab zalutumumabandand nimotuzumab nimotuzumab antigen-binding antigen-binding sites. sites.

Bispecific antibodies Bispecific antibodiescan can also also be used asas mediators be used mediatorsto toretarget retargeteffector effector 20 mechanisms 20 mechanisms to disease-associatedtissues, to disease-associated tissues, e.g. e.g. tumors. tumors. Thus, Thus, inin a afurther further embodiment, embodiment, said said firstororsaid first saidsecond second epitope epitope is located is located on a on a tumor tumor cell, as cell, such such a as a tumorcell tumor cell protein protein or or tumor tumorcell cellcarbohydrate, carbohydrate,andand the the other other epitope epitope is located is located on on an an effectorcell. effector cell. In one In embodiment, one embodiment, the the effector effector cellis isa aT Tcell. cell cell. 25 25 Possible targets Possible targets ononeffector effectorcells cellsinclude includethe thefollowing: following:FcgammaRI FcgammaRI (CD64): (CD64):

expressed on expressed on monocytes monocytesand andmacrophages macrophages andand activated activated neutrophils;FcgammaRIII neutrophils; FcgammaRIII (CD16): expressed (CD16): expressed on on natural natural killer killer and and macrophages; macrophages; CD3: expressed CD3: expressed on circulating on circulating

T cells; CD89: T cells; expressedononPMNPMN CD89: expressed (polymorphonuclear (polymorphonuclear neutrophils), neutrophils), eosinophils, eosinophils, monocytes and monocytes andmacrophages; macrophages; CD32a: CD32a: expressed expressed on macrophages, on macrophages, neutrophils, neutrophils, 30 eosinophils;FcERI 30 eosinophils; FcRIexpressed expressedon on basophils basophils andand mast mast cells.InInone cells. oneembodiment embodiment the the epitope is epitope is located on CD3 located on CD3expressed expressed oncells. on T T cells. In another In another embodiment, embodiment,thethe firstantibody first antibody hashas binding binding specificityforfora specificity a pathogenic microorganism pathogenic microorganism and andthe thesecond secondantibody antibody hashas binding binding specificity for specificity for an an effector cell effector cellprotein, protein,such suchasasCD3, CD3, CD4, CD4, CD8, CD40,CD25, CD8, CD40, CD25,CD28, CD28, CD16, CD16, CD89,CD89, 35 CD32, 35 CD32, CD64, CD64, FcERIor FcERI CD1. or CD1.

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bispecificantibodies Furthermore,bispecific Furthermore, antibodiescancan usedused be be to target to target a chemotherapeutic a chemotherapeutic

agent more agent more specificallyto to specifically thethe cells cells on which on which the agent the agent should should act.inThus, act. Thus, one in one embodiment,one embodiment, oneofofthe thehomodimeric homodimeric proteins proteins is is an an antibody antibody which which recognizes recognizes a a small molecule small moleculeororpeptide, peptide,or or is isable ableto toform form bondbond a covalent a covalent with with such asuch a molecule, molecule,

5 e.g.e.g. 5 according according to the to the principle principle described described in Rader in Rader et al,et(2003) al, (2003) PNAS 100:5396. PNAS 100:5396. In a In a further embodiment further embodiment of the of the method method of theof the invention, invention, theantibody the first first antibody has has binding binding specificity for specificity for(i.e. binds (i.e. bindstotoananepitope epitope on) on) aa tumor cell or tumor cell or tumor tumor cell surface protein, cell surface protein, such as such as erbB1, erbB1, erbB2, erbB2, erbB3, erbB3, erbB4, erbB4,EGFR3vIII, CEA,MUC-1, EGFR3vIII,CEA, MUC-1, CD19, CD19, CD20, CD20, CD4, CD4, CD38, EPCAM, CD38, EPCAM,c-Met, c-Met,AXL, AXL,L1-CAM, Tissue L-CAM,Tissue Factor,CD74 Factor, CD74or or CXCR5 CXCR5 and and the the second second 10 antibody 10 antibody hashas a binding a binding specificityfor specificity for a achemotherapeutic chemotherapeuticagent, agent,such such as as a toxin a toxin (including radiolabelled peptide), a radiolabelled (including a peptide), aa drug drug or or aa prodrug. prodrug. Bispecific antibodies Bispecific mayalso antibodies may alsobe be usedused to target to target a vesicle, a vesicle, e.g.electron e.g. an an electron densevesicles, dense vesicles,ororminicell minicell containing containinga atoxin, toxin,drug drug or or prodrug prodrug to atotumor. a tumor. See See e.g. e.g. MacDiarmidet et MacDiarmid al.al.(2009) (2009) Nature Nature Biotech Biotech 27:643. 27:643. Minicells Minicells are achromosomal are achromosomal cells cells 15 that 15 that areare products products ofofaberrant aberrantcell cell division division which which do do not not contain contain chromosomal DNA. chromosomal DNA. Thus, in Thus, in another anotherembodiment, embodiment, wherein wherein said first said first or said or said second second epitope epitope is located is located on on tumorcell, a tumor a cell, such suchasasa atumor tumor proteinor or cellprotein cell tumor tumor cellcell carbohydrate, carbohydrate, andother and the the other epitope is epitope is located located on on an an electron electron dense densevesicle vesicleororminicell. minicell. Furthermore,serum Furthermore, serum half-lifeofofan an half-life antibody antibody may may be altered be altered by including by including in a in a 20 bispecific 20 bispecific antibody antibody a binding a binding specificity specificity forfor a serum a serum protein. protein. For instance, For instance, serumserum half- half life may life beprolonged may be prolongedby by including including in ainbispecific a bispecific antibody, antibody, a binding a binding specificity specificity for for serumalbumin. serum albumin. Thus, Thus, in ainfurther a further embodiment embodiment of the of the of method method of the invention, the invention, the the first antibody first has binding antibody has bindingspecificity specificity for for aa tumor tumorcell cellorortumor tumor cellcell protein, protein, such such as as erbB1 (EGFR), erbB1 (EGFR), erbB2 erbB2 (HER2), (HER2), erbB3, erbB3, erbB4, erbB4, MUC-1, MUC-1, CD19, CD20, CD4, CD19, CD20, CD4, CD38, CD38, 25 CD138, 25 CD138, CXCR5, CXCR5, c-Met, c-Met, HERV-envelope HERV-envelope protein, protein, periostin, periostin, Bigh3, Bigh3, SPARC, SPARC, BCR, BCR, CD79, CD79, CD37, EGFrvIII, CD37, EGFrvIII, L1-CAM, AXL, Tissue Li-CAM, AXL, Tissue Factor Factor (TF), (TF), CD74, EpCAMororMRP3, CD74, EpCAM MRP3, CEA CEA andand the second the secondantibody antibody has has a binding a binding specificity specificity for a for a blood blood protein, protein, such assuch serumas serum albumin. A second albumin. A second binding binding specificity specificity can alsocanbealso usedbe to used to an target target an antibody antibody to a to a specific tissue, specific tissue, such as the such as the central central nervous nervous system system or brain or brain (across (across the blood the blood brain brain 30 barrier). 30 barrier).Thus, Thus,inina afurther further embodiment embodimentof of thethe method method of the of the invention, thethe invention, first first antibody has antibody hasbinding bindingspecificity specificityfor for aa brain-specific brain-specific target, target, such as amyloid-beta such as amyloid-beta(e.g. (e.g. for treatment for treatmentofofAlzheimer's Alzheimer's disease), disease), Her-2 Her-2 (e.g. (e.g. for treatment for treatment of cancer of breast breast cancer metastasesininbrain), metastases brain),EGFr EGFr(e.g. (e.g.for fortreatment treatmentof of primary primary brain brain cancer), cancer), NogoNogo A (e.g. A (e.g.

for treatment for treatmentofofbrain braininjury), injury),TRAIL TRAIL (e.g. (e.g. for treatment for treatment of HIV), of HIV), alpha-synuclein alpha-synuclein

35 (e.g. 35 (e.g.forfortreatment treatmentof ofParkinson), Parkinson),Htt Htt(e.g. (e.g. for for treatment treatment ofof Huntington), Huntington), aa prion prion

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(e.g. (e.g. for for treatment treatment ofofmad madCOWcow disease), disease), a West a West Nile virus Nile virus protein, protein, andsecond and the the second antibody has antibody hasa abinding bindingspecificity specificity for for aa blood blood brain brain barrier barrier protein, protein, such suchasastransferrin transferrin receptor (TfR), receptor (TfR),insulin insulin receptor, receptor, melanotransferrin melanotransferrin receptor receptor (MTfR), lactoferrin (MTfR), lactoferrin

receptor (LfR), receptor (LfR), Apolipoprotein Apolipoprotein E Ereceptor receptor2 2(ApoER2), (ApoER2), LDL-receptor- LDL-receptor- related related protein protein 1 1 5 andand 5 2 (LRP1 2 (LRP1 and and LRP2), LRP2), receptor receptor for for advanced advanced glycosylation glycosylation end-products end-products (RAGE), (RAGE), diphtheria toxin diphtheria toxin-receptor -receptor= heparin-binding = heparin-binding epidermal epidermal growth factor-like growth factor-like growth growth factor (DTR factor (DTR = =HB-EGF), HB-EGF), gp190 gp190 (Abbott (Abbott et al,etNeurobiology al, Neurobiology of Disease of Disease 37 13- 37 (2010) (2010) 13 25). 25).

bindingspecificity AA binding specificityfor for a ablood blood brain brain barrier barrier protein protein can be can also alsoused be toused to 10 target 10 target another, another, non-antibody, non-antibody, molecule, molecule, to to a specifictissue, a specific tissue,such suchasasthe thecentral central nervous systemororbrain nervous system brain(across (across thethe blood blood brain brain barrier). barrier). Thus, Thus, in ainfurther a further embodiment,one embodiment, oneofofthethehomodimeric homodimeric proteins proteins is full-length is a antibodyhaving a full-length antibody havinga a binding specificity binding specificity for for aa blood bloodbrain brainbarrier barrierprotein protein (such (such as TfR, as TfR, insulin insulin receptor, receptor,

MTfR, LfR, MTfR, LfR, ApoER2, LRP1, LRP2, ApoER2, LRP1, LRP2,RAGE, RAGE,DTRDTR (= (= HB-EGF) HB-EGF) or gp190) or gp190) and other and the the other 15 homodimeric 15 homodimeric protein protein is Fc is an an region Fc region linked linked at the at the N- orN-C-terminus or C-terminus to another to another protein, such protein, asaa cytokine, such as cytokine,a asoluble solublereceptor receptor or or other other protein, protein, e.g. e.g. VIPVIP (vasoactive (vasoactive

intestinal peptide), intestinal peptide), BDNF (brain-derivedneurotrophic BDNF (brain-derived neurotrophic factor), factor), FGF FGF (fibroblast (fibroblast growth growth

factor), multiple factor), multiple FGFs, FGFs,EGF EGF (epidermal (epidermal growth growth factor), factor), PNA (peptide PNA (peptide nucleic nucleic acid), acid), NGF(Nerve NGF (Nervegrowth growth factor), factor), Neurotrophin Neurotrophin (NT)-3, (NT)-3, NT-4/5, NT-4/5, glial derived glial derived neurotrophic neurotrophic

20 factor, 20 factor, ciliary ciliary neurotrophic neurotrophic factor, factor, neurturin, neurturin, neuregulins, neuregulins, interleukins, interleukins, transforming transforming

growth factor growth factor (TGF)-alpha, (TGF)-alpha, TGF-beta, TGF-beta,erythropoietin, erythropoietin, hepatocyte hepatocytegrowth growth factor, factor, platelet derived platelet growthfactor, derived growth factor,artemin, artemin,persephin, persephin, netrins,cardiotrophin-1, netrins, cardiotrophin-1, stem stem cellcell

factor, midkine, factor, midkine,pleiotrophin, pleiotrophin,bone bone morphogenic morphogenic proteins, proteins, saposins, saposins, semaphorins, semaphorins,

leukocyte inhibitory leukocyte inhibitoryfactor, factor,alpha-L-iduronidase, alpha-L-iduronidase, iduronate-2-sulfatase, iduronate-2-sulfatase, N-acetyl N-acetyl-

25 galactosamine-6-sulfatase, arylsulphatase 25 galactosamine-6-sulfatase, arylsulphatase B, B, acid acidalpha-glucosidase, alpha-glucosidase,or or sphingomyelinase sphingomyelinase (Pardridge, (Pardridge, Bioparmaceutical Bioparmaceutical drug targeting drug targeting to the to the Journal brain, brain, Journal of Drug of Drug Targeting Targeting 2010, 2010,1-11; 1-11;Pardridge, Pardridge,Re-engineering Re-engineeringBiopharmaceuticals Biopharmaceuticalsforfor delivery to delivery to brain brain with withmolecular molecular Trojan Trojan horses. horses. Bioconjugate Bioconjugate Chemistry Chemistry 2008, 19:2008, 19: 1327-1338. 1327-1338. 30 30 Moreover,a asecond Moreover, second binding binding specificity specificity can can be to be used used to target target blood clotting blood clotting

factors to factors to aaparticular desired particulardesired site site of action. of action. For example, For example, a bispecific a bispecific antibody antibody

having having a a firstbinding first binding specificity specificity for for a tumor a tumor cella second cell and and a binding secondspecificity binding specificity for a for a blood clotting blood clotting factor factor could coulddirect directblood blood clotting clotting to to a tumor, a tumor, and stop and thus thustumor stop tumor growth. Thus, growth. Thus, in in aa further further embodiment embodimentofofthe themethod method of of thethe invention,thethe invention, first first 35 antibody 35 antibody has binding has binding specificity specificity for afor a tumor tumor celltumor cell or or tumor cell protein, cell protein, such such as erbB1, as erbB1,

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erbB2, erbB3, erbB2, erbB3, erbB4, erbB4, MUC-1, MUC-1, CD19, CD20,CD4 CD19,CD20, CD4 or or CXCR5 CXCR5 and and the the second second antibody antibody hasaabinding has binding specificity specificity for for a protein a protein involved involved in clotting, in blood blood clotting, such factor. such as tissue as tissue factor. Further particularly Further particularly interesting interesting binding bindingspecificity specificity combinations combinations include: include: CD3 CD3 2022201608 08

+ HER2, + HER2, CD3 CD3+ +CD20, CD20, IL-12 IL-12 + IL18, + IL18, IL-la IL-1a + IL-1b, + IL-1b, VEGF VEGF + EGFR, + EGFR, EpCAM EpCAM + CD3, + CD3, 5 GD2 5 GD2+ CD3,GD3+ + CD3, CD3, GD3 + CD3, HER2+ HER2 CD64, + CD64, EGFR+ EGFR CD64,CD30 + CD64, CD30+ CD16,NG2 + CD16, NG2+ CD28, + CD28, HER2 ++HER3, HER2 HER3,CD20 CD20 + CD28, + CD28, HER2 HER2 + CD16, + CD16, Bcl2 Bcl2 + CD3, + CD3, CD19 CD19 CD3,+ CEA + CD3,+ CEA CD3, + CD3, EGFR ++ CD3, EGFR CD3,IgE IgE ++ CD3, CD3,EphA2 EphA2+ +CD3, CD3, CD33 CD33 + CD3, + CD3, MCSPMCSP + CD3, + CD3, PSMA PSMA CD3, TF + CD3,+ TF

+ CD3, CD19 + CD3, CD16, CD19 CD19 ++ CD16, CD19 ++ CD16a, CD30 ++ CD16a, CD16a, CD30 CD16a,CEA CEA+ +HSG, HSG,CD20 CD20+ +HSG, HSG, MUC1 + +HSG, MUC1 HSG,CD20 CD20 + CD22, + CD22, HLA-DR HLA-DR + CD79, + CD79, PDGFRPDGFR + VEGF, + VEGF, IL17a IL17a + IL23, IL23, CD32b + CD32b 10 + CD25, 10 + CD25, CD20 CD20 + CD38, + CD38, HER2HER2 + AXL, + AXL, CD89CD89 + HLA+ class HLA class II, II, CD38+CD138, CD38+CD138, TF +TF

+ cMet, Her2 cMet, Her2 ++ EpCAM, EpCAM,HER2 HER2 + HER2, + HER2, EGFR EGFR + EGFR, + EGFR, EGFR + EGFR + c-Met c-Met, c-Met, + c-Met non- + non binding arm binding armand andcombinations combinations of G-protein of G-protein coupled coupled receptors. receptors.

In aa futher In futher embodiment, embodiment,the the bispecific bispecific antibodies antibodies according according to thetoinvention the invention may be may be used usedtotoclear clear pathogens, pathogens, pathogenic pathogenic autoantibodies autoantibodies or or harmful harmful compounds compounds 15 such 15 such as venoms as venoms and toxins and toxins from from the the circulation circulation by targeting by targeting to erythrocytes to erythrocytes essentially as essentially described inin Taylor as described Tayloretetal. al. J. Immunol.158:842-850 J. Immunol. 158:842-850 (1997) (1997) and Taylor and Taylor

and Ferguson, and Ferguson,J. J.Hematother. Hematother. 4:357-362, 4:357-362, 1995.first 1995. Said Saidepitope first epitope is located is located on an on an erythrocyte (red erythrocyte (redblood blood cell)protein cell) protein including, including, but but not limited not limited to, erythrocyte to, the the erythrocyte complementreceptor complement receptor1 1and and said said second second epitope epitope is located is located on on thethe compound compound or or 20 organism 20 organism to to be be targetedfor targeted forclearance. clearance. In aa further In further embodiment, thesecond embodiment, the secondFab-arm Fab-arm comprises comprises a fusion a fusion protein protein representingananautoantigen representing autoantigen orconjugation or a a conjugation site site to attach to attach an autoantigen an autoantigen such assuch as dsDNA. Targeting dsDNA. Targeting ofofpathogens, pathogens, autoantibodies autoantibodies or harmful or harmful compounds compounds by the by the bispecific antibodies bispecific of the antibodies of theinvention invention followed followed by erythrocyte-mediated by erythrocyte-mediated clearance clearance

25 maymay 25 thusthus havehave therapeutic therapeutic utility in utility in the the treatment treatment of of various various diseases diseases and and syndromes. syndromes.

Conjugation Conjugation In further In embodiments further embodiments of of thethe invention, invention, thethe first first and/or and/or second second homodimeric homodimeric

30 proteinis islinked 30 protein linkedtotoa compound a compound selected selected fromfrom the group the group consisting consisting of: aof: a toxin toxin (including aa radioisotope) (including radioisotope) aa prodrug prodrugor or a drug. a drug. Such Such compound compound may may make makeof killing killing of target cells target cells more effective, e.g. more effective, e.g. in in cancer cancer therapy. therapy. The resulting heterodimeric The resulting heterodimeric protein is thus protein is an immunoconjugate. thus an immunoconjugate.The The compound compound may alternatively may alternatively be tocoupled to be coupled

the resulting the resulting heterodimeric heterodimericprotein, protein,i.e. i.e. after after the Fab-armexchange the Fab-arm exchange has has taken taken place. place.

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2022201608 08 Mar Suitable compounds Suitable forming immunoconjugates for forming compounds for thepresent immunoconjugatesof ofthe presentinvention invention include taxol, include taxol, cytochalasin cytochalasin B,B,gramicidin gramicidinD, D, ethidium ethidium bromide, bromide, emetine, emetine, mitomycin, mitomycin,

etoposide, tenoposide, etoposide, tenoposide,vincristine, vincristine,vinblastine, vinblastine,colchicin, colchicin,doxorubicin, doxorubicin, daunorubicin, daunorubicin,

dihydroxy anthracin dihydroxy anthracin dione, dione, mitoxantrone, mitoxantrone, mithramycin, actinomycin D, mithramycin, actinomycin D, 1-dehydro- 1-dehydro 5 testosterone, 5 testosterone,glucocorticoids, glucocorticoids, procaine, procaine,tetracaine, tetracaine,lidocaine, lidocaine, propranolol, propranolol,and and puromycin, antimetabolites (such puromycin, antimetabolites (such as methotrexate, as methotrexate, 6-mercaptopurine, 6-mercaptopurine, 6-thioguanine,cytarabine, 6-thioguanine, cytarabine, fludarabin, fludarabin, 5-fluorouracil, 5-fluorouracil, decarbazine, decarbazine, hydroxyurea, hydroxyurea,

asparaginase,gemcitabine, asparaginase, gemcitabine, cladribine), cladribine), alkylating alkylating agents agents (such (such as mechlorethamine, as mechlorethamine,

thioepa, chlorambucil, thioepa, chlorambucil, melphalan, carmustine (BSNU), melphalan, carmustine (BSNU), lomustine lomustine (CCNU), (CCNU), 10 cyclophosphamide, 10 cyclophosphamide, busulfan, busulfan, dibromomannitol, dibromomannitol, streptozotocin, streptozotocin, dacarbazine dacarbazine (DTIC), (DTIC), procarbazine, mitomycin procarbazine, mitomycin C,C,cisplatin cisplatin and andother other platinum platinum derivatives, derivatives, such such as as carboplatin), antibiotics carboplatin), antibiotics (such (such asasdactinomycin dactinomycin (formerly (formerly actinomycin), actinomycin), bleomycin, bleomycin,

daunorubicin (formerly daunorubicin (formerlydaunomycin), daunomycin), doxorubicin, doxorubicin, idarubicin, idarubicin, mithramycin, mithramycin, mitomycin, mitoxantrone, mitomycin, mitoxantrone, plicamycin, plicamycin, anthramycin anthramycin(AMC)), (AMC)),diphtheria diphtheriatoxin toxinandand 15 related 15 relatedmolecules molecules (such (such as diphtheria as diphtheria A chain A chain and and active active fragments fragments thereof thereof and and hybrid molecules), hybrid molecules),ricin ricin toxin toxin (such (such asasricin ricin AA or or aa deglycosylated ricin AA chain deglycosylatedricin chain toxin), toxin), cholera toxin, cholera toxin, aa Shiga-like Shiga-liketoxin toxin(SLT-I, (SLT-I,SLT-II, SLT-II, SLT-IIV), SLT-IIV), LT toxin, LT toxin, C3 toxin, C3 toxin, ShigaShiga

toxin, pertussis toxin, pertussis toxin, toxin, tetanus tetanus toxin, toxin, soybean Bowman-Birk soybean Bowman-Birk protease protease inhibitor, inhibitor, Pseudomonas Pseudomonas exotoxin, exotoxin, alorin, alorin, saporin, saporin, modeccin, modeccin, gelanin, gelanin, abrinabrin A chain, A chain, modeccin modeccin A A 20 chain, 20 chain, alpha-sarcin, alpha-sarcin, Aleurites Aleurites fordii fordii proteins, proteins, dianthin dianthin proteins, proteins, Phytolacca Phytolacca americana americana

proteins (PAPI, proteins (PAPI, PAPII, PAPII,andand PAP-S), PAP-S), momordica momordica charantia charantia inhibitor, inhibitor, curcin, curcin, crotin, crotin, sapaonariaofficinalis sapaonaria officinalis inhibitor, inhibitor, gelonin, gelonin,mitogellin, mitogellin,restrictocin, restrictocin,phenomycin, phenomycin, and and enomycintoxins. enomycin toxins.Other Other suitable suitable conjugated conjugated molecules molecules include include ribonuclease ribonuclease (RNase), (RNase),

DNaseI,I,Staphylococcal DNase Staphylococcal enterotoxin-A, enterotoxin-A, pokeweed pokeweed antiviral antiviral protein, protein, diphtherin diphtherin toxin,toxin,

25 Pseudomonas 25 Pseudomonas endotoxin, endotoxin, Maytansinoids, Maytansinoids, Auristatins Auristatins (MMAE, (MMAE, MMAF), MMAF), Calicheamicins Calicheamicins and Duocarmycin and Duocarmycinanalogs analogs(Ducry (Ducryand andStump, Stump, Bioconjugate Bioconjugate Chem. Chem. 2010, 2010, 21: 21: 5-13), 5-13), Dolostatin-10, Dolostatin-10, Dolostatin-15, Dolostatin-15,Irinotecan Irinotecanoror its active metabolite its active metabolite SN38, SN38, pyrrolobenzodiazepines(PBD's). pyrrolobenzodiazepines (PBD's).

In aa further In further embodiment embodiment ofofthe theinvention, invention, the thefirst first and/or and/or second second 30 homodimeric 30 homodimeric protein protein is linked is linked to alpha to an an alpha emitter, emitter, including including but but not not limited limited to to Thorium-227, Radium-223, Thorium-227, Radium-223,Bismuth-212, Bismuth-212,and andActinium-225. Actinium-225. In aa further In further embodiment embodiment ofofthe theinvention, invention, the thefirst first and/or and/or second second homodimeric homodimeric protein protein is linked is linked to a to beta emitting a emitting beta radionuclide, radionuclide, includingincluding but not but not limited to limited to Iodium-313, Iodium-313, Yttrium-90, Yttrium-90, Fluorine-18, Fluorine-18, Rhenium-186, Rhenium-186, Gallium-68, Gallium-68, 35 Technetium-99, 35 Technetium-99, Indium-111, Indium-111, and and Lutetium-177. Lutetium-177.

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another embodiment, In another In the compound embodiment, the conjugatedcomprises compoundtotobebeconjugated comprises a nucleic a nucleic acid or acid or nucleic nucleic acid-associated acid-associatedmolecule. molecule.In Inoneone such such facet facet of the of the present present invention, invention,

the conjugated the conjugatednucleic nucleicacid acidisisa acytotoxic cytotoxicribonuclease, ribonuclease, an an antisense antisense nucleic nucleic acid, acid, an an inhibitory RNA inhibitory RNAmolecule molecule (e.g., (e.g., a siRNA a siRNA molecule) molecule) or an or an immunostimulatory immunostimulatory nucleic nucleic 5 acid 5 acid(e.g., (e.g., an an immunostimulatory immunostimulatoryCpG CpGmotif-containing motif-containing DNA DNAmolecule). molecule). Any method Any method known known in the in the art for art for conjugating conjugating may may be be employed, employed, including including the the methods described methods describedby by Hunter Hunter et al., et al., Nature Nature 144, 144, 945 (1962), 945 (1962), David David et al., et al., Biochemistry 13, Biochemistry 1014 (1974), 13, 1014 (1974), Pain Pain et et al., al., J.J.Immunol. Meth. 40, Immunol. Meth. 40, 219 (1981) and 219 (1981) and Nygren, J. Nygren, J. Histochem. Histochem. and and Cytochem. 30, 407 Cytochem. 30, 407 (1982). (1982). Conjugates Conjugates may maybebeproduced produced 10 by chemically 10 by chemically conjugating conjugating the other the other moiety moiety to the to the N-terminal N-terminal side or C-terminal side or C-terminal side side of the of the protein protein (see, (see, e.g., e.g., Antibody AntibodyEngineering EngineeringHandbook, Handbook, edited edited by by Osamu Osamu Kanemitsu, published Kanemitsu, published by Chijin Shokan by Chijin Shokan (1994)). (1994)). Such Such conjugated conjugated antibody antibody derivatives may derivatives mayalso also be generated be generated by conjugation by conjugation at internal at internal residues residues or sugars, or sugars,

where appropriate. where appropriate. The The agents agentsmay may be be coupled coupled either either directlyororindirectly directly indirectly to to aa 15 protein 15 protein of the of the present present invention. invention. One example One example of indirect of indirect coupling coupling of a agent of a second second agent is coupling is by aa spacer coupling by spacermoiety. Linkingtechnologies moiety.Linking technologiesfor fordrug-conjugates drug-conjugateshave have recently been recently been summarized by Ducry summarized by Ducry and and Stump Stump(2010) (2010)Bioconjugate Chem.21:21:5. 5. Bioconjugate Chem.

Compositions and Compositions and uses uses 20 In In 20 a further a further main main aspect, aspect, the the invention invention relates relates to to a pharmaceutical a pharmaceutical composition composition comprisinga aheterodimeric comprising heterodimeric protein protein according according to thetoinvention the invention as described as described herein herein and aa pharmaceutically-acceptable and pharmaceutically-acceptable carrier. carrier.

The pharmaceutical The pharmaceutical compositions compositionsmaymay be formulated be formulated in accordance in accordance with with conventional techniques conventional techniques such such as as those those disclosed disclosed in in Remington: Remington: The TheScience Science andand 25 Practice 25 PracticeofofPharmacy, Pharmacy, 19th 19th Edition,Gennaro, Edition, Gennaro,Ed., Ed.,Mack MackPublishing PublishingCo., Co., Easton, Easton, PA, PA, 1995. AA pharmaceutical 1995. pharmaceuticalcomposition compositionof of thethe present present invention invention may may e.g. e.g. include include diluents, fillers, diluents, fillers, salts, salts, buffers, buffers, detergents (e. g.,g.,a nonionic detergents (e. a nonionic detergent, detergent, such such as as Tween-20ororTween-80), Tween-20 Tween-80), stabilizers(e. stabilizers (e. g., g., sugars sugars ororprotein-free protein-free amino aminoacids), acids), preservatives, tissue preservatives, tissuefixatives, fixatives,solubilizers, solubilizers,and/or and/or otherother materials materials suitable suitable for for 30 inclusion 30 inclusion in ainpharmaceutical a pharmaceutical composition. composition.

Pharmaceuticallyacceptable Pharmaceutically acceptable carriers carriers include include anyall any and andsuitable all suitable solvents, solvents,

dispersion media, dispersion media,coatings, coatings, antibacterial antibacterial andand antifungal antifungal agents, agents, isotonicity isotonicity agents, agents,

antioxidants and antioxidants andabsorption absorption delaying delaying agents, agents, andlike and the thethat likeare that are physiologically physiologically

compatiblewith compatible witha acompound compound of present of the the present invention. invention. Examples Examples of suitable of suitable aqueous aqueous

35 andand 35 nonaqueous nonaqueous carriers carriers which which maymay be employed be employed in the in the pharmaceutical pharmaceutical compositions compositions

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of the of the present presentinvention include inventioninclude water, water, saline, saline, phosphate phosphate buffered saline,saline, buffered ethanol, ethanol,

dextrose, polyols dextrose, polyols (such (such as as glycerol, glycerol, propylene propylene glycol, glycol, polyethylene polyethylene glycol). glycol). Pharmaceuticallyacceptable Pharmaceutically acceptable carriers carriers include include sterileaqueous sterile aqueous solutions solutions or dispersions or dispersions

and sterile and sterile powders powdersfor forthetheextemporaneous extemporaneous preparation preparation of sterile of sterile injectable injectable 5 solutions 5 solutions or dispersion. or dispersion. Proper Proper fluidity fluidity maymay be maintained, be maintained, for example, for example, by the by usethe of use of 2022201608 coating materials, coating materials, such suchasaslecithin, lecithin,bybythe themaintenance maintenance of required of the the required particle particle size size in in the the case of dispersions, case of dispersions, and bythe and by theuse useofofsurfactants. surfactants. Pharmaceutical compositions Pharmaceutical compositions of of the the present presentinvention invention may may also also comprise comprise pharmaceuticallyacceptable pharmaceutically acceptable antioxidants antioxidants for for instance instance (1) (1) water water soluble soluble antioxidants, antioxidants,

10 suchsuch 10 as ascorbic as ascorbic acid,acid, cysteine cysteine hydrochloride, hydrochloride, sodium sodium bisulfate, bisulfate, sodiumsodium metabisulfite, metabisulfite,

sodiumsulfite sodium sulfite and andthe thelike; like;(2) (2)oil-soluble oil-solubleantioxidants, antioxidants,such such as as ascorbyl ascorbyl palmitate, palmitate,

butylated hydroxyanisole, butylated hydroxyanisole, butylated butylated hydroxytoluene, hydroxytoluene, lecithin, lecithin, propyl propyl gallate, gallate, alpha-alpha

tocopherol, and tocopherol, the like; and the like; and and (3) (3) metal metalchelating chelatingagents, agents, such such as citric as citric acid, acid, ethylenediaminetetraacetic ethylenediamine tetraaceticacid acid(EDTA), (EDTA), sorbitol, sorbitol, tartaric tartaric acid,phosphoric acid, phosphoric acid, acid, and and

15 thethe 15 like. like. Pharmaceutical compositions Pharmaceutical compositions of of the the present presentinvention inventionmay may also also comprise comprise isotonicity agents, isotonicity such asassugars, agents, such sugars,polyalcohols, polyalcohols, such such as mannitol, as mannitol, sorbitol, sorbitol, glycerol glycerol

or sodium or sodiumchloride chlorideinin the thecompositions. compositions. The pharmaceutical The pharmaceutical compositions compositions of of the the present present invention invention may may also also contain contain 20 20 oneone or more or more adjuvants adjuvants appropriate appropriate for for thethe chosen chosen route route of administration of administration such such as as preservatives, wetting preservatives, wettingagents, agents, emulsifying emulsifying agents, agents, dispersing dispersing agents, agents, preservatives preservatives

or buffers, or buffers, which which may may enhance enhance the shelf the shelf life life or effectiveness or effectiveness of pharmaceutical of the the pharmaceutical composition.The composition. Thecompounds compounds of present of the the present invention invention may bemay be prepared prepared with with carriers carriers that will that will protect protect the the compound compound against against rapidrapid release, release, such such as as a controlled a controlled release release 25 formulation,including 25 formulation, including implants, implants, transdermal transdermal patches, patches, and microencapsulated and microencapsulated delivery systems. delivery systems.Such Such carriers carriers maymay include include gelatin, gelatin, glyceryl glyceryl monostearate, monostearate, glyceryl glyceryl

distearate, biodegradable, distearate, biodegradable,biocompatible biocompatible polymers polymers such such as as ethylene ethylene vinyl vinyl acetate, acetate, polyanhydrides,polyglycolic polyanhydrides, polyglycolicacid, acid,collagen, collagen,polyorthoesters, polyorthoesters,andand polylactic polylactic acidalone acid alone or with or with aa wax, wax,ororother othermaterials wellknown materialswell known in the in the art.art. Methods Methods for preparation for the the preparation 30 of such 30 of such formulations formulations are generally are generally known known to skilled to those those skilled in the inart. the art. Sterile injectable Sterile injectablesolutions solutionsmay may be prepared bybyincorporating be prepared incorporating the theactive active compound inin the compound the required required amount amount inin ananappropriate appropriate solvent solvent with with one one or or a a combination of combination of ingredients ingredients e.g. e.g. as as enumerated enumeratedabove, above, as as required, required, followed followed by by sterilization microfiltration. sterilization microfiltration.

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2022201608 08 Mar The actual The levels ofof the dosagelevels actual dosage theactive active ingredients in the ingredients in pharmaceutical the pharmaceutical compositionsmay compositions may be be varied varied so to so as as obtain to obtain an amount an amount of theofactive the active ingredient ingredient which which is effective is to achieve effective to achievethethe desired desired therapeutic therapeutic response response for a particular for a particular patient,patient,

composition,and composition, and mode mode of administration, of administration, without without beingtotoxic being toxic to the patient. the patient. The The 5 selected 5 selected dosage dosage levellevel will will depend depend upon upon a a variety variety of pharmacokinetic of pharmacokinetic factors factors including including

the activity the activity of of the the particular compositions particular compositions of of the the present present invention invention employed, employed, the the route of route of administration, administration,thethe time time of administration, of administration, the ofrate the rate of excretion excretion of the of the particular compound particular being employed, compound being employed,the theduration duration ofofthe thetreatment, treatment, other other drugs, drugs, compoundsand/or compounds and/ormaterials materialsused usedinincombination combinationwith withthe theparticular particular compositions compositions 10 employed, 10 employed, the sex, the age, age,weight, sex, weight, condition, condition, general general health health and priorand prior history medical medical history of the of the patient being treated, patient being treated, and andlike like factors factors well well known knownininthe themedical medicalarts. arts. The pharmaceutical The pharmaceutical composition composition may maybebeadministered administeredbybyanyany suitableroute suitable route and mode. and mode.In Inoneone embodiment, embodiment, a pharmaceutical a pharmaceutical composition composition of the of the present present invention isis administered invention administeredparenterally. parenterally. "administered "administered parenterally" parenterally" as herein as used used herein 15 means 15 means modesmodes of administration of administration other other than than enteral enteral and administration, and topical topical administration, usually usually by injection, by injection, and and include includeepidermal, epidermal,intravenous, intravenous, intramuscular, intramuscular, intraarterial, intraarterial, intrathecal, intracapsular, intrathecal, intracapsular, intraorbital, intraorbital,intracardiac, intracardiac,intradermal, intradermal, intraperitoneal, intraperitoneal,

intratendinous, transtracheal, intratendinous, transtracheal, subcutaneous, subcutaneous, subcuticular, subcuticular, intraarticular,subcapsular, intraarticular, subcapsular, subarachnoid,intraspinal, subarachnoid, intraspinal, intracranial, intracranial, intrathoracic, intrathoracic, epidural epidural and intrasternal and intrasternal

20 injection 20 injectionand andinfusion. infusion. In one In one embodiment embodimentthatthat pharmaceutical pharmaceutical composition composition is administered is administered by by intravenousororsubcutaneous intravenous subcutaneous injection injection or or infusion. infusion.

In aa main In main aspect, aspect,the theinvention relatestotoa aheterodimeric inventionrelates heterodimeric protein protein according according to to the invention, the invention, such such asasaabispecific bispecific antibody antibodyaccording accordingtotothe theinvention, invention,for foruse useasasa a 25 medicament. 25 medicament. The The heterodimeric heterodimeric protein protein of of theinvention the invention may maybebeused usedfor for aa number number of purposes. of In particular, purposes. In particular, as explained above as explained abovethe theheterodimeric heterodimeric proteins proteins of the of the

invention may invention maybebeused used forfor the the treatment treatment of various of various forms forms of cancer, of cancer, including including

metastatic cancer metastatic cancerand andrefractory refractorycancer. cancer. Thus, inin one Thus, oneaspect, aspect,thethe invention invention relates relates to atomethod a method for inhibiting for inhibiting growthgrowth

30 and/or 30 and/or proliferation proliferation of and/or of and/or for for killing killing of of a tumor a tumor cellcell comprising comprising administration administration of a of a heterodimericprotein heterodimeric proteinaccording according to to thethe invention invention as described as described herein herein to antoindividual an individual in need in thereof. need thereof.

In another In embodiment another embodiment the the heterodimeric heterodimeric proteins proteins of theofinvention the invention are are used used for the for the treatment ofimmune treatment of immuneandand autoimmune autoimmune diseases, diseases, inflammatory inflammatory diseases,diseases,

35 infectious 35 infectious diseases, diseases, cardiovascular cardiovascular diseases, diseases, CNSmusculo-skeletal CNS and and musculo-skeletal diseases. diseases.

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Dosageregimens Dosage regimensin in thethe above above methods methods of treatment of treatment andareuses and uses are adjusted adjusted to to provide the optimum provide the optimum desired desired response response (e.g., (e.g., a therapeutic a therapeutic response). response). For example, For example, a a single bolus single bolus may be administered, may be several divided administered, several divideddoses may be doses may administered over be administered over time or time or the the dose dose may maybebeproportionally proportionally reduced reduced ororincreased increased asasindicated indicated by by the the 5 exigencies 5 exigencies of the of the therapeutic therapeutic situation. situation.

The efficient The efficient dosages andthe dosages and thedosage dosage regimens regimens for heterodimeric for the the heterodimeric proteins proteins

depend on depend on the thedisease diseaseororcondition condition to to be be treated treated and and may maybebedetermined by by determined the the personsskilled persons skilled inin the theart. art.AnAn exemplary, exemplary, non-limiting non-limiting range range for a therapeutically for a therapeutically

effective amount effective amountofofa bispecific a bispecificantibody antibody of the of the present present invention invention is about is about 0.1-100 0.1-100

10 mg/kg, 10 mg/kg, suchsuch as about as about 0.1-50 0.1-50 mg/kg, mg/kg, for example for example about 0.1-20 about 0.1-20 mg/kg, mg/kg, such as such as about 0.1-10 about 0.1-10mg/kg, mg/kg, for for instance instance aboutabout 0.5, about 0.5, about such assuch 0.3, as 0.3,1,about about about 1, 3, about 3, about 5, about 5, or or about about8 8mg/kg. mg/kg. physician or A physician A or veterinarian having ordinary veterinarian having ordinary skill skill in in the the art art may mayreadily readily determine and determine and prescribe prescribe the the effective effective amount amountofofthe thepharmaceutical pharmaceuticalcomposition composition 15 required. 15 required.ForFor example, example, the the physician physician or veterinarian or veterinarian couldcould startstart dosesdoses of theof the heterodimericprotein heterodimeric proteinemployed employed in pharmaceutical in the the pharmaceutical composition composition at levelsat levels lower lower than that than that required requiredininorder order to to achieve achieve the the desired desired therapeutic therapeutic effecteffect and gradually and gradually

increase the increase the dosage dosage untilthe until the desired desired effect effect is is achieved. achieved. In general, In general, a suitable a suitable daily daily

dose ofof aa composition dose compositionof ofthethepresent present invention invention willwill be be thatthat amount amount of compound of the the compound 20 which 20 which is the is the lowest lowest dose dose effectivetotoproduce effective produce a therapeuticeffect. a therapeutic effect. Administration Administration maye.g. may e.g. bebeparenteral, parenteral,such suchasas intravenous, intravenous, intramuscular intramuscular or subcutaneous. or subcutaneous.

A heterodimeric A heterodimeric protein protein of the invention of the invention may may also also bebeadministered administered prophylactically in prophylactically in order order totoreduce reducethethe risk risk of of developing developing disease, disease, such such as cancer, as cancer,

delay the delay the onset onsetofofthe theoccurrence occurrenceof of an an event event in disease in disease progression, progression, and/or and/or reducereduce

25 the the 25 riskrisk of of recurrence recurrence whenwhen a disease, a disease, such such as as cancer cancer is in remission. is in remission.

Heterodimericproteins, Heterodimeric proteins,such such as as bispecificantibodies, bispecific antibodies,of ofthe thepresent present invention invention

may also may alsobebeadministered administered in combination in combination therapy, therapy, i.e.,i.e., combined combined with with other other therapeutic agents therapeutic agentsrelevant relevantfor forthe thedisease diseaseor orcondition condition to to be be treated. treated. Accordingly, Accordingly, in in one embodiment, one embodiment,thethe Heterodimeric-protein-containing medicament Heterodimeric-protein-containing medicament is isforfor 30 combination 30 combination withwith one one or more or more further further therapeutic therapeutic agents, agents, such such as as a cytotoxic, a cytotoxic, chemotherapeutic or chemotherapeutic or anti-angiogenic anti-angiogenic agents. agents. Such Such combined administration may combined administration may be be simultaneous, separate simultaneous, separate ororsequential. sequential. InIna further a further embodiment, embodiment, the present the present invention provides invention method for provides aa method for treating treating or or preventing preventing disease, disease, such such asas cancer, cancer, which method which method comprises comprises administration administration to to aa subject subject in in need need thereof thereof of of aa

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2022201608 08 Mar effective amount therapeutically effective therapeutically amountof aofheterodimeric a heterodimeric protein, protein, such assuch as a bispecific a bispecific

antibody of antibody of the the present presentinvention, invention,inincombination combination with with radiotherapy radiotherapy and/or and/or surgery. surgery.

Heterodimericproteins, Heterodimeric proteins,such such as as bispecificantibodies, bispecific antibodies,of ofthe thepresent present invention invention

mayalso may alsobebeused usedforfordiagnostic diagnosticpurposes. purposes. 5 5 EXAMPLES EXAMPLES Example 1: Example 1: Expression Expression vectors vectors for for the the expression expression of of human IgG1-2F8and human IgG1-2F8 and IgG1-7D8 IgG1-7D8 The VH The VH and andVLVLcoding codingregions regionsofofHuMab HuMab 2F82F8 (WO (WO 02/100348) 02/100348) and HuMab and HuMab 10 7D87D8 10 (WO (WO 04/035607) 04/035607) were were clonedcloned in expression in the the expression vector vector pConGlf pConG1f (containing (containing the the genomicsequence genomic sequence of the of the human human IgG1fIgGlf allotype allotype constant constant region region (Lonza (Lonza Biologics)) Biologics)) for for the production the production of of the the human humanIgG1 IgG1 heavy heavy chain chain and and pConKappa pConKappa (containing (containing the the humankappa human kappa light light chain chain constant constant region, region, LonzaLonza Biologics) Biologics) forproduction for the the production of the of the kappalight kappa light chain. chain. For ForIgG4 IgG4antibodies antibodies thethe VH regions VH regions were were inserted inserted in the inpTomG4 the pTomG4 15 vector 15 vector (containingthe (containing thegenomic genomicsequence sequence of of thethe human human IgG4IgG4 constant constant region region in the in the pEE12.4vector pEE12.4 vector (Lonza (Lonza Biologics)). Biologics)). Alternatively, Alternatively, in follow-up in follow-up constructs, constructs, vectors vectors

were used were usedcontaining containing the the fully fully codon-optimized codon-optimized codingcoding regionsregions of thechain of the heavy heavy chain (IgG1 or IgG4) (IgG1 or IgG4)in inthe thepEE12.4 pEE12.4 vector vector or the or the human human light of kappachain kappa light chain HuMabof2F8 HuMab 2F8 or HuMab or 7D8 HuMab 7D8 in in thethe pEE6.4 pEE6.4 vector vector (Lonza (Lonza Biologics). Biologics).

20 20 Example 22 Expression Example Expression vectors vectors for for the the expression expression hinge-deleted-IgG1-2F8, hinge-deleted-IgG1-2F8, and human and humanIgG1 IgGI and and IgG4 IgG4 CH2-CH3 CH2-CH3 fragments fragments containing containing specific specific mutations mutations To introduce To introduce mutations in the mutations in the hinge hinge and and CH3 regions of CH3 regions of the the antibody antibody heavy heavy 25 chains, 25 chains, Quickchange Quickchange site-directedmutagenesis site-directed mutagenesis kit kit (Stratagene,La La (Stratagene, Jolla,CA) Jolla, CA)waswas used according used accordingtotothe themanufacturer's manufacturer's recommendations. recommendations. Alternatively Alternatively the constructs the constructs

were fully were fully synthesized synthesizedororVHVH regions regions were were cloned cloned in a vector in a vector already already containing containing the the specific amino specific acid encoding amino acid encodingsubstitutions. substitutions. Constructs encoding Constructs encoding the the CH2 and CH3 CH2 and CH3fragments fragmentswere were constructedeither constructed eitherbyby 30 30 PCR PCR or synthesized or synthesized fully fully codoncodon optimized. optimized. These constructs These constructs had an N-terminal had an N-terminal signal signal peptide and peptide anda a6 6amino amino acid acid HisHis tagtag andand contained contained amino amino acids acids 341-447 341-447 of the of the human human IgG1/4constant IgG1/4 constantregion. region.The The constructs constructs were were cloned cloned in pEE12.4. in pEE12.4.

To construct hinge-deleted-IgG1 To construct hinge-deleted-IgG1(Uni-G1) (Uni-G1) molecules, molecules, a synthetic a synthetic DNA DNA construct were construct were was made encoding was made encoding the the Uni-G1 Uni-G1 format format for for human humanIgG1 IgG1isotypes isotypeswith with 35 35 EGFREGFR specificity. specificity. In this In this construct construct the natural the natural hingehinge regionregion (as defined (as defined by the hinge by the hinge

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exon) was exon) was deleted. deleted. An An extra Ser to extra Ser to Cys Cys mutation mutation at position 158 at position 158 was madeinin the was made the IgG1 construct IgG1 construct to to salvage salvage the the Cys Cysbond bondbetween between the the HC LC HC and andchains LC chains in in this this subtype. The subtype. protein sequence The protein sequence is is shown shown below. below. The Theconstruct constructwas wasinserted insertedinin the the 2022201608 08

pEE6.4 vector pEE6.4 vector and and named pHG1-2F8. named pHG1-2F8. 5 5 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYG QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYG DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTV DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTV SSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL SSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVAPELLGGPSVFLFPPKPKDTLMISRTPEV YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVAPELLGGPSVFLFPPKPKDTLMISRTPEV 10 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC 10 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 3: Example 3: Expression Expression vectors vectors for for the the expression expression of of rhesus rhesus IgG4-2F8 and IgG4-2F8 and 15 IgG4-7D8 15 IgG4-7D8 Vectors containing Vectors containing the the coding regions for coding regions for the the IgG4 heavy and IgG4 heavy andkappa kappalight light chains Chinese chains Rhesus monkey Chinese Rhesus monkeyand andthetheVHVH andand VL VL regions regions of of Humab Humab 2F8 2F8 and and 7D8 7D8 were synthesized, were synthesized, fullycodon-optimized fully codon-optimizedand and inserted inserted in pEE12.4 in pEE12.4 (heavy (heavy chain) chain) and and pEE6.4(light pEE6.4 (light chain). chain). The Theheavy heavy chain chain constant constant region region sequence sequence as usedas used on(based (based on 20 thethe 20 sequences sequences described described by by Scinicariello et Scinicariello et al., al., Immunology 111: 66-74, Immunology 111: 66-74, 2004) 2004) was was the following the following (aligned (aligned to to the the human human sequence): sequence):

Human IgG4 Human IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYEPEPVTVSWNSGALTSGVH Rhesus (Ch) IgG4 -STKGPSVFPLASCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH Rhesus (Ch) IgG4 -STKGPSVFPLASCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVF 25 25 Human IgG4 Human IgG4 TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG TFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG Rhesus (Ch) Rhesus (Ch) IgG4 IgG4 FFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVVHEPSNTKVDKRVEFT- TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVVHEPSNTKVDKRVEFT-

Human IgG4 Human IgG4 PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV 30 Rhesus 30 Rhesus (Ch) (Ch) IgG4 IgG4 PPCPACPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV PPCPACPAPELLGGPSVELFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV

Human IgG4 Human IgG4 QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV Rhesus (Ch) Rhesus (Ch) IgG4 IgG4 QFNWYVDGAEVHHAQTKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKV QFNWYVDGAEVHHAQTKPRERQFNSTYRVVSVLTVTHQDWLNGKEYTCKV

35 Human 35 Human IgG4 IgG4 SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY Rhesus (Ch) Rhesus (Ch) IgG4 IgG4 SNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNOVSLTCLVTGFY SNKGLPAPIEKTISKAKGQPREPQVYILPPPQEELTKNQVSLTCLVTGFY

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Human IgG4 Human IgG4 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF Rhesus (Ch) Rhesus (Ch) IgG4 IgG4 PSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIF PSDIAVEWESNGQPENTYKTTPPVLDSDGSYLLYSKLTVNKSRWQPGNIF

5 Human 5 IgG4 HumanIgG4 SCSVMHEALHNHYTQKSLSLSLGK SCSVMHEALHNHYTQKSLSLSLGK Rhesus (Ch) IgG4 TCSVMHEALHNHYTQKSLSVSPGK Rhesus (Ch) IgG4 TCSVMHEALHNHYTQKSLSVSPGK

The Rhesus The Rhesuslight lightchain chainconstant constant region region (CL) (CL) sequence sequence used used was: was: AVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKTGNSQESVTEQDSKDN AVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKTGNSQESVTEQDSKDN 10 TYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVTKSFNRGEC 10 TYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVTKSFNRGEC

Example4:4:Antibody Example Antibody production production by transient by transient expression expression in HEK-293F in HEK-293F cells cells Antibodies were Antibodies were produced, produced, under underserum-free serum-freeconditions, conditions,bybycotransfecting cotransfecting relevant heavy relevant heavyandand light light chain chain expression expression vectors vectors in HEK-293F in HEK-293F cells (Invitrogen), cells (Invitrogen),

15 using 15 using 293fectin 293fectin (Invitrogen), (Invitrogen), according according to manufacturer's to the the manufacturer's instructions. instructions.

Example5:5:Purification Example Purificationof of IgG1 IgGIand andIgG4 IgG4 antibodies antibodies IgG1 and IgG1 andIgG4 IgG4 antibodies antibodies were were purified purified by protein by protein A affinity A affinity chromatography. chromatography.

The cell culture The cell culture supernatants supernatantswere were filtered filtered over over a 0.20 a 0.20 pM dead-end µM dead-end filter,filter, followed followed

20 20 by by loading loading on on mL Protein a 5 amL5 Protein A column A column (rProtein (rProtein GE Healthcare, A FF,AGEFF,Healthcare, Uppsala, Uppsala, Sweden)andand Sweden) elution elution of the of the IgG IgG with with 0.1 M0.1 M citric citric acid-NaOH, acid-NaOH, pH eluate pH 3. The 3. Thewas eluate was immediately neutralized immediately neutralized with with M Tris-HCI, 2 M2 Tris-HCI, pH 9pH and dialyzed and9 dialyzed overnight overnight to 12.6tomM12.6 mM

sodium phosphate, sodium phosphate, 140 140mMmM NaCl, NaCl, pH pH 7.4 7.4 (B. (B. Braun, Braun, Oss,Oss, The The Netherlands). Netherlands). After After dialysis, samples dialysis, weresterile samples were sterilefiltered filtered over overa a0.20 0.20µM pM dead-end dead-end filter. filter. Concentration Concentration

25 25 of of thethe purifiedIgGs purified IgGswas was determined determined by by nephelometry nephelometry and and absorbance absorbance at nm. at 280 280 nm. Purified Purifiedproteins proteinswere wereanalyzed analyzedbyby SDS-PAGE, mass spectrometry IEF, mass SDS-PAGE, IEF, spectrometry and and glycoanalysis. glycoanalysis.

Example Example 6:6:Purification Purificationof of CH2-CH3 CH2-CH3 fragments fragments 30 30 The CH2-CH3 His-tagged CH2-CH3 The His-tagged proteins proteins were were purifiedby by purified immobilized immobilized metal metal ion ion (Ni2 affinity chromatography (Ni²)) affinity chromatography (Macherey-Nagel GmbH,Düren, (Macherey-Nagel GmbH, DOren, Germany), Germany), desalted desalted

using PD-10 using PD-10columns columns (GE Healthcare) (GE Healthcare) equilibrated equilibrated with PBSwith PBS and filtered-sterilized and filtered-sterilized

over 0.2 over 0.2 µMpMdead-end dead-end filters.The filters. The concentration concentration of the of the purified purified proteins proteins was was determined bybyabsorbance determined absorbanceat at280280 nm. nm. The The quality quality of purified of the the purified proteins was was proteins 35 analyzed 35 analyzedbybySDS-PAGE. SDS-PAGE.

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2022201608 08 Mar Generation ofofbispecific Example 7:7: Generation Example antibodies by bispecific antibodies GSH-inducedFab-arm by GSH-induced Fab-arm exchange between exchange betweenhuman humanandand rhesus rhesus IgG4 IgG4 antibodies antibodies As mentioned As mentioned above, above, WO WO2008119353 2008119353 (Genmab) (Genmab) describes describes an an in vitro in vitro methodfor method forproducing producing bispecificantibodies bispecific antibodies wherein wherein a bispecific a bispecific antibody antibody is formed is formed by by 5 "Fab-arm" 5 "Fab-arm" or "half-molecule"exchange or "half-molecule" exchange (swapping (swapping of a ofheavy a heavy chainchain and attached and attached light chain) light chain) between twomonospecific between two monospecific IgG4- IgG4- or IgG4-like or IgG4-like antibodies antibodies upon incubation upon incubation

under reducing under reducing conditions. conditions. This This Fab-arm Fab-armexchange exchange reaction reaction is the is the result result of aof a disulfide-bond isomerization disulfide-bond isomerizationreaction reactionwherein whereinthethe inter inter heavy-chain heavy-chain disulfide disulfide bonds bonds in in the hinge the hinge regions regions of of monospecific monospecific antibodies antibodies are are reduced reduced and andthe theresulting resulting free free 10 cysteines 10 cysteinesform form a new a new interinter heavy-chain heavy-chain disulfide disulfide bondbond withwith cysteine cysteine residues residues of of anotherantibody another antibody molecule molecule with with a different a different specificity. specificity. The resulting The resulting productproduct is a is a bispecific antibody bispecific having two antibody having twoFab Fabarms arms with with different different sequences. sequences.

To test To test for for Fab-arm exchangebetween Fab-arm exchange betweenhuman human and and rhesus rhesus igG4 igG4 antibodies, antibodies, humanIgG4-2F8 human IgG4-2F8(anti-EGFR), (anti-EGFR),Human Human IgG4-7D8 IgG4-7D8 (anti-CD20), (anti-CD20), Rhesus Rhesus IgG4-2F8 IgG4-2F8 and and 15 Rhesus 15 Rhesus IgG4-7D8 IgG4-7D8 werewere usedused to make to make all possible all possible combinations combinations of of twoantibodies. two antibodies. For For the in the in vitro vitro Fab-arm exchange, Fab-arm exchange, thethe antibody antibody mixtures, mixtures, containing containing each antibody each antibody at a at a final concentration final concentration of of4 4 pg/mL in 0.5 µg/mL in 0.5 mL mLPBS PBS with with 0.50.5 mM mM reduced reduced glutathione glutathione (GSH), were incubated (GSH), were incubatedatat37°C 37°Cfor for24h. 24h.To To stop stop thethe reduction reduction reaction,0.5 reaction, 0.5mL mL PBS/0.05 %%Tween PBS/0.05 Tween2020(PBST) (PBST)was was added added to to thereaction the reaction mixture. mixture. 20 20 The presence The presenceofofbispecific bispecificantibodies antibodieswas was tested tested by by determination determination of bispecific of bispecific

binding using binding using aa sandwich sandwichenzyme-linked enzyme-linked immunosorbent immunosorbent assay (ELISA). assay (ELISA). ELISA ELISA plates plates (Greiner bio-one, Frickenhausen, (Greiner bio-one, Frickenhausen, Germany) werecoated Germany) were coated overnight overnight with with 2 pg/mL 2 µg/mL (100 pL/well) of (100 µL/well) of recombinant recombinant extracellular extracellular domain domain of EGFR of EGFR in at in PBS PBS4 °C. at 4The The plates °C. plates were washed were washed once once withwith PBST. PBST. Dilution Dilution series series of antibody of the the antibody samples samples (0-1inpg/mL (0-1 µg/mL in 25 3-fold 25 3-folddilutions) dilutions) in in PBST/0.2 BSA PBST/0.2 %%BSA (PBSTB) (PBSTB) were were transferred transferred to to thethe coated coated ELISA ELISA plates (100 plates (100 µL/well) pL/well)and andincubated incubated onplate on a a plate shaker shaker (300 (300 rpm)60 for rpm) for min 60 at min room at room temperature (RT). temperature (RT). Samples Sampleswere werediscarded discardedand andthe theplates plateswere werewashed washed once once withwith PBS/0.05 %%Tween PBS/0.05 Tween 20 20 (PBST). (PBST). Next, Next, thethe plates plates were were incubated incubated on on a plate a plate shaker shaker (300 rpm) with (300 rpm) with2 2µg/mL pg/mL mouse mouse anti-idiotypic anti-idiotypic monoclonal monoclonal antibody antibody 2F2 SAB1.1 2F2 SAB1.1 30 (directed 30 (directedagainst against7D8; 7D8;Genmab) Genmab) in PBTB in PBTB (100(100 pL/well) µL/well) forfor 60 60 min. TheThe min. plates plates were were washed once washed oncewith with PBS/0.05 PBS/0.05 %%Tween Tween 20 20 (PBST).Next, (PBST). Next, theplates the plates were were incubated incubated on on plate shaker a plate a shaker (300 (300 rpm) rpm)with with an an goat goat HRP-conjugated HRP-conjugated anti-mouse anti-mouse IgG IgG (15G; (15G; Jackson ImmunoResearch Jackson ImmunoResearch Laboratories, Laboratories, Westgrove, Westgrove, PA, PA, USA;USA; 1:5.000) 1:5.000) in PBSTB in PBSTB (100 pL/well) for (100 µL/well) for 60 at RT. min at 60 min RT. The Theplates plates were werewashed washed once once with with PBS/0.05 PBS/0.05 % %

35 Tween 35 Tween 20 (PBST). 20 (PBST). ABTS ABTS (50 (50 mg/mL; mg/mL; RocheRoche Diagnostics Diagnostics GmbH,GmbH, Mannheim, Mannheim, Germany) Germany)

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was added was added(100 (100 and and pL/well) µL/well) incubated incubated protected protected fromforlight from light for at 30 min 30 RT. at minThe RT. The reaction was reaction stopped with was stopped with 2%2%oxalic oxalicacid acid(100 (100µL/well; pL/well;Riedel RiedeldedeHaen Haen Seelze, Seelze, Germany). After Germany). After 10 10 min min atatRT, RT,absorbance absorbanceatat405 405nm nm waswas in aninELISA measured measured an ELISA plate reader. plate reader. 5 5 Figure 11 shows Figure shows that that aa combination combination ofofhuman humanandand rhesus rhesus IgG4IgG4 resulted resulted in in more bispecific more bispecific binding binding(a (ahigher higherODOD 405 nm) compared 405 nm) comparedwith witheach eachof ofthethe combinationsofofIgG4 combinations IgG4 molecules molecules of the of the samesame species. species. These These datathat data show show that Fab-arm Fab-arm exchange occurs exchange occursbetween between human human IgG4 IgG4 and rhesus and rhesus IgG4. Moreover, IgG4. Moreover, the the higher higher bispecific binding bispecific binding suggests suggests that humanIgG4 that human IgG4 halfhalf molecules molecules showshow preferential preferential 10 dimerisation 10 dimerisationto torhesus rhesus IgG4 IgG4 halfhalf molecules molecules (heterodimerization), (heterodimerization), resultingin inan an resulting equilibrium of equilibrium of the the Fab-arm Fab-arm exchange exchange reaction reaction that that is is shifted shifted towards towards the bispecific the bispecific

heterodimer instead heterodimer instead of of aa stochastic stochastic exchange exchangewith with50%50% heterodimer heterodimer and and 50% 50% homodimers. homodimers.

15 Example 15 Example 8: 8: Sequence Sequence analysis analysis ofofhuman humanandand rhesus rhesus IgG4 IgG4 The ability The ability of of an antibody to an antibody to engage engagein inFab-arm Fab-arm exchange exchange has described has been been described to involve to involve the the third third constant constantdomain domain (CH3) (CH3) in addition in addition to atoso-called a so-called permissive permissive (for (for example CPSC example CPSCcontaining-) containing-) hinge hinge region region that that only only requires requires aa reducing reducing environment environment to be to be activated activated(Van (Vanderder Neut Neut Kolfschoten, Kolfschoten, 2007,2007, Science). Science). Forantibodies, For human human antibodies, 20 Fab-arm 20 Fab-arm exchange exchange was was found found to beto an beinherent an inherent feature feature of of IgG4, IgG4, characterizedbybyanan characterized arginine (R) arginine (R) at at position position 409 409ininthe theCH3 CH3 domain domain and aand a permissive permissive hinge (226-CPSC hinge (226-CPSC-

229) (see WO 229) (see WO2008145142 2008145142 (Genmab)). (Genmab)). In contrast, In contrast, human human IgG1, IgG1, which which does not does not engageininFab-arm engage Fab-arm exchange, exchange, has ahas a Lysine Lysine (K)position (K) at at position 409a and 409 and a stable stable (i.e. (i.e. non- non permissive) hinge permissive) hinge(226-CPPC-229) (226-CPPC-229) (EU numbering, (EU numbering, seeFigure see also also Figure 16). 16). 25 25 In an In an attempt to elucidate attempt to elucidatethe theincreased increasedFab-arm Fab-arm exchange between human exchange between human and rhesus and rhesus IgG4 IgG4 compared comparedtoto the the Fab-arm Fab-armexchange exchangebetween between IgG4 IgG4 molecules molecules of of thethe same species, same species, the the core core hinge hinge and and CH3-CH3 CH3-CH3 interfaceamino interface amino acids acids of human of human and and rhesus antibodies rhesus antibodies were were analyzed analyzed (see (see e.g. e.g. Dall'Acqua, et al Dall'Acqua, et al (1998) (1998) Biochemistry Biochemistry 37:9266 forananoverview 37:9266 for overview of the of the residues residues of the of the human human CH3-CH3 CH3-CH3 interface). interface). Figure 2 Figure 2

30 shows 30 shows thatthat thethe core core hinge hinge sequence sequence in Chinese in Chinese rhesus rhesus IgG4IgG4 is 226-CPAC-229 is 226-CPAC-229 and and that the that the CH3 CH3domain domain contains contains a Lysine a Lysine (K) at(K) at position position 409. In409. In addition, addition, sequence sequence

alignment showed alignment showedthat thatrhesus rhesusIgG4 IgG4 is characterizedby by is characterized three three moremore aminoamino acid acid substitutions in substitutions in the CH3-CH3interface the CH3-CH3 interface as as compared compared to human to human IgG4: isoleucine IgG4: isoleucine (I) at (I) at position 350 position 350 inin rhesus rhesusversus versus threonine threonine (T) (T) in human; in human; threonine threonine (T) at(T) at position position 370 370

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2022201608 08 Mar in rhesus in rhesus versus lysine (K) versus lysine (K) in in human; leucine (L) andleucine human; and (L) at at position position 405 405 inin rhesus rhesus versus phenylalanine versus phenylalanine(F)(F)ininhuman. human.

Example9:9:Generation Example Generation of of bispecific bispecific antibodies antibodies using using GSH-induced GSH-induced Fab-armFab-arm 5 exchange 5 exchangebetween betweenhuman human IgG4 IgG4 andand human human IgG1IgGI containing containing rhesusIgG4 rhesus IgG4 CH3 sequences CH3 sequences It has It has been beendescribed describedforforhuman human antibodies antibodies thatthat for allowing for allowing Fab-arm Fab-arm exchangetotooccur exchange occurininIgG1 IgG1 molecules, molecules, replacing replacing the the IgG1IgG1 core core hingehinge sequence sequence (CPPC) (CPPC) with the with the human IgG4sequence human IgG4 sequence (CPSC) (CPSC) by by a P228S a P228S substitution substitution hadhad no effect,butbut no effect, 10 that 10 that mutating mutating CH3CH3 to IgG4-like to an an IgG4-like sequence sequence was required was required for Fab-arm for Fab-arm exchange exchange activity (Van activity (Van der NeutKolfschoten, der Neut Kolfschoten,2007, 2007, Science). Science).

Based on Based on the the Fab-arm Fab-arm exchange exchangebetween between human human and and rhesus rhesus IgG4IgG4 described described in Example in Example 7,7,itit was wasanalyzed analyzed whether whether the the Chinese Chinese rhesus rhesus IgG4 IgG4 CH3 CH3 sequence sequence could could engage human engage humanIgG1 IgG1 forfor Fab-arm Fab-arm exchange. exchange. Therefore, Therefore, the the triple triple mutation mutation T3501 T350I- 15 K370T-F405L 15 K370T-F405L (referred (referred to ITL to as as ITL hereafter) hereafter) waswas introduced introduced in human in human IgG1-2F8 IgG1-2F8 in in addition to addition to the the P228S P228Smutation mutation that that results results in in thethe hinge hinge sequence sequence CPSC.CPSC. The The human human IgG1-2F8 mutantswere IgG1-2F8 mutants werecombined combined with with human human IgG4-7D8 IgG4-7D8 for inforvitro in vitro GSH-induced GSH-induced Fab-arm exchange.TheThe Fab-arm exchange. antibody antibody mixtures, mixtures, containing containing eacheach antibody antibody at a at a final final concentration of concentration of 44 pg/mL in 0.5 µg/mL in 0.5 mL PBS with mL PBS with 0.5 0.5 mM GSH,were mM GSH, wereincubated incubatedatat37°C 37°C 20 forfor 20 0-3-6-24h.ToTo 0-3-6-24h. stopthe stop thereduction reduction reaction, reaction, 0.5 0.5 mL PBS/0.05 %%Tween mL PBS/0.05 Tween20 20 (PBST) (PBST) was added was addedto to thethe reaction reaction mixture. mixture. Measurements Measurements of bispecific of bispecific bindingbinding in an in an ELISA ELISA were performed were performedas as described described in Example in Example 7. 7. Figure 33 confirms Figure confirms that that introduction introductionofofa aCPSC CPSC hinge hinge alone alone does does not not engage engage humanIgG1-2F8 human IgG1-2F8forforGSH-induced GSH-induced Fab-arm Fab-arm exchange exchange when when combined combined with with human human 25 IgG4-7D8. 25 IgG4-7D8. AlsoAlso the the introduction introduction of the of the rhesus rhesus IgG4-specific IgG4-specific CH3CH3 interface interface amino amino acids (ITL) acids (ITL) into into human humanIgG1-2F8, IgG1-2F8, while while preserving preserving the wild the wild type type IgG1 hinge, IgG1 hinge, did notdid not result ininengagement result for Fab-arm engagement for exchangewhen Fab-arm exchange whencombined combined with with human human IgG4-7D8 IgG4-7D8 under these under these conditions. conditions. InIncontrast, variant contrast,a a human variant humanIgG1-2F8 IgG1-2F8 backbone sequence backbone sequence that harbors that harbors both both aa CPSC sequenceinin the CPSC sequence the hinge hinge and andthe therhesus rhesusIgG4-specific CH3 IgG4-specific CH3 30 interface 30 interfaceamino amino acids acids (ITL)showed (ITL) showed increased increased bispecificbinding bispecific binding after after GSH-induced GSH-induced Fab-arm exchange Fab-arm exchangewith with human human IgG4-7D8 IgG4-7D8 compared compared to two to two humanhuman IgG4 antibodies. IgG4 antibodies. These data These data show showthat thataa CPSC-containing CPSC-containinghinge hingeinin combination combinationwith witha aCH3 CH3domain domain containing I,I, TT and containing andL at L at positions positions 350, 350, 370 405, 370 and and respectively, 405, respectively, is sufficient is sufficient to to engage human engage humanIgG1 IgG1for forGSH-induced GSH-induced Fab-arm Fab-arm exchange exchange and and thatthat the the equilibrium equilibrium of of

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the exchange the exchangereaction shifted towards reaction isis shifted towards the the exchanged bispecific product exchangedbispecific when product when combined with combined with human humanIgG4. IgG4.

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Example 10: Example 10:Generation Generation of bispecific of bispecific antibodies antibodies by vivo by in in vivo Fab-arm Fab-arm 5 exchange 5 exchange between between human human IgG4 IgG4 and IgGI and IgG1 or IgG4 or IgG4 mutants mutants To further To further identify identify the the required requiredcharacteristics characteristics for for Fab-arm Fab-arm exchange exchange engagement,human engagement, human IgG4 IgG4 and and IgG1 IgG1 variants variants were were analyzed analyzed in vivo. in vivo. Four Four female female SCIDmice SCID mice(Charles (Charles River, River, Maastricht, Maastricht, The The Netherlands) Netherlands) per group per group were were i.v. i.v. injected injected

with antibody with antibodymixtures, mixtures,containing containing 600600 tg antibody µg antibody (500 (500 µg 7D8tg+ 7D8 + 2F8) 100 µg 100 intg a2F8) in a 10 total 10 totalvolume volume of of 300 300 µL. Bloodsamples pL.Blood sampleswere were drawn drawn from from thethe saphenal saphenal vein vein at at 3,3,24, 24, 48 and 48 and7272hours hours after after injection.Blood injection. Blood was was collected collected in heparin-containing in heparin-containing vials vials and and centrifuged at centrifuged at 10,000 10,000g for g for5 min 5 min to separate to separate plasma plasma from cells. from cells. The generation The generation of of bispecific antibodies bispecific antibodies was followed bybyassessing was followed assessingCD20 CD20 and and EGFREGFR bispecific bispecific reactivity reactivity in in an ELISA an ELISAusing usingserial serialdiluted dilutedplasma plasma samples samples in PBSTB in PBSTB as described as described in 7. in Example Example 7. 15 Bispecific 15 Bispecificantibodies antibodies inin plasma plasmasamples samples were were quantified quantified by by non-linear non-linear regression regression curve-fitting (GraphPad curve-fitting Software, San (GraphPad Software, San Diego, Diego,CA) CA)using using an an in vitro in vitro exchanged exchanged antibody mixture antibody mixtureasasreference. reference. Figure 44 shows Figure that human shows that humanIgG4-2F8, IgG4-2F8,ininwhich whicheither either the the hinge hinge or or the the CH3 CH3 sequence is sequence is converted converted to to the corresponding human the corresponding IgG1 sequence human IgG1 sequence(CPPC (CPPCororR409K, R409K, 20 respectively), 20 respectively),does doesnot notengage engage in inFab-arm Fab-arm exchange exchange anymore anymore in vivo. in vivo. ViceVice versa, versa, humanIgG1, human IgG1,ininwhich whichboth boththe thehinge hingeregion regionand andthe theCH3 CH3 interfacesequences interface sequencesareare convertedtotothe converted thecorresponding corresponding human human IgG4 IgG4 sequences sequences (CPSC (CPSC and andisK409R), K409R), able to is able to participate ininFab-arm participate Fab-arm exchange in vivo. exchange in vivo. These These data show that data show that aa CPSC-containing CPSC-containing hinge (S hinge (S atatposition position 228) 228)in incombination combination withwith CH3 domain a domain a CH3 containing containing an arginine an arginine

25 (R)(R)at at 25 position 409 position 409isis enough enough to to enable enable Fab-arm exchangebybyhuman Fab-arm exchange humanIgG1 IgG1in invivo. vivo.

Example11: Example 11:Generation Generation of bispecific of bispecific antibodies antibodies by 2-MEA-induced by 2-MEA-induced Fab-armFab-arm exchange:bypass/disruption exchange: bypass/disruption ofstabilized of a a stabilized hinge hinge 2-Mercaptoethylamine.HCI (2-MEA) isis aa mild 2-Mercaptoethylamine-HCl (2-MEA) mild reducing reducing agent agentthat thathas hasbeen been 30 described 30 described to selectively to selectively cleave cleave disulphide disulphide bonds bonds in in theregion the hinge hingeof region of antibodies, antibodies,

while preserving while preserving the thedisulphide disulphidebonds bonds between between the the heavyheavy and light and light chains. chains. Therefore, Therefore,

concentrationseries a concentration a seriesofof2-MEA 2-MEAwaswas tested tested for for its its ability ability to to induce induce thethe generation generation of of bispecific antibodies bispecific by Fab-arm antibodies by Fab-armexchange exchange between between two antibodies two antibodies containing containing CPSC CPSC or CPPC or CPPChinge hinge regions. regions. TheThe antibody antibody mixtures, mixtures, containing containing each antibody each antibody at at a final a final 35 concentration 35 concentration of mg/mL, of 0.5 0.5 mg/mL, were incubated were incubated with a concentration with a concentration series of series of 2-MEA 2-MEA (0, (0,

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2022201608 08 Mar 0.5, 1.0, 0.5, 2.0, 5.0, 1.0, 2.0, 5.0, 7.0, 7.0, 10.0, 15.0, 25.0 10.0, 15.0, 25.0 and 40.0mM)mM) and40.0 in ain total a total volume volume of 100 of 100 tL µL TE TE at 37°C at 37°Cfor for9090min. min.To To stop stop the the reduction reduction reaction, reaction, the reducing the reducing agentwas agent 2-MEA 2-MEA was removedby by removed desalting desalting the the samples samples using using spin columns spin columns (Microcon (Microcon centrifugal centrifugal filters, filters, 30k, Millipore) 30k, Millipore) according according totothe themanufacturer's manufacturer's recommendations. recommendations. Bispecific bindingbinding Bispecific 5 waswas 5 measured measured in an in an ELISA ELISA as described as described in inExample Example 7. 7. 2-MEA-induced Fab-arm 2-MEA-induced Fab-armexchange exchange was was testedfor tested forthe the combination combination IgG4-2F8 IgG4-2F8X x IgG4-7D8, containing IgG4-7D8, containing CPSC CPSC hinge hinge regions regions and and known knowntotoparticipate participate in in GSH-induced GSH-induced Fab-arm exchange, Fab-arm exchange,and andfor forthe thecombination combinationIgG1-2F8-ITL IgG1-2F8-ITL x IgG4-7D8-CPPC, X IgG4-7D8-CPPC, not not participating in participating in GSH-induced Fab-arm GSH-induced Fab-arm exchange exchange due to due to the stabilized the stabilized hinge hinge regions regions 10 (described 10 (describedin in Example Example 9, Figure 9, Figure 3). Surprisingly, 3). Surprisingly, 2-MEA 2-MEA was to was found found to induce induce separation of separation of light lightchains chainsfrom fromheavy heavy chains chains as as determined determined by non-reducing SDS- by non-reducing SDS PAGE(data PAGE (datanotnotshown). shown). Nonetheless, Nonetheless, functional functional bispecific bispecific antibodies antibodies were were generated generated

as shown as shownininFigure Figure5.5.The Themaximal maximal level level of bispecific of bispecific binding binding after after Fab-arm Fab-arm exchange exchange

between wild between wild type type human IgG4-2F8and human IgG4-2F8 andIgG4-7D8 IgG4-7D8 was was reached reached at at concentrationofof a aconcentration 15 2.0 15 2.0mMmM 2-MEA 2-MEA and and was was comparable comparable to the to the level level reachedwith reached with0.5 0.5mMmM GSHGSH as as described in described in Example (Figure 3). Example 99 (Figure 3). However, However,2-MEA 2-MEAwaswas able able to induce to induce Fab-arm Fab-arm exchange between exchange betweenthe thehuman human antibodies antibodies IgG1-2F8-ITL IgG1-2F8-ITL and and IgG4-7D8-CPPC IgG4-7D8-CPPC (with (with stabilized hinge stabilized hinge regions) regions)inina adose-dependent dose-dependent manner. manner. While or While little little or no bispecific no bispecific

antibodies were antibodies wereformed formed at low at low 2-MEA 2-MEA concentrations, concentrations, probably probably duepresence due to the to the presence 20 of of 20 a CPPC a CPPC sequence sequence in thein hinge the hinge of bothofantibodies, region region both antibodies, the generation the generation of of bispecific antibodies bispecific wasvery antibodies was veryefficient efficientatathigher higherconcentrations concentrations of 2-MEA. of 2-MEA. Maximal Maximal

bispecific binding bispecific binding was reachedatat2525 was reached mM mM 2-MEA 2-MEA and exceeded and exceeded maximal maximal binding binding after after Fab-arm exchange Fab-arm exchangebetween between the the two two wild wild type type IgG4 IgG4 antibodies. antibodies. These These maximal maximal binding levels binding levels were werecomparable comparableto to what what is described is described in Example in Example 9 (Figure 9 (Figure 3) for3)GSH for GSH 25 treatment 25 treatment of of thethe antibodywith correspondingantibody corresponding CPSChinge with aa CPSC hinge(IgG1-2F8-CPSC-ITL). (IgG1-2F8-CPSC-ITL). AsAs IgG1-2F8-ITL and IgG1-2F8-ITL and IgG4-7D8-CPPC IgG4-7D8-CPPC both both contain contain a CPPC a CPPC hinge, hinge, these these datadata indicate indicate that 2-MEA that 2-MEA could could bypass bypassthe therequirement requirement of of a CPSC a CPSC hinge hinge for vitro for in in vitro Fab-arm Fab-arm exchange. exchange.

30 Example 30 Example 12: 12: MassMass spectrometry spectrometry to follow to follow the generation the generation of bispecific of bispecific antibodies by antibodies by 2-MEA-induced Fab-arm exchange 2-MEA-induced Fab-arm exchange The generation The generation of of bispecific bispecific antibodies antibodiesby by 2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange is described is in Example described in Example11,11, where where bispecific bispecific binding binding was was shown shown by an by an (Figure ELISA ELISA (Figure 5). To 5). confirmthat To confirm thatbispecific bispecificantibodies antibodiesare areformed, formed, the the samples samples were were analyzed analyzed by by 35 electrospray 35 electrosprayionization ionizationmass mass spectrometry spectrometry (ESI-MS) (ESI-MS) to determine to determine the molecular the molecular

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First, samples weights. First, weights. weredeglycosylated samples were deglycosylated by incubating by incubating 200antibody 200 µg tg antibody overnight overnight

at 37°C at with 0.005 37°C with 0.005 UU N-Glycanase N-Glycanase(cat.no. (cat.no. GKE-5006D; GKE-5006D; Prozyme) Prozyme) in 180 in 180 tL PBS. µL PBS. Samples were Samples weredesalted desalted on on ananAquity Aquity UPLC UPLC" (Waters, (Waters, Milford, Milford, USA) USA) with with a BEH300 a BEH300 C18, 1.7µm, C18, 1.7pm,2.12.1X 50 x 50 mm mm column column at and at 60°C 60°C and with eluted eluted with a gradient a gradient of a of of a mixture mixture of 5 MQMQ 5 water water (Eluens (Eluens A) LC-MS A) and and LC-MS grade acetonitrile grade acetonitrile (eluens (eluens B) (Biosolve, B) (Biosolve, Valkenswaard, The Valkenswaard, TheNetherlands) Netherlands)containing containing0.05% 0.05% formic formic acidacid (Fluka (Fluka Riedel-de Riedel-de Hain, Buchs, Haën, Buchs, Germany). Germany).Time-of-flight Time-of-flight electrospray electrospray ionization ionization mass spectra were mass spectra were recorded on-line recorded on-line on micrOTOFTM on aa micrOTOF mass mass spectrometer spectrometer (Bruker, (Bruker, Bremen, Bremen, Germany) Germany) operating in operating in the the positive positive ion ion mode. Prior to mode. Prior to analysis, analysis,a a500-4000 500-4000 m/z scale was m/z scale was 10 calibrated 10 calibrated withwith ES tuning ES tuning mix (Agilent mix (Agilent Technologies, Technologies, Santa Clara, Santa Clara, USA). USA). Mass Mass spectra spectra were deconvoluted were deconvoluted by by using usingMaximal MaximalEntropy Entropy thatthat is provided is provided withwith the the DataAnalysisT software DataAnalysis softwarev.v. 3.4 3.4 (Bruker, (Bruker, Bremen, Bremen, Germany). Germany).Based Basedon on themolecular the molecular massofofthe mass theantibodies antibodiesused used forfor Fab-arm Fab-arm exchange exchange in thisin experiment, this experiment, the bispecific the bispecific

antibodies could antibodies couldbebe discriminated discriminated from from the original the original antibodies antibodies (also described (also described in in 15 Example 15 Example 15, 15, Figure Figure 9C 9C forfor IgG1-2F8-ITLxIgG4-7D8-CPPC). IgG1-2F8-ITLxIgG4-7D8-CPPC). For the For the peakpeak of bispecific of bispecific antibody, the antibody, the area area under the curve under the curve was was determined determinedand anddivided dividedbybythe thetotal total area area under the under the curves curvestotocalculate calculatethe thepercentage percentage bispecific bispecific antibody antibody in in each each sample. sample.

Figure 6A Figure 6A shows showsthree three representative representative mass massspectrometry spectrometryprofiles profiles ofof the the Fab-arm Fab-arm exchange reaction exchange reaction between betweenIgG1-2F8-ITL IgG1-2F8-ITL andand IgG4-7D8-CPPC IgG4-7D8-CPPC with with 0 0 mM mM 2-MEA 2-MEA 20 (two 20 (two peaks peaks corresponding corresponding to the to the parental parental antibodies), antibodies), 7 mM 7 mM 2-MEA2-MEA (three(three peaks peaks correspondingtotothe corresponding theparental parentalandand thethe bispecific bispecific antibodies), antibodies), andand 40 2-MEA 40 mM mM 2-MEA (one (one peak corresponding peak corresponding totothe thebispecific bispecific antibody). antibody). The Thehomogenous homogenous peak peak of theof the bispecific product bispecific indicates that product indicates no light that no light chain mispairing occurred, chain mispairing occurred,which whichwould would have have

resulted in resulted in subdivided subdividedpeaks. peaks. TheThe quantified quantified data data are presented are presented in 6B in Figure Figure and 6B and 25 show 25 show thatthat Fab-arm Fab-arm exchange exchange between between IgG1-2F8-ITL IgG1-2F8-ITL and IgG4-7D8-CPPC and IgG4-7D8-CPPC resulted resulted in in nearly 100% nearly 100% bispecificantibody. bispecific antibody. In contrast, In contrast, Fab-arm Fab-arm exchange exchange between between wild type wild type IgG4antibodies IgG4 antibodiesresulted resultedininless lessthan than50%50% bispecific bispecific product. product. These These data data confirm confirm the the results from results the bispecific from the bispecific binding ELISAdescribed binding ELISA describedininExample Example 11 (Figure 11 (Figure 5). 5).

30 Example 30 Example 13: 13: Stabilityofofbispecific Stability bispecific antibodies antibodies generated generated by by 2-MEA-induced 2-MEA-induced Fab-arm exchange Fab-arm exchange The stability of The stability of bispecific bispecificantibodies antibodies generated by2-MEA-induced generated by 2-MEA-induced in vitro in vitro Fab Fab-

arm exchange arm exchangewas was tested.Therefore, tested. Therefore,2 2µgpgofofa abispecific bispecific sample sample generated from generatedfrom IgG1-2F8-ITL and IgG1-2F8-ITL and IgG4-7D8-CPPC IgG4-7D8-CPPCwith with7.0 7.0mMmM 2-MEA 2-MEA (as (as described described in in Example Example 11, 11, 35 Figure 35 Figure 5) 5)was was used used in ina aGSH-induced GSH-inducedFab-arm Fab-arm exchange exchange reaction reaction in inthe thepresence presenceofof aa

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2022201608 08 Mar series(0,(0,2,2, 20, concentration series concentration 20, 100 pg) irrelevant 100 µg) irrelevant IgG4 (IgG4-MG against IgG4 (IgG4-MG against acetylcholine receptor), acetylcholine receptor), representing representinga a0,0,1,1,10, 10,50x excess 50xexcess of of IgG4-MG IgG4-MG compared compared to to the 22 µgpgbispecific the bispecifictest testsample. sample.Fab-arm Fab-arm exchange exchange in thisinreaction this reaction would inresult would result in loss of loss of bispecific bispecific EGFR/CD20 binding. EGFR/CD20 binding. TheThe conditions conditions for the for the GSH reduction GSH reduction reaction reaction

5 were 5 were thethe same same as as described described in inExample Example 7 (24hat at37°C 7 (24h 37°C in in0.5 0.5mLmLPBS/0.5 PBS/0.5mMmM GSH). GSH). To stop To stop the the reduction reduction reaction, reaction,0.5 0.5 mL mL PBSTB wasadded PBSTB was addedtotothe thereaction reaction mixture. mixture. Bispecific binding Bispecific wasmeasured binding was measured in ELISA in an an ELISA as described as described in Example in Example 7. Bispecific 7. Bispecific

binding after binding after the theGSHGSH reduction reduction reaction reaction is presented is presented relative relative to the bispecific to the bispecific

binding measured binding measuredin in the the startingmaterial starting material(control), (control),which which waswas set set to 100%. to 100%.

10 10 Figure 7A Figure shows that 7A shows that for for the the IgG1-2F8-ITL IgG1-2F8-ITL Xx IgG4-7D8-CPPC IgG4-7D8-CPPC derived derived bispecific sample, bispecific EGFR/CD20 sample, EGFR/CD20 bispecific bispecific binding binding is notissignificantly not significantly changed changed after after GSH-induced Fab-arm GSH-induced Fab-armexchange exchange in the in the presence presence of of irrelevantIgG4. irrelevant IgG4.This Thisindicates indicates that the that the bispecific bispecific product is stable, product is stable, i.e. does not i.e.does not participate participate ininGSH-induced Fab-arm GSH-induced Fab-arm

exchange. As exchange. As a acontrol, control, Figure Figure 7B 7Bshows showsthat thatan an IgG4-2F8 IgG4-2F8 x IgG4-7D8 X IgG4-7D8 derived derived 15 sample 15 sample shows shows diminished diminished EGFR/CD20 EGFR/CD20 bispecific bispecific binding binding after after GSH-induced GSH-induced Fab-arm Fab-arm exchangein inthethe exchange presence presence of irrelevant of irrelevant IgG4, IgG4, indicating indicating that that this this is product product not is not stable. These stable. These data show that data show that the the heterodimer consisting of heterodimer consisting of aa human humanIgG1 IgG1 heavy heavy chain containing chain containing the the triple triplemutation mutationT3501-K370T-F405L T350I-K370T-F405L in in the the CH3 domain, and CH3 domain, and aa humanIgG4 human IgG4 heavy heavy chainchain containing containing the substitution the S228P S228P substitution resulting resulting in a stabilized in a stabilized

20 hinge 20 hinge (CPPC),is isstable. (CPPC), stable.

Example 14: Example 14:InInvivo analysisof of vivoanalysis the the pharmacokinetics pharmacokinetics and and stability stability of of bispecific antibodies bispecific antibodies generated by2-MEA-induced generated by 2-MEA-induced Fab-arm Fab-arm exchange exchange The bispecific The bispecific antibody antibody generated generatedby by in vitro in vitro 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm 25 exchange 25 exchange between between IgG1-2F8-ITLX xIgG4-7D8-CPPC IgG1-2F8-ITL IgG4-7D8-CPPCwas wasinjected injected in in SCID SCID mice mice to to analyze its analyze its stability stability(in(invivo vivoFab-arm Fab-arm exchange) and pharmacokinetic exchange) and pharmacokineticproperties properties (plasma clearancerate) (plasma clearance rate)in incomparison comparison to parental to the the parental antibodies antibodies IgG1-2F8-ITL IgG1-2F8-ITL and and IgG4-7D8-CPPC. Three IgG4-7D8-CPPC. Three groups groups ofofmice mice(3 (3 micemice per per group) group) were were injected injected intravenouslyinin the intravenously thetail tail vein veinwith with200200 pL purified µL purified antibody: antibody: (1) µg100 (1) 100 pg bispecific bispecific

30 antibody; 30 antibody; (2)(2)100100 µg pg bispecificantibody bispecific antibody+ +1,000 1,000 µg pg irrelevantIgG4 irrelevant IgG4(natalizumab, (natalizumab, anti-a4-integrin); (3)(3)5050pgµgIgG1-2F8-ITL anti-a4-integrin); IgG1-2F8-ITL ++ 50 50 pg IgG4-7D8-CPPC. Blood µg IgG4-7D8-CPPC. Bloodsamples samples (50-100 pL)were (50-100 µL) were collected collected by by cheek cheek puncture puncture at pre-determined at pre-determined time intervals time intervals after after

antibody administration antibody administration(10(10min, 3h,3h, min, 1, 7, 1, 2, 2, 14, 7, 14, 21 days). 21 days). BloodBlood was collected was collected into into heparin containing heparin containingvials vialsand andcentrifuged centrifuged forfor 10 10 minmin at 14,000 at 14,000 g. Plasma g. Plasma was was stored stored 35 at -20°C 35 at -20°C before before further further analysis. analysis.

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2022201608 08 Mar Total IgG Total IgG concentrations concentrations ininthe theplasma plasmasamples samples were were assayed by ELISA. assayed by ELISA. The The conditionsofofthe assay conditions assay the succeeding succeedingsteps steps were were the the samesame as the as for for ELISA the ELISA described described in in Example7.7.Specific Example compounds Specificcompounds used used for total for total IgG measurement were the were IgG measurement the following: following:

coat with coat with 2 pg/mL mouse 2 µg/mL mouseanti-human anti-human IgGIgG (clone (clone CLB; CLB; MH16-1; MH16-1; cat. cat. no. M1268); no. M1268); 5 serum 5 serum samples samples dilutions dilutions (1:500 (1:500 andand 1:2,500 1:2,500 forfor groups groups 1 and 1 and 3) 3) andand (1:2,500 (1:2,500 andand 1:10,000for 1:10,000 forgroup group2);2);conjugate: conjugate: HRP-conjugated HRP-conjugated goat anti-human goat anti-human IgG11H; IgG (clone (clone 11H; Jackson;cat. Jackson; cat. no. no.109-035-098; 109-035-098; 1:10,000). 1:10,000). The presence The presence of bispecific of bispecific antibodies antibodies in in the plasma the plasma samples sampleswaswas assayed assayed and quantified and quantified by and by CD20 CD20EGFR andbispecific EGFR bispecific reactivity in reactivity in an an ELISA as described ELISA as describedinin Example Example10.10.

10 10 Figure 8A Figure 8A shows showstotal total antibody antibodyplasma plasmaconcentrations. concentrations.The The shape shape of the of the plasma clearance curves plasma clearance curves was wasidentical identical inin all all groups, groups, indicating indicating that the plasma that the plasma clearance ofofthe clearance thebispecific bispecificantibody antibody was was the as the same same for as the for the parental parental antibodiesantibodies

IgG1-2F8-ITL and IgG1-2F8-ITL and IgG4-7D8-CPPC IgG4-7D8-CPPC over over thethe analyzed analyzed timeinterval. time interval. Figure Figure 8B shows 8B shows the plasma the plasmaconcentrations concentrations of bispecific of bispecific antibodies antibodies overover time.time. The addition The addition of of a 10- a 10 15 foldfold 15 excess excess irrelevant irrelevant IgG4IgG4 to bispecific to the the bispecific antibody antibody did affect did not not affect bispecific bispecific antibody antibody

concentrations,indicating concentrations, indicating that that nonoFab-arm Fab-arm exchange exchange occurred occurred in vivo. in vivo. After After injection injection

of the of parental antibodies the parental antibodies(IgG1-2F8-ITL (IgG1-2F8-ITL + IgG4-7D8-CPPC), + IgG4-7D8-CPPC), no bispecific no bispecific antibodies antibodies

were detectable were detectableininthe theplasma, plasma, confirming confirming thatthat these these antibodies antibodies doparticipate do not not participate in in Fab-armexchange Fab-arm exchange in vivo. in vivo. These These data data indicate indicate thatbispecific that the the bispecific antibody antibody product, product,

20 generated 20 generated by vitro by in in vitro 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange between between IgGl-2F8-ITL IgG1-2F8-ITL X x IgG4-7D8-CPPC,was IgG4-7D8-CPPC, wasstable stableinin vivo vivo (no (no Fab-arm Fab-arm exchange) exchange)and andshowed showed comparable comparable pharmacokinetic properties pharmacokinetic properties (plasma (plasma clearance clearance rate) rate) asasthe theparental parentalmonovalent monovalent antibodies. antibodies.

25 Example 25 Example 15: 15: Purity Purity of of thebispecific the bispecific antibody antibody generated generated by by2-MEA-induced 2-MEA-induced Fab-arm exchange Fab-arm exchange between betweentwo twoantibodies antibodies A batch A batchof ofbispecific bispecificantibody, antibody,generated generated by 2-MEA-induced by 2-MEA-induced Fab-armFab-arm exchange between exchange betweenhuman human IgGl-2F8-ITL IgG1-2F8-ITL x IgG4-7D8-CPPC, X IgG4-7D8-CPPC, was purified was purified on aonPD-10 a PD-10 desalting column desalting (cat.no. 17-0851-01; column (cat.no. 17-0851-01;GEGEHealthcare). Healthcare).Next, Next, thethe purity purity of of thethe 30 bispecific 30 bispecific product product was was analyzed analyzed by bysodium sodium dodecyl dodecyl sulfate sulfate polyacrylamide polyacrylamide gelelectrophoresis (SDS-PAGE), gelelectrophoresis high performance (SDS-PAGE), high performancesize sizeexclusion exclusionchromatography chromatography (HP-SEC) andmass (HP-SEC) and mass spectrometry. spectrometry. The The functionality functionality of the of the generated generated bispecific bispecific antibody was antibody wasconfirmed confirmed by by bispecific bispecific binding binding in in an an ELISA ELISA (data (data not not shown). shown).

SDS-PAGEwas SDS-PAGE wasperformed performed under under reducing reducing andand non-reducing non-reducing conditions conditions on on 4- 4 35 12%12% 35 NuPAGE NuPAGE Bis-Tris Bis-Tris gels gels (Invitrogen, (Invitrogen, Breda, Breda, The The Netherlands) Netherlands) usingusing a modified a modified

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2022201608 08 Mar Laemli method Laemli (Laemli 1970 method (Laemli Nature 227(5259): 1970 Nature 227(5259): 680-5), 680-5), where the samples where the samples were were run run at neutral at pH. The neutral pH. TheSDS-PAGE SDS-PAGEgels gels were were stained stained with Coomassie with Coomassie and digitally and digitally imaged imaged using the using the GeneGenius GeneGenius(Synoptics, (Synoptics,Cambridge, Cambridge, UK).UK). Figure Figure 9A shows 9A shows that that the the antibody sample antibody sample after after Fab-arm Fab-arm exchange exchange consists consists of intact of intact IgG,a with IgG, with trace tracea of half of half 5 molecules 5 molecules (H111) (H1L1) detectableononthe detectable thenon-reduced non-reducedgel gel(Figure (Figure 9A-b). 9A-b). HP-SECfractionation HP-SEC fractionationwaswas performed performed usingusing a Waters a Waters Alliance Alliance 2695 separation 2695 separation

unit (Waters, unit Etten-Leur, The (Waters, Etten-Leur, Netherlands) connected The Netherlands) connected totoa aTSK TSK HP-SEC HP-SEC column column (G3000SWx Toso (G3000SWxl; Toso Biosciences, Biosciences, via via Omnilabo, Omnilabo, Breda,Breda, The Netherlands) The Netherlands) and a and a Waters Waters 2487 dual 2487 dual 2k absorbance absorbancedetector detector(Waters). (Waters).The Thesamples samples were were run run at 1atmL/min. 1 mL/min. 10 Results 10 Results were were processed processed using using Empower Empower software software version version 2002 2002 and expressed and expressed per per peak aspercentage peak as percentageof of totalpeak total peak height. height. Figure Figure 9B shows 9B shows that of>98% that >98% of the sample the sample

consists of consists of intact intact IgG, IgG, with practically no with practically no aggregates formed. aggregates formed.

Mass spectrometry Mass spectrometry was wasperformed performedas as described described in in Example Example 12. Figure 12. Figure 9C 9C shows the shows the mass massspectrometry spectrometryprofiles profilesofofthe the starting starting materials materials IgG1-2F8-ITL IgG1-2F8-ITL and and 15 IgG4-7D8-CPPC 15 IgG4-7D8-CPPC and bispecific and the the bispecific product product generated generated by by Fab-arm Fab-arm exchange exchange between between IgG1-2F8-ITL Xx IgG4-7D8-CPPC. IgG1-2F8-ITL IgG4-7D8-CPPC.TheThe product product in in theFab-arm the Fab-arm exchanged exchanged sample sample is is 145,901kDa, 145,901 kDa,which which perfectly perfectly matches matches with with the bispecific the bispecific product product derived derived from from IgG1- IgG1 2F8-ITL (146,259.5/2=73,130) 2F8-ITL (146,259.5/2=73,130) + +IgG4-7D8-CPPC IgG4-7D8-CPPC (145,542.0/2=72,771). (145,542.0/2=72,771). Moreover, Moreover, the bispecific the bispecific antibody productshowed antibody product showed a homogenous a homogenous peak, indicating peak, indicating that no that lightno light 20 chain 20 chain mispairing mispairing occurred,which occurred, which would would havehave resulted resulted in subdivided in subdivided peaks. peaks. These These data show data show that that Fab-arm Fab-arm exchange resultedinin100% exchangeresulted 100% bispecificantibody. bispecific antibody. The The small small peaks detected peaks detected inin addition addition to to the themain mainpeak peak (K0)(KO) of the of the IgG4-7D8-CPPC IgG4-7D8-CPPC and and bispecific sample bispecific sample can can be attributed to be attributed to the the presence of one presence of one (K1) (K1) oror two two(K2) (K2)C-C terminal lysines. terminal lysines. 25 25 These data show These data showthat thata a~100% ~100% functional functional bispecificantibody bispecific antibodysample sample waswas generated by generated by 2-MEA-induced 2-MEA-induced Fab-arm Fab-armexchange exchange between between IgG1-2F8-ITL IgG1-2F8-ITL x IgG4-7D8 X IgG4-7D8- CPPC. CPPC.

Example 16: Unraveling Example 16: Unraveling the therequirement requirementofofthe theT350I, T3501,K370T K370T andand F405L F405L 30 substitutionsfor 30 substitutions for Fab-arm Fab-arm exchange exchangeengagement engagement of of human human IgG1IgGI To further identify To further identify the determinantsininthe the determinants theIgG1 IgG1 CH3CH3 domain domain that that are required are required

for IgG1 for to be IgG1 to engaged inin Fab-arm be engaged Fab-armexchange, exchange,IgG1 IgG1 containingthe containing thetriple triple mutation mutation T3501-K370T-F405L (ITL) was T350I-K370T-F405L (ITL) wascompared compared to to thethe double double mutants mutants T3501-K370T T350I-K370T (IT),(IT), T3501-F405L (IL) and T350I-F405L (IL) andK370T-F405L K370T-F405L (TL). (TL). Also Also thethe single single mutant mutant F405L F405L (L) was (L) was 35 tested.2-MEA 35 tested. 2-MEA waswas usedused as aasreductant a reductant to to induce induce in in vitroFab-arm vitro Fab-armexchange exchange (50(50 µg pg

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2022201608 08 Mar of each of antibody in each antibody 100 pL in 100 PBS/25 mM µL PBS/25 mM2-MEA 2-MEA forfor 90 90 minmin at 37C). at 37°C). ForFor thethe single single mutant F405L mutant F405Lantibody, antibody, unpurifiedantibody unpurified antibody fromfrom supernatant supernatant of a transient of a transient transfection was transfection wasused used after after buffer-exchange buffer-exchange to using to PBS PBS Amicon using Ultra Amicon Ultra centrifugal centrifugal

devices (30k, devices (30k,Millipore, Millipore,cat. cat.no. no.UFC803096). UFC803096). To the To stop stop the reduction reduction reaction,reaction, the the 5 reducing 5 reducing agent agent 2-MEA 2-MEA waswas removed removed by desalting by desalting the the samples samples using using spinspin columns columns as as described inin Example described Example11.11. TheThe generation generation of bispecific of bispecific antibodies antibodies was determined was determined by by bispecific binding bispecific binding measured measured ininananELISA ELISA as as described described in Example in Example 7. 7. The triple (ITL), The triple (ITL), double doublemutations mutations (IT,(IT, IL TL) IL and and and TL)single and single mutation mutation (L) (L) were introduced were introduced in in IgG1-2F8. IgG1-2F8. These mutants were These mutants were combined combined with with IgG4-7D8, IgG4-7D8, 10 containing 10 containing a CPSC a CPSC hinge hinge (wild or (wild type) type) or a stabilized a stabilized hinge (IgG4-7D8-CPPC), hinge (IgG4-7D8-CPPC), for Fab- for Fab arm exchange exchangeusing using 25 25 mMmM2-MEA 2-MEA 0 arm forfor 90 90 at at minmin 37 C. 37°C. Figure10A-B Figure 10A-B shows shows that that the the IgG1-2F8-IL and IgG1-2F8-IL and -TL -TL mutants mutantsshowed showed Fab-arm Fab-arm exchange exchange to the to the samesame levellevel as as the the triple mutant triple ITL, irrespective mutant ITL, irrespective ofofthe thecombined combined IgG4-7D8 IgG4-7D8 (CPSC (CPSC or CPPC or CPPC In hinge). hinge). In contrast, no contrast, no bispecific bispecific binding binding was wasfound found for for thethe combination combination withIgG1-2F8-IT with the the IgG1-2F8-IT 15 mutant. 15 mutant. Figure Figure 10C10C shows shows that that alsoalso the the IgG1-2F8-F405L IgG1-2F8-F405L mutant mutant showedshowed Fab-armFab-arm exchange,irrespective exchange, irrespectiveofofthe thecombined combined IgG4-7D8 IgG4-7D8 (CPSC (CPSC or CPPCor CPPC These hinge). hinge). These data data indicate that indicate thatthe theF405L F405L mutation is sufficient mutation is sufficientto to engage engagehuman human IgG1 for Fab-arm IgG1 for Fab-arm exchangeunder exchange under thethe conditions conditions mentioned mentioned above. above.

20 Example 20 Example 17:17: Generation Generation of of bispecificantibodies bispecific antibodies by by 2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchangeatatdifferent exchange differenttemperatures temperatures The ability of The ability of 2-MEA 2-MEA totoinduce induce thethe generation generation of bispecific of bispecific antibodies antibodies by Fab by Fab-

arm exchange arm exchangebetween betweentwo two different different antibodies,waswas antibodies, tested tested at different at different temperatures. The temperatures. The Fab-arm Fab-armexchange exchangereactions reactionswere werestarted startedbybyincubating incubating160 160µgpg 25 human 25 human IgG1-2F8-ITL IgG1-2F8-ITL with with 160 160 tg IgG4-7D8-CPPC µg IgG4-7D8-CPPC in 320inµl 320 pl PBS/25 PBS/25 mM (final mM 2-MEA 2-MEA (final concentration of 0 0 concentration of 0.5 0.5 mg/mL for each mg/mL for each antibody) antibody) at at either either 0°C, 20 C (RT) 0°C, 20°C (RT) or or 37°C. 37 C. From these From thesereactions, reactions,20 20 tL samples µL samples were were taken taken at different at different time points time points (0,5,2.5, (0, 2.5, 5, 10, 15, 10, 15, 30, 30, 45, 45, 60, 60, 75, 75, 90, 90, 120, 120, 150, 150, 180 180 and 240 min). and 240 20 µL min). 20 tL PBS PBS was wasadded addedto to each sample each samplebefore beforethethereducing reducing agent agent 2-MEA 2-MEA was removed was removed by desalting by desalting the the 30 samples 30 samples using using a Zeba a Zeba 96-well 96-well spinspin desalting desalting plate plate (7k,cat# (7k, cat#89808 89808 Thermo Thermo Fisher Fisher Scientific), according Scientific), to the according to themanufacturer's manufacturer's recommendations. recommendations. The The total total antibody antibody concentrations were concentrations determined bybymeasuring were determined measuringabsorbance absorbance at at 280280 nm wavelength nm wavelength using using aa Nanodrop ND-1000spectrophotometer Nanodrop ND-1000 spectrophotometer (Isogen (Isogen LifeScience, Life Science,Maarssen, Maarssen,The The Netherlands). Dilution Netherlands). Dilution series series of of the the antibody antibodysamples samples (totalantibody (total antibody concentration concentration 0- 0-

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2022201608 08 Mar 20 µg/mL 20 pg/mLin in 25-fold 25-fold dilutions) dilutions) were were usedused in anin ELISA an ELISA to measure to measure bispecific bispecific binding binding

as described as described inin Example Example7. 7. Figure 11 Figure 11 shows showsthat thatthe thegeneration generation of of bispecificantibodies bispecific antibodies by by 2-MEA-induced 2-MEA-induced

Fab-arm exchange Fab-arm exchangebetween betweenhuman human IgG1-2F8-ITL IgG1-2F8-ITL and and IgG4-7D8-CPPC IgG4-7D8-CPPC was to was found found to be most 5 be most 5 efficient efficient at 37°C, at 37°C, with maximal with maximal bispecific bispecific bindingbinding reached reached after 45 after 45 min. At min. At room temperature, room temperature,the thegeneration generationof of bispecific antibodies bispecific antibodies was wasslower, slower,reaching reaching maximal bispecificbinding maximal bispecific bindingafter after240 240 At At min. min. 0°C, 0°C, no generation no generation of bispecific of bispecific binding binding

wasobserved was observedduring during thethe analyzed analyzed timetime course. course.

10 Example 10 Example 18: Analysis 18: Analysis of different of different reducing reducing agentsagents for their for their ability ability to induce to induce the generation the generationofofbispecific bispecific antibodies antibodies by byin in vitro vitro Fab-arm exchange Fab-arm exchange It has It been shown has been shownabove above that that 0.50.5 mM mM GSHinduce GSH can can induce in Fab-arm in vitro vitro Fab-arm exchange between exchange betweenhuman human IgG4 IgG4 and and IgG1-CPSC-ITL, IgG1-CPSC-ITL, but between but not not between human human IgG4 IgG4 and IgG1-ITL and IgG1-ITLcontaining containing a stable a stable hinge hinge (Figure (Figure 3). addition, 3). In In addition, 2-MEA2-MEA wastofound was found to 15 be be 15 ableable to induce to induce Fab-arm Fab-arm exchange exchange betweenbetween antibodies antibodies with stabilized with stabilized hinge hinge regions, such regions, such as IgG1-ITL Xx IgG4-CPPC as IgG1-ITL IgG4-CPPC (Figure (Figure 5). 5). To To test test whether whether other other concentrationsofofGSH concentrations GSHor or 2-MEA 2-MEA or other or other reducing reducing agentsagents are capable are capable of inducing of inducing in in vitro Fab-arm vitro exchange Fab-arm exchange between between two different two different antibodies, antibodies, concentration concentration series series of 2- of 2 MEA, GSH MEA, GSHand andDTTDTT (dithiothreitol) were (dithiothreitol) were tested. tested. Therefore, Therefore, combinations combinations of of 10 10 µg pg 20 human 20 human IgG1-2F8-ITL IgG1-2F8-ITL andµg10IgG4-7D8-CPPC and 10 pg IgG4-7D8-CPPC in 20 in 20 µl PBS (final PBS pl(final concentration concentration of of 0.5 mg/mL 0.5 mg/mLforfor each each antibody) antibody) were were incubated incubated 370concentration at 37°Catwith C with concentration series of series of the different the different reducing reducing agents agents(0.0, (0.0,0.04, 0.04,0.1, 0.1, 0.2, 0.2, 0.5, 0.5, 1.0, 1.0, 2.5, 2.5, 5.0,12.5, 5.0, 12.5, 25.0 25.0 and and

50.0 mM). 50.0 mM). After After 90 90 min, min, 2020µLpLPBS PBS waswas added added to each to each sample sample and reducing and the the reducing agent was agent wasremoved removed by desalting by desalting the samples the samples usingdesalting using spin spin desalting plate plate as as described described

25 in in 25 Example Example 17. Total 17. Total antibody antibody concentrations concentrations were were determined determined as described as described in in Example17.17.Dilution Example Dilutionseries seriesofofthe theantibody antibody samples samples (total (total antibody antibody concentration concentration 0- 0 20 µg/mL 20 pg/mLin in3-fold 3-folddilutions) dilutions)were wereused used in in an an ELISA ELISA to measure to measure bispecific bispecific binding binding as as described inin Example described Example7. 7. Figure 12 Figure 12 confirms confirms that that2-MEA 2-MEA induces induces maximal maximal bispecific bispecific binding binding at aat a 30 concentration 30 concentration ofof2525mMmM 2-MEA. 2-MEA. DTT DTT was was found found to very to be be very effectiveininthe effective the generation generation of bispecific of bispecific antibodies antibodies with maximalbispecific with maximal bispecificbinding bindingreached reached at at 2.52.5 mM DTT. mM DTT. GSH GSH concentrations in concentrations in the range 0-5 the range 0-5 mMmMwere were not not ableable to induce to induce the the generation generation of of bispecific antibodies bispecific antibodiesbybyFab-arm Fab-arm exchange betweenthe exchange between theIgG1-ITL IgG1-ITLandand IgG4-CPPC IgG4-CPPC antibodies, both antibodies, containing stabilized both containing stabilized hinge hinge regions. regions. Higher Higher GSH concentrations GSH concentrations (12.5-50 35 (12.5-50 35 mM)mM) resulted resulted in the in the formation formation of of antibodyaggregates, antibody aggregates,asaswas wasdetermined determined

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by non-reducing by non-reducing SDS-PAGE SDS-PAGE (data (data not shown). not shown). Therefore, these these Therefore, samplessamples were were excluded from excluded from the theanalysis. analysis. These Thesedata datashow show thatthat the the generation generation of bispecific of bispecific antibodies by antibodies byFab-arm Fab-arm exchange exchange between between two different two different antibodies antibodies can be can be induced induced by by different reducing different agents. reducing agents.

5 5 Example 19: Determinants Example 19: Determinantsatatthe the IgG1 IgGI409 409position positionfor forengagement engagementin in2-2 MEA-induced Fab-arm exchange MEA-induced Fab-arm exchangeinincombination combinationwith withIgG1-ITL IgGI-ITL 2-MEA can 2-MEA caninduce induceFab-arm Fab-arm exchange exchange between between humanhuman IgG1-ITL IgG1-ITL and and IgG4- IgG4 CPPC, asasdescribed CPPC, describedin inExample Example 11 (Figure 11 (Figure 5). CH3 5). The Theinterface CH3 interface residuesresidues of humanof human 10 IgG1IgG1 10 and IgG4 and IgG4 differdiffer at position at position 409 only: 409 only: lysinelysine (K) in(K)IgG1 in IgG1 and arginine and arginine (R) in (R) IgG4in IgG4 (described inin Example (described Example8, 8, Figure Figure 2).2). Therefore, Therefore, it was it was tested tested whether whether substitution substitution of of lysine at lysine at position position 409 by arginine 409 by arginine or or any anyother otheramino amino acid acid (K409X) (K409X) could could enable enable IgG1 IgG1 to engage to in 2-MEA-induced engage in 2-MEA-induced Fab-arm Fab-armexchange exchange with with IgG1-ITL. IgG1-ITL. Combinations Combinations of of 10 10 pg human µg human IgG1-2F8-ITL IgG1-2F8-ITL and and 10 10 µg pg IgG1-7D8-K409X IgG1-7D8-K409X in in 20 20 µl pl PBS/25 PBS/25 mM 2-MEA mM 2-MEA 15 (final 15 (finalconcentration concentration of of 0.5 0.5 mg/mL mg/mLfor foreach eachantibody) antibody)were wereincubated incubatedfor for9090min minatat 37°C. Unpurified 37°C. Unpurifiedantibodies antibodies from from supernatants supernatants of transient of transient transfections transfections were were used used after buffer-exchange after buffer-exchangetotoPBSPBS using using Amicon Amicon Ultra Ultra centrifugal centrifugal devices devices (30k, Millipore, (30k, Millipore,

cat. no. cat. UFC803096).After no. UFC803096). After thethe Fab-arm Fab-arm exchange exchange reaction, reaction, 20 pL 20 µL PBS was PBS was added to added to each sample each sample and andthe thereducing reducing agent agentwas wasremoved removed by desalting by desalting thethe samples samples using using 20 spin 20 spin desaltingplate desalting plateas asdescribed describedin inExample Example 17. 17. Dilution Dilution series series of of thethe antibody antibody samples(total samples (totalantibody antibodyconcentration concentration 0-200-20 µg/mLpg/mL in 3-fold in 3-fold dilutions) dilutions) werein used were used in an ELISA an ELISAtotomeasure measure bispecific bispecific binding binding as as described described in Example in Example 7. 7. Figure 13A Figure 13Ashows shows thethe results results of bispecific of bispecific binding binding uponupon 2-MEA2-MEA inducedinduced Fab- Fab arm exchange arm exchange between between IgG1-2F8-ITL IgG1-2F8-ITL X xIgG1-7D8-K409X. IgG1-7D8-K409X.In InFigure Figure13B, 13B,thethe 25 exchange 25 exchange is presented is presented as bispecific as bispecific bindingbinding relative relative to a purified to a purified batch batch of of bispecific bispecific

antibody derived antibody derived from from aa 2-MEA-induced 2-MEA-inducedFab-arm-exchange Fab-arm-exchange between between IgG1-2F8-ITL IgG1-2F8-ITL and IgG4-7D8-CPPC, and IgG4-7D8-CPPC,which whichwas wasset settoto100%. 100%.These These data data were were alsoscored also scoredasas(-) (-) no no Fab-arm exchange, Fab-arm exchange,(+/-) (+/-) low, low, (+) intermediate or (+) intermediate or (++) high Fab-arm (++) high exchange,as Fab-arm exchange, as presentedininTable presented Table1.1.NoNoFab-arm Fab-arm exchange exchange (-)found (-) was was when foundthewhen the 409 inposition 409 position in 30 IgG1-7D8 30 IgG1-7D8 waswas K (=wildtype K (=wild typeIgG1), IgG1), L L or or M. M. Fab-arm Fab-arm exchange exchange was was found found to to be be intermediate(+) intermediate when (+) when thethe 409 409 position position in IgG1-7D8 in IgG1-7D8 was F,was I, NF,orI,Y Nand or high Y and high (++) (++) whenthe when the409 409 positionin inIgG1-7D8 position IgG1-7D8 was was A, D,A,E,D,G,E,H,G,Q,H,R,Q,S,R,T,S,V T, or VW.or W.

Table 1: Table 1: 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange between between IgG1-2F8-ITL IgG1-2F8-ITL andand 35 IgG1-7D8-K409X 35 IgG1-7D8-K409X mutants. mutants. The generation The generation of bispecific of bispecific antibodies antibodies after 2-MEA- after 2-MEA-

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2022201608 08 Mar induced in induced in vitro vitro Fab-arm exchangebetween Fab-arm exchange between IgG1-2F8-ITL IgG1-2F8-ITL and IgG-7D8-K409X and IgG1-7D8-K409X mutants was mutants was determined determined by a by a sandwich sandwich ELISA. ELISA. (-) no, (-)no, (+-)low, (+/-) low, ()intermediate, (+) intermediate,

high Fab-arm (++) high (++) Fab-arm exchange. exchange. Fab-arm exchange Fab-arm exchange IgGl-7D8-K409X IgG1-7D8-K409X x IgGl-2F8-ITL X IgG1-2F8-ITL A A ++ ++ D ++ E ++ F + G ++ H ++ I + K I

L - M -

N + Q ++ R ++ S ++ T ++ V ++ W ++ Y +

5 Example 5 Example 20: Antibody 20: Antibody deglycosylation deglycosylation does does not not influence influence the generation the generation of of bispecific bispecific antibodies antibodies by by 2-MEA-induced Fab-arm 2-MEA-induced Fab-arm exchange exchange IgG4-7D8and IgG4-7D8 andIgG4-7D8-CPPC IgG4-7D8-CPPC samples samples were were deglycosylated deglycosylated by incubating by incubating 200 µg 200 antibody overnight pg antibody overnight at at37°Cwith 37°C with 0.005 0.005 UN-Glycanase U N-Glycanase (cat.no. (cat.no.GKE-5006D; GKE-5006D; Prozyme) Prozyme) in in 180 180 µL PBS.These pLPBS. Thesesamples sampleswere wereused used directlyin directly inaa Fab-arm exchange Fab-arm exchange 10 reaction. 10 reaction.Fab-arm Fab-arm exchange exchange waswas performed performed by incubating by incubating 50 of 50 µg pg each ofeach antibody antibody in in 100 µl 100 plPBS/25 mM2-MEA PBS/25 mM 2-MEA (finalconcentration (final concentrationofof0.5 0.5 mg/mL mg/mL foreach for eachantibody) antibody)for for 90min 90 at 37°C. min at The reducing 37°C. The reducing agent agent 2-MEA 2-MEAwas was removed removed by by desalting desalting thethe samples samples using spin using spin columns asdescribed columns as describedininExample Example11.11. Dilutionseries Dilution seriesofofthe theantibody antibody (totalantibody samples(total samples concentration antibodyconcentration 0-20 0-20 pg/mL µg/mL in 3-fold in 3-fold dilutions) dilutions) were were used used in a in a 15 sandwich 15 sandwich ELISAELISA to measure to measure bispecific bispecific bindingbinding as described as described in Example in Example 7. 7. Mass spectrometry Mass spectrometry analysis analysis showed showed that that the the deglycosylation deglycosylation reaction reaction resulted resulted

in in 100%/ deglycosylatedantibody 100% deglycosylated antibodyproduct product(data (datanot notshown). shown).Figure Figure1414shows showsthat that Fab-arm exchange Fab-arm exchange involving involving deglycosylated deglycosylated antibodies antibodies did notdid not from differ differFab-arm from Fab-arm exchangewith exchange with the the corresponding correspondingglycosylated glycosylated antibodies antibodies (IgG4-2F8 (IgG4-2F8XxIgG4-7D8 IgG4-7D8- 20 deglycosylated 20 deglycosylated versus versus IgG4-2F8x IgG4-2F8 IgG4-7D8 X IgG4-7D8 and andIgG1-2F8-ITL xIgG4-7D8-CPPC- IgG1-2F8-ITL X IgG4-7D8-CPPC-

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deglycosylated versus deglycosylated IgG1-2F8-ITL Xx IgG4-7D8-CPPC). versus IgG1-2F8-ITL IgG4-7D8-CPPC). These These datadata indicate indicate thatthat deglycosylationdid deglycosylation didnotnot affect affect the the generation generation of bispecific of bispecific antibodies antibodies by by 2-MEA- 2-MEA induced Fab-arm induced exchange. Fab-arm exchange. 2022201608 08

5 Example 5 Example 21: Quantification 21: Quantification of the of the non-covalent non-covalent CH3-CH3 CH3-CH3 interaction interaction The strengthofofthe The strength theinteractions interactions at at thethe CH3CH3 interface interface should should be that be such suchitthat is it is

possible that possible that both both heavy heavychains chains in in thethe parental parental antibodies antibodies dissociate dissociate in the in the Fab-arm Fab-arm

exchangereaction exchange reaction andand thatthat they they subsequently subsequently associate associate in the heterodimerization in the heterodimerization

reaction. Therefore, reaction. Therefore,the thecorrelation correlationbetween between the ability the ability to participate to participate in Fab-arm in Fab-arm

10 exchange 10 exchange and and the strength the strength of the of the non-covalent non-covalent CH3-CH3 CH3-CH3 interaction interaction (dissociation (dissociation constant, KD) constant, KD) was was analyzed. analyzed. GSH-induced GSH-induced Fab-arm Fab-arm exchange exchange was performed was performed as as described in described in Example (0.5 mM Example 99 (0.5 mMGSHGSH at 37°C) at 37°C) for for the the following following combinations combinations of of humanantibodies: human antibodies: IgG1-2F8 Xx IgG1-7D8 IgG1-2F8 IgG1-7D8 15 15 IgG1-2F8-CPSCX xIgG1-7D8-CPSC IgG1-2F8-CPSC IgG1-7D8-CPSC IgG1-2F8-CPSC-T350IX xIgG1-CPSC-7D8-T350I IgG1-2F8-CPSC-T350I IgG1-CPSC-7D8-T350I IgGl-2F8-CPSC-K370T x IgGl-7D8-CPSC-K370T IgG1-2F8-CPSC-K370T X IgG1-7D8-CPSC-K370T IgG1-2F8-CPSC-ITL X xIgG1-7D8-CPSC-ITL IgG1-2F8-CPSC-ITL IgG1-7D8-CPSC-ITL IgG1-2F8-CPSC-K409R X IgG1-2F8-CPSC-K409R x IgGl-7D8-CPSC-K409R IgG1-7D8-CPSC-K409R 20 20 IgG4-2F8 Xx IgG4-7D8 IgG4-2F8 IgG4-7D8 IgG4-2F8-R409KX xIgG4-7D8-R409K IgG4-2F8-R409K IgG4-7D8-R409K IgG4-2F8-R409AX xIgG4-7D8-R409A IgG4-2F8-R409A IgG4-7D8-R409A IgG4-2F8-R409LX xIgG4-7D8-R409L IgG4-2F8-R409L IgG4-7D8-R409L IgG4-2F8-R409M X IgG4-2F8-R409M x IgG4-7D8-R409M IgG4-7D8-R409M 25 25 IgG4-2F8-R409TX xIgG4-7D8-R409T IgG4-2F8-R409T IgG4-7D8-R409T IgG4-2F8-R409W x IgG4-7D8-R409W IgG4-2F8-R409W X IgG4-7D8-R409W IgG4-2F8-F405AX xIgG4-7D8-F405A IgG4-2F8-F405A IgG4-7D8-F405A IgG4-2F8-F405L Xx IgG4-7D8-F405L IgG4-2F8-F405L IgG4-7D8-F405L IgG4-2F8-Y349DX xIgG4-7D8-Y349D IgG4-2F8-Y349D IgG4-7D8-Y349D 30 30 IgG4-2F8-L351K IgG4-7D8-L351K IgG4-2F8-L351KX xIgG4-7D8-L351K IgG4-2F8-E357T IgG4-7D8-E357T IgG4-2F8-E357T X xIgG4-7D8-E357T IgG4-2F8-S364DX xIgG4-7D8-S364D IgG4-2F8-S364D IgG4-7D8-S364D IgG4-2F8-K370Q IgG4-2F8-K370Q xX IgG4-7D8-K370Q IgG4-7D8-K370Q IgG4-2F8-K370EX xIgG4-7D8-K370E IgG4-2F8-K370E IgG4-7D8-K370E

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generationofofbispecific The generation The antibodieswaswas bispecificantibodies measured measured by determination by determination of bispecific of bispecific

binding in binding in aa sandwich sandwich ELISA as described ELISA as described in in Example 7. Figures Example 7. Figures 15A/B/C show the 15A/B/C show the results of results of the the bispecific bispecificbinding binding after afterthe the Fab-arm exchange Fab-arm exchange reaction. reaction.

To measure To measurethethe effectof ofthe effect theabove above mentioned mentioned CH3 mutations CH3 mutations on the strength on the strength

5 of of 5 thetheCH3-CH3 CH3-CH3 interaction,fragments interaction, fragmentscomposed composedof of onlythe only theCH2-CH3 CH2-CH3 domains domains were were made.The made. Thelack lack ofof a a hinge hinge region region in these in these fragments fragments prevented prevented covalent covalent inter-heavy inter-heavy

chain disulfide chain disulfidebonds. bonds.The The fragments fragments were analyzed by were analyzed by native native mass mass spectrometry. spectrometry. Samples were Samples werebuffer-exchanged buffer-exchanged toto 100 100mMmM ammonium ammonium acetate acetate pH 7,pHusing 7, using 10 10 kDa kDa MWCO MWCO spin-filtercolumns. spin-filter columns. Aliquots Aliquots (~1(1µL) pL)of serial of serial diluted diluted samples samples (20 (20 pMnM; µM 25 - 25 nM; 10 monomer 10 monomer equivalent) equivalent) were into were loaded loaded into gold-plated gold-plated borosilicate borosilicate capillaries capillaries for analysis for analysis

on aa LCT on LCT mass mass spectrometer spectrometer(Waters). (Waters). The The monomer monomer signal,M,M,waswas signal, defined defined as as the the area of area of the themonomer monomerpeakspeaks as a fraction as a fraction of the of theofarea area of all inpeaks all peaks in the spectrum the spectrum

(M,/(M+D,)where (M/(M+D) where = dimer D =D,the the dimer signal). signal). TheThe concentrationof ofmonomer concentration monomer at at equilibrium, [M]eq, equilibrium, [M]eq, was defined asasM.[M] was defined Ms.[M]o wherewhere

[M] is[M]o is the overall the overall protein protein 15 concentration 15 concentration in interms termsof ofmonomer. monomer. The The dimer dimer concentration concentration at equilibrium, at equilibrium, [D]q,

[D]eq, was defined was definedasas([M]-[M])/2. ([M]o-[M]e)/2. Thewas The KD, wasextracted KD,then then extracted from from the the gradient gradient of a plotof a plot 2 of [D]eq of [D]eq versus The KDKDofofthe versus [M]eq . The

[M]eq². thenon-covalent non-covalent CH3-CH3 CH3-CH3 interactions interactions is presented is presented

in Table in Table 2.2.

The correlation between The correlation the ability between the abilitytotoengage engageininFab-arm Fab-arm exchange and the exchange and the 20 strength 20 strength of of thethe non-covalent non-covalent CH3-CH3 CH3-CH3 interactions interactions was analyzed. was analyzed. Figures Figures 15D/E 15D/E showthe show thepercentage percentage bispecific bispecific binding binding after after Fab-arm Fab-arm exchange exchange plotted plotted against against the the measured KDKDofof the measured the corresponding corresponding CH2-CH3 CH2-CH3fragment fragment (Figure15D15D (Figure forfor IgG1;Figure IgG1; Figure for IgG4). 15E for 15E IgG4). These Thesedata datasuggest suggest that that under under the the tested tested conditions conditions therethere is a is a specific specific

range of range of apparent apparentKDKDvalues values of of thethe CH3-CH3 CH3-CH3 interaction interaction that that allows allows efficient efficient Fab-arm Fab-arm

25 exchange. 25 exchange. Table 2: Table 2: The of the KD of The KD the non-covalent non-covalentCH3-CH3 CH3-CH3 interactions interactions CH2-CH3 construct CH2-CH3 construct KD(M) KD (M) fold-difference* fold-difference* G1 G1 3.0 xX 10-9 3.0 10 1.0000 1.0000 G1-T350I G1-T350I 7.0 xX 10-9 7.0 10 0.4000 0.4000 G1-K370T G1-K370T 4.5 xX 10-8 4.5 10 0.0700 0.0700 G1-ITL G1-ITL 1.0 1.0 xX 10-6 10 0.0030 0.0030 G1-K409R G1-K409R 1.1 1.1 xX 10-7 10 0.0300 0.0300 G4 G4 4.8 xX 10-8 4.8 10 1.0000 1.0000 G4-R409K G4-R409K 8.0 xX 10-9 8.0 10 6.0000 6.0000 G4-R409A G4-R409A 1.6 xX 10-7 1.6 10 0.3000 0.3000 G4-R409L G4-R409L 1.5 1.5 xX 10-8 10 3.2000 3.2000 G4-R409M G4-R409M 3.0 xX 10-9 3.0 10 16.0000 16.0000

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G4-R409T G4-R409T 7.2 xX 10-7 7.2 10 0.0700 0.0700 2022201608 08 Mar

G4-R409W G4-R409W 3.4 xX 10-5 3.4 10 0.0014 0.0014 G4-F405A G4-F405A 1.9 1.9 xX 10-5 10 0.0025 0.0025 G4-F405L G4-F405L 2.5 xX 10-5 2.5 10 0.0019 0.0019 G4-L351K G4-L351K 7.4 xX 10-7 7.4 10 0.0600 0.0600 G4-E357T G4-E357T 4.1 xX 10-5 4.1 10 0.0012 0.0012 G4-S364D G4-S364D 4.7 xX 10-" 4.7 10 1.0200 1.0200 G4-K370Q G4-K370Q 1.1 1.1 xX 10-8 10 4.3000 4.3000 G4-K370E G4-K370E 2.0 xX 10-9 2.0 10 24.0000 24.0000

* Compared to the Compared to the corresponding CH2-CH3fragments corresponding CH2-CH3 fragments wild type ofofwild type IgG1 IgG1 or or IgG4 IgG4

Example22: Example 22:Analysis Analysis of of different different reductantia reductantia forfor their their ability ability toto induce induce thethe generation ofofbispecific generation bispecificantibodies antibodiesby by in in vitro vitro Fab-arm-exchange Fab-arm-exchange between between 5 IgG1-2F8-F405L 5 IgG1-2F8-F405Land andIgG1-7D8-K409R IgG1-7D8-K409R 2-MEA andDTT 2-MEA and DTT were were found found to induce to induce in vitro in vitro Fab-arm-exchange Fab-arm-exchange between between human IgG1-ITL and human IgG1-ITL andIgG4-CPPC IgG4-CPPC (Figure12). (Figure 12).It Itwaswas tested tested whether whether these these reductantia can reductantia can also also induce induce in in vitro vitroFab-arm-exchange between human Fab-arm-exchange between humanIgG1-2F8- IgG1-2F8 F405L and IgG1-7D8-K409R. F405L and IgG1-7D8-K409R. Concentration Concentration series series of of 2-MEA, 2-MEA, DTT,DTT, GSH GSH and TCEP and TCEP 10 (tris(2-carboxyethyl)phosphine) 10 (tris(2-carboxyethyl)phosphine)were weretested. tested.Fab-arm-exchange Fab-arm-exchange was was performed performed as as described inin Example described Example18. 18. The The tested tested concentration concentration series series of the of the different different reducing reducing

agents were agents wereas as follows: follows: 0.0, 0.0, 0.04, 0.04, 0.1,0.1, 0.2, 0.2, 1.0, 1.0, 0.5, 0.5, 5.0, 25.0, 5.0, 25.0, 50.0 50.0 mM mM 2-MEA, 2-MEA, GSH, DTT GSH, DTTor or TCEP. TCEP. Figure 17 Figure 17 confirms confirms that that 2-MEA 2-MEAinduces induces maximal maximal Fab-arm-exchange Fab-arm-exchange at a at a 15 concentration 15 concentration of of 2525mM mM 2-MEA, 2-MEA, which which persisted persisted at the at the higher higher concentrationofof50.0 concentration 50.0 mM 2-MEA.DTTDTT mM 2-MEA. was was foundfound to betovery be effective very effective in thein generation the generation of bispecific of bispecific antibodies with antibodies with maximal Fab-arm-exchange maximal Fab-arm-exchange reached reached at 0.5 at 0.5 mM which mM DDT, DDT, which also also persisted over higher persisted over higherconcentrations concentrationsof of DTTDTT (1.0-50.0 (1.0-50.0 mM). mM). Also was Also TCEP TCEP wastofound to found

be veryeffective be very effectiveinin the thegeneration generation of bispecific of bispecific antibodies antibodies withwith maximal maximal Fab-arm Fab-arm-

20 exchange 20 exchange reached reached at 0.5 at 0.5 mM. mM. At aAt a concentration concentration > 25.0 25.0 mM, Fab-arm-exchange mM, Fab-arm-exchange by by TCEP wasdisturbed. TCEP was disturbed. GSH GSHconcentrations concentrations in in the the range range 0.0-5.0 0.0-5.0 mM mMwere werenot notable abletoto induce the induce the generation generation of of bispecific bispecific antibodies antibodiesby by Fab-arm-exchange. Higher GSH Fab-arm-exchange. Higher GSH concentrations (25.0-50.0 concentrations mM) resulted (25.0-50.0 mM) resulted inin the the formation formationofofantibody antibodyaggregates aggregates (data not (data not shown). shown).Therefore, Therefore, these these samples samples were were excluded excluded from from the the analysis. analysis. These These 25 data 25 data show show that that thethe generationofofbispecific generation bispecific antibodies antibodiesbybyFab-arm-exchange Fab-arm-exchange between between two different two different antibodies antibodies can canbebeinduced inducedby by different different reducing reducing agents. agents.

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Example23: Example Generation 23:Generation of of bispecific bispecific antibodies by by antibodies 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm- exchange between exchange between IgG1-2F8-F405L IgG1-2F8-F405L and and IgG1-7D8-K409R IgG1-7D8-K409R To confirm To confirmthe theformation formation of bispecific of bispecific antibodies antibodies by 2-MEA-induced by 2-MEA-induced Fab-armFab-arm 2022201608 08

exchange between exchange between human human IgG1-2F8-F405L IgG1-2F8-F405Land andIgG1-7D8-K409R, IgG1-7D8-K409R,the themolecular molecular 5 weights 5 weights of of samples samples fromthethe from Fab-arm-exchange Fab-arm-exchange reactions reactions witha aconcentration with concentration series series of 2-MEA of 2-MEA were weredetermined determined by ESI-MS. by ESI-MS. The tested The tested concentration concentration seriesseries was was as as follows: 0.0, follows: 0.0, 0.5, 0.5, 1.0, 1.0, 2.0, 2.0, 5.0, 5.0, 7.0, 7.0, 10.0, 10.0, 15.0, 15.0, 25.0 25.0 and and40.0 40.0mM mM 2-MEA. 2-MEA. Fab-arm Fab-arm-

exchange (in exchange (in PBS) and sandwich PBS) and sandwich ELISA ELISA were wereperformed performedasasdescribed described in in Example Example 11. 11. ESI-MS was ESI-MS wasperformed performedasas described described in in Example 12. Example 12. 10 10 Figure 18A Figure 18A shows that 2-MEA shows that 2-MEA induced induced Fab-arm-exchange Fab-arm-exchangebetween between IgG1-2F8 IgG1-2F8- F405L and F405L and IgG1-7D8-K409R IgG1-7D8-K409Rin in a dose-dependent a dose-dependent manner, manner, efficientlyleading efficiently leadingtoto the the generation ofofbispecific generation bispecificantibodies antibodieswith with a maximal a maximal level level of bispecific of bispecific binding binding at a at a concentration ofof15.0 concentration 15.0mMmM 2-MEA. 2-MEA. The The quantified quantified ESI-MS ESI-MS data data are are presented presented in in Figure Figure 18B and 18B and show show that that Fab-arm-exchange Fab-arm-exchange between between IgG1-2F8-F405L IgG1-2F8-F405L and and IgG1-7D8- IgGl-7D8 15 K409R 15 K409R resulted resulted in nearly in nearly 100% 100% bispecificantibody, bispecific antibody,confirming confirmingthe theresults results from from the the bispecific-binding ELISA. bispecific-binding ELISA.

Example24: Example 24:Purity Purity of of thethe bispecific bispecific antibody antibody generated generated by 2-MEA-induced by 2-MEA-induced Fab-arm-exchange between Fab-arm-exchange betweenhuman human IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R X IgG1-7D8-K409R 20 20 A batch A batch ofofbispecific bispecific antibody, antibody,generated generatedby 2-MEA-induced by 2-MEA-induced Fab-arm Fab-arm- exchange between exchange betweenhuman human IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R, X IgG1-7D8-K409R, was purified was purified usingusing a a PD-10desalting PD-10 desaltingcolumn column (cat.no. (cat.no. 17-0851-01; 17-0851-01; GE Healthcare). GE Healthcare). Next, Next, the the of purity purity the of the bispecific product bispecific product was analyzedbybymass was analyzed mass spectrometry spectrometry as described as described in Example in Example 12. 12. Figure 19 Figure 19 shows showsthe themass mass spectrometry spectrometry profilesof ofthethestarting profiles startingmaterials materials 25 IgG1-2F8-F405L 25 IgG1-2F8-F405L and and IgGl-7D8-K409R IgG1-7D8-K409R and and the the bispecific bispecific product product generated generated by by Fab- Fab arm-exchangebetween arm-exchange betweenIgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R. X IgG1-7D8-K409R. The The product product in the in the Fab Fab- arm-exchanged arm-exchanged sample sample is 146,160.7 is 146,160.7 kDa, matches kDa, which which matches with the bispecific with the bispecific product product derived from derived from IgG1-2F8-F405L IgG1-2F8-F405L(146,606.8/2=73,303.3) (146,606.8/2=73,303.3)X IgG1-7D8-K409R x IgGl-7D8-K409R (146,312.2/2=73,156.1) = 146,459.4 (146,312.2/2=73,156.1) = 146,459.4 kDa. Moreover, kDa. Moreover, the bispecific the bispecific antibodyantibody product product

30 showed 30 showed a homogenous a homogenous peak, indicating peak, indicating that that no no chain light light chain mispairing mispairing occurred, occurred, which would which would have haveresulted resultedinin subdivided subdividedpeaks. peaks.These Thesedata data show show thatthat Fab-arm Fab-arm- exchangeresulted exchange resultedininapproximately approximately 100%100% bispecific bispecific antibody. antibody.

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Example 25:InInvivo Example 25: analysisof of vivoanalysis the the stability andpharmacokinetics stabilityand of of pharmacokinetics bispecific antibodies bispecific antibodiesgenerated from generated IgG1-2F8-F405L from IgG1-2F8-F405L xXIgG1-7D8-K409R by IgG1-7D8-K409R by 2-MEA-induced Fab-arm-exchange 2-MEA-induced Fab-arm-exchange The bispecific The bispecific antibody antibody generated generatedbyby in in vitro vitro 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm- 5 exchange 5 exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R X IgG1-7D8-K409R was injected was injected in mice in SCID SCID to mice to analyze its analyze its stability stability (in (in vivo Fab-arm-exchange) vivo Fab-arm-exchange) and and pharmacokinetic pharmacokinetic properties properties as as described inin Example described Example14.14. TwoTwo groups groups of mice of mice (3 per (3 mice micegroup) per group) were analyzed: were analyzed: (1) (1) 100 µg 100 pg bispecific bispecific antibody; antibody; (2) (2) 100 100µgpgbispecific bispecificantibody antibody + 1,000 + 1,000 pg irrelevant µg irrelevant IgG4IgG4

(IgG4-637, described in (IgG4-637, described in WO2007068255). Total W02007068255).Total IgGIgG concentrations concentrations in in thethe plasma plasma 10 samples 10 samples were were assayed assayed by ELISA by ELISA as described as described in Example in Example 14, 14, with with thethe exceptionthat exception that in this in thisexample, example, HRP-conjugated goat anti-human HRP-conjugated goat anti-human IgG IgG(Jackson, (Jackson, cat. cat. no. no. 109-035- 109-035 098, 1/10,000)waswas 098, 1/10,000) usedused as a as a conjugate conjugate for detection. for detection. The presence The presence of bispecific of bispecific

antibodies inin the antibodies the plasma plasma samples wasassayed samples was assayedand andquantified quantified bybyCD20 CD20andand EGFREGFR bispecific reactivity bispecific reactivityinina asandwich sandwich ELISA as described ELISA as describedininExample Example14.14.

15 15 Figure 20A Figure 20Ashows shows totalantibody total antibody plasma plasma concentrations concentrations over The over time. time. The shape shape of the of plasmaclearance the plasma clearance curves curves was was identical identical in both in both groups. groups. FigureFigure 20Bthe 20B shows shows the plasmaconcentrations plasma concentrations of bispecific of bispecific antibody antibody over over time. time. The addition The addition of a of a 10-fold 10-fold excess irrelevant excess irrelevant IgG4 IgG4to to thethe bispecific bispecific antibody antibody did affect did not not affect bispecific bispecific antibody antibody

concentrations, indicating concentrations, indicatingthat thatnono Fab-arm-exchange Fab-arm-exchange occurred occurred in vivo.inThese vivo.data These data 20 indicate 20 indicatethat thatthe thebispecific antibody product, bispecific antibody product, generated by in generated by in vitro vitro 2-MEA-induced 2-MEA-induced Fab-arm-exchangebetween Fab-arm-exchange between IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R, X IgG1-7D8-K409R, was stable was stable in vivo in vivo (no (no Fab-arm-exchange). Fab-arm-exchange).

Example26: Example 26:CDC-mediated CDC-mediated cell cell kill kill by bispecific by bispecific antibody antibody generated generated by 2- by 2 25 MEA-induced 25 MEA-inducedFab-arm-exchange Fab-arm-exchange between betweenhuman human IgG1-2F8-F405L IgG1-2F8-F405L x IgG1 X IgG1- 7D8-K409R 7D8-K409R The CD20 The CD20antibody antibodyIgG1-7D8 IgG1-7D8 can can kill CD20-expressing efficientlykill efficiently CD20-expressingcells cellsbyby complement-dependent complement-dependent cytotoxicity cytotoxicity (CDC). (CDC). In contrast, In contrast, the antibody the EGFR EGFR antibody IgG1-2F8 IgG1-2F8

does not does not mediate mediate CDC CDConontarget targetcells cells expressing expressing EGFR. EGFR. It It was was tested tested whether whether the the 30 mutant 30 mutant IgGl-7D8-K409R IgG1-7D8-K409R andbispecific and the the bispecific antibody antibody generated generated by 2-MEA-induced by 2-MEA-induced Fab-arm-exchangebetween Fab-arm-exchange between IgG1-2F8-F405L IgG1-2F8-F405L x IgGl-7D8-K409R X IgG1-7D8-K409R were able were still still to able to induce CDC induce CDConon CD20-expressing CD20-expressing cells.cells. 105 Daudi 10 Daudi or Rajiorcells Raji were cells pre-incubated were pre-incubated for for 15 min 15 witha aconcentration min with concentration series series of of antibody antibody in 80 in 80 pL RPMI µL RPMI medium medium supplemented supplemented

with 0.1% with BSAin 0.1% BSA in aa shaker shaker at at room temperature. 20 room temperature. 20 µL pL normal normal human humanserum serum (NHS) (NHS) 35 waswas 35 added added as aassource a source of of complement complement (20%(20% NHS final NHS final concentration) concentration) and and incubated incubated

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0 BSA 2022201608 08 Mar for 45 for at 37 min at 45 min C. 30 37°C. 30 pL µL ice coldRPMI ice cold RPMI medium medium supplemented with0.1% supplementedwith 0.1%BSA waswas addedtotostop added stopthe theCDC CDC reaction. reaction. DeadDead and viable and viable cells cells were were discriminated discriminated by by adding adding 10 µL 10 pL 1010µg/mL pg/mL propidium propidium iodide iodide (PI) (PI) (1 (1 pg/mL µg/mL finalfinal concentration) concentration) and and FACS FACS analysis. analysis.

5 5 Figure 21 Figure 21 shows showsthat thatCDC-mediated CDC-mediated cell cell kill kill of of CD20-expressing CD20-expressing Daudi Daudi (Figure (Figure

21A) and 21A) andRaji Raji(Figure (Figure21B) 21B) cellsbyby cells IgG1-7D8 IgG1-7D8 wasinfluenced was not not influenced by the by the introduction introduction

of the of the K409R K409Rmutation. mutation. Both Both Daudi Daudi and cells and Raji Raji cells do notdoexpress not express EGFR, resulting EGFR, resulting in in monovalentbinding monovalent binding of of thethe bispecificantibody bispecific antibody generated generated by 2-MEA-induced by 2-MEA-induced Fab-arm-Fab-arm

exchange between exchange betweenIgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R. X IgG1-7D8-K409R. Nonetheless, Nonetheless, the bispecific the bispecific 10 antibody 10 antibody still still induced induced CDC-mediated CDC-mediated cell of cell kill killthe of the CD20-expressing CD20-expressing cells. cells. These These data data indicate that indicate that CDC CDCcapacity capacity of aofparental a parental antibody antibody was retained was retained in the bispecific in the bispecific

format. format.

Example27: Example 27:ADCC-mediated ADCC-mediatedcell cell killkill by by thethe bispecific bispecific antibody antibody generated generated by by 15 2-MEA-induced 15 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between humanhuman IgG1-2F8-F405L IgG1-2F8-F405L X IgG1-x IgG1 7D8-K409R 7D8-K409R The EGFR The EGFR antibody antibody IgG1-2F8 IgG1-2F8 can EGFR-expressing can kill kill EGFR-expressing cells, cells, such such as as by A431, A431, by antibody-dependent antibody-dependent cellular cellular cytotoxicity(ADCC). cytotoxicity (ADCC). A431 A431 cellscells do not do not express express CD20 CD20 and and therefore the therefore the CD20 CD20 antibody antibody IgG1-7D8 IgG1-7D8 does does not not induce induce ADCC on ADCC on these these cells. cells. It was It was 20 tested 20 tested whether whether thethe mutant mutant and and IgG1-2F8-F405L IgG1-2F8-F405L the the bispecificantibody bispecific antibodygenerated generatedbyby 2-MEA-induced Fab-arm-exchange 2-MEA-induced Fab-arm-exchange between between IgG1-2F8-F405L IgG1-2F8-F405L Xx IgG1-7D8-K409R IgG1-7D8-K409R were still were still able to induce able to induceADCC ADCC on A431 on A431 cells.cells. For effector For effector cell isolation, cell isolation, peripheral peripheral

blood mononuclear blood mononuclear cells cells (PBMCs) (PBMCs) werewere isolated isolated from whole from whole blood blood of of a healthy a healthy donor donor using Leucosep® using Leucosep® tubes tubes (Greiner (Greiner Bio-one, Bio-one, cat.# cat.# 227290) 227290) according according totothethe 25 manufacturer's 25 manufacturer's recommendations. recommendations. Target Target cells cells were were labelledbybyadding labelled adding100100 µCipCi 51 ¹Cr Cr 6 to 5x10 to A431 cells 5x10 A431 cells in in 11 mL mL RPMI RPMI medium mediumsupplemented supplementedwith with0.1% 0.1% BSABSA andand incubating for incubating for 60 60 min 370 shaking mininina a37°C C shaking water water bath.bath. Labelled Labelled cellscells were were washedwashed and and resuspended in resuspended in RPMI RPMIsupplemented supplementedwith with 0.1% 0.1% BSA.BSA. 5x105x10 4 labelled labelled target target cells cells in in RPMI supplemented RPMI supplementedwith with 0.1% 0.1%BSA BSA were were preincubated preincubated in in100 100µLpL for1515min for withthe minwith the 30 antibody 30 antibody concentrationsseries concentrations series(range (range 0-10 0-10µg/mL pg/mLfinal final concentration concentration in in ADCC assay ADCC assay in 3-fold in 3-fold dilutions) dilutions) at at room temperature.TheThe room temperature. ADCC ADCC assayassay was started was started by 50 by adding adding 50 pL effector µL effector cells cells (5x10 6 cells) (5x10cells) in in an an E:T E:T ratioratio 100:1. 100:1. After After 4 at 4 hours 370 37°C,at¹Cr hours C,5 1 Cr release from release from triplicate triplicate experiments was experiments was measured measured in a in a scintillation scintillation counter counter as counts as counts

per min per (cpm).The min (cpm). The percentage percentage of cellular of cellular toxicity toxicity waswas calculated calculated using using the following the following

35 formula: 35 formula: percentage percentage of specificlysis of specific lysis == (experimental (experimental cpm cpm- basal - basalcpm)/(maximal cpm)/(maximal

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cpm -- basal cpm basal cpm) cpm) XX 100. 100. Maximal 51Crrelease Maximal¹Cr releasewas adding5050µLpL5%5% determinedbybyadding wasdetermined 2022201608 08 Mar

Triton X-100 Triton X-100toto5050µL pL target target cells cells (5x10 (5x10 4 cells), cells), and and basalbasal release release was measured was measured in in the absence the absenceofofsensitizing sensitizing antibody antibodyand andeffector effectorcells. cells. Figure 22 Figure 22 shows shows that that the the CD20-specific CD20-specific antibody antibody IgG1-7D8 IgG1-7D8diddidnotnotinduce induce 5 ADCC 5 ADCC on the on the CD20-negative CD20-negative A431 A431 cells.cells. Both Both IgG1-2F8 IgG1-2F8 andmutant and the the mutant IgGl-2F8 IgG1-2F8- F405L wereable F405L were able to to induce induce ADCCADCC oncells, on A431 A431 indicating cells, indicating that introduction that introduction of the of the F405L mutationinin IgG1-2F8 F405L mutation IgG1-2F8diddidnotnot affectitsitsADCC affect ADCC effectorfunction. effector function.Also Alsothethe bispecific antibody bispecific antibodyderived derivedfrom fromIgG1-2F8-F405L IgG1-2F8-F405L xX IgG-7D8-K409R IgG1-7D8-K409R induced ADCC inducedADCC on A431 on A431cells cells inin a adose-dependent dose-dependent manner, manner, indicating indicating thatthat the the ADCC ADCC effector effector 10 function 10 function was was retained retained in bispecific in the the bispecific format. format.

Example 28: Determinants Example 28: Determinantsatatthe the IgG1 IgGI405 405position positionfor forengagement engagementin in2-2 MEA-induced Fab-arm-exchangeinincombination MEA-induced Fab-arm-exchange combinationwith withIgG1-K409R IgG1-K409R In Example In Example16 16 it it isisdescribed described that that thethe F405L F405L mutation mutation is sufficient is sufficient to enable to enable

15 human 15 human IgG1 IgG1 to to engage engage in in Fab-arm-exchangewhen Fab-arm-exchange when combined combined with IgG4-7D8.ToTo withIgG4-7D8. further test further testthe thedeterminants determinants at at the the IgG1 IgG1 405 position for 405 position for engagement in 2-MEA- engagement in 2-MEA induced Fab-arm-exchange induced Fab-arm-exchangein incombination combination with with human human IgG1-K409R, IgG1-K409R, all possible all possible IgG1-2F8-F405Xmutants IgG1-2F8-F405X mutants(with (withthe theexception exception of of CC and and P) P) were were combined combined with with IgG1- IgG1 7D8-K409R. The 7D8-K409R. Theprocedure procedurewaswas performed performed with with purifiedantibodies purified antibodiesasasdescribed describedinin 20 Example 20 Example 19. 19. Figure 23 Figure shows the 23 shows the results results of of bispecific bispecific binding bindingupon upon2-MEA-induced Fab 2-MEA-induced Fab- arm-exchangebetween arm-exchange betweenIgG1-2F8-F405X IgG1-2F8-F405X x IgG1-7D8-K409R. X IgG1-7D8-K409R. These These data also data were were also scored asas(-) scored (-) nonoFab-arm Fab-arm exchange, exchange, (+/-) (+/-) low,intermediate low, (+) (+) intermediate or (++) or high Fab- high (++) Fab arm exchange, arm exchange,asaspresented presented inin Table Table 3. 3. No No Fab-arm Fab-armexchange exchange(-)(-)was wasfound foundwhen when 25 25 thethe 405405 positionininIgG1-2F8 position IgG1-2F8was wasF F(=(=wild wild type type IgG1). IgG1). Fab-arm exchangewas Fab-arm exchange wasfound found to be to low (+/-) be low (+/-) when the 405 when the 405 position position in in IgG1-2F8 was GG or IgG1-2F8 was or R. R. Fab-arm Fab-arm exchange exchange wasfound was foundtotobebehigh high(++) (++) when when the position the 405 405 position in IgG1-2F8 in IgG1-2F8 was A, was A,H,D,I,E, K, D, E, H,L,I, K, L, M, N, M, N, Q, Q, S,S,T,T,V,V,W Wor or Y. Y. These These datadata thatthat indicate indicate particular particular mutations mutations at theatIgG1 the IgG1 405 position 405 position allow allow IgG1 IgG1totoengage engage in 2-MEA-induced in 2-MEA-induced Fab-arm-exchange Fab-arm-exchange when when 30 combined 30 combined withIgG1-K409R. with IgG1-K409R.

Table 3: Table 3: 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1-2F8-F405X IgG1-2F8-F405X mutants and IgG1-7D8-K409R. mutants and IgG1-7D8-K409R. The generation ofofbispecific The generation bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-inducedin invitro vitroFab-arm- Fab-arm 35 exchange 35 exchange betweenIgG1-2F8-F405X between IgG1-2F8-F405Xmutants mutantsand andIgG1-7D8-K409R IgG1-7D8-K409Rwas wasdetermined determined

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by aa sandwich by sandwichELISA. ELISA.(-) (-) no, no,(+/-) (+/-)low, low,(+) ()intermediate, (++) intermediate, (++) high high Fab-arm Fab-arm- exchange. exchange.

Fab-arm-exchange Fab-arm-exchange IgGl-2F8-F405X IgG1-2F8-F405X x X IgGl-7D8-K409R IgG1-7D8-K409R A A ++ 2022201608 ++ D ++ E ++ F -

G +/-

H ++ I ++ K ++ DL ++ ++ M E ++ ++ N ++ Q ++ R +/-

S ++ T ++ V ++ W ++ Y ++

5 Example 5 Example 29: 29: Determinants Determinants at the at the IgG1i407 IgG1 position 407 position forfor engagement engagement in 2-in 2 MEA-induced Fab-arm-exchange MEA-induced Fab-arm-exchange in in combinationwith combination withIgG1-K409R IgG1-K409R In Example In 28, Example 28, it itisisdescribed thatcertain described that certainsingle single mutations mutations at position at position F405F405

are sufficient are sufficientto to enable human enable IgG1 human toengage IgG1 inFab-arm-exchange to engage in Fab-arm-exchangewhen whencombined combined with IgG1-K409R. with IgG1-K409R. To To test test whether whether other other determinants determinants implicated implicated in Fc:Fc in the the Fc:Fc 10 interface 10 interface positionsininthe positions theCH3 CH3domain domain could could also also mediate mediate thetheFab-arm-exchange Fab-arm-exchange mechanism, mutagenesis mechanism, mutagenesis of of theIgG1 the IgG1i407 positionwas 407 position wasperformed performedand andthethemutants mutants were tested were tested for for engagement engagement inin 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange in combination in combination with human with IgG1-K409R.All human IgG1-K409R. Allpossible possible IgG1-2F8-Y407X IgG1-2F8-Y407X mutants mutants (with (with theexception the exception of Cand of C and P)were P) were combinedwith IgG-7D8-K409R. combined with IgG1-7D8-K409R. The The procedure procedure was was performed performed 15 withwith 15 purified purified antibodies antibodies as described as described in Example in Example 19. 19.

24 shows Figure 24 Figure the results showsthe resultsof binding upon 2-MEA-induced bispecificbindingupon of bispecific Fab-arm 2-MEA-induced Fab-arm- exchangebetween exchange betweenIgG1-2F8-Y407X IgG1-2F8-Y407X xIgG1-7D8-K409R. X IgG1-7D8-K409R. These These datadata werewere alsoalso scored scored as (-) as (-) no no Fab-arm Fab-arm exchange, (+/-)low, exchange,(+/-) intermediate oror (+)intermediate low,(+) (+)high (++) Fab-arm high Fab-arm 20 exchange, 20 exchange, as presented as presented in Table in Table 4. 4. No NoFab-arm exchange Fab-arm exchange (-) was (-) was found found when when the the

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407 position 407 IgG1-2F8waswas position inin IgG1-2F8 Y (= Y (= wildwild typetype E, K,E,Q,K,orQ,R.orFab-arm IgG1), IgG1), R. Fab-arm exchange exchange

was found was foundtotobebelowlow(+/-) (+/-)when when the the 407 407 position position in IgG1-2F8 in IgG1-2F8 was D,was D, SF,orI,TSand F, I, or T and intermediate(+) intermediate when (+) when thethe 407407 position position in IgG1-2F8 in IgG1-2F8 was was A, H, A, H, V, N or N or and(++) andV,high high (++) 2022201608 08

when the when the 407 407position position in in IgG1-2F8 was IgG1-2F8was G, G, L, L, M W. M or or These W. These data data indicate indicate that that 5 particular 5 particular single single mutations mutations at the at the IgG1 IgG1 407 position 407 position allowtoIgG1 allow IgG1 to in engage engage 2-MEA-in 2-MEA induced Fab-arm-exchange induced whencombined Fab-arm-exchange when combined withIgG1-K409R. with IgGl-K409R.

Table 4: Table 4: 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1-2F8-Y407X IgG1-2F8-Y407X mutants and mutants and IgG1-7D8-K409R IgG1-7D8-K409R 10 10 The generation The generationofofbispecific bispecificantibodies antibodiesafter after 2-MEA-induced 2-MEA-induced in vitro in vitro Fab-arm Fab-arm

exchange between exchange betweenIgG1-2F8-Y407X IgGl-2F8-Y407X mutants mutants and and IgGl-7D8-K409R IgG1-7D8-K409R was determined was determined by aa sandwich by sandwichELISA. ELISA.(-) (-) no, no,(+/-) low,(+) (+/-)low, ()intermediate, (++) intermediate, (++) high high Fab-arm Fab-arm- exchange. exchange.

Fab-arm-exchange Fab-arm-exchange IgGl-2F8-Y407X IgG1-2F8-Y407X xX IgGl-7D8-K409R IgG1-7D8-K409R A A + +

D +/- E -

F +/-

G ++ H + I +/-

K -

L ++ M ++ N + Q =

R -

S +/- T +/-

V + W ++ Y I 15 15 Example 30:Quantification Example 30: Quantificationofof thenon-covalent the non-covalent CH3-CH3 CH3-CH3 interaction interaction in IgG1 in IgG1 heterodimers heterodimers It It isdescribed in Example is described in Example 2121 that that there there is is a aspecific range specific range in in the the strength strength of of

the interaction the interaction of of the the CH3-CH3 homodimers CH3-CH3 homodimers that allows that allows efficient efficient Fab-arm-exchange. Fab-arm-exchange.

20 TheThe 20 strength strength of the of the interactions interactions at the at the CH3CH3 interface interface should should be such be such that that it isit is

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possible that possible that both both heavy heavychains theparental chainsininthe parentalantibodies antibodies (homodimers) (homodimers) dissociate dissociate in in the Fab-arm-exchange the Fab-arm-exchangereaction reaction and and that that they they subsequently subsequently associate associate in the in the heterodimerizationreaction. heterodimerization reaction.ToTo generate generate a stable a stable heterodimer, heterodimer, the strength the strength of the of the heterodimer interaction heterodimer interaction should be greater should be greater than thanthe thestrength strengthofofthethehomodimer homodimer 5 interaction, 5 interaction, such such thatthat it favors it favors heterodimerization heterodimerization over over homodimerization. homodimerization. To confirm To confirm

this, the this, the strength of the strength of the CH3-CH3 CH3-CH3 interaction interaction in in thethe heterodimers heterodimers was measured was measured and and compared toto the compared thestrength strength inin the the homodimers. homodimers.TheThe of of KD KD thethe CH2-CH3 CH2-CH3 fragments fragments derived from derived from IgGl-K409R, IgGl-F405Land IgG1-K409R, IgG1-F405L andIgG1-ITL IgGl-ITLhomodimers homodimers were were measured measured as as described inin Example described Example21.21. ForFor thethe determination determination of KD of the theinKDheterodimers, in heterodimers, CH2-CH3CH2-CH3

10 domain 10 domain fragments fragments (G1-F405L (G1-F405L and and G1-ITL) G1-ITL) werewere mixed mixed with with the the IgGlAhinge IgG1Ahinge fragment fragment of IgG1-7D8-K409R, of IgG1-7D8-K409R, which which contain contain all antibody all antibody domains domains except except the theThehinge. hinge. lack The lack of hinge of hinge regions regionsininboth both fragments fragments prevented prevented covalent covalent inter-heavy inter-heavy chain disulfide chain disulfide

bonds. The fragments bonds. The fragmentswere weremixed mixed and and analyzed analyzed afterafter 24 hours 24 hours by native by native mass mass spectrometry as spectrometry as described described in in Example 21. The Example 21. The KD values of KD values of the the non-covalent non-covalent CH3- CH3 15 CH3CH3 15 interactions interactions in the in the indicated indicated CH2-CH3 CH2-CH3 fragments fragments or mixtures or mixtures of CH2-CH3 of CH2-CH3 fragmentswith fragments withIgG1Ahinge IgGlAhinge are are presented presented in Table in Table 5. These 5. These data suggest data suggest that that under under the tested the tested conditions, conditions,the thestrength strength of of thethe heterodimer heterodimer interaction interaction is greater is greater (lower(lower

than the KD) than the corresponding correspondinghomodimer homodimer interactions. interactions.

20 Table 20 Table55 CH2-CH3 CH2-CH3 construct/(IgG1Ahinge) Interaction construct/(IgG1Ahinge) Interaction (M) KDKD(M) G1-F405L/G1-K409R G1-F405L/G1-K409R Heterodimer 1.2 Heterodimer 1.2 Xx 10 10-8 G1-ITL/G1-K409R G1-ITL/G1-K409R Heterodimer 1.7 Heterodimer 1.7 Xx 10 10-8 G1-K409R G1-K409R Homodimer 1.1 Xx 10 Homodimer 1.1 10-7

G1-F405L G1-F405L Homodimer 8.5 Xx 10 Homodimer 8.5 10

G1-ITL G1-ITL Homodimer 10-6 1.2 Xx 10 Homodimer 1.2

Example31: Example 31:Biochemical Biochemical analysis analysis of of a bispecific a bispecific antibody antibody generated generated by by 2- 2 MEA-induced Fab-arm MEA-induced Fab-arm exchange exchange A batch A batchof ofbispecific bispecificantibody, antibody,generated generated by 2-MEA-induced by 2-MEA-induced Fab-armFab-arm 25 exchange 25 exchange betweenhuman between human IgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R, X IgG1-7D8-K409R, waswas purified onon a a purified PD-10desalting PD-10 desaltingcolumn column (cat.no. (cat.no. 17-0851-01; 17-0851-01; GE Healthcare). GE Healthcare). Next, Next, the the of purity purity the of the bispecific product bispecific was analyzed product was analyzed by sodium by sodium dodecyldodecyl sulfate sulfate polyacrylamide polyacrylamide gelelectrophoresis (SDS-PAGE), gelelectrophoresis High Performance (SDS-PAGE), High PerformanceSize SizeExclusion ExclusionChromatography Chromatography (HP-SEC), mass spectrometry, (HP-SEC), mass spectrometry, HPLC HPLCcation cation exchange exchangechromatography chromatography (HPLC-CIEX), (HPLC-CIEX), 30 capillary 30 capillary isoelectrofocussing isoelectrofocussing (cIEF). (cIEF).

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2022201608 08 Mar SDS-PAGEwaswas SDS-PAGE performed performed under under non-reducing non-reducing (Figure (Figure 25A) 25A) and reducing and reducing (Figure (Figure 25B) conditions as 25B) conditions as described described in in Example Example15. 15.Figure Figure25A25A show show thatthat the the antibody sample antibody sample after after 2-MEA 2-MEA induced induced Fab-arm Fab-arm exchange exchange consists consists of intactofIgG, intact withIgG, with a trace a trace of of half half molecules (H111)detectable molecules (H1L1) detectableon on thethe non-reduced gel. gel. non-reduced 5 5 HP-SEC was HP-SEC wasperformed performedasasdescribed describedinin Example Example15. 15.Figure Figure 26(B) 26(B)and andFigure Figure 26(A) show 26(A) showthethe HP-SEC HP-SEC profiles profiles of the of the starting starting materials materials IgG1-2F8-F405L IgG1-2F8-F405L and and IgG1- IgG1 7D8-K409R, 7D8-K409R, respectively. respectively. The The mixture mixture (1:1) (1:1) of bothofantibodies both antibodies and the bispecific and the bispecific

product product generated by 2-MEA generated by 2-MEA induced induced Fab-arm Fab-armexchange exchange between between IgG1-2F8-F405L IgG1-2F8-F405L X x IgG1-7D8-K409R IgG1-7D8-K409R are shown are shown in Figure in Figure 26C 26C and and 26D, Figure Figure 26D, respectively. respectively. In addition, In addition,

10 Figure 10 Figure 26D 26D showsshows thatof>99% that >99% of theconsists the sample sample ofconsists of intact intact IgG IgG with practically with practically no no aggregates formed. aggregates formed. Mass spectrometry Mass spectrometry (ESI-MS) (ESI-MS)waswas performed performed as described as described in Example in Example 12. 12. Figure 27(B) Figure 27(B)and andFigure Figure 27(A) 27(A) showshow the spectrometry the mass mass spectrometry profiles profiles of of the the starting starting materials materials IgG1-2F8-F405L and IgG1-7D8-K409R, IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. The respectively. Themixture mixture(1:1) (1:1) ofof 15 both 15 both antibodies antibodies andand the the bispecificproduct bispecific productgenerated generated by by 2-MEA 2-MEA induced induced Fab-arm Fab-arm exchange between exchange betweenIgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R X IgG1-7D8-K409R are are shown shown in Figure in Figure 27C 27C and and Figure 27D, Figure 27D, respectively. respectively. The product in The product in the the 2-MEA 2-MEAinduced inducedFab-arm Fab-arm exchanged exchanged sampleisis 146,159.7 sample 146,159.7 kDa, kDa, which which perfectly perfectly matches matches withbispecific with the the bispecific product product derived derived

from from IgG1-2F8-F405L IgG1-2F8-F405L (146,289.0/2=73,145) (146,289.0/2=73,145) x X IgG1-7D8-K409R IgG1-7D8-K409R 20 (146,028.0/2=73,014). 20 (146,028.0/2=73,014). Moreover, Moreover, the thebispecific bispecific antibody antibody product product showed showeda a homogenous homogenous peak, peak, indicating indicating that that no light no light chain chain mispairing mispairing occurred, occurred, which which would would have resulted have resulted in in subdivided subdivided peaks. peaks. These data show These data show that that 2-MEA 2-MEAinduced Fab-arm inducedFab-arm exchangeresulted exchange resultedin inbispecific bispecificIgG. IgG.TheThe small small peaks peaks indicated indicated by (*)byresulted (*) resulted from from incomplete deglycosylation incomplete deglycosylation prior prior to analysis. These to analysis. data show These data showthat thata bispecific a bispecific 25 antibody 25 antibody sample sample waswas generated generated by 2-MEA-induced by 2-MEA-induced Fab-arm Fab-arm exchange exchange between between IgG1- IgG1 2F8-F405L Xx IgG1-7D8-K409R. 2F8-F405L IgG1-7D8-K409R. Capillary isoelectrofocussing Capillary isoelectrofocussing (cIEF) (cIEF) was wasperformed performed usingusing an iCE280 an iCE280 Analyzer Analyzer

(Convergent Biosciences). (Convergent Biosciences). Figure Figure 28A and Figure 28A and Figure 28B 28Bshows shows cIEF cIEF profilesof ofthethe profiles starting materials starting materialsIgG1-2F8-F405L IgG1-2F8-F405L and IgG1-7D8-K409R,respectively. and IgG1-7D8-K409R, respectively. The The mixture mixture 30 (1:1) 30 (1:1) of of bothantibodies both antibodiesand andthe thebispecific bispecific product product generated by Fab-arm generated by exchange Fab-arm exchange between IgG1-2F8-F405LX IgG1-7D8-K409R between IgG1-2F8-F405L x IgGl-7D8-K409R are shown are shown in Figure in Figure 28CFigure 28C and and Figure 28D, respectively. 28D, respectively.All All samples sampleswere were desalted desalted before before use. use. FinalFinal concentrations concentrations in thein the assay mix assay mix were were 0.3 0.3 mg/mL mg/mLIgG IgG(0.35% (0.35% Methyl Methyl Cellulose;2%2% Cellulose; CarrierAmpholytes Carrier Ampholytes3-3 10; 6% 10; Carrier Ampholytes 6%Carrier Ampholytes8-10.5; 8-10.5; 0.5% 0.5% pI marker pI marker 7.65 7.65 and pI and 0.5% 0.5% pI marker marker 35 10.10). 35 10.10). Focusing Focusing was was performed performed for 7 for 3000atV 3000 min 7atmin and the and the whole-capillary V whole-capillary

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absorption image absorption imagewaswas captured captured by a by a charge-coupled charge-coupled device device camera.camera. After calibration After calibration

of the of peakprofiles, the peak profiles, the thedata datawere were analyzed analyzed by EZChrom by the the EZChrom software. software. pI pI markers markers are indicated are indicated by by (*). (*). These Thesedata datashow show thatthat a bispecific a bispecific antibody antibody sample sample was was 2022201608 08

generated by generated by 2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange between between IgG1-2F8-F405L IgG1-2F8-F405L x X IgG1- IgG1 5 7D8-K409R. 5 7D8-K409R. Anothertechnique Another techniqueto to study study the the charged charged isoforms isoforms of monoclonal of monoclonal antibodies antibodies is is High Pressure High Pressure Liquid LiquidChromatography Cation Exchange Chromatography Cation (HPLC-CIEX). Figure Exchange (HPLC-CIEX). Figure 29A 29A and and Figure 29B Figure show HPLC-CIEX 29B show HPLC-CIEXprofiles profiles of of the the starting starting materials materials IgG1-2F8-F405L and IgG1-2F8-F405L and IgG1-7D8-K409R,respectively. IgG1-7D8-K409R, respectively. The Themixture mixture (1:1) (1:1) of both of both antibodies antibodies and and the the 10 bispecific 10 bispecificproduct productgenerated generatedbyby2-MEA 2-MEA induced induced Fab-arm Fab-arm exchange exchange between between IgG1- IgG1 2F8-F405L 2F8-F405L X x IgG1-7D8-K409R IgG1-7D8-K409R are shown are shown in 29C in Figure Figure and 29C and29D, Figure Figure 29D, respectively. respectively.

Samples werediluted Samples were diluted to to 11 mg/mL mg/mLin inmobile mobilePhase Phase A (10 A (10 mM NaPO4, mM NaPO4, pH for pH 7.0) 7.0) for injection onto injection the HPLC. onto the HPLC.TheThe differently differently charged charged IgG molecules IgG molecules were separated were separated by by using aa ProPac@ using WCX-10,4 4mmmm ProPac® WCX-10, x 250 X 250 mm, mm, analytical analytical column column withwith a flow a flow rate rate of of1 1 15 mL/min. 15 mL/min. Elution Elution waswas performed performed with with a gradient a gradient of of MobilePhase Mobile PhaseA AtotoMobile MobilePhase PhaseB B (10 mMNaPO, (10 mM NaPOpH 4, pH 7.0, 7.0, 0.25M M 0.25 NaCl)andand NaCl) detectionoccurred detection occurredatat280 280nm. nm.These Thesedata data show that show that aa bispecific bispecific antibody antibody sample sample was generated by was generated by2-MEA-induced Fab-arm 2-MEA-inducedFab-arm exchange between exchange betweenIgG1-2F8-F405L IgG1-2F8-F405L x IgG1-7D8-K409R. X IgG1-7D8-K409R. It also It also showsshows that cation that cation exchangeisisa apowerful exchange powerful toolto to tool separate separate residual residual homodimers homodimers from from the the heterodimer. heterodimer.

20 Another 20 Another application application of cation of cation exchange exchange chromatography chromatography is therefore is therefore the of the polishing polishing of bispecific heterodimer, a bispecific a i.e. to heterodimer, i.e. to purify purify away any residual away any residual homodimers homodimers after after exchange. exchange.

Example 32: Example 32: Recombinant Recombinantexpression expression of of heterodimers heterodimers by by simultaneous co simultaneous co- expression of expression of both both homodimers homodimers 25 25 To illustrate To illustrate that that heterodimer formationalso heterodimer formation alsooccurs occurswhen when the the two two

homodimers homodimers areare co-expressed co-expressed recombinantly, recombinantly, HEK-293F HEK-293F cells cells were were co-transfected co-transfected

with the with the four four expression expressionvectors vectors(see (seeExample Example 1) encoding 1) encoding the heavy the heavy and chain and light light chain of IgG1-7D8-K409R of IgG1-7D8-K409R and and IgG1-2F8-F405 IgG1-2F8-F405 in a 1:1:1:1 in a 1:1:1:1 ratio. Antibodies ratio. Antibodies were were transiently produced transiently underserum-free produced under serum-free conditions conditions as described as described in Example in Example 4. 4. Next, Next, 30 IgG IgG 30 was was purified purified by Protein by Protein A chromatography A chromatography as described as described in 5. in Example Example 5. Purified Purified

IgG was IgG wasdeglycosylated deglycosylatedandand subsequently subsequently analyzed analyzed by electrospray by electrospray ionization ionization mass mass spectrometryasasdescribed spectrometry described in in Example Example 12. 12. The theoretic The theoretic mass massofofheavy heavy andand light light chain chain of of IgG1-7D8-K409R IgG1-7D8-K409R and and IgG1- IgG1 2F8-F405 areshown 2F8-F405 are shown in Table in Table 6. 6.

35

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2022201608 08 Mar Table 6: Table 6: Theoretical massofofthe Theoretical mass theheavy heavyand and light light chainofofIgG1-7D8-K409R chain IgG1-7D-K409R and IgG1-2F8-F405 and IgG1-2F8-F405 Homodimer Homodimer L-chain (Da) L-chain (Da) H-Chain (Da) H-Chain (Da) IgG1-2F8-F405 IgG1-2F8-F405 23252.8 23252.8 49894.6 49894.6 IgGl-7D8-K409R IgG1-7D8-K409R 23438.1 23438.1 49579.0 49579.0

On the On the basis basisof of these thesemasses, masses, the the following following IgGIgG molecules molecules could could theoretically theoretically

5 be detected 5 be detected (Table (Table 7). measured 7). The The measured masses masses (Figure (Figure 30) 30) are indicated are indicated in the in the final final column. column.

Table 7: Table 7: Theoretical detection of Theoretical detection of heavy heavyand andlight lightchain chainofofIgG1-7D8-K409R IgG1-7D-K409R and IgG1-2F8-F40 and IgG1-2F8-F40 IgG1-2F8-F405 IgG1-2F8-F405 IgGl-7D8-K409R IgG1-7D8-K409R Theoretic Theoretic Mass Mass H-chain H-chain L-chain L-chain H-chain H-chain L-chain L-chain mass measured measured mass (Da) (Da) (Da) (Da) 2 2 22 146287 146287 146284 146284 2 2 2 2 146026 146026 146026 146026 2 2 2 2 146657 146657 146664 146664 22 2 2 145656 145656 145660 145660 2 2 1 1 1 1 146472 146472 146477 146477 1 1 2 2 11 145841 145841 145846 145846 11 1 1 1 1 1 1 146157 146157 146159 146159 11 22 11 145971 145971 145972 145972 11 1 1 2 2 146342 146342 146345 146345 10 10 The two The two most most abundant abundantpeaks peaksofof 146345 146345and and146159 146159DaDa represented represented heterodimerswith heterodimers witha asingle single(from (fromIgG1-7D8-K409R) IgG1-7D8-K409R) orlight or both both chains light chains incorporated, incorporated,

respectively. Homodimers respectively. Homodimers of of both both thethe heavy heavy chainchain of IgG-7D8-K409R of IgG1-7D8-K409R or IgGl-2F8 or IgG1-2F8-

F405 were F405 weredetected, detected, butbut only only in in minor minor amounts. amounts. TheseThese data that data show showhetero- that hetero 15 dimerization 15 dimerizationalso alsooccurs occurs when whenthe thetwo twohomodimers homodimers areco-expressed. are co-expressed.

Example33: Example 33:Monitoring Monitoring thethe kinetics kinetics of of 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange and quantifying and quantifyingresidual residualhomodimers homodimers after after exchange exchange by using by using HPLC-CIEX HPLC-CIEX The generation The generationofofbispecific bispecific antibodies antibodiesbyby2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange

20 is described 20 is described in Example in Example 11.this 11. In In this example example the exchange the exchange reactionreaction was monitored was monitored by by

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conducting High conducting High Pressure Pressure Liquid Chromatography Cation LiquidChromatography Exchange (HPLC-CIEX; Cation Exchange as (HPLC-CIEX; as described inin Example described Example31)31) at at various various time time points points during during the the exchange exchange reaction. reaction.

HomodimersIgG1-2F8-F405L Homodimers IgG1-2F8-F405Landand IgG1-7D8-K409R IgG1-7D8-K409R were were mixedmixed in the in the molar molar 2022201608 08

ratio 1:1 ratio 1:1 at at aa concentration of 11 mg/mL concentration of mg/mL each. each. After thethe After addition addition of of 25 25 mM 2-MEA, mM 2-MEA,

5 thethe 5 sample sample waswas placed placed in inthe theautosampler autosamplerofofthe the HPLC, HPLC, prewarmed prewarmedatat25°C. 25°C.Figure Figure 31Ato 31A to 31H 31Hshows shows eight eight consecutive consecutive injections injections at different at different time time intervals intervals obtained obtained by by HPLC-CIEXranging HPLC-CIEX ranging from from t = t0 =min to t to 0 min t = 450 = 450 min, min, respectively, respectively, afterafter the addition the addition of of 2-MEA.The 2-MEA. Thedata data show show thatthat bispecific bispecific IgGIgG waswas formed formed ratherrather quickly quickly and of and most most the of the homodimerwas homodimer wasexchanged exchanged after135 after 135min. Theheterogeneous min.The heterogeneousheterodimer heterodimerpeaks peaks 10 appearing 10 appearing after4545min after resolvedinto minresolved into more more homogeneous homogeneous peaks peaks afterapproximately after approximately 180 min, 180 suggestingthat min, suggesting thatexchange exchange occurs occurs in different in different phases. phases. Furthermore, FigureFigure Furthermore, 32Ashows 32A showsthat thatapproximately approximately 3% 3% of of residual residual homodimers homodimers was detected was detected with the with the CIEX method CIEX method (indicated (indicated by by arrows). arrows). As shown As shown this method this method is suitable is suitable for quantitating for quantitating

the remaining the remaininghomodimer homodimer content content (elution (elution of homodimers of the the homodimers is in is shown shown in Figure Figure 15 32B) 15 32B) when when exchange exchange reaction reaction waswas almost almost complete). complete).

Example 34:Generation Example 34: Generation of of bispecific bispecific antibodies antibodies by by 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchangeatathigh exchange highantibody antibody concentrations concentrations at various at various 2-MEA 2-MEA concentrations, concentrations, temperaturesand temperatures and incubation incubation times times 20 20 2-MEA induced 2-MEA induced Fab-arm Fab-armexchange exchangewas was performedat athigh performed highIgG IgGconcentrations. concentrations. The influence The influence ofof 2-MEA 2-MEAconcentration, concentration, incubation incubation temperature temperature and on and time time theon the amount of amount of exchange exchange was wasstudied. studied. The exchange The exchange process process was was performed performed using using the the combination combination of of IgG1-7D8 IgG1-7D8- K409RX xIgG1-2F8-F405L. K409R IgG1-2F8-F405L. Both Both materials materials were purified were purified with affinity with affinity chromatography chromatography

25 using 25 using protein protein A. After A. After concentration concentration of material of the the material to mg/mL, to >20 >20 mg/mL, a successive a successive

anion exchange anion exchange step step waswas performed performed (in flow (in flow through through mode) mode) using QHiPrep using HiPrep Q FF FF 16/10 16/10 (GE HealthCare, (GE Health Care,#28-9365-43). #28-9365-43). The The finalfinal purified purified material material was was buffer-exchanged buffer-exchanged to to PBS. PBS. The bispecific The bispecific exchange was exchange was studied studied at at finalIgG final IgG concentrations concentrations of mg/mL of 20 20 mg/mL 30 (each 30 (each homodimer homodimer at aatfinal a finalconcentration concentrationof of 10 10 mg/mL) mg/mL)and and1010mg/mL mg/mL (each (each homodimer homodimer at at a finalconcentration a final concentration of of 5 mg/mL) 5 mg/mL) in PBS. in PBS. Separate Separate mixtures mixtures were were preparedfor prepared forboth bothIgG IgGconcentrations concentrations including including 2-MEA 2-MEA at final at final concentrations concentrations of of 10,10, 25, 50 25, 50 and and100 100mM.mM. The The mixtures mixtures were were divided divided intoµL100 into 100 pL aliquots aliquots in eppendorf in eppendorf

tubes and tubes andstored storedatat15, 15,2525and and 37cC. 37°C. Separate Separate tubes tubes were were useddifferent used for for different 35 incubation 35 incubation timesofof9090min, times hours and min,55 hours and 24 24hours hours at at each each temperature. temperature.

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The mixture The mixturewas was also also prepared prepared without without 2-MEA 2-MEA for IgG for both IgG concentrations bothconcentrations and stored and stored atat 4°C 4CCasasananuntreated untreated control.After control. Afterthetheappropriate appropriate incubation incubation times, times, the the 90 min 90 and5 5hours minand hours samples samples werewere collected collected for desalting for desalting to remove to remove the 2-MEA the 2-MEA (the (the 2022201608 08

90 min 90 samples minsamples were were initiallyput initially putononiceicetotostop stopthe theexchange exchange reaction). reaction). The The samples samples

5 werewere 5 desalted desalted usingusing a Zeba a Zeba 96-well 96-well desalting desalting platecat# plate (7k, (7k,89808, cat# 89808, Thermo Thermo Fisher Fisher Scientific). The Scientific). The 24 hours samples 24 hours sampleswere were desalted desalted separately separately after after 24 hours 24 hours incubation. incubation.

Serial dilutions Serial dilutions of ofthe theantibody antibody samples (total antibody samples (total antibodyconcentration concentration10 10 -

0.123 pg/mL 0.123 µg/mL in in 3-folddilutions 3-fold dilutionsfor forthe the9090min minandand 5 hours 5 hours samples; samples; 10 - 0.041 10 0.041

pg/mLinin3-fold µg/mL 3-folddilutions dilutions for for the 24 hours the 24 hourssamples) samples) were were usedused in ainsandwich a sandwich ELISAELISA to to 10 measure 10 measure bispecific bispecific binding binding as described as described in Example in Example 7. For 7. Forplate, each each aplate, a control control was was included of included of aa purified purified batch of bispecific batch of bispecific antibody derived from antibody derived froma a2-MEA-induced 2-MEA-inducedFab-Fab

arm exchange arm exchangebetween betweenIgG1-2F8-ITL IgG1-2F8-ITLand andIgG4-7D8-CPPC IgG4-7D8-CPPC (as (as described described in inExample Example 15). Figure 15). Figure 34(A)-(F) 34(A)-(F) shows showsthethe resultsofofthe results thebispecific bispecificbinding bindingasasmeasured measured in the in the

individual ELISA individual plates. The ELISA plates. The top topOD405 OD405 values values (as (as determined determined for 10 for the theµg/mL 10 pg/mL 15 concentrations 15 concentrations in the in the ELISA) ELISA) were were used used to to calculate calculate the bispecific the bispecific binding binding in in comparisontotothe comparison thecontrol, control,which whichwaswas arbitrarilysetsetatat100%. arbitrarily 100%. ThisThis resulted resulted in the in the

percentageofofcontrolled percentage controlledFab-arm Fab-arm exchange exchange (% cFAE) (% cFAE) compared compared to the control to the control as is as is shownininFigure shown Figure34(A)-(D) 34(A)-(D)forfor each each 2-MEA 2-MEA concentration. concentration.

The data The datashow show that that maximal maximal level level of bispecific of bispecific binding binding (89-109% (89-109% with respect with respect

20 to to 20 control)was control) wasreached reachedatataa concentration concentration of of 100 100 mM 2-MEAfor mM 2-MEA for both both IgG IgG concentrationsatatall concentrations all temperature-time conditions. temperature-time conditions. At At 50 50 mM mM 2-MEA, 2-MEA, maximal maximal binding binding

(88-10 7 0 and 37C and also at 15 0 C after 24 hours (88-107%)%)waswas achieved achieved at 25 at 25°C andC 37°C and also at 15°C after 24 hours

incubation. For incubation. For the the lower lower concentrations concentrationsofof2525mM mM and and 10 mM102-MEA, mM 2-MEA, the exchange the exchange

was more was moreefficient efficientatathigher highertemperatures temperaturesand and increased increased with with prolonged prolonged incubation incubation

25 time, 25 time, leadingtotomaximal leading maximalexchange exchange at at 37Cupon 37°C upon 2424 hoursincubation hours incubationat at 25 25 mM mM2-2 MEA. None MEA. Noneof ofthe theconditions conditions tested tested at at 10 10 mM mM 2-MEA 2-MEA generated generated 100% bispecific 100% bispecific

product. The product. Theexchange exchange process process was was slightly slightly faster faster at IgG at IgG concentrations concentrations of 10of 10 mg/mLcompared mg/mL comparedtoto2020mg/mL mg/mL totalIgG. total IgG. To confirm To confirmthat thatbispecific bispecific antibodies antibodies were wereformed formedandand to study to study the the bispecific bispecific

30 products 30 products in inmore more detail, samples detail, sampleswere wereanalyzed analyzed with with Cation Cation Exchange Exchange (HPLC-CIEX) (HPLC-CIEX) analysis. The analysis. HPLC-CIEX The HPLC-CIEX analysis analysis waswas performed performed as described as described in Example in Example 31 for 31 for the the sampleswith samples withIgG IgGconcentrations concentrations of 20 of 20 mg/mL mg/mL after after 5 hours 5 hours and 24and 24 incubation hours hours incubation and all and all 2-MEA concentrations. 2-MEA concentrations.

The CIEX The CIEXchromatograms chromatograms in Figure in Figure 35(A)-(D) 35(A)-(D) showthe show that that the highest highest yield yield of of 35 bispecific 35 bispecific product product was was obtained obtained at 50at 50100 and andmM100 mMconfirming 2-MEA 2-MEA confirming the the results of results of

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2022201608 08 Mar the bispecific the bispecific ELISA. minor However,minor ELISA. However, amounts amounts of residual of residual homodimer homodimer were were still still detected at detected at 50 50 and and 100 100 mM 2-MEA(2(2 3.5% mM 2-MEA - 3.5% of each of each homodimer homodimer for for samples samples incubatedatat 25°C incubated 25Cand 37 0C).Exchange and37°C). Exchange at higher at higher temperature, temperature, longerlonger (24 (24 hours) hours) incubation time incubation time and andincreasing increasing2-MEA 2-MEA concentration concentration result result in the in the appearance appearance of of 5 additional 5 additional peaks peaks at 22-24 at 22-24 thein CIEX min min in the profile. CIEX profile. Minimal amounts Minimal amountsofofadditional additional peaks peakswere were obtained obtained when when exchange exchange was was concludedwithin concluded within5 5hours. hours.To To identify identify thethe nature nature of these of these peaks, peaks, SDS-PAGE SDS-PAGE analysisanalysis

and HP-SEC and HP-SEC analysis analysis was was performed. performed. HP-SEC HP-SECshowed showed thatthe that theamount amountof ofaggregates aggregates was below was below1%1% for for all all conditions,suggesting conditions, suggestingthat that thethe additionalpeaks additional peaks do do not not 10 represent 10 represent aggregates. aggregates. However, However, non-reduced non-reduced SDS-PAGE SDS-PAGE indicated indicated that that the the extra extra peaksmay peaks may represent represent heterodimer heterodimer lacking lacking one orone twoorlight two chains. light chains. Minor amounts Minor amounts of of half-moleculeswere half-molecules weredetected detected as as well. well.

The experiment The experimentshows shows thatthat the the exchange exchange reaction reaction takes takes place place at at high high homodimer homodimer concentrations, concentrations, which which makesmakes the process the process attractive attractive for commercial for commercial scale, scale, 15 and 15 andthat that thethe yieldof of yield bispecific antibody bispecific antibody depends depends onon2-MEA 2-MEA concentration, concentration, temperatureand temperature and incubation incubation time. time.

Example 35: Example 35: Determinants Determinants atatthe the IgG1 IgG1368 368position positionfor for engagement engagementin in2-2 MEA-induced Fab-arm MEA-induced Fab-arm exchange exchangeinincombination combinationwith withIgG1-K409R IgG1-K409R 20 20 Example 28 Example 28and and2929show show thatcertain that certain single single mutations mutations at at position position F405 F405 and and Y407 are Y407 are sufficient sufficient toto enable enable human IgG1totoengage human IgG1 engage in in Fab-arm Fab-arm exchange exchange when when combined with combined withIgG1-K409R. IgGl-K409R. As As illustratedin inthis illustrated thisexample example further further determinants determinants implicated in implicated in the the Fc:Fc Fc:Fcinterface interfacepositions positionsininthe theCH3 CH3 domain domain may mediate may also also mediate the the Fab-arm exchange Fab-arm exchangemechanism. mechanism.To To thiseffect this effect mutagenesis mutagenesisofofthe the IgG1 IgG1368 368position position 25 waswas 25 performed performed and and the the mutants mutants werewere tested tested for for engagement engagement in 2-MEA-induced in 2-MEA-induced Fab- Fab arm-exchangeinin combination arm-exchange combination with with human humanIgG1-K409R. IgG-K409R. AllAllpossible IgG-2F8-L368X possible IgG1-2F8-L368X mutants (with mutants (with the the exception exception of of CC and and P) P) were were combined with IgG1-7D8-K409R. combined with IgG1-7D8-K409R.The The procedurewas procedure wasperformed performed withwith purified purified antibodies antibodies as described as described in Example in Example 19. 19. Figure 36 Figure 36 shows showsthe theresults resultsofofbispecific bispecific binding bindingupon upon2-MEA-induced 2-MEA-induced Fab-arm Fab-arm

30 exchange 30 exchange between between IgGl-2F8-L368X IgG1-2F8-L368X x IgG1-7D8-K409R. X IgG1-7D8-K409R. Thesewere These data dataalso werescored also scored as (-) as (-) no no Fab-arm Fab-arm exchange, exchange,(+/-) low,(+) (+/-)low, intermediateor or(++) (+)intermediate (++)highhigh Fab-arm Fab-arm exchange, as exchange, as presented presented in in Table Table 8. 8. No Fab-arm exchange No Fab-arm exchange(-)(-)was wasfound foundwhen when the the 368 position 368 position ininIgG1-2F8 IgG1-2F8 was L (= was L wild type (= wild type IgG1), IgG1), FF or orM.M. Fab-arm Fab-arm exchange was exchange was found to found to be be low (+/-) when low (+/-) the 368 when the 368 position position ininIgG1-2F8 IgG1-2F8 was was Y. Y. Fab-arm exchange Fab-arm exchange 35 waswas 35 found found to to be be intermediate(+)(+)when intermediate when thethe 368 368 positioninin IgG1-2F8 position IgG1-2F8was wasK Kand andhigh high

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(++) when (++) when 368368 thethe position position in IgG1-2F8 in IgG1-2F8 was was A, E, D, D, A, G, E, H, G, I, H, N, I, R, Q, Q, N, S, R, T, S, V, T, W. or W. or V,

Thesedata These dataindicate indicatethat thatparticular particularmutations mutations at at thethe IgG1 IgG1 368 position 368 position allowallow IgG1 IgG1 to to engage in engage in 2-MEA-induced Fab-armexchange 2-MEA-induced Fab-arm exchangewhen when combined combined with with IgG-K409R. IgG1-K409R. 2022201608 08

5 Table 5 Table8:8:2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange between between IgG1-2F8-L368X IgG1-2F8-L368X mutants and mutants and IgG1-7D8-K409R IgG1-7D8-K409R The generation The generation ofofbispecific bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-inducedin vitro in vitro Fab-arm Fab-arm exchange between exchange betweenIgG1-2F8-L368X IgGl-2F8-L368X mutants mutants and and IgGl-7D8-K409R IgG1-7D8-K409R was determined was determined by aa sandwich by sandwich ELISA. ELISA.(-) (-) no, no, (+/-) (+/-) low, low, (+) intermediate oror (++) (+) intermediate highFab-arm (++)high Fab-arm 10 exchange. 10 exchange.

Fab-arm exchange Fab-arm exchange Fab-arm exchange Fab-arm exchange IgGl-2F8-L368X IgG1-2F8-L368X xX IgGl-7D8-K409R IgG1-7D8-K409R A A ++ ++ D D ++ ++ E ++ F -

G ++ H ++ I ++ K + L -

M -

N ++ Q ++ R ++ S ++ T ++ V ++ W ++

36:Determinants Example36: Example Determinants theIgG1 atatthe IgG1i370 370 position for engagement positionfor engagement in in2 2- MEA-induced Fab-armexchange MEA-induced Fab-arm exchangeinincombination combinationwith withIgG1-K409R IgG1-K409R 15 15 Examples Examples 28,28, 29 29 andand 35 35 show show thatthat certain certain single single mutations mutations at positions at positions F405,F405,

Y407or Y407 or L368 L368are are sufficient sufficient totoenable enablehuman human IgG1 IgG1 toengage to engage inFab-arm exchange in Fab-arm exchange whencombined when combined with with IgG1-K409R. IgG1-K409R. As illustrated As illustrated in this in this example example further further determinants determinants

implicated the Fc:Fc inthe implicated in Fc:Fcinterface positionsininthe interfacepositions theCH3 CH3 domain domain may also also mediate maymediate the the Fab-arm exchangemechanism. Fab-arm exchange mechanism. To To this this effectmutagenesis effect mutagenesisofofthe the IgG1 IgG1i370 position 370 position 20 waswas 20 performed performed and and the the mutants mutants were were tested tested forforengagement engagement in in2-MEA-induced 2-MEA-induced Fab-Fab-

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2022201608 08 Mar arm-exchangeinin combination arm-exchange combination with humanIgG1-K409R. with human IgG-K409R.AllAllpossible possible IgG1-2F8-K370X IgG1-2F8-K370X mutants (with mutants (with the the exception exception of of CC and and P) P) were were combined with IgG1-7D8-K409R. combined with The IgG1-7D8-K409R.The procedurewas procedure wasperformed performed withwith purified purified antibodies antibodies as described as described in Example in Example 19. 19. Figure 37 Figure 37 shows showsthe theresults resultsofofbispecific bispecific binding binding upon upon2-MEA-induced 2-MEA-induced Fab-arm Fab-arm

5 exchange 5 exchangebetween between IgG1-2F8-K370X IgG1-2F8-K370X x IgG1-7D8-K409R. X IgG1-7D8-K409R. These These data data were were also also scored asas(-) scored (-) nonoFab-arm Fab-arm exchange, exchange, (+/-)(+/-) low,intermediate low, (+) (+) intermediate or (++) or high Fab-high (++) Fab arm exchange, arm exchange,asaspresented presented inin Table Table 9. 9. No No Fab-arm Fab-armexchange exchange(-)(-)was wasfound foundwhen when the 370 the 370position positioninin IgG1-2F8 IgG1-2F8waswas (= wild K (=K wild type type IgG1), IgG1), A, D, A, E, D, F, E, G, F, H, G, I, H, I, L, L, M, N, M, N, Q, R, Q, R, S, S, T, T, V or Y. V or Y. Only substitution of Only substitution of K370 withW W K370with in in resulted resulted intermediate intermediate Fab-arm Fab-arm

10 exchange 10 exchange (+).(+). These These datadata indicate indicate thatonly that onlyone onemutation mutationatatthe theIgG1 IgG1370 370 position position (K370W) allows IgG1 (K370W) allows IgG1 toto engage engagein in2-MEA-induced 2-MEA-inducedFab-arm Fab-arm exchange exchange whenwhen combined with combined with IgG1-K409R. IgGl-K409R.

Table 9: Table 9: 2-MEA-induced 2-MEA-induced Fab-armFab-arm exchange exchangebetween between IgG1-2F8-K370X IgG1-2F8-K370X 15 mutants 15 mutantsandandIgG1-7D8-K409R IgG1-7D8-K409R The generation The generation ofofbispecific bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-inducedin vitro in vitro Fab-arm Fab-arm exchange between exchange betweenIgG1-2F8-K370X IgG1-2F8-K370X mutants mutants and and IgGl-7D8-K409R IgG1-7D8-K409R was determined was determined by aa sandwich by sandwich ELISA. ELISA.(-) (-) no, no, (+/-) low, (+) (+/-) low, intermediate oror (++) (+) intermediate highFab-arm (++)high Fab-arm exchange. exchange. 20 20 Fab-arm exchange Fab-arm exchange IgG1-2F8-K370X IgG1-2F8-K370X xX IgGl-7D8-K409R IgG1-7D8-K409R A A -

D - E -

F 1

G - H - I - K - L - M I N - Q - R - S - T - V -

W +

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Y Y

Determinants atatthe 37: Determinants Example 37: Example the IgG1 399position IgGI399 positionfor engagementin in2-2 for engagement MEA-induced Fab-arm MEA-induced Fab-arm exchange exchangeinincombination combinationwith withIgG1-K409R IgG1-K409R Examples28,28, Examples 29,29, 35 35 and and 36 show 36 show that certain that certain single single mutations mutations at positions at positions

5 F405, 5 F405, Y407, Y407, L368 L368 or or K370 K370 areare sufficient to sufficient to enable enable human humanIgG1 IgG1toto engage engageinin Fab-arm Fab-arm exchange when exchange whencombined combined with with IgGl-K409R. IgG1-K409R. As illustrated As illustrated in in thisexample this example further further determinantsimplicated determinants implicatedin inthethe Fc:Fc Fc:Fc interface interface positions positions in in thethe CH3CH3 domain domain may may also also mediate the Fab-arm mediate the Fab-arm exchange exchangemechanism. mechanism. To To this this effectmutagenesis effect mutagenesisofofthe theIgG1 IgG1 399 position 399 position was was performed and the performed and the mutants mutants were were tested tested for for engagement in 2-MEA- engagement in 2-MEA 10 induced 10 induced Fab-arm-exchange Fab-arm-exchange in combination in combination with human with human IgG1-K409R. IgG1-K409R. All possible All possible IgG1-2F8-D399X mutants(with IgG1-2F8-D399X mutants (withthe theexception exception of of CC and and P) P) were combinedwith were combined with IgG1- IgG1 7D8-K409R. Theprocedure 7D8-K409R. The procedurewaswas performed performed with with purifiedantibodies purified antibodiesasasdescribed described inin Example 19. Example 19. Figure 38 Figure 38 shows showsthe theresults resultsofofbispecific bispecific binding binding upon upon2-MEA-induced 2-MEA-induced Fab-arm Fab-arm

15 exchange 15 exchangebetween between IgG1-2F8-D399X IgG1-2F8-D399X x IgG1-7D8-K409R. X IgG1-7D8-K409R. These These data data were were also also scored as scored as (-) (-) no, no, (+/-) (+/-) low, (+) intermediate low, (+) intermediateoror(++) (++) high high Fab-arm exchange, asas Fab-arm exchange, presentedinin Table presented Table10. 10.NoNoFab-arm Fab-arm exchange exchange (-) found (-) was was found when when the 399 the 399 in position position in IgG1-2F8 was IgG1-2F8 was D (= D (= wildwild typetype IgG1), IgG1), E and E and Q. Fab-arm Q. Fab-arm exchange exchange was was found to found be low to be low

(+/-) when (+/-) the 399 when the 399position position in in IgG1-2F8 wasV,V,intermediate IgG1-2F8 was intermediate (+)(+) when when the the 399 399 20 position 20 position in IgG1-2F8 in IgG1-2F8 was was G, I, G, L, I, M,L,N,M,S,N, T S, W.or or T W. Fab-arm Fab-arm exchange exchange was foundwas found to be to be high (++) high whenthe (++) when the399 399position position inin IgG1-2F8 IgG1-2F8was wasA,A,F, F,H,H,K,K,R RororY.Y.These Thesedata data indicate that indicate that particular particular mutations mutationsatatthe theIgG1 IgG1 399399 position position allow allow IgG1 IgG1 to engage to engage in in 2-MEA-induced Fab-arm 2-MEA-induced Fab-armexchange exchangewhen when combined combined with with IgG1-K409R. IgG1-K409R.

25 Table 25 Table10: 10:2-MEA-induced 2-MEA-inducedFab-arm Fab-armexchange exchange between between IgG1-2F8-D399X IgG1-2F8-D399X mutants and mutants and IgG1-7D8-K409R IgG1-7D8-K409R The generation The generation ofofbispecific bispecific antibodies antibodies after after 2-MEA-induced 2-MEA-inducedin vitro in vitro Fab-arm Fab-arm exchange between exchange betweenIgG1-2F8-D399X IgG1-2F8-D399X mutants mutants and and IgG1-7D8-K409R IgG1-7D8-K409R was determined was determined by aa sandwich by sandwich ELISA. ELISA.(-) (-) no, no, (+/-) low, (+) (+/-) low, intermediate oror (++) (+) intermediate highFab-arm (++)high Fab-arm 30 exchange. 30 exchange.

Fab-arm exchange Fab-arm exchange IgG1-2F8-D399X IgG1-2F8-D399X xX IgG1-7D8-K409R IgG1-7D8-K409R A A ++ ++ D D -

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E E - FF ++ ++ G G +

+ HH ++

+ 2022201608 08

II +

+ K K ++ ++ L L +

+ M__ _ M +

+ N N +

+ Q Q I R R ++ ++ SS + +

T T + +

VV +/ +/- w + W +

Y ++

Example 38: Example Determination of 38: Determination of the condition range the condition range in in which which 2-MEA-induced 2-MEA-induced Fab-arm exchange Fab-arm exchangeoccurs occurs suboptimally suboptimally to discriminate to discriminate between between highlyhighly efficient IgGI efficient mutants IgG1 mutants 5 5 The process The process of of 2-MEA-induced 2-MEA-induced Fab-arm Fab-armexchange exchange occurs occurs efficiently atat 37°C efficiently 37°C when2525mMmM when 2-MEA 2-MEA is used. is used. UnderUnder these these conditions, conditions, the majority the majority of permissive of permissive IgG1 IgG1 mutants (IgG1with mutants (IgG1 with certain certain singlemutations single mutations at positions at positions 368,368, 370,370, 399, 399, 405407 405 and and 407 and/or 409 and/or 409asasdescribed described in in Examples Examples 19,29, 19, 28, 28,and 29,35-37) and 35-37) show show high highof levels levels 2- of 2 MEA-induced Fab-arm MEA-induced Fab-armexchange exchange (80% (80% - 100%). 100%). To identify To identify experimental experimental conditions conditions 10 that 10 thatwould wouldallow allowdiscrimination discrimination between between the the IgG1 IgG1 mutants mutantswith withthe thehighest highest efficiency, 2-MEA-induced efficiency, 2-MEA-induced Fab-arm for four Fab-arm for four different different mutant combinations (IgG1- mutant combinations (IgG1 2F8-F405S Xx IgG1-7D8-K409A, 2F8-F405S IgG1-7D8-K409A, IgG1-2F8-D399R IgG1-2F8-D399RX xIgG1-7D8-K409G, IgG1-7D8-K409G,IgG1-2F8- IgG1-2F8 L368R Xx IgG1-7D8-K409H L368R andIgG1-2F8-F405L IgG1-7D8-K409H and IgG1-2F8-F405L X xIgG1-7D8-K409R) IgG1-7D8-K409R)was wasstudied studied over time over 150 C time atat15°C andand 200 respectively. 20°C, C, respectively. Apart Apart from from changes changes in temperature, in temperature, time time 15 period 15 period andand antibody antibody dilution dilution (20,(20, 2, 0.2 2, 0.2 and 0.02 and 0.02 µg/mL)pg/mL) the procedure the procedure was was performedasasdescribed performed described in in Example Example 19. 19. At 20 0 C At 20°C 2-MEA-induced 2-MEA-inducedFab-arm Fab-armexchange exchange of the of the four four mutant mutant combinations combinations occurred occurred atatdifferent differentrates ratescompared compared to maximal to the the maximal exchangeexchange (positive (positive control). control).

After 105 After 105 min incubation IgG1-2F8-L368R min incubation IgG1-7D8-K409H IgG1-2F8-L368R Xx IgG1-7D8-K409H reached reached the the maximal maximal 20 level 20 levelofofexchange, exchange,whereas whereas IgG1-2F8-F405S IgG1-2F8-F405S x IgG1-7D8-K409A, X IgG1-7D8-K409A, IgG1-2F8-D399R IgG1-2F8-D399R X x IgG1-7D8-K409G and IgG1-7D8-K409G and IgG1-2F8-F405L IgG1-2F8-F405L Xx IgG1-7D8-K409R IgG1-7D8-K409R reached reached aa maximum maximumofof 90%,85% 90%, 85%and and 85%,85%, respectively, respectively, afterafter 200 200 min. min.

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0 C showed most Incubation of Incubation of the the different different IgG1 IgG1mutant combinations at mutant combinations at 15 15°C showed most prominentdifferences prominent differencesininexchange exchange rates rates (shown (shown in Figure in Figure 39). After 39). After 60105 60 and andmin105 min incubations, 2-MEA-induced incubations, Fab-armexchange, 2-MEA-induced Fab-arm exchange, thethe differencesbetween differences between thethe four four mutant combinations mutant combinations were were most most extreme. extreme. Fab-arm Fab-armexchange exchange after200200 after minmin 5 incubation 5 incubationshowed showed efficiencies of efficiencies of 100% 100%(IgG1-2F8-L368R (IgGl-2F8-L368R x IgG1-7D8-K409H), X IgG1-7D8-K409H), 85% 85% (IgG1-2F8-F405L xX IgGl-7D8-K409R (IgG1-2F8-F405L IgG1-7D8-K409R and and IgGl-2F8-D399R IgG1-7D8-K409G) or x IgG1-7D8-K409G) IgG1-2F8-D399R X or 65%(IgG1-2F8-F405S 65% (IgG1-2F8-F405SX IgG1-7D8-K409A) x IgG1-7D8-K409A) compared compared to the to the positive positive control. control.

Example 39: Analyzing Example 39: 2-MEA-induced Analyzing 2-MEA-induced Fab-arm Fab-arm exchange exchange efficiencies efficiencies of of 10 mutants 10 mutants at at suboptimal suboptimal conditions conditions The process of The process of 2-MEA-induced 2-MEA-induced Fab-arm Fab-armexchange exchange occurs occurs 370 C efficientlyatat 37°C efficiently when2525mMmM when 2-MEA 2-MEA is used. is used. UnderUnder these these conditions, conditions, the majority the majority of permissive of permissive IgG1 IgG1 mutants (IgG1with mutants (IgG1 with certain certain singlemutations single mutations at positions at positions 368,368, 370,370, 399, 399, 405407 405 and and 407 and/or 409 and/or 409asasdescribed described in in Examples Examples 19,29, 19, 28, 28,and 29,35-37) and 35-37) show show high highof levels levels 2- of 2 15 MEA-induced 15 MEA-induced Fab-arm Fab-arm exchange exchange (80-100%). (80-100%). In Example In Example 38 it is 38 it is described described that that differences inin 2-MEA-induced differences Fab-arm exchange 2-MEA-induced Fab-arm exchangeefficiencies efficiencies are are most most pronounced pronounced after incubation at socalled socalled suboptimal suboptimalconditions, conditions,namely namely 0 C for 60 to 105 min. after incubation at at at 15for 15°C 60 to 105 min.

In total In total 24 IgG1-2F8mutants 24 IgG1-2F8 mutants at the at the L368, L368, D399, D399, F405Y407 F405 and and(see Y407 (see11)Table Table that 11) that show >90% show >90% 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange with with IgG1-7D8-K409R IgG1-7D8-K409R (Example (Example 28, 28, 20 29,29, 20 andand 35-37) 35-37) werewere selected selected and subjected and subjected to Fab-arm to Fab-arm exchange exchange analysis analysis with with IgG1-7D8-K409A, G, IgG1-7D8-K409A, G, HH ororR R(based (basedononresults results reported reported in in Example Example 19). 19). To To categorize these categorize thesemutant mutant combinations combinations upon efficiencies upon their their efficiencies to generate to generate bispecific bispecific

antibodies, 2-MEA-induced Fab-arm exchange exchange 0 antibodies, 2-MEA-induced Fab-arm waswas performed performed at 15forC 90 at 15°C for min 90 min (suboptimal conditions). (suboptimal conditions). Two IgG1-2F8 mutants Two IgG1-2F8 mutantsY407Q Y407Q and and D399Q) D399Q) that showed that showed 25 weak 25 weak 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm exchange exchange after after incubation incubation withIgG1-7D-K409R with IgG1-7D-K409R (Example (Example 2929 andand 37) 37) werewere takentaken along along as additional as additional negative negative controlscontrols and used and to used to

study whether study whetherincubation incubation with with another another amino amino acidtheatK409 acid at the position K409 position (G, H, (G, H, or W) or W) leads to leads different result. a different to a result.Apart Apartfrom from aa change in temperature change in temperature and and changes changesin in antibody dilution antibody dilution (20, (20, 2, 2, 0.2 0.2 and and 0.02 the procedure ug/mL), the 0.02 ug/mL), procedurewas wasperformed performed as as 30 described 30 described in inExample Example 19.19. Incubation of all Incubation of all different different IgG1 mutantscombinations IgG1 mutants combinations (as (as becomes becomes clear clear from from

Table 11) 11) at at 15°C 0 15 C for for 90 showeda arange min showed rangeofofdifferent different 2-MEA-induced 2-MEA-induced Fab-arm Fab-arm Table 90 min exchangeefficiencies. exchange efficiencies. The Theresult resultofofbispecific bispecific binding binding atat an anantibody antibodyconcentration concentration of of 20 µg/mL, 20 pg/mL,is isshown shown in Table in Table 11. Results 11. Results were were categorized categorized in 4 classes; in 4 classes; no (-), no low(-), low 35 (+/-) 35 (+/-) intermediate intermediate and(++) (+)high (+) and highbispecific (++) bispecific binding efficiency binding efficiency as is specified as is specified in in

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below for legend below the legend for Table 11. From Table 11. From these these resultsit itbecomes results becomes clear clear that that under under 2022201608 08 Mar

suboptimalconditions suboptimal conditionssome some combinations combinations of amino of amino acid mutations acid mutations in IgG1 inmolecules IgG1 molecules

will be will be favorable favorable for for 2-MEA-induced Fab-arm 2-MEA-induced Fab-arm exchange. exchange.

5 Table 5 Table 11:11: Bispecificbinding Bispecific binding (% (0/o relative relative to positive to positive control) control) between between permissive IgGI permissive IgG1 mutants (20pg/mL)atat15°C mutants (20µg/mL) 150Cfor for 90 90 min min

Fab-arm Fab-arm IgGl-7D8- IgG1-7D8- IgGl-7D8- IgG1-7D8- IgGl-7D8- IgG1-7D8- IgGl-7D8 IgG1-7D8-

exchange K409A K409A K409G K409G K409R K409R K409H K409H IgG1-2F8-L368A IgG1-2F8-L368A 33 33 33 33 25 25 37 37 ...... : : : : : : :: : : : : : : : : : : : : : : :A --- ::i:i:i:i: ___ _______ ------- ___ --- --- -_ ---------- - _ _ _ _ -- _ _ _ _ _ _ -- _ _ _ _ _ -X ............................................ X .................................... ..... X X X ............................. X : ..... ......... . . . . . . . .... ......... . IgG1-2F8-L368D ........ ::........ I g G 1 - 2 F 8 - L 3 6 8 D ...... ...... ... .:::::::::::::::::::::::::::::::::::::::::::::: - 49 9 - --- -------- __ 50 --- __ __ __ __ __ __ __ __ __41 5 0 ____ _______ __ 4 l __ __ __ ..... _--- ....... ...... ... .................................. . ...... . ................... ............................................................ .............................................. . . . . . . . . . . . . . . .............................................. . . . . ::::::::: . . . . . . . .....4 .S.- 54. . . . . ........... :.......... .. ......... ... ...... .. .. .. . . . . . . . . . . .. . . . . . . . . . . . . . . ................................ ...... ...... . . . . . . . . . . . ..... . . . . . . .. . . . .

.. _ _ _ _ _ _ _ _ _ _ _---- _ .............................................. . . . . . . . . . . . . . ,.,.,.,.,.,.,.,.,.,.::::: IgG l-2F8-L368E IgG1-2F8-L368E- 32 32 38 38 37 37 ..... ..... ............................. .............................................. .............................................. ........................... ........................... ................................................. ....... ............................................. ............................................. ............................................. :.:.:.:.4 42 .2:.,.,.,.,.,.,.,.,.,. . ..:.......... ......... ......... ........ ............

. ............................................. ............................................. ............................................. ............................................. - - - - - __ __ - - - - - - ....- - .............................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . ....... :':i:i:i:i: : :__: : :__: : : : : : : : : : : : : ::::::- '::...... : : : :A :6 ::::: --- ---------- 3 ---- - - - - - - - - - - 53 -_ -_ -_- -- -- ---: -------- -__-_-_-- -- --44 : ..... . . . . . . . . . . . . . . . . . . . . . . . .... . . . . ...................... -- .................................... ........................ 4-- --_---..."...........................................................- . ,..,.,.,..,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,::::::::: . . .-:::::s . 53 .......... ........... ....'3.. 1.11::.:.......... ......... ........

. IIgG1-2F8-L368G g G l - 2 F 8 - L 3 6 8 G :....... ....... ...... 46 - ..... .............................................. .................................................... ...... ....... _ _ __---- - .....- - .............................................. ............................................ -............................................ IgG1-2F8-L368H IgG1-2F8-L368H 26 26 25 25 21 21 29 29 ........................................................................................................................................................................................ . . : ......... ..................... ....................... ...................................... . . " ......... ..................... . . . . . . . . . . . . . . . . . ::::::::... ... ............ .......... . . . . . . . . " IgG IgG1-2F8-L368N ...... ....... ........ .. ..................... ..................... . . . . . : ............................... l-2F8-L368N :::::::::.:.:.:.:."::::::::::::::: ........... :::::::::::::::..:..:..:..:..:..:..:..:..:..:..:..:..:.4 ..7 .......... .......... ......... .......... .....:.:.............. ........... ......... ..................... .. ....................... .............................. . .::::::::: ........................................................................................................................................................................................ ........................................................................................................................................................................................ 47 ..::::::::.5 .. .. .. .. . . . 52 .2 "...:: .......... ......... .......... .:................ -.-. '........... ............ ..................... ...... ..... ................................................... ..................... .............................. ...... .4 ....... ::::::::.I.3A .... 43 .............................. ,:.......... .................... . . . . . . . . . . . .............................. ......................................... .. . ........................... . . . . . . . . . . . . . . . . . . . . :::::::::-5 ......... . . . . . . 54 -4.......... ............ ................ :........... ....... ... ........................................................................................................................................................................................ ........................................................................................................................................................................................ ........................................................................................................................................................................................ ........................................................................................................................................................................................ ......................................................................................................................................................................................... ...... ...... . .. .. '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,. ............... :.:.:.:.:.:.:....:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.S .,.5 . : . , . . . . . . . . . ,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.",""."",." ............... .............................. " " """""""""""""""", :::::::. . . .-"..4 -:.......... """""""""""""""""""""" " " " '-'-'-" - - ' ,,,I', ........................ ................. .... .......... ..................... .......... ........... ............................. . . . . . . . . . . . . . . . . . . . . . . . ::.:.:.:.:.:.:.6 . . . . . . . . . . . ...................... '.......... '.'. .'.'. .'.'. .'. " . . . . ..... .. ..................... ....... .................. ................... ..... ............. .. . . . . . . . . i -.52 .'. '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.' . . . . . . . . .i. . . . . . . . . . . . . .: --S ..2. . i i i i i i i i i 91 I g G l - 2 F 8 - L 3 6 8 R :........................................ ............................................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55 . . . . . . ........... ............................. . . . . . . . . ..64 . . . . . . . .. .... . .......................................... :.:.:.:.:.:.:.:..:'..:'..:'..:'..:'..:.'.:.':. .':. ::::::::::::: .

IgG1-2F8-L368R ...................................................................................................................

, ................................................................................................................................... .............................................. ................................................................................................................................... .................................................................................................. "."

, ....................... ...................... ...................... ::::::: .. : ......... :::::::::::::::::::::::::::: ............................................. .. :........... .......... ......... IgG l-2F8-L368S IgG1-2F8-L368S 39 39 ............................. ....................... ................................ ............................................. ....................... .............................................. ............................................. .............................................. ....... . 45 :.:.:.:.:.:.4 5. . ........... :.......... . .... .". . " ......... . ..'.'.'.'.' . . . .. 37 37 ............................. .............................. ::::::::::::::::::::::::::::: ................................................. ........ ............................................. ............................................. s 53.-3 .......... ': -:.:.............. ........... -.. ............................................. .............................................. ............................................. .............................................. ............................................. ............................................. ......... ............................................. ........................... ............................................. IgG1-2F8-L368T ......... . . . . . . : .................. ',',',"",:::::::: ....... ..s .......... ....... Ig G l - 2 F 8 - L 3 6 8T ............:::::::::::::.::::::::::::::::::42.:.:.:.:.:.:.:.:.:.:.: :::::::::::: .............................................. .............................................. .............................................. 42 b_ .......... ....... ...... ::::::::::::::::::::::::... ::::::::::::::: _ _ i.i.i.i.i.i.................................. .............................. ...... ::::::::s ......................................... ............................................. .............................................. ............................................. ....... 51 .: ...''.'.'.'.'.'.'.'.'.'. :........... ......... . . . . . . ... - 339 9 ..................................... ..... :.,.":.:.:::::::::::::::::::::::::.s .:.:....................... :.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:. . ............................. ............................ ............................................. ............................................. ....................... . . 6. '. :.:............ 56 .......... ,..,......... .,..,..,..,..,..,..,. .............................................. .. ......-. ....................................... ............................................... ....... ............................... .......... .............................................. .............................................. ............................. ............................................. ......... ............................................. ............................................. ............. : ...... ......... ::.-.-.-.-.-.-.-.-.-.................. . . . . . . . . . . .................................................. ............................................. I g G l - 2 F 8 - L 3 6 8V .:',',',"",:::.:.:.:.:.:.::::::::.............. .............. ................... :.4 .- :..:. :. :. :. :.42 2.- . .' .'..'..'. .' .'..'. .'. '. '.............................. ':........... :. :.4 -9.'.:. . .'..'..'. .'..'. ...'.'.,'.' .'. .'. '. .'..'. .'. '. .'. '. .'. '. .'. '..'..'. .' .'..'. .'. '. .:..:. :. .:. :. :.49 3 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::. _............................ . . . . . . . . . . . :.:.:.:.:B .............................................. . . . . . . . . . . . ............................. .I .51 : ,--- '.:,..:,........... .,...,..,..,...,..,..,..,..., IgG1-2F8-L368V . . . . . . :................................... .............................................. .............................................. .............................................. .............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. 333 ............................................. .............................................. ...... .............................................. ........ .............................................. ;;;;;;;;;;;;;I_ _ _ _ _ _ ............................................. . ............ I g G l - 2 F8 - :........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................... ......................................................................................................................................................................................... . . . . . . . . . . . . . . . ............................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................. .............................................................................................................................................. ........................................................................................................................................ .............................................. .................................................................................................................................................................................... ........................................................................................................................................................................................ ........... . . . . .............................................. .............................. IgG1-2F8- ........ ................................................................................................................................ ........................................................................................................................................................................................ ......................................................................................................................................................................................... ........................................................................................................................................................................................ ......................................................................................................................................................................................... ........................................................................................................................................................................................ ......................................................................................................................................................................................... ........................................................................................................................................................................................ ......................................................................................................................................................................................... ........................................................................................................................................................................................ ......................................................................................................................................................................................... :::::: .. ..: .. ........... L3 6 8 W ....... ...... ...... ....... . . . . . :.:.:.:::: ...................... ::::::::::::::: ................... .................................................... ....................... ...................................... 5..6 ........... ........... M .......... ............ ....................... . . . . . . . . . . . . . ...... ............................... ............................. :.,..,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.......... . . .i. . ..... . . . ,::::::: ........... ....... .,.,. . . . .565. .......... .................. :.......... .............................................. .......... ..... ......................................... .................... :::::::::::::::::::::: "::::::::::::::::::::::' . . .6....................................................................... ........... . . . . . . ......... ...................4 '- '-. -.'I .. .'. ........................ .'.'.'. '. '. .'......................... ::.:......................................... .... ."...... '.'.'.'." . ...........................................................:.:..:..:..:..:..:...:..:..:..:..:...:..:..:..:..:........... ..... .. . . 6 .0 .............. . ..................... ........... .. . L368W ......................................................................................................................................................................................... 56 ........................................................................................................................................................................................ ......................................................................................................................................................................................... ........................................................................................................................................................................................ ......................................................................................................................................................................................... 41 60 ........................................................................................................................................................................................ g G l - 2 F8 - IIgG1-2F8- .............................................. ...........................................................................................................................................

D399F D399F 13 13 15 15 14 14 15 IgGl-2F8 IgG1-2F8- D399H D399H 12 12 14 14 10 10 19 19 ............................................. .............................................. ............................................ ............................................ ............................................. .............................................. ............................................. ....................... ........ ............................................. ............................................. ....................... ,.,.,.,. . . . . . . . . . . . . . . . . . I g G l - 2 F8 - ..................................... ........ """".............................. ............................................. .............................................. ............................................. .................... ........................ ............................................. ............................................. IgG1-2F8- .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. ............................................. ............................................. .............................................. ............................................. .............................................. ............................................. ............................................. ............................................. ............................................. ....................... . . . . . . . . . . . . . . . . . . . . . . . ,...... ,". . . 3 ......... W:............ .......... . . . .'.. '.. '..'.', ::::::::::::::::::::::::::::::::::: ............................ .......... ........ D 399 K D399K 4 40 0 :::::::::::::4 43-.- :..'. '. '. '. ..... 3 344 ....... . . . . . :....................... ............................ ............................................. . :.4 .6 . :. :. :. :. :. :. :. :. :. :. :. :. :. :. :. :. :. :. :. :46 .......... --':::.................. ......... .......... ............................................. .............................................. ............................................. ............................................. ............................................. ............................................. IIgG1-2F8- g G l - 2 F8 - .-............................................. . . .-. . . .-........ . . ._. . . . . _. . . .-. . .-. . . -. . . .-. . . -................. :............................................... ............................................. ...................................... . . _. ,. . _. . . . . . - - .............................................. ............................................... ............................................. ............................................. .............................................. ...................................................................................................................................................................... ............................................. .............................................. . . . . . . ........................................ ..................................... ................................................... ............................................. .............................................. .............................................. .............................................. .............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. ............................................. ............................................. ............................................. .............................................. .............................................. .............................................. .............................................. .............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. ............................................. .............................................. .................................................... ............................................. .::::::S ...2. ... D 399 R .:..:.. :.. .:..:..:................ .,.............................................. ..,..,..,..,..,.: :......................... .. .. .. .. .. .. .............................................. ............ ...................... ........................ ::::::: .5. .:::................ ::::::4 .. ..:..:.. :.. .:..:..:.. :.. .:..:.. :.. :.. :. :. :. :.47 :.............................................................................. ...................... ............................. ............................ .........- .......... ........... 3 ...................... .................................. ..... ::::::::::::::::: ....................... ...................... . . . . . . . ....... ........... .:........... .. .. .. .. .. .. .. . ........... ......... 388 ................................... ............................. ............................ 52 ........... D399R - 45 .............................................. .............................................. .............................................. .............................................. 47 .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ............................................. .............................................. ....................... .............................................. IgGl-2F8 IgG1-2F8- D399Q D399Q 0 0 0 0 00 0 0 ............................................. .............................................. ....................... . . . . . . . . . . . . . . . . . . . . . . ::::::: ....................... : - ......... -.'.'.'.'.'.'.'.'. '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.':.:.:.:.:. ..... .B .. .......... IgG 1-2F8-F405I IgG1-2F8-F405I 32 32 . . . . . . . . . . . . . . . . . . . . . . :::::::'4'..9.: .............................................. ............................. ............................................. .............................................. ...... :: ..... 49 .......... ...... . . . . ......... ......... ......... . . . . . , 39 39 ...... ..... :. : . : . : . : . : . : . : . : .............................................. . . . . . . ...................... ............................ ............................. . : . : . ....................................... : . : . : . : . : . : . : ....... ..... 6 60 . ........... .......... .......... .......... ........... .......... ............................................. .............................................. .

.............................................. ............................................. .............................................. ............................................. ............................................. ............................................. .............................................. .............................................. - ---- - - _ _ _ _ _ _ _ _ ..................................................... .............................................. ..-_--------- ............................................... ....................... ...................... ::::::: ...B.::............. """"""""'.." . .. .. .. .. .. .. ...... . .............................. ,............. ...... ........................... ::::::::: .::..""","","", :... ... .. ....... ..... .. IIgG1-2F8-F405K g G 1 - 2 F8 - F40 5 K 29 29 ....................... .......................... ....................... ................................ ............................................... .............................................. ............................................... :.:.:.:.:.:.4 .......................................................... .............................................. 48 .......... . . _ _ _ _ __ _ _ _ _4 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 47 _ _ 7__ ___--___ ,."""'........................................................ ..... ..... ..... .. ............................. ....................... .............. .................................... ::::::::::::::: .............................................. ..................................................... .............................................. ..................................................... :.:.:.:.:.:.:.:.4 ....... 40 . ........... .0 .............................................. .............................................. .............................................. ._ _ _ _ _ _ __ _ _ _ _ _ _.....__ ............................................... .............................................. ............................................., ............................................. ................................. ........... IgG 1-2F8-F405L IgG1-2F8-F405L 31 31 ............................. ...................... """""""""""':::::::::::::4.4:.............'.."' ................................... ............................................. :x......... ....................... .......... ......... . . . . . . ._, 39 39 :.......................................... ...... .................................... 46 ...........:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.4 ..... ::::::::::::::::::::::: ............................................ -............................................. - ,.,::........... .6 .,.,.,.,.,.,.,.,.I.,.,.,.,.,.,.,.,.,. .............................................. 44 -............................................ -............................................. .

............................................. ............................................. .............................................. -............................................ -............................................. -............................................ ...................... ................. -------- ------- ..... - ....... ........ ...................... IgG 1-2F8-F405S IgG1-2F8-F405S 34 34 1:........................ ............................... ............................. :....... . . . . . ... . . . . . . . . . . .. .. .. ..:. :....:::::: X....... X X 5 .".I.". ,","""""""",""""'......................... ....................... : .......................................... . . . . . .3. . . . . . . . 45 .. .. .. .. .. ... 5:::::::::::::::::::::,4 .............................. 39 9 ............................. 51 ............................. .............................. .............................. ............................... ................................ ............................... .............................. .

I - ........................................................... i!!!!Ili iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii.' .'.'.'.'.'.'.,....,.,.,.,.,.,...............""""""""", I

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IgG1-2F8-F405T IgG1-2F8-F405T 35 45 47 4. . . . . . 42 4. . . . . ...... 46 ...... .. .. .. .. .. .. .. .. .. ..46. . . . .. . IgG1-2F8-F405V F.36 .. .. ... ... ... ... ... 46... ... 37 43

. 20. .......... .. ... ... .. ... .. ...

. IgG1-2F8- IgG1-2F8...07L........4.... 17 20 16 .. .. .... .. .. .... ..18 F405W

. .. .. ... ... ... . .. .... 4... .. ...

. IgG1-2F8-F 0 IgG1-2F8-Y407L V3.......3 44 41 49 ........ 49 2022201608

IgG1-2F8-

Y407W 48 53 47 62 Low(4-39..................................... . . .

. IgG1-2F8-

4 9 1 44 Y407Q

Legend for Table 11 L38, gg2F-451 gl2840Ln I22FY0W)2Einue No (0-3%) bispecific binding (-) Fa-recagefceclogiF80W 07Qasnayedorscodtm Low (4-39%) bispecific binding (+/-)

Intermediate (40-69%) bispecific binding (+)

High (70-100%) bispecific binding (++) mutated8Yg0L-2F.oleclesith. 78...9Rtatarreprtedn.xaple .- 28,2,an35-7asegaiveaestllointresasptentallprootigth.g. 15~~ EA.dueda mutated..IgG1-2F8 ~... From the .a.ecane molecules tested (Table 11), six were selected Example40:.Usingthebspec.f.cformattoremveundesiredagon.sti for a second analysis to confirm the results obtained before (Table 11). Several activityofantagonisticc-Metantibodiesconvertingthemintoamonovalent,................................. bispecififormat................ 5 mutants were selected for their high (IgG1-2F8-L368R) and intermediate (IgG1-2F8- 20...Severalbiva entantibodiesdevelopedformonoc..........lona......antibodytherapyshow....... ...... uneiabegnstccivtuobidntohitrgtTissloheaeo L368W, IgG1-2F8-F405I, IgG1-2F8-F405L and IgG1-2F8-Y407W) 2-MEA-induced Fab-arm exchange efficiency. Also IgG1-2F8-Y407Q was analyzed for a second time

since it showed an unexpected positive 2-MEA-induced Fab-arm exchange reaction with IgG1-7D8-K409H. In general, these results, presented in Figure 40, confirmed

10 the primary analysis (Table 11) and show that 2-MEA-induced Fab-arm exchange

reactions of mutated IgG1-2F8 molecules with IgG1-7D8-K409H showed highest

efficiency. Furthermore, 2-MEA-induced Fab-arm exchange reactions between

mutated IgG1-2F8 molecules with IgG1-7D8-K409R that are reported in Examples 28, 29, and 35-37 as negative are still of interest as potentially promoting the IgG1

15 2-MEA-induced Fab-arm exchange.

Example 40: Using the bispecific format to remove undesired agonistic activity of antagonistic c-Met antibodies converting them into a monovalent,

bispecifi format

20 Several bivalent antibodies developed for monoclonal antibody therapy show

undesirable agonistic activity upon binding to their target. This is also the case for

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mostIgG1 most IgG1based based antibodies antibodies targeting targeting the the receptor receptor tyrosine tyrosine kinase kinase c-Met. c-Met. TheseThese

agonistic agonistic antibodies inducereceptor antibodies induce receptordimerization dimerizationfollowed followed by by activation activation of of several several

downstream downstream signaling signaling pathways. pathways. As aAs a result result growth growth and differentiation and differentiation of (tumor) of (tumor)

cells isisinduced. cells induced. The The use of monovalent use of antibody monovalent antibody formats formats can can prevent prevent induction induction of of 5 receptor 5 receptor dimerization. dimerization. Combination Combination of an of an anti-c-Met anti-c-Met antibody antibody Fab-arm Fab-arm with with a Fab- a Fab arm of arm of an anirrelevant irrelevant antibody antibodyresults resultsinin aa bispecific bispecific antibody antibodythat that is is functionally functionally monovalentandand monovalent therefore therefore completely completely antagonistic. antagonistic. Here Here we combined we combined a partial a partial-

(IgGl-069) (IgG1-069) orora afull full (IgG1-058) (IgGl-058)agonistic agonistic antibody, antibody, with with IgG1-b12 IgG1-b12 (first (first described described in in

Burton DR, Burton DR,etetal, al, "Efficient "Efficient neutralization neutralization of of primary isolates of primary isolates of HIV-1 by aa HIV-1 by

10 recombinant 10 recombinant human human monoclonal monoclonal antibody", antibody", Science. Science. 1994 1994 Nov Nov 11; 11; 266(5187):1024 266(5187): 1024- 1027) inin bispecific 1027) bispecific antibodies. antibodies. IgG1-b12 was IgG1-b12 was regarded regarded as irrelevant as an an irrelevant non-binding non-binding

antibody since antibody since it it is is raised raised against against aa viral viralprotein protein(HIV-gp120). Theanti-c-Met (HIV-gp120). The anti-c-Met antibodies used antibodies usedinin this this example exampleare arefully fullyhuman human monoclonal monoclonal antibodies antibodies generated generated in in transgenic mice. transgenic mice. IgG1-058 IgG1-058andand IgG1-069 IgG1-069 bind bind to to different different epitopes epitopes on c-Met. on c-Met.

15 15 The two The anti-c-Metantibodies twoanti-c-Met antibodiesused used areare IgG1,K IgG1,k antibodies antibodies being being modified modified in in their Fc their Fc regions as further regions as further disclosed. Theyhave disclosed. They havethe thefollowing followingheavy heavy chain chain andand light light

chain variable chain variable sequences. sequences. 058: 058: VH 058 VH 058 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYW EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYW VRQTPEKRLEWVATISDDGSYTYYPDSVKGRFTISRD VRQTPEKRLEWVATISDDGSYTYYPDSVKGRFTISRD NAKNNLYLQMSSLKSEDTAMYYCAREGLYYYGSGSY YNQDYWGQGTLVTVSS YNQDYWGQGTLVTVSS VL 058 VL 058 AIQLTQSPSSLSASVGDRVTITCRASQGLSSALAWYR QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQFTSYPQITFGQGTRLEIK SSLQPEDFATYYCQQFTSYPQITFGQGTRLEIK

20 20 069: 069: VH 069 VH 069 QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISW VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY WGQGTLVTVSS WGQGTLVTVSS VL 069 VL 069 DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWF DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWF QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK

Receptorphosphorylation Receptor phosphorylation Monovalent bispecific Monovalent bispecific c-Met c-Met antibodies antibodies were were generated generated by a by a Fab-arm Fab-arm exchange reaction exchange reaction with with IgG1-058-F405L or IgG1-069-F405L IgG1-058-F405L or IgG1-069-F405Land andIgG1-b12-K409R IgG1-b12-K409Ras as 25 described 25 described in inExample Example 23 23 using using 25mM 25mM 2-MEA. 2-MEA. The effect The effect of the of the bispecific antibody bispecific antibody on on c-Met phosphorylation c-Met phosphorylation was wasevaluated. evaluated.Upon Upon dimerizationof of dimerization twotwo adjacent adjacent c-Met c-Met

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receptors by receptors byeither eitherthe thenatural ligandHGFHGF naturalligand or agonistic or by by agonistic bivalent bivalent antibodies, antibodies, threethree

tyrosine residues tyrosine residues (position (position 1230, 1230,1234 1234 andand 1235) 1235) in the in the intracellular intracellular domain domain of c-Met of c-Met

are cross are cross phosphorylated. phosphorylated.This Thisleads leads to to phosphorylation phosphorylation of several of several other other aminoamino acids acids in the in the intracellular intracellulardomain of c-Met domain of c-Met and andactivation activationofofa anumber number of signaling of signaling cascades. cascades.

5 TheThe 5 dimerization dimerization andand activation activation of of c-Met c-Met can can be monitored be monitored by using by using antibodies antibodies 2022201608 specific for specific for the the phosphorylated receptoratatthese phosphorylated receptor these positions, positions, which which functions functions asread as a a read out for out for the the potential potential agonism agonism ofofanti-c-Met anti-c-Metantibodies. antibodies. A549 cells, A549 cells, CCL-185 obtained from CCL-185 obtained from ATCC, ATCC,were were grown grown in serum in serum containing containing DMEMmedium DMEM medium until until 70%70% confluency confluency was was reached. reached. CellsCells werewere trypsinized, trypsinized, washed washed 10 and and 10 plated plated in a in a 6 well 6 well culture culture plateplate at 1*10e6 at 1*10e6 cells/well cells/well in serum in serum containing containing culture culture medium. After medium. After overnight overnight incubation incubation the the cells cells were treated with were treated with either either HGF HGF (R&D (R&D systems; cat. systems; cat. 294-HG) 294-HG)(50 (50ng/mL) ng/mL) or the or the panel panel of antibodies of antibodies (30 (30 pg/mL) µg/mL) and and incubatedfor incubated for 15 15minutes minutesatat37°C. 37°C. TheThe cells cells were were washed washed twice twice withcold with ice ice PBS coldand PBS and lysed with lysed with lysis lysis buffer buffer(Cell (CellSignaling; Signaling;cat. cat. 9803) 9803) supplemented supplemented with a with a protease protease 15 inhibitor 15 inhibitorcocktail cocktail (Roche; (Roche; cat. cat. 11836170001). 11836170001). Cell Cell lysate lysate samples samples were were stored stored at at 80 0 C. Receptor 80°C. Receptoractivation activationwaswas determined determined by detection by detection of c-Met of c-Met phosphorylation phosphorylation on on Westernblot Western blotusing usingphospho phospho c-Met c-Met specific specific antibodies. antibodies. The The proteins proteins present present in cell in the the cell lysate were lysate were separated on aa 4-12% separated on SDS-PAGE 4-12%SDS-PAGE gel gel and and transferred transferred to to nitrocellulose nitrocellulose membrane that membrane thatwaswas subsequently subsequently stained stained withwith an antibody an antibody specific specific for for 20 phosphorylated 20 phosphorylated c-Met c-Met (Y1234/1235) (Y1234/1235) (Cell Signaling, (Cell Signaling, cat:As3129). cat: 3129). As for a control a control gel for gel loading total loading total B-actin ß-actin and c-Met levels and c-Met levels were weredetermined determinedusing using anti anti c-Met c-Met (Cell (Cell Signaling, Cat. Signaling, Cat. No. No. 3127) 3127)and and antiß-actin anti B-actin(Cell (CellSignaling, Signaling,Cat. Cat.No.No. 4967) 4967) antibodies. antibodies.

Results of Results of the Westernblots the Western blotsare areshown shownin in Figure Figure 41.41.

Tissue culture Tissue culture medium medium controls controls and and cells cells treated treated withwith the monovalent the monovalent format format 25 UniBody® 25 UniBody®(Genmab, (Genmab,WO2007059782 W02007059782 andand W02010063785) WO2010063785) of antibody of antibody 5D5 5D5 (Genentech; WO96/38557) (Genentech; WO 96/38557) diddid notnot show show anyany c-Met c-Met receptor receptor phosphorylation. phosphorylation. TheThe monovalentUniBody monovalent UniBody format format as used as used hereinherein is an is an IgG4, IgG4, wherein wherein theregion the hinge hinge has region has been deleted been deleted and and wherein wherein the the CH3 CH3region regionhas hasbeen been mutated mutated at at positions405405 positions andand 407. In 407. In contrast, contrast, Western Westernblot blotanalysis analysis of of cellstreated cells treatedwith with thethe positive positive control control HGFHGF

30 or or 30 agonisticantibody agonistic antibodyIgG1-058 IgG-058 showed showed a clear a clear band band at the at the expected expected height height of of thethe phosphorylated c-Met. phosphorylated c-Met. Partial Partial agonistic agonistic antibody antibodyIgG1-069 IgGl-069 showed showed less, less, but but detectable receptor detectable receptorphosphorylation phosphorylation indicating indicating that that some some cross cross linking linking of the of the receptor receptor

takes place. takes place. However, However,both both bispecificIgG1 bispecific IgG1 058/b12 058/b12 and bispecific and bispecific 069/b12 069/b12 antibodies did antibodies did not notinduce induce c-Met c-Met phosphorylation phosphorylation at showing at all, all, showing that that the the agonistic agonistic

35 activity 35 activity associated associated withwith the the parent parent antibodies antibodies was completely was completely absent absent (Figure (Figure 41). 41).

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of c-Met Effect of Effect c-Met antibodies on NCI-H441 antibodies on NCI-H441 proliferationin invitro proliferation vitro The potential proliferative The potential proliferative agonistic activity of agonistic activity of c-Met antibodieswas c-Met antibodies was tested tested in in

vitro in vitro in the lung adenocarcinoma the lung cellline adenocarcinoma cell lineNCI-H441 NCI-H441 (ATCC, (ATCC, HTB-174TM).Thiscellline HTB-174 This cell line

expresseshigh expresses highlevels levelsofofc-Met, c-Met,butbut does does not not produce produce its ligand its ligand HGF. HGF. NCI-H441 NCI-H441 cells cells 5 were 5 were seeded seeded in a in96-wells a 96-wells tissue tissue culture culture plate plate (Greiner (Greiner bio-one, bio-one, Frickenhausen, Frickenhausen, Germany)(5,000 Germany) (5,000 cells/well) cells/well) in in RPMI RPMI (Lonza) (Lonza) without without serum. serum. Anti c-Met Anti c-Met antibody antibody was was diluted to diluted to 66,7 66,7 nM in RPMI nM in RPMI medium medium without without serum serum and and added added to cells. to the the cells. After After seven days seven days incubation incubation at at 37°C/5% 37°C/5%CO, 2 , the C0the amount amount of viable of viable cells cells waswas quantified quantified with Alamarblue with Alamarblue (BioSource (BioSource International, International, San SanFrancisco, Francisco,US) US)according according to to thethe 10 manufacturer's 10 manufacturer's instruction.Fluorescence instruction. Fluorescencewas was monitored monitored using using the the EnVision 21012101 EnVision Multilabel reader Multilabel (PerkinElmer,Turku, reader (PerkinElmer, Turku,Finland) Finland)with withstandard standard Alamarblue Alamarblue settings. settings.

In contradiction In to IgG1-069, contradiction to IgGl-069,no no proliferationwaswas proliferation induced induced uponupon incubation incubation of of NCI-H441cells NCI-H441 cellswith withthethe bispecificIgG1-069/b12, bispecific IgG1-069/b12, as isasshown is shown in Figure in Figure 42.the 42. Also Also the UniBody-069 UniBody-069 control control diddid notnot induce induce proliferation, proliferation, which which was was comparable comparable to thetonone- the none 15 or or 15 IgG1-b12 IgG1-b12 treated. treated.

Example41: Example 41:CDC-mediated CDC-mediated cell cell killing killing by by bispecific bispecific antibodies antibodies generated generated by by 2-MEA-induced Fab-arm-exchange 2-MEA-induced Fab-arm-exchange between between human human IgG1-2F8-F405L IgG1-2F8-F405L or or IgG1-7D8-F405Land IgG1-7D8-F405L IgG1-7D8-K409R and IgG1-7D8-K409R 20 20 The CD20 The CD20antibody antibodyIgG1-7D8 IgG1-7D8 can can efficientlykill efficiently kill CD20-expressing CD20-expressingcells cellsbyby complement-dependent complement-dependent cytotoxicity cytotoxicity (CDC). (CDC). In contrast, In contrast, the antibody the EGFR EGFR antibody IgG1-2F8 IgG1-2F8

does not does not mediate mediate CDC CDConontarget targetcells cells expressing expressing EGFR. EGFR. Both Both IgG1-7D8-K409R IgG1-7D8-K409Randand the bispecific the bispecific antibody antibody generated by 2-MEA-induced generated by 2-MEA-inducedFab-arm-exchange Fab-arm-exchange between between IgG1-2F8-F405L IgG1-7D8-K409R IgG1-2F8-F405L X xIgG1-7D8-K409R are are able able to induce to induce CDCCD20-expressing CDC on on CD20-expressing 25 cells 25 cells (as(as is described is described in Example in Example 26). 26). It wasIttested was tested whether whether the bispecific the bispecific antibody antibody

generated by generated by 2-MEA-induced 2-MEA-induced Fab-arm-exchange Fab-arm-exchange between between IgG1-7D8-F405L IgG1-7D8-F405Land and IgG1-7D8-K409Rcould IgG1-7D8-K409R couldalso alsoinduce induce CDC CDCononCD20 CD20 expressing expressing cells. 10 cells. DaudiororRaji 105Daudi Raji cells were cells pre-incubatedfor were pre-incubated for1515min min with with a concentration a concentration series series of antibody of antibody in 100 in 100 µL pL RPMI medium RPMI mediumsupplemented supplemented with with 0.1% 0.1% BSABSA in ain shaker a shaker at at room room temperature. temperature. 25 25 µL pL 30 normal 30 normal human human serumserum (NHS)(NHS) was added was added as a source as a source of complement of complement (20% (20% NHS NHS final final concentration) and concentration) andincubated incubated forfor 45 45 min min at 37°C. at 37°C. AfterAfter incubation, incubation, plate plate was placed was placed

on ice on ice to to stop stop the the CDC CDCreaction. reaction.Dead Dead and and viable viable cellscells werewere discriminated discriminated by adding by adding

10 µL 10 pL 10 10 µg/mL pg/mLpropidium propidium iodide(PI) iodide (PI)(0.6 (0.6µg/mL pg/mL finalconcentration) final concentration)and andFACS FACS analysis. analysis.

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2022201608 08 Mar Figure 43 Figure that IgG1-7D8 shows that 43 shows andthethebispecific IgG1-7D8and bispecific product generated byby2-2 product generated MEA-induced Fab-arm-exchange MEA-induced Fab-arm-exchange between between IgG1-7D8-F405L IgG1-7D8-F405L and and IgG1-7D8-K409R IgG1-7D8-K409R have the have the same samepotency potencytotoinduce induceCDC-mediated CDC-mediatedcell kill of cellkill of CD20-expressing Daudi CD20-expressing Daudi (Figure 43A)and (Figure 43A) andRaji Raji(Figure (Figure43B). 43B). Both Both Daudi Daudi and cells and Raji Raji cells do notdoexpress not express EGFR, EGFR,

5 resulting 5 resultingininmonovalent monovalent binding binding of the of the bispecificantibody bispecific antibody generated generated by 2-MEA by 2-MEA- induced Fab-arm-exchange induced Fab-arm-exchange between between IgG2-2F8-F405L IgG2-2F8-F405L X xIgG1-7D8-K409R IgG1-7D8-K409R ThisThis bispecific product bispecific also induced product also inducedCDCCDC mediated mediated cell kill, cell kill, albeit albeit slightly slightly lessless effecient. effecient.

These data indicate These data indicate that that CDC capacity of CDC capacity of aa parental parental antibody antibody was was retained retained in in the the bispecific format. bispecific InductionofofCDCCDC format. Induction mediated mediated cell killing cell killing bybivalent by the the bivalent bispecific bispecific

10 product 10 product (IgG1-7D8-F405L (IgG1-7D8-F405L x IgG1-7D8-K409R) X IgG1-7D8-K409R) was slightly was slightly more more efficient efficient compared compared to the to the monovalent monovalentbispecific bispecific product product (IgG2-2F8-F405L (IgG2-2F8-F405LX IgG1-7D8-K409R). x IgG1-7D8-K409R). The The CD20 targeting CD20 targeting 11B8 11B8antibody antibodyis isnotnotable ableto toinduce CDCCDC induce mediated mediated cell cell killkill andand functions as functions as aa negative negativecontrol. control.

15 Example 15 Example 42: HER2 42: HER2 HER2 bispecific X HER2x bispecific antibodies antibodies tested tested in an ininvitro an inkappa- vitro kappa directed ETA' directed ETA' killing killing assay assay The example The exampleshows shows that that HER2 HER2 x HER2 X HER2 bispecific bispecific antibodies antibodies can can deliver deliver a a cytotoxic agent cytotoxic agentinto intotumor tumor cellsafter cells afterinternalization internalizationin ina ageneric generic in in vitro vitro cell-based cell-based

killing assay killing using kappa-directed assay using kappa-directedpseudomonas-exotoxin pseudomonas-exotoxin A (anti-kappa-ETA'). A (anti-kappa-ETA'). This This 20 assay 20 assay makes makes usea of use of high affinity a affinity high anti-kappa anti-kappa domain domain antibody antibody conjugated conjugated to a to a truncated form truncated formofofthethe pseudomonas-exotoxin pseudomonas-exotoxin A. Similar A. Similar fusion proteins fusion proteins of antibody of antibody

binding proteins binding proteins (IgG-binding (IgG-bindingmotif motif from from Streptococcal Streptococcal protein protein A or protein A or protein G) and G) and diphtheria toxin diphtheria toxin or or Pseudomonas Pseudomonas exotoxin exotoxin A have A have previously previously been (Mazor been (Mazor Y. et Y. et al., J. al., J. Immunol. Methods Immunol. Methods2007; 2007; 321:41-59); 321:41-59); Kuo Kuo SR.al., SR. et et al., 20092009 Bioconjugate Bioconjugate Chem.Chem. 25 2009; 25 2009; 20:1975-1982). 20:1975-1982). These These molecules molecules in contrast in contrast to to anti-kappa-ETA'bound anti-kappa-ETA' bound thethe Fc Fc part of part of complete complete antibodies. antibodies. UponUpon internalization internalization and endocytic and endocytic sorting sorting the anti-the anti kappa-ETA' domain kappa-ETA' domainantibody antibodyundergoes undergoes proteolysis proteolysis andand disulfide-bondreduction, disulfide-bond reduction, separating the separating the catalytic catalytic from the binding from the binding domain. domain. The Thecatalytic catalytic domain domainis isthen then transported from transported fromthe the Golgi Golgi to to thethe endoplasmic endoplasmic reticulum reticulum via a via KDEL KDEL retention a retention motif, motif, 30 and and 30 subsequently subsequently translocated translocated to the to the cytosol cytosol where itwhere it inhibits inhibits protein protein synthesis synthesis and and induces apoptosis induces apoptosis(Kreitman (KreitmanRJ.RJ. et.et.al., al.,BioDrugs BioDrugs 2009; 2009; 23:1-13). 23:1-13).

The anti-HER2 The anti-HER2antibodies antibodies used used in this in this example example and and the following the following Examples Examples

43-45are 43-45 arefully fully human human monoclonal monoclonal antibodies antibodies generated generated in transgenic in transgenic mice. mice. They They bind to bind to different different epitopes on HER2. epitopes on HER2.

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Theyare They areall all IgG1,k IgG1,Kantibodies beingmodified antibodies being modified in in theirFcFcregions their regionsas as further further

disclosed. They disclosed. havethe They have thefollowing followingheavy heavy chain chain and and light light chain chain variable variable sequences. sequences.

005: 005: VH 005 VH 005 EVQLVQSGAEVKKPGESLKISCKASGYSFHFYWIGWVRQMPGKGLEWMGS EVQLVQSGAEVKKPGESLKISCKASGYSFHFYWIGWVRQMPGKGLEWMGS 2022201608 IYPGDSDTRYRPSFQGQVTISADKSISTAYLQWTSLKASDTAIYYCARQRGD IYPGDSDTRYRPSFQGQVTISADKSISTAYLQWTSLKASDTAIYYCARQRGD YYYFYGMDVWGQGTTVTVSS YYYFYGMDVWGQGTTVTVSS VL005 VL 005 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGA EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGA SSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS LTFGGGTKVEIK LTFGGGTKVEIK 5 5 025: 025: VH 025 VH 025 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE IHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYDS GVYYFDYWAQGTLVTVSS GVYYFDYWAQGTLVTVSS VL025 VL 025 DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAA DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAA SSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTR SSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTR LEIK LEIK

153: 153: VH 153 VH 153 QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYVIHWVRQAPGKGLEWVTV QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYVIHWVRQAPGKGLEWVTV ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAMYYCARGGI ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAMYYCARGGI TGTTGVFDYWGQGTLVTVSS TGTTGVFDYWGQGTLVTVSS VL153 VL 153 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYDA DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYDA SSLQSGVPSRFSGSGYGTDFSLTISSLQPEDFAIYYCQQYKSYPITFGQGTRL SSLQSGVPSRFSGSGYGTDFSLTISSLQPEDFAIYYCQQYKSYPITFGQGTRL EIK EIK

10 169: 10 169: VH 169 VH 169 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMG QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMG WLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDR WLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDR IVVRPDYFDYWGQGTLVTVSS IVVRPDYFDYWGQGTLVTVSS VL169 VL 169 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTK NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTK VEIK VEIK

HER2 Xx HER2 HER2 HER2bispecific bispecific antibodies antibodies were were pre-incubated pre-incubated with with the the anti-kappa- anti-kappa ETA' before ETA' before incubation incubationwith withA431 A431 cells.A431 cells. A431 cells cells express express -15,000 ~15,000 HER2 antibodies HER2 antibodies

per cell (determined per cell via Qifi (determined via Qifi analysis) analysis) and andare arenot notsensitive sensitivetototreatment treatment with with 'naked' 'naked'

15 HER2-antibodies. 15 HER2-antibodies. First, First, the the optimal concentrationofofanti-kappa-ETA' optimal concentration anti-kappa-ETA' was was determined determined for each for each

cell line, cell line, i.e. i.e.the the maximally tolerated dose maximally tolerated dosethat that does does not not leadlead to induction to induction of of non- non specific cell specific cell death. A431cells death. A431 cells(2500 (2500cells/well) cells/well)were were seeded seeded in normal in normal cell culture cell culture

medium medium in in 96-wells a a96-wells tissue tissue culture culture plate(Greiner plate (Greiner bio-one) bio-one) and and allowed allowed to adhere to adhere for for

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at least at least 44 hours. hours. These Thesecells cellswere wereincubated incubated withwith an anti-kappa-ETA' an anti-kappa-ETA' dilution dilution series, series,

10, 1, 100, 10, 100, 1, 0.1, 0.01, 0.001 0.1, 0.01, 0.001 and pg/mLinin normal and 00 µg/mL normalcell cell culture culture medium. After 33 medium. After days, the days, the amount amount of viable of viable cells cells was was quantified quantified with with Alamarblue Alamarblue (BioSource (BioSource International, San International, Francisco, US) San Francisco, US) according accordingtotothethe manufacturer's manufacturer's instruction. instruction. 5 Fluorescence 5 Fluorescence waswas monitored monitored using using thethe EnVision 2101 EnVision2101 Multilabel reader (PerkinElmer, Multilabel reader (PerkinElmer, Turku, Finland)with Turku, Finland) withstandard standard Alamarblue Alamarblue settings. settings. The highest The highest concentration concentration anti- anti kappa-ETA'that kappa-ETA' thatdiddidnotnot kill the kill thecells cells by byitself itself (1 (1 µg/mL pg/mLfor forA431 A431 cells) cells) waswas usedused for for following experiments. following experiments. Next, the Next, the effect effect of of HER2 HER2X xHER2 HER2 bispecific bispecific antibodies antibodies and and HER2 HER2 monospecific monospecific

10 antibodies 10 antibodies pre-incubated pre-incubated with anti-kappa-ETA' with anti-kappa-ETA' wasfor was tested tested their for theirtoability ability induceto induce cell kill. cell kill.A431 A431 cells cells were seededasasdescribed were seeded described above. above. A dilution A dilution series series of HER2 of the the HER2 specific antibodies specific antibodies (monospecific (monospecific and bispecific antibodies) and bispecific antibodies)was was made andpre- made and pre incubated for 30 incubated for 30min withthe minwith thepredetermined predetermined concentration concentration of anti-kappa-ETA' of anti-kappa-ETA' before before

adding them adding themto tothethe cells.After cells. After3 3days days at at incubation incubation 370the 37°C, C, the amount amount of viable of viable cells cells

15 was was 15 quantified quantified as described as described above.above. The Alamarblue The Alamarblue signal of signal of cells with cells treated treated with anti- anti kappa-ETA'pre-incubated kappa-ETA' pre-incubated with with the the antibodies antibodies was plotted was plotted compared compared to cellsto cells treated treated

without antibody without antibody treatment. treatment. EC EC values 5 0 values andand maximal maximal cell cell death death were were calculated calculated using GraphPad using Prism 55 software. GraphPad Prism software. Staurosporin Staurosporin (23.4 (23.4 µg/mL) pg/mL) was wasused usedasaspositive positive control for control for cell cellkilling. killing.An Anisotype isotypecontrol controlantibody antibody (IgG1/kappa; IgG1-3G8-QITL) (IgG1/kappa; IgG1-3G8-QITL) was was 20 used 20 used as as negative negative control. control. Figure 44 Figure 44 shows showsthat thatallallanti-kappa-ETA' anti-kappa-ETA'pre-incubated pre-incubated HER2HER2 bispecific bispecific antibodies were antibodies wereable ableto to killA431 kill A431 cells cells in in a dose-dependent a dose-dependent manner. manner. These These results results demonstratethat demonstrate that most most HER2 HER2 bispecific bispecific antibodies antibodies tested tested were were more more effective effective than than the monospecific the monospecificantibody antibody present present in the in the combination combination in this in this anti-kappa-ETA' anti-kappa-ETA' assay. assay.

25 In In 25 addition,thethe addition, efficacyofofbispecific efficacy bispecific antibody antibody 005X169, 005X169,025X169 025X169 and and 153X169 153X169 showedthat showed thatthethe efficacy efficacy of of a monospecific a monospecific antibody antibody which activity which lacks lacks activity in this inin this in vitro kappa-directed vitro kappa-directed ETA' killing, HER2 ETA' killing, HER2 specific specific antibody antibody 169, 169, can be increased can be increased throughbispecific through bispecific combination combinationwith withanother another HER2 HER2 specific specific antibody. antibody.

30 Example 30 Example 43:43: HER2 HER2 receptor receptor downmodulation downmodulation by incubation by incubation with bispecific with bispecific antibodies targeting antibodies targeting different different HER2 HER2epitopes epitopes HER2 X xHER2 HER2 HER2bispecific bispecific antibodies antibodies may maybind bindtwo twodifferent different epitopes epitopes on on two two spatially different spatially differentHER2 HER2 receptors. receptors.This Thismay may allow allow other other HER2 HER2 HER2 XxHER2 bispecific bispecific antibodies to antibodies to bind bind to to the theremaining remaining epitopes epitopes on these on these receptors. receptors. This This couldcould resultresult in in 35 multivalent 35 multivalent receptorcross-linking receptor cross-linking (compared (compared to to dimerization dimerization induced induced by by monovalent monovalent

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antibodies) and antibodies) enhancereceptor consequently enhance and consequently receptor downmodulation. downmodulation. To investigate To investigate whether HER2 whether HER2X HER2 x HER2 bispecific bispecific antibodiesinduce antibodies induce enhanced enhanced downmodulation downmodulation of of HER2,AU565 HER2, AU565 cells cells were were incubated incubated with with antibodies antibodies and bispecific and bispecific antibodies antibodies for three for three

days. Total days. Total levels levels of of HER2 andlevels HER2 and levelsofofantibody antibodybound bound HER2HER2 were were determined. determined.

5 5 AU565cells AU565 cellswere were seeded seeded in a in a 24-well 24-well tissue tissue cultureculture plate (100.000 plate (100.000 cells / cells/ well) in well) in normal cell culture normal cell culture medium andcultured medium and cultured for for three three days 370 C daysatat37°C in inthe the presence of 10 presence of 10 µg/mL pg/mLHER2 HER2 antibody antibody or or HER2 HER2 x HER2 X HER2 bispecific bispecific antibodies. antibodies. After After washing with washing with PBS, PBS,cells cellswere were lysed lysed by incubating by incubating themthem for 30formin30at min roomat room temperaturewith temperature with 25 25 pL Surefire µL Surefire LysisLysis buffer buffer (Perkin (Perkin Turku, Turku, Elmer,Elmer, Finland). Finland). Total Total 10 protein 10 protein levels levels werewere quantified quantified using using bicinchoninic bicinchoninic acid protein acid (BCA) (BCA) assay proteinreagent assay reagent (Pierce) following manufacturer's (Pierce) following manufacturer'sprotocol. protocol. HER2 HER2 protein protein levels levels in lysates in the the lysates were were

analyzed using analyzed using a a HER2-specific HER2-specific sandwich sandwich ELISA. ELISA.Rabbit-anti-human Rabbit-anti-humanHER2 HER2 intracellular domain intracellular domain antibody (Cell Signaling) antibody (Cell Signaling) was was used usedto tocapture capture HER2 HER2 and and biotinylated goat-anti-human biotinylated goat-anti-human HER2 polyclonal antibody HER2 polyclonal antibody R&D R&D systems, systems,Minneapolis, Minneapolis, 15 USA), 15 USA), followed followed by streptavidin-poly-HRP, by streptavidin-poly-HRP, were were usedused to detect to detect boundbound HER2. HER2. The The reaction was reaction wasvisualized visualized using using 2,2'-azino-bis 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic 3-ethylbenzothiazoline-6-sulfonic acid acid (one ABTS (one ABTStablet tablet diluted diluted in in 50 50 mL mLABTS ABTS buffer[Roche buffer [Roche Diagnostics,Almere, Diagnostics, Almere, TheThe Netherlands]) and Netherlands]) and stopped stoppedwith with oxalic oxalic acidacid (Sigma-Aldrich, (Sigma-Aldrich, Zwijndrecht, Zwijndrecht, The The Netherlands). Fluorescence Netherlands). Fluorescence at at 405 nm was 405 nm wasmeasured measured on on a microtiter a microtiter platereader plate reader 20 (BiotekInstruments, 20 (Biotek Instruments, Winooski, Winooski, USA) USA) andand the the amount amount of HER2 of HER2 was expressed was expressed as a as a percentagerelative percentage relativeto to untreated untreatedcells. cells. The results are The results are shown in Figure shown in Figure 45 45 which which demonstrates demonstratesthat thatall all the the tested tested HER2 X xHER2 HER2 HER2 bispecificantibodies bispecific antibodies induced induced 40%>40% HER2 downmodulation. HER2 downmodulation. Interestingly, all Interestingly, all HER2 HER2 HER2 X xHER2 bispecific bispecific antibodies antibodies demonstrated demonstrated increased increased HER2 HER2 25 downmodulation 25 downmodulation compared compared to both to both of their of their monospecific monospecific counterparts. counterparts.

Example 44: Example 44:Colocalization Colocalization ofof HER2 HER2 x HER2 X HER2 bispecific bispecific antibodies antibodies withwith lysosomal marker lysosomal marker LAMP1 LAMPIanalyzed analyzed bybyconfocal confocal microscopy microscopy The HER2 The HER2downmodulation downmodulation assay assay as as described described in in Example Example 43 indicated 43 indicated that that 30 HER2 30 HER2 x HER2 X HER2 bispecific bispecific antibodies antibodies were were able able to to increase increase lysosomal lysosomal degradation degradation of of HER2.ToToconfirm HER2. confirmthese these findings, findings, confocal confocal microscopy microscopy technology technology was applied. was applied. AU565 AU565 cells were cells grownglass were grown glass coverslips coverslips (thickness (thickness 1.5 micron, 1.5 micron, ThermoThermo Fisher Scientific, Fisher Scientific,

Braunschweig,Germany) Braunschweig, Germany) in standard in standard tissue tissue culture culture medium medium for 3atdays for 3 days 37°C.atCells 37C. Cells were pre-incubated were pre-incubated for for 1 hour 1 hour with with (Sigma)(Sigma) leupeptin leupeptin to blocktolysosomal block lysosomal activity activity 35 after 35 afterwhich which 10 ug/mL 10 ug/mL HER2 HER2 monospecific monospecific antibodies antibodies or HER2orX HER2 x HER2 bispecific HER2 bispecific

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2022201608 08 Mar antibodies were antibodies added. wereadded. TheThe cells cells werewere incubated incubated for anfor an additional additional or 18 athours 3 hours 3 or 18 at 37cC. Hereafter 37°C. Hereafter they they were were washed washedwith withPBSPBS andand incubated incubated for for 30 min. 30 min. at room at room temperature with temperature with 4% formaldehyde 4%formaldehyde (Klinipath). Slides (Klinipath). Slides were werewashed washed withblocking with blocking buffer (PBS buffer supplemented with (PBS supplemented with 0.1% 0.1%saponin saponin [Roche]

[Roche] andand 2% BSA BSA [Roche]) 2% [Roche]) and and 5 incubated 5 incubatedfor for 20 20min with blocking minwith blocking buffer buffer containing containing2020mM NH 4 CI to mM NHCI to quench quench formaldehyde. Slides formaldehyde. Slides were washed again were washed againwith with blocking blocking buffer buffer and incubated for and incubated for 45 45 at room min at min temperature with room temperature with mouse-anti-human mouse-anti-humanCD107a CD107a (LAMP1) (LAMP1) (BD(BD Pharmingen) Pharmingen) to stain to stain lysosomes. lysosomes.Following Followingwashing washing withwith blocking blocking buffer buffer the slides the slides werewere incubated incubated

30 min 30 min atat room roomtemperature temperature with with a cocktail a cocktail of of secondary secondary antibodies; antibodies; goat-anti goat-anti- 10 mouse 10 mouse IgG-Cy5 IgG-Cy5 (Jackson) (Jackson) and goat-anti-human and goat-anti-human IgG-FITC IgG-FITC (Jackson). (Jackson). Slides Slides were were washedagain washed again with with blocking blocking buffer buffer andand mounted mounted overnight overnight on microscope on microscope slides slides using using 20 µL 20 pL mounting mounting medium medium (6(6 gram gramGlycerol [Sigma] and Glycerol [Sigma] and 2.4 2.4 gram gram Mowiol 4-88 Mowiol 4-88

[Omnilabo]was

[Omnilabo] was dissolved dissolved in 6in mL mL distilled 6 distilled water water to which to which 12 mL 12 mLTris 0.2M 0.2M Tris

[Sigma] [Sigma] pH8.5 was was added addedfollowed followedbybyincubation incubation for for 10 10 min 0 min atat 50-60°C. 50-60 C. Mounting Mountingmedium medium pH8.5 15 was was 15 aliquoted aliquoted and stored and stored at -20C.). at -20°C.). SlidesSlides were imaged were imaged a Leicaconfocal with SPE-II with a Leica SPE-II confocal microscope (Leica microscope (Leica Microsystems) Microsystems)equipped equipped with with a 1.32-0.6 a 63x 63x 1.32-0.6 oil immersion oil immersion objective lens objective lens and andLAS-AF LAS-AF software. software. To allow To allow for quantification for quantification of overlapping of overlapping pixel pixel intensities, saturation intensities, saturation of of pixels pixels should be avoided. should be avoided. Therefore Thereforethethe FITC FITC laser laser intensity intensity

was decreased was decreased to to 10%, 10%,smart smartgain gainwas wassetsetatat830 830V Vand andsmart smart offsetwas offset wasset setatat 20 9.48 20 9.48 By using %. using %. By thesethese settings, settings, the the bispecificantibodies bispecific antibodieswere were clearlyvisualized clearly visualized without pixel without pixel saturation, saturation, but butthe themonospecific monospecific antibodies antibodies werewere sometimes sometimes difficult difficult to to detect. To detect. compare lysosomal To compare lysosomalcolocalization colocalization between betweenmonospecific and monospecificand bispecific bispecific antibodies, these antibodies, these settings settings were werekept keptthe thesame same for for allall analyzed analyzed confocal confocal slides. slides.

12-bit images 12-bit were analyzed images were analyzedfor for colocalisation colocalisation using MetaMorph* software using MetaMorph® software 25 (version 25 (versionMeta Meta Series Series 6.1, 6.1, MolecularDevices Molecular Devices Sunnyvale Inc,Sunnyvale Inc, California, USA). California, USA).FITC FITC and Cy5 and Cy5 images imageswere wereimported importedasasstacks stacksand andbackground background was was subtracted.Identical subtracted. Identical thresholds settings thresholds settings were wereused used (manually (manually set)set) for for all all FITC FITC images images and Cy5 and all all Cy5 images. images.

Colocalisation was Colocalisation wasdepicted depicted as as thethe pixel pixel intensity intensity of FITC of FITC in region in the the region of overlap of overlap

(ROI), were (ROI), werethe theROIROI is composed is composed of allofCy5 all positive Cy5 positive regions. regions. To compare To compare different different

30 slides 30 slides stained stained withwith several several HER2HER2 antibodies antibodies or XHER2 or HER2 x HER2 bispecific HER2 bispecific antibodies, antibodies, the the imageswere images werenormalized normalized using using the pixel the pixel intensity intensity of Cy5. of Cy5. Goat-anti-mouse Goat-anti-mouse IgG-Cy5 IgG-Cy5 was used was used toto stain stain the the lysosomal lysosomal marker markerLAMP1 LAMP1 (CD107a). (CD107a). The pixel The pixel intensity intensity of of LAMP1 should LAMP1 shouldnot notdiffer differ between betweenvarious variousHER2 HER2 antibodies antibodies or the or the HER2HER2 x X HER2 HER2 bispecific antibodies bispecific antibodies tested tested (one cell had (one cell pixel intensity a pixel had a intensity of of Cy5 Cy5 of of roughly 200.000). roughly 200.000).

35

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Normalizedvalues Normalized forcolocalization valuesfor FITCand colocalizationofofFITC andCy5Cy5= =

[(TPI-FITC Xx percentage

[(TPI-FITC percentageFITC-Cy5 FITC-Cy5 colocalization)/100] colocalization)/100] x [200.000/TPI-Cy5] X [200.000/TPI-Cy5]

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In this In this formula, formula,TPITPI stands stands for Total for Total Pixel Pixel Intensity. Intensity.

5 5 presents percentage presents percentageof of viable viable cells, cells, as as measured measured byFITC by the the pixel FITC intensity pixel intensity overlapping with overlapping with Cy5 for various Cy5 for various monospecific HER2 antibodies monospecific HER2 antibodies and and HER2 HER2X xHER2 HER2 bispecific antibodies. bispecific antibodies.For For each antibody or each antibody or bispecific bispecific molecule molecule depicted, depicted, three three different images different were analyzed images were analyzedfrom fromoneone slide slide containing~ 1, containing ~ 31,or3 >5or cells. >5 cells. 10 Significant 10 Significantvariation variation was was observed observedbetween betweenthethe differentimages different imageswithin withineach eachslide. slide. However, it was However, it was evident evident that that all all HER2 HER2X xHER2 HER2 bispecificantibodies bispecific antibodies demonstrate demonstrate increased colocalisation with increased colocalisation with the the lysosomal lysosomalmarker marker LAMP1, LAMP1, when when compared compared with their with their

monospecific counterparts. monospecific counterparts. These These results results indicate indicate that internalized, that once once internalized, HER2 X HER2 x

HER2bispecific HER2 bispecificantibodies antibodiesareare efficientlysorted efficiently sorted towards towards lysosomal lysosomal compartments, compartments,

15 making 15 making them them suitable suitable for a for a bispecific bispecific antibody antibody drug conjugate drug conjugate approach. approach.

Example 45:Inhibition Example 45: Inhibition ofof proliferationofofAU565 proliferation AU565 cells cells upon upon incubation incubation with with HER2 monospecific HER2 monospecific or or HER2 HER2 X HER2 X HER2 bispecific bispecific antibodies antibodies HER2bispecific HER2 bispecificantibodies antibodieswere were tested tested for for their their abilityto to ability inhibitproliferation inhibit proliferation 20 of of 20 AU565 AU565 cells cells in vitro.DueDue in vitro. to to thethe highhigh HER2HER2 expression expression levels levels on AU565 on AU565 cells cells (-1.000.000copies (~1.000.000 copies perper cellasasdetermined cell determined withwith Qifi-kit), Qifi-kit), HER2 HER2 is constitutively is constitutively active active

in these in cells and these cells thus not and thus not dependent dependenton on ligand-induced ligand-induced heterodimerization. heterodimerization. In a In a 96- 96 wells tissue wells tissue culture culture plate plate (Greiner (Greinerbio-one, bio-one,Frickenhausen, Frickenhausen, Germany), Germany), 9.000 9.000 AU565 AU565 cells were cells were seeded per well seeded per well in in the the presence presence of of 10 10 pg/mL HER2 antibody µg/mL HER2 antibody ororHER2 HER2X x 25 25 HER2HER2 bispecific bispecific antibodies antibodies in serum-free in serum-free cell culture cell culture medium. medium. As a control, As a control, cells were cells were

seededininserum-free seeded serum-free medium medium without without antibody antibody or bispecific or bispecific antibodies. antibodies. After After three three days, the days, the amount amount of viable of viable cells cells was was quantified quantified with with Alamarblue Alamarblue (BioSource (BioSource International, San International, Francisco, US) San Francisco, US) according according toto the themanufacturer's manufacturer'sinstructions. instructions. Fluorescencewas Fluorescence was monitored monitored using using the EnVision the EnVision 2101 Multilabel 2101 Multilabel reader reader (PerkinElmer, (PerkinElmer,

30 Turku, 30 Turku, Finland) Finland) withwith standard standard Alamarblue Alamarblue settings. settings. The Alamarblue The Alamarblue signal signal of of antibody-treatedcells antibody-treated cells was wasplotted plottedasasa apercentage percentage relative relative to to untreated untreated cells. cells.

Figure 47 Figure 47 depicts depicts the thefluorescent fluorescentintensity intensityofofAlamarblue Alamarblue of AU565 of AU565 cells cells afterafter

incubation with incubation with HER2 antibodies and HER2 antibodies HER2XHER2 and HER2XHER2 bispecificantibodies. bispecific antibodies. Herceptin® Herceptin® (trastuzumab) was (trastuzumab) wasincluded includedas aspositive positivecontrol controlandand demonstrated demonstrated inhibition inhibition of of 35 proliferation 35 proliferation as described as described by Juntilla by Juntilla TT.al., TT. et et al., Cancer Cancer Cell 2009; Cell 2009; 15: 429-440. 15: 429-440. All All

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HER2X xHER2 HER2 HER2 bispecific bispecific antibodies antibodies werewere able able to inhibit to inhibit proliferation proliferation of AU565 of AU565 cells. cells. Bispecific antibodies: Bispecific antibodies:IgG1-005-ITL IgG1-005-ITLx X IgG1-169-K409R andIgG1-025-ITL IgG1-169-K409R and IgG1-025-ITL x IgG1 X IgG1- 005-K409R 005-K409R wereweremore more effectivecompared effective compared to their to their monospecific monospecific antibody antibody counterpartsinin this counterparts this assay. assay. 5 5 Example 46:InInvitro Example 46: vitroand andininvivo analysis vivoanalysis ofof FcRn FcRn binding binding by bispecific by bispecific IgGI IgG1 antibodies and antibodies andhinge-deleted hinge-deleted IgG1IgGI bispecific bispecific antibodies antibodies containing containing one orone or two FcRn two FcRnbinding binding sitesininthe sites theFcFcregion. region. The present example The present exampleillustrates illustrates the the generation generation ofof asymmetrical asymmetricalbispecific bispecific 10 molecules, 10 molecules, i.e. i.e. molecules molecules with with different different characteristics characteristics in each in each Fab-arm Fab-arm according according to to the invention. the invention. The neonatalFcFcreceptor The neonatal receptor (FcRn) (FcRn) is responsible is responsible for for the the longlong plasma plasma half-life half-life of of

IgG by IgG byprotecting protectingIgGIgG from from degradation. degradation. AfterAfter internalization internalization of antibody, of the the antibody, FcRn FcRn binds to binds to antibody antibodyFc Fc regions regions in endosomes, in endosomes, where where the interaction the interaction is stableis in stable the in the 15 mildly 15 mildly acidicenvironment acidic environment(pH (pH6.0). 6.0). Upon Uponrecycling recycling to to the the plasma plasma membrane, membrane,where where the environment the environment isis neutral neutral (pH (pH 7.4), 7.4), the theinteraction interaction is is lost lost and the antibody and the is antibody is released backinto released back intothethe circulation.TheThe circulation. Fc region Fc region of an of an antibody antibody containscontains 2 FcRn 2 FcRn

binding sites, binding sites, one in each one in each heavy heavychain chainat at thethe CH2-CH3 CH2-CH3 interfaces. interfaces. An H435A An H435A mutationinin the mutation the FcFcregion regionofofthe theantibody antibodyabrogates abrogates binding binding to FcRn to FcRn (Shields, (Shields, R.L.,R.L., et et 20 al, al, 20 J BiolChem, J Biol Chem, 2001, 2001, Firan, Firan, M.,al, M., et et Int al, Int Immunol, Immunol, 2001) 2001) andthealso and also theregion hinge hinge region is thought is to influence thought to influence FcRn FcRn binding binding (Kim, (Kim,J.K., J.K.,etetal., al., Mol MolImmunol., Immunol., 1995). 1995). Furthermore,a arole Furthermore, rolefor forbivalent bivalentover overmonovalent monovalent antibody antibody binding binding to has to FcRn FcRn has been been suggestedininefficient suggested efficient recycling recycling (Kim, (Kim, J.K., J.K., et et al., al.,Scand Scand JJ Immunol., 1994). Immunol., 1994).

In this In this example example the theinfluence influenceofofFcRn FcRn binding binding valency valency is evaluated is evaluated by by 25 asymmetric 25 asymmetric bispecific bispecific IgG1 IgG1 molecules, molecules, containing containing a single a single FcRn FcRn binding binding site.TheThe site. additional contribution additional contribution ofofthe thehinge hinge region region is evaluated is evaluated by asymmetric by asymmetric bispecificbispecific

hinge-deletedIgG1 hinge-deleted IgG1(Uni-G1) (Uni-G1) molecules. molecules.

FcRn binding FcRn binding of of bispecific bispecific IgG1 IgG1 or hinge-deleted IgG1 or hinge-deleted IgG1 (Uni-G1) (Uni-G1) molecules molecules containing no, containing no, 11 or or 22 FcRn FcRn binding binding sites siteswas wasmeasured measured by humanand by human andmouse mouse FcRn FcRn 30 ELISA. 30 ELISA.Antibodies Antibodies IgG1-2F8-ITL, IgG1-2F8-ITL, IgG1-7D8-K409R IgG1-7D8-K409R and andIgG1-7D8-K409R-H435A IgG1-7D8-K409R-H435A monospecificmolecules monospecific molecules were were produced produced as described as described in example in example 2, 3, 42, 3, 5. and 4 and 5. Hinge- Hinge deleted IgG1 deleted molecules Uni-G1-2F8-ITL, IgG1 molecules Uni-G1-2F8-ITL, Uni-G1-7D8-K409R and Uni-G1-7D8- Uni-G1-7D8-K409R and Uni-G1-7D8 K409R-H435Amonospecific K409R-H435A monospecificmolecules molecules were were produced produced as described as described in example in example 11. 11. Bispecific IgG1 Bispecific IgG1 molecules were generated molecules were generatedbyby2-MEA 2-MEA induced induced Fab-arm Fab-arm exchange exchange 35 between 35 between IgG1-2F8-ITL IgG1-2F8-ITL and and IgG1-7D8-K409R IgG1-7D8-K409R or IgG1-7D8-K409R-H435A or IgG1-7D8-K409R-H435A molecules. molecules.

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2022201608 08 Mar Bispecific hinge-deleted Bispecific IgG1molecules hinge-deleted IgG1 molecules were were produced produced by incubation by incubation of Uni-G1-2F8 of Uni-G1-2F8-

ITL with ITL with Uni-G1-7D8-K409R or Uni-G1-7D8-K409R-H435A. Uni-G1-7D8-K409R or Uni-G1-7D8-K409R-H435A. A 3-fold A 3-fold dilutionseries dilution series of of monospecificand monospecific andbispecific bispecificIgG1 IgG1 molecules molecules and hinge-deleted and hinge-deleted IgG1 molecules IgG1 molecules were were addedtotobiotinylated added biotinylatedhuman- human- or mouse-FcRn or mouse-FcRn captured captured on a streptavidin-coated on a streptavidin-coated elisa elisa 5 plate 5 plate followed followed by incubation by incubation at pHat6.0 pH and 6.0 7.4 andfor 7.41 for 1 hour. hour. Bound Bound antibody antibody and and hinge- hinge deleted IgG1 deleted IgG1molecules molecules werewere visualized visualized using using horseradishperoxidase-labeled horseradishperoxidase-labeled goat- goat anti-human (Fab') anti-human (Fab') 2as as conjugate conjugate and and ABTS ABTSasassubstrate. substrate. Results Results were were measured measuredasas optical density optical density at at aa wavelength wavelength ofof405 405nmnm using using the the EL808-Elisa-reader. EL808-Elisa-reader.

Figure 48 Figure 48 shows showsthe thebinding binding results results of of monovalent monovalent or bivalent or bivalent IgG1IgG1 antibodies antibodies

10 andand 10 hinge-deleted hinge-deleted IgG1 IgG1 molecules molecules to to human human FcRnFcRn (A) (A) andand mouse mouse FcRnFcRn (B) pH (B) at at 6.0 pH 6.0 and pHpH7.4. and 7.4.As As expected, expected, all antibodies all antibodies tested, tested, both (bispecific) both (bispecific) IgG1 IgG1 and and hinge- hinge deleted IgG1 deleted IgG1molecules, molecules, do do not not bindbind efficiently efficiently to to FcRn FcRn (both (both humanhuman and at and mouse) mouse) at pH 7.4. pH 7.4. At At slightly slightly acidic acidic condition condition (pH 6.0) monospecific (pH 6.0) monospecificIgG1-2F8-ITL IgG-2F8-ITLand and bispecific bispecific

IgG1 generated IgG1 generatedfrom fromIgG1-2F8-ITL IgG-2F8-ITLand and IgG-7D8-K409R IgG1-7D8-K409R show bivalent show bivalent binding binding 15 efficiencies 15 efficiencies to to FcRn, FcRn, albeit albeit for formouse mouse FcRn FcRn 33 fold fold higher highercompared compared to to human, which human, which mimicsthe mimics thepositive positivecontrol control(IgG1-2F8) (IgG1-2F8) for for FcRnFcRn binding. binding. This This indicates indicates thatITL that the the ITL mutationand mutation andthe theK409R K409R do not do not disturb disturb binding binding to FcRn. to FcRn.

clear effect A clear A effect of of2 2 vs vs 11 vs vs 00 FcRn interaction sites FcRn interaction sitescan canbebeseen seen when the when the binding of binding of the the IgG1 molecules to IgG1 molecules to human humanand andmouse mouse FcRnFcRn is compared is compared at pHat6.0 pH 6.0 20 (Figure 20 (FigureXXAXXA andand B, left B, , pH6, panel). left panel). IgG1-2F8-ITL, IgG1-2F8-ITL, IgG1-7D8-K409R IgG1-7D8-K409R and IgG1-2F8 and IgG1-2F8- ITL/IgG1-7D8-K409R(2(2FcRn ITL/IgG1-7D8-K409R FcRn bindingsites) binding sites) bind bind comparable comparabletotothe thecontrol control (IgG1- (IgG1 2F8). The 2F8). molecules with The molecules with 00 FcRn FcRnbinding bindingsites, sites, IgG1-7D8-K409R-H435A IgG1-7D8-K409R-H435Ashowshow no no binding at binding all. The at all. The molecules with 11 FcRn molecules with FcRn binding binding site, site, IgG1-2F8-ITL/IgG1-7D8- IgG1-2F8-ITL/IgG1-7D8 K409R-H435A,show K409R-H435A, show intermediatebinding intermediate bindingwhen when compared compared to the to the molecules molecules withwith 2 2 25 FcRn 25 FcRn binding binding sites. sites. Figure 48(A), Figure 48(A), pHpH6.0, 6.0,right rightpanel panelshows shows the the binding binding to human to human FcRn FcRn of of hinge- hinge deleted IgG1 deleted IgG1molecules molecules (Uni-G1). (Uni-G1). All hinge-deleted All hinge-deleted molecules molecules are impaired are impaired in their in their interaction to interaction to human FcRn human FcRn when when compared compared to theto the control control IgG1 molecules IgG1 molecules (IgG1-2F8) (IgG1-2F8)

indicating that indicating that the the hinge hingeis isindeed indeed of of influence influence in the in the interaction interaction with with FcRn FcRn when when 30 evaluated 30 evaluated in aninFcRn an binding FcRn binding ELISA. ELISA. No clearNo clearofeffect effect ofvs2 0vsFcRn 2 vs 1 1 vsinteraction 0 FcRn interaction sites can sites can be be seen seen when the binding when the to human binding to humanFcRn FcRnatatpH6.0 pH6.0isiscompared comparedof of these these hinge-deletedmolecules. hinge-deleted molecules. However,since However, sincethethebinding binding of of human human IgG IgG to to mouse mouse FcRn is FcRn is stronger, stronger, a clear a clear effect of effect vs 11 vsvs0 0FcRn 2 vs of 2 FcRn sites interactionsites interaction cancan be seen be seen when when the binding the binding of theseof these 35 hinge-deleted 35 hinge-deleted IgGIgG molecules molecules to to mouse mouse FcRn FcRn at at pH pH 6.06.0 is iscompared compared (Figure48(B), (Figure 48(B),pHpH

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6.0, 6.0, right rightpanel). panel).The Thebinding of of binding Uni-G1-7D8-K409R-H435A/Uni-G1-2F8-ITL (1 FcRn Uni-G1-7D8-K409R-H435A/Uni-G1-2F8-ITI (1 FcRn binding site) binding site) is is intermediate when intermediate when compared compared to binding to the the binding of Uni-G1-2F8-ITL, of Uni-G1-2F8-ITL, Uni- Uni G1-7D8-409Randand G1-7D8-409R Uni-G1-2F8-ITL/Uni-G1-7D8-K409R Uni-G1-2F8-ITL/Uni-G1-7D8-K409R (2 binding (2 FcRn FcRn binding sites) sites) and and Uni-G1-2F8-ITL-H435A Uni-G1-2F8-ITL-H435A (0 FcRn (0 FcRn binding binding sites, sites, no binding). no binding).

5 5 Example Example 47:47:Her2 Her2 x CD3 X CD3 bispecific bispecific antibodies antibodies tested tested in an inin an in vitro vitro cytotoxicity assay cytotoxicity assay CD3isis aa co-receptor CD3 co-receptorinin the theTTcell cell receptor receptor complex complexexpressed expressed on mature on mature T T cells. Combination cells. ofaa CD3 Combination of CD3specific specificantibody antibodyFab-arm Fab-arm withwith a tumor a tumor antigen antigen specific specific

10 antibody 10 antibody Fab-arm Fab-arm in a bispecific in a bispecific antibody antibody would would result result in theinspecific the specific targeting targeting of T of T cells to cells to tumor cells, leading tumor cells, leading to to TT cell cellmediated tumorcell mediated tumor cell lysis. lysis. Likewise, Likewise, CD3 positive CD3 positive

T cells could T cells couldbebe targeted targeted to other to other derailed derailed cells cells in in thetobody, the body, to cells infected infected or cells or directly to directly to pathogens. pathogens.

Her2 Xx CD3 Her2 CD3bispecific bispecificantibodies antibodieswere were generated. generated. Heavy Heavy and light and light chainchain

15 variable 15 variable region region sequences sequences forHer2 for the the specific Her2 specific Fab-arm Fab-arm were aswere as indicated indicated for for antibody 153 antibody 153and and169169 in in Example Example 42. following 42. The The following heavyheavy and chain and light light chain variable variable

region sequences region sequencesforforthe theCD3 CD3 specific specific Fab-arm Fab-arm werewere used:used:

YTH12.5 YTH12.5 (Sequence (Sequence as described as described by Routledge by Routledge et al.,etEur al.,J Eur J Immunol. Immunol. 1991, 1991, 20 21(11):2717-25.) 20 21(11):2717-25.) VH VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTI EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTI YTH12.5 YTH12.5 STSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQY SGGFDYWGQGTLVTVSS SGGFDYWGQGTLVTVSS VL VL DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDD DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDD YTH12.5 YTH12.5 DKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGG DKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGG TKLTVL TKLTVL

huCLB-T3/4 huCLB-T3/4 (Sequence (Sequence as as described described by by Parrenetetal., Parren al., Res Immunol. 1991, Immunol. 1991, 142(9):749-63.Minor 142(9):749-63. Minor amino amino acidacid substitutions substitutions werewere introduced introduced to the to make make the sequenceresemble sequence resemble thethe closest closest human human germline.) germline.)

VH VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAPGKGLEWVAT EVQLVESGGGLVKPGGSLRLSCAASGFTESSYGMFWVRQAPGKGLEWVAT huCLB- huCLB- ISRYSRYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRPLY T3/4 T3/4 GSSPDYWGQGTLVTVSS GSSPDYWGQGTLVTVSS VL VL EIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQAPRLLIYDTSK EIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQAPRLLIYDTSK huCLB- huCLB- LASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGSGTKLEM LASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGSGTKLEM T3/4 T3/4 R R 25 25 All antibodies All antibodies were expressedasasIgG1,k were expressed IgG1,K being being modified modified in their in their Fc regions Fc regions as as described as described as follows: follows:IgGl-Her2-153-K409R IgG1-Her2-153-K409R and IgG1-Her2-153-N297Q-K409R, and IgG1-Her2-153-N297Q-K409R,

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IgGl-Her2-169-K409R, IgGl-hu-CLB-T3/4-F405L IgG1-Her2-169-K409R, IgG1-hu-CLB-T3/4-F405L andand IgG-hu-CLB-T3/4-N297Q IgG1-hu-CLB-T3/4-N297Q- F405L, IgG1-YTH12.5-F405L F405L, IgGl-YTH12.5-F405Land andIgG1-YTH12.5-N297Q-F405L. IgG1-YTH12.5-N297Q-F405L. Bispecific antibodies Bispecific antibodiesfrom from these Her2 and these Her2 andCD3CD3 specific specific antibodies antibodies were were generatedasasdescribed generated described in Example in Example 11 and11tested and tested in an ininvitro an incytotoxicity vitro cytotoxicity assay assay 5 using 5 using AU565 AU565 cells. cells. AU565cells AU565 cellswere werecultured culturedtotonear near confluency. confluency. Cells Cells were were washed washed twice twice with with PBS, and PBS, andtrypsinized trypsinizedfor for55minutes minutesatat37°C. 370C. 12 12 mL culture mL culture medium medium was to was added added to inactivate trypsin inactivate trypsin and cells were and cells spundown were spun downforfor 800800 5 min, 5 min, rpm.rpm. Cells Cells werewere

resuspendedin in1010mL mL resuspended culture culture medium medium and aand a single single cell suspension cell suspension wasbymade was made by 10 passing 10 passing the the cells cells through through a cellstrainer. a cellstrainer. 100 100 pLa of µL of a 5x10 5x10 5 cells/mL cells/mL suspension suspension was was addedtotoeach added eachwell wellofofa a96-well 96-wellculture cultureplate, plate,and andcells cellswere wereincubated incubated at at least least 3 hrs 3 hrs

at 370C, at C02 5%CO2 37°C, 5% to to allow allow adherence adherence to plate. to the the plate. Peripheral blood Peripheral blood mononuclear mononuclear cells cells (PBMC) (PBMC) werewere isolated isolated from from bloodblood from from healthy volunteersusing healthy volunteers usingLeucosep Leucosep 30 30 mL tubes, mL tubes, according according to thetomanufacturer's the manufacturer's 15 protocol 15 protocol (Greiner (Greiner Bio-one). Bio-one). T cells T cells werewere isolated isolated from from PBMC preparations PBMC preparations by negative by negative

selection using selection using the the Untouched UntouchedHuman Human T-cells T-cells Dynabead Dynabead kit (Dynal). kit (Dynal). Isolated Isolated cells cells were resuspended were resuspended in culture in culture medium medium to a to a final final concentration concentration 7x10 6 cells/mL. op cells/mL. op 7x10

Culture medium Culture wasremoved medium was removedfrom fromthe theadhered adheredAU565 AU565 cells,and cells, and replaced replaced with 50 with 50 µl/well 2x concentrated pl/well 2x concentratedantibody-dilution antibody-dilution andand 50 50 pl/well µl/well 7x10 7x10 6 T cells/mL T cells/mL

20 (ratio 20 (ratio effector:target effector:target = 7:1). = 7:1). Plates Plates werewere incubated incubated for 3for 3 days days at 37 at 37°C, 0 C, 5% CO.5% CO 2 Supernatantswere Supernatants were removed removed and plates and plates were washed were washed twice twice with with PBS. To PBS. To each each well . well 150 µL 150 pLculture culture medium mediumandand 15 Alamar 15 µL pL Alamar blueadded. blue was was added. Platesincubated Plates were were incubated for for 4 hours 0 4 hours at at 37 C, 5% 37°C, C02 ,and 5% CO, andabsorbance absorbancewas wasmeasured measured (Envision,Perkin (Envision, Perkin Elmer). Elmer). Figure 49 Figure 49 shows showsthat thatwhereas whereas control control antibodies antibodies (Her2 (Her2 monospecific monospecific IgG1- IgG1

25 Herceptin, 25 Herceptin, CD3CD3 monospecific monospecific IgG1-YTH12.5 IgG1-YTH12.5 andand monospecific monospecific IgG1-huCLB-T3/4, IgG1-huCLB-T3/4, irrelevant antigen irrelevant monospecificIgG1-b12, antigen monospecific IgG1-b12, andand CD3 CD3 b12 bispecific X b12x bispecific antibodies) antibodies) did did not induce not induce TT cell cell mediated cytotoxicity,bispecific mediated cytotoxicity, bispecific (Duo) (Duo) Her2 Her2X xCD3 CD3 antibodies antibodies

huCLB/Her2-153,huCLB/Her2-169, huCLB/Her2-153, huCLB/Her2-169,YTH12.5/Her2-153 YTH12.5/Her2-153 andand YTH12.5/Her2-169 YTH12.5/Her2-169 induced dose induced dosedependent dependent T cell T cell mediated mediated cytotoxicity cytotoxicity of AU565 of AU565 cells. cells. Bispecific Bispecific

30 antibodies 30 antibodiescontaining containingHer2-169 Her2-169were weremore more potentthan potent thanthose thosecontaining containing Her2-153. Her2-153. Mutants of Mutants of IgG1-hu-CLB-T3/4, IgG1-YTH12.5and IgG1-hu-CLB-T3/4, IgG1-YTH12.5 andHer2-153 Her2-153were weremade made containing aa N297Q containing N297Q mutation mutation to remove to remove a glycosylation a glycosylation site; site; glycosylation glycosylation at this at this site site

is critical is criticalforfor IgG-Fcgamma receptorinteractions IgG-Fcgamma receptor interactions(Bolt (BoltS Setetal., al., Eur EurJJImmunol 1993, Immunol 1993,

23:403-411).Figure 23:403-411). Figure 49 49 shows shows thatthat N297Q N297Q mutation mutation and therefore and therefore absence absence of Fc of Fc 35 glycosylation 35 glycosylation of Her2 of Her2 CD3 bispecific X CD3x bispecific antibodies antibodies YTH12.5/Her2-153 YTH12.5/Her2-153 and and

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huCLB/Her2-153 huCLB/Her2-153 diddid notnot impact impact the the potential potential to to induce induce dose dose dependent dependent T cell T cell

mediated cytotoxicity of mediated cytotoxicity of AU565 AU565cells. cells. It It is is to to be understood be understood that, that, if any if any prior prior art art publication publication is referred is referred to herein, to herein,

such referencedoes such reference doesnot notconstitute constitutean anadmission admission that that the the publicationforms publication forms a part a part ofof

5 5 thethe common common general general knowledge knowledge in theinart, in the art, in Australia Australia or anyorother any other country. country. 2022201608

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CLAIMS CLAIMS

1. 1. An in An in vitro vitro method methodfor forgenerating generatinga a bispecific antibody, bispecific antibody, said said method method comprising thefollowing comprising the followingsteps: steps: 5 5 a) providingaafirst a) providing first monospecific monospecificantibody antibody comprising comprising an immunoglobulin an immunoglobulin

Fc region, said Fc region, said Fc Fc region regioncomprising comprising a firstCH3 a first CH3 region, region, wherein wherein said said firstfirst 2022201608

monospecific antibody comprises monospecific antibody comprises aaPhe Pheatatposition position405 405and and an an ArgArg at at position position409 409 (EU (EU numbering) and aa Cys-Pro-Pro-Cys numbering) and Cys-Pro-Pro-Cys sequence sequencein in the the core core 10 10 hinge region, hinge region,

b) providing a b) providing a second second monospecific monospecific antibody antibody comprising comprising anan immunoglobulin immunoglobulin Fc Fc region, region, said said Fc Fc region region comprising comprising a second a second CH3 region, CH3 region,

wherein said wherein said second second monospecific monospecific antibody antibodycomprises comprisesa aLeu Leu atat position position 15 15 405 anda aLys 405 and Lysatatposition position 409 409(EU (EU numbering) numbering) and and a Cys-Pro-Pro-Cys a Cys-Pro-Pro-Cys sequence sequence ininthe thecore corehinge hinge region, region,

c) incubatingsaid c) incubating saidfirst first monospecific monospecific antibody antibody together together with with said second said second

monospecific antibody monospecific antibody under under reducing reducing conditions conditions sufficient sufficient to the to allow allow the 20 20 cysteines in the cysteines in theCys-Pro-Pro-Cys Cys-Pro-Pro-Cys sequences sequences of theofcore the hinge core regions hinge regions to to undergo disulfide-bond undergo disulfide-bond isomerization, isomerization, andand

d) d) obtaining said bispecific obtaining said bispecific antibody; antibody;

wherein thesequences wherein the sequences of said of said first first and and second second CH3 CH3 regions regions are different are different and are and are

25 25 suchsuch thatthat the the heterodimeric heterodimeric interaction interaction between between said and said first firstsecond and second CH3 regions CH3 regions

is is stronger stronger than eachof than each of the thehomodimeric homodimeric interactions interactions of of said said firstand first and second second CH3 CH3

regions, andwherein regions, and whereinsaid saidbispecific bispecificantibody antibody has has binding binding specificity specificity fora afirst for first and and second epitope, second epitope,

30 wherein 30 wherein said said firstfirst and and second second epitope epitope are located are located on a cell on a tumor tumorandcell and wherein wherein the the bispecific bispecific antibody hasbinding antibody has binding specificityforfora target specificity a target selected selected fromfrom the group the group

consisting consisting of: of:erbB1 erbB1(EGFR), (EGFR), erbB2 erbB2 (HER2), (HER2), erbB3, erbB3, erbB4, erbB4, MUC-1, CD19,CD20, MUC-1, CD19, CD20, CD4, CD38,CD138, CD4, CD38, CD138, CXCR5, CXCR5, c-Met, c-Met, HERV-envelop HERV-envelop protein, protein, periostin, periostin, Bigh3,Bigh3, SPARC, BCR,CD79, SPARC, BCR, CD79, CD37, CD37, EGFrvIII, EGFrvIII, L1-CAM, L1-CAM, AXL, AXL, Tissue Tissue Factor Factor (TF),(TF), CD74, CD74, 35 EpCAM 35 EpCAM andand MRP3; MRP3; oror

whereinsaid wherein saidfirst first and andsecond second epitope epitope are located are located on a cell on a tumor tumor andcell and wherein wherein said bispecific antibody said bispecific hasbinding antibody has binding specificityfor specificity fora atarget targetcombination combination selected selected

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from the from the group group consisting consisting of: of: erbB1 + erbB2, erbB1 + erbB2, erbB2 erbB2+ +erbB3, erbB3,erbB1 erbB1 + erbB3, + erbB3, CD19 CD19 ++ CD20, CD20, CD38 CD38++CD34, CD34,CD4 CD4+ +CXCR5, CXCR5, CD38 CD38 + RANKL, + RANKL, CD38 CD38 + CXCR4, + CXCR4, CD20 CD20 ++ CXCR4, CXCR4, CD20 CD20++CCR7, CCR7,CD20 CD20+ + CXCR5, CXCR5, CD20 CD20 + RANKL, + RANKL, erbB2 erbB2 + AXL, + AXL, erbB1 + cMet, erbB1 + cMet, erbB2 + c-Met, erbB2 + c-Met, erbB2 + EpCAM, erbB2 + EpCAM,c-Met c-Met++AXL, AXL,c-Met c-Met++TF, TF,CD38 CD38+ + 5 CD20, 5 CD20, CD38 CD38 + + CD138; CD138; oror

whereinsaid wherein saidfirst firstororsecond second epitope epitope is located is located on a on a cell tumor tumorandcell the and otherthe other 2022201608

epitope is located epitope is located on onananeffector effectorcell, cell, wherein whereinthe thebispecific bispecificantibody antibodyhashas binding binding

specificity specificityfor fora atarget targetselected selected from from the groupconsisting the group consistingof: of:FcgammaRI FcgammaRI (CD64), (CD64),

10 FcgammaRIII 10 FcgammaRIII (CD16),CD3, (CD16), CD3,CD89, CD89,CD32a, CD32a, FcRI; FcεRI; or or

wherein saidbispecific wherein said bispecificantibody antibodyhashas binding binding specificity specificity for for a target a target combination combination

selected selected from: from: CD3 + HER2, CD3 + HER2,CD3 CD3+ + CD20, CD20, IL-12 IL-12 + IL18, + IL18, IL-1a IL-1a + + IL-1b,VEGF IL-1b, VEGF + + EGFR, EGFR, EpCAM EpCAM ++ CD3, CD3, GD2 GD2+ +CD3, CD3,GD3 GD3+ + CD3, CD3, HER2 HER2 + CD64, + CD64, EGFR EGFR + CD64, + CD64, 15 CD30 15 CD30 + CD16, + CD16, NG2 NG2 + CD28, + CD28, HER2 HER2 + HER3, + HER3, CD20 CD20 + + CD28, CD28, HER2 HER2 + + CD16, CD16, Bcl2++ Bcl2 CD3, CD19++CD3, CD3, CD19 CD3,CEA CEA + CD3, + CD3, EGFR EGFR + CD3, + CD3, IgE IgE + CD3, + CD3, EphA2EphA2 + CD33 + CD3, CD3, +CD33 + CD3, MCSP++CD3, CD3, MCSP CD3,PSMA PSMA + CD3, + CD3, TF TF + CD3, + CD3, CD19 CD19 + CD16, + CD16, CD19 CD19 + CD16a, + CD16a, CD30 CD30 + CD16a, CEA + CD16a, CEA ++ HSG, HSG, CD20 CD20+ +HSG, HSG,MUC1 MUC1 + HSG, + HSG, CD20 CD20 + CD22, + CD22, HLA-DR HLA-DR + + CD79, CD79, PDGFR PDGFR ++ VEGF, VEGF, IL17a IL17a ++ IL23, IL23, CD32b CD32b ++ CD25, CD25, CD20 CD20+ +CD38, CD38,HER2 HER2+ + 20 AXL, 20 AXL, CD89 CD89 + HLA + HLA classII, class II, CD38+CD138, CD38+CD138,TFTF+ +cMet, cMet,Her2 Her2++EpCAM, EpCAM,HER2 HER2+ + HER2, EGFR+ +EGFR, HER2, EGFR EGFR, EGFR EGFR + c-Met, + c-Met, c-Met c-Met + non-binding + non-binding armarm and and combinations combinations of of G-protein coupledreceptors. G-protein coupled receptors.

2. 2. The in The in vitro vitro method according method according to to claim claim 1, 1, wherein wherein the the Fc region Fc region of the of the first first

25 monospecific 25 monospecific antibody antibody is of is anofisotype an isotype selected selected from from the the consisting group group consisting of IgG1,of IgG1,

IgG2, IgG3 and IgG2, IgG3 andIgG4 IgG4 andand wherein wherein the the Fc region Fc region of the of the second second monospecific monospecific antibody is of antibody is of an an isotype isotypeselected selectedfrom from thethe group group consisting consisting of IgG1, of IgG1, IgG2,IgG2, IgG3 IgG3

and IgG4. and IgG4.

3. 3. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

30 30 the the Fc regions Fc regions of both of both said said firstfirst and and saidsaid second second monospecific monospecific antibody antibody are of the are of the

IgG1 isotype. IgG1 isotype.

4. 4. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

the heterodimeric the heterodimericinteraction interactionininthe theresulting resultingbispecific bispecific antibody antibodyis is

i) such that i) such that no no Fab-arm exchange can Fab-arm exchange can occur occur at at 0.5 0.5 mM GSH; mM GSH;

35 35 ii) ii) such that no such that no Fab-arm Fab-arm exchange exchange occurs occurs in vivo in vivo in mice; in mice; and/or and/or

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iii) iii)more more than than two times stronger, two times stronger, such such as as more more than than three three times times stronger, e.g. more stronger, e.g. morethan than five five times times stronger stronger thanthan the strongest the strongest of of the two homodimeric the two homodimeric interactions. interactions.

5. 5. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

5 5 thethe sequences sequences of said of said firstfirst and and second second CH3 regions CH3 regions arethat are such such that

i) the dissociation i) the dissociation constants of the constants of the heterodimers heterodimersininthe theresulting resulting 2022201608

bispecific bispecific antibody antibody is is below 0.05 micromolar; below 0.05 micromolar; and/or and/or

ii) ii) the dissociation constants the dissociation constantsof of both both homodimeric homodimeric interactions interactions are are above 0.01micromolar, above 0.01 micromolar, such such as above as above 0.05 0.05 micromolar, micromolar, preferably preferably

10 10 between 0.01and between 0.01 and1010micromolar, micromolar, such such as as between between 0.050.05 and and 10 10 micromolar, micromolar, more preferably between more preferably 0.01 and between 0.01 and 5, 5, such such as as between between 0.05 0.05 and and 55 micromolar, micromolar, even even more morepreferably preferably between between0.01 0.01and and1 1 micromolar, micromolar, such such as as between between 0.05 0.05 and and 1 1 micromolar, micromolar, between 0.01 between 0.01 and 0.5or and 0.5 or between between 0.01 0.01 andand 0.1 0.1 micromolar. micromolar.

15 15 6. 6. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

said said first firsthomodimeric homodimeric monospecific monospecific antibody antibody has has no no more than one more than one amino aminoacid acid substitution substitution in in the CH3region, the CH3 region,and and thethe second second homodimeric homodimeric monospecific monospecific antibody hasnonomore antibody has more than than one one amino amino acid substitution acid substitution in theinCH3 theregion CH3 region relative relative

to the to the wild-type CH3regions. wild-type CH3 regions.

20 7. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein 20 7. said first and said first secondCH3 and second CH3 regions, regions, except except for specified for the the specified mutations, mutations, comprise comprise

the sequence the sequenceset setforth forthininSEQ SEQIDID NO:1. NO:1.

8. 8. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

said said first firstand and second homodimeric second homodimeric monospecific monospecific antibody, antibody, except except forspecified for any any specified 25 mutations, 25 mutations, areare human human antibodies. antibodies.

9. 9. The in The in vitro vitro method according method according to to anyany oneone of the of the preceding preceding claims, claims, wherein wherein

both said first both said first and said second and said secondhomodimeric homodimeric monospecific monospecific antibody antibody further further comprise comprise a alight light chain. chain.

10. 10. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

30 said 30 said firstand/or first and/orsaid saidsecond second homodimeric homodimeric monospecific monospecific antibody antibody comprises comprises a a mutation removing mutation removing thethe acceptor acceptor sitesite forfor Asn-linked Asn-linked glycosylation. glycosylation.

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11. 11. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

said first and said first and second homodimeric second homodimeric monospecific monospecific antibody antibody provided provided in stepina)step and a) and

b) arepurified. b) are purified.

12. 12. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

5 said 5 said first and/or first and/orsecond secondhomodimeric homodimeric monospecific monospecific antibody antibody is is conjugated conjugated to to a a drug, drug, aa prodrug prodrugorora atoxin toxinororcontains containsananacceptor acceptor group group for for thethe same. same. 2022201608

13. 13. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

the first the first antibody binds to antibody binds toan anepitope epitopeon on a tumor a tumor cell, cell, and and the other the other antibody antibody is is an irrelevant or an irrelevant or inactive inactive antibody antibodywithout withoutany any relevant relevant in in vivo vivo binding binding activity activity forfor

10 10 thethe applicationintended. application intended.

14. 14. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

the reducing the reducingconditions conditionsininstep stepc)c)comprise comprisethethe addition addition ofreducing of a a reducing agent, agent, e.g. e.g.

a reducingagent a reducing agent selected selected from from the the groupgroup consisting consisting of: 2-mercaptoethylamine, of: 2-mercaptoethylamine,

dithiothreitol dithiothreitoland and tris(2-carboxyethyl)phosphine tris(2-carboxyethyl)phosphine oror chemical chemical derivatives derivatives thereof. thereof.

15 15 15.15. The inThe in vitro vitro method method according according to any to any one one of the of the preceding preceding claims, claims, wherein wherein step c) is step c) is performed performed under under reducing reducing conditions conditions with awith a potential redox redox potential between between

-150 -150 and -600 mV, and -600 mV, such such as as between between -250 -250and and-400 -400mV. mV.

16. 16. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

step c) comprises step c) incubation comprises incubation foratatleast for least9090min min at at a temperature a temperature of least of at at least 20°C20°C

20 20 in in the the presence presence ofofatatleast least 25 25 mM mM2-mercaptoethylamine 2-mercaptoethylamineor or ininthe thepresence presenceofofat at least least 0.5 0.5 mM dithiothreitol. mM dithiothreitol.

17. 17. The The in vitro in vitro method method according according to anyto any one of one the of the preceding preceding claims, claims, wherein wherein

step d) comprises step d) comprisesremoval removalof of a reducing a reducing agent, agent, e.g.e.g. by desalting. by desalting.

25 25 18.18. A method A method for the for the selection selection of a bispecific of a bispecific antibody antibody having having a desired a desired property, said method property, said method comprising comprising the the steps steps of: of:

a) providinga afirst a) providing first set set ofofhomodimeric homodimeric antibodies antibodies comprising comprising antibodies antibodies

with different variable with different variable regions anda aCys-Pro-Pro-Cys regions and Cys-Pro-Pro-Cys sequence sequence in core in the the core hinge region,wherein hinge region, wherein said said antibodies antibodies of said of said first first set set comprise comprise identical identical

30 30 first CH3 first CH3 regions, whereinsaid regions, wherein saidCH3 CH3 regions regions comprise comprise a Phe a Phe at position at position 405 405 and an Arg and an Arghave haveanan amino amino acid acid substitution atat position substitution position 409 409 (EU (EU numbering), numbering),

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b) b) providing providing aa second secondset setofofhomodimeric homodimeric antibodies antibodies comprising comprising antibodies antibodies

with different variable with different regions or variable regions or identical identical variable variable regions regionsand anda a Cys-Pro- Cys-Pro-

Pro-Cys sequence Pro-Cys sequence in in the the core core hinge hinge region, region, wherein wherein said said antibodies antibodies of said of said

second set comprise second set compriseidentical identical second secondCH3 CH3 regions, regions, wherein wherein saidsaid CH3 CH3 5 5 regions regions comprise comprise aa Leu Leu at at position position 405 405 and anda aLys Lysatatposition position409 409(EU (EU numbering), numbering), 2022201608

c) c) incubating combinations incubating combinations ofof antibodies antibodies of of saidfirst said first set set and andofofsaid saidsecond second set set under reducingconditions under reducing conditions sufficienttotoallow sufficient allowthe thecysteines cysteines in in the the hinge hinge

10 10 region to undergo region to undergo disulfide-bond disulfide-bond isomerization, isomerization, thus thus generating generating a set of a set of

bispecific antibodies, bispecific antibodies,

d) d) optionally optionally restoring restoring the conditions to the conditions to non-reducing, non-reducing,

15 15 e) assayingthe e) assaying theresulting resulting setset of of bispecific bispecific antibodies antibodies for for a given a given desired desired

property, and property, and

f) selecting f) selecting aa bispecific bispecificantibody antibody having the desired having the desired property; property; whereinthe wherein thesequences sequences of said of said first first and and second second CH3 CH3 regions regions are different are different and and are are 20 20 suchsuch thatthat the the heterodimeric heterodimeric interaction interaction between between said and said first firstsecond and second CH3 regions CH3 regions

is is stronger stronger than eachof than each of the thehomodimeric homodimeric interactions interactions of of said said firstand first and second second CH3CH3

regions, andwherein regions, and whereinsaid saidbispecific bispecificantibody antibody has has binding binding specificity specificity fora afirst for first and and second epitope, second epitope,

25 wherein 25 wherein said said firstfirst and and second second epitope epitope are located are located on a cell on a tumor tumorandcell and wherein wherein the the bispecific bispecific antibody hasbinding antibody has binding specificityforfora target specificity a target selected selected fromfrom the group the group

consisting consisting of: of:erbB1 erbB1(EGFR), (EGFR), erbB2 erbB2 (HER2), (HER2), erbB3, erbB3, erbB4, erbB4, MUC-1, CD19,CD20, MUC-1, CD19, CD20, CD4, CD38,CD138, CD4, CD38, CD138, CXCR5, CXCR5, c-Met, c-Met, HERV-envelop HERV-envelop protein, protein, periostin, periostin, Bigh3,Bigh3, SPARC, BCR,CD79, SPARC, BCR, CD79, CD37, CD37, EGFrvIII, EGFrvIII, L1-CAM, L1-CAM, AXL, AXL, Tissue Tissue Factor Factor (TF),(TF), CD74, CD74, 30 EpCAM 30 EpCAM andand MRP3; MRP3; oror

whereinsaid wherein saidfirst first and andsecond second epitope epitope are located are located on a cell on a tumor tumor andcell and wherein wherein said bispecific antibody said bispecific hasbinding antibody has binding specificityfor specificity fora atarget targetcombination combination selected selected

from the from the group group consisting consisting of: of: erbB1 erbB1 + erbB2, erbB2 + erbB2, erbB2+ +erbB3, erbB3,erbB1 erbB1 + erbB3, + erbB3, 35 CD19 35 CD19 + CD20, + CD20, CD38 CD38 + CD34, + CD34, CD4 CD4 + CXCR5, + CXCR5, CD38CD38 + RANKL, + RANKL, CD38 CD38 + CXCR4, + CXCR4, CD20 CD20 ++ CXCR4, CXCR4, CD20 CD20++CCR7, CCR7,CD20 CD20+ + CXCR5, CXCR5, CD20 CD20 + RANKL, + RANKL, erbB2 erbB2 + AXL, + AXL, erbB1 + cMet, erbB1 + cMet, erbB2 erbB2 ++ c-Met, c-Met, erbB2 + EpCAM, erbB2 + EpCAM,c-Met c-Met++AXL, AXL,c-Met c-Met++TF, TF,CD38 CD38+ + CD20, CD38+ +CD138; CD20, CD38 CD138;oror

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whereinsaid wherein saidfirst firstororsecond second epitope epitope is located is located on a on a cell tumor tumorandcell the and otherthe other epitope is located epitope is located on onananeffector effectorcell, cell, wherein whereinthe the bispecificantibody bispecific antibodyhashas binding binding

specificity specificityfor fora atarget targetselected selected from from the groupconsisting the group consistingof: of:FcgammaRI FcgammaRI (CD64), (CD64),

5 FcgammaRIII 5 FcgammaRIII (CD16),CD3, (CD16), CD3,CD89, CD89,CD32a, CD32a, FcRI; FcεRI; or or

whereinsaid wherein saidbispecific bispecificantibody antibodyhashas binding binding specificity specificity for for a target a target combination combination 2022201608

selected selected from: from: CD3 + HER2, CD3 + HER2,CD3 CD3+ + CD20, CD20, IL-12 IL-12 + IL18, + IL18, IL-1a IL-1a + + IL-1b,VEGF IL-1b, VEGF + + EGFR, EGFR, EpCAM EpCAM ++ CD3, CD3, GD2 GD2+ +CD3, CD3,GD3 GD3+ + CD3, CD3, HER2 HER2 + CD64, + CD64, EGFR EGFR + CD64, + CD64, 10 CD30 10 CD30 + CD16, + CD16, NG2 NG2 + CD28, + CD28, HER2 HER2 + HER3, + HER3, CD20 CD20 + + CD28, CD28, HER2 HER2 + + CD16, CD16, Bcl2++ Bcl2 CD3, CD19++CD3, CD3, CD19 CD3,CEA CEA + CD3, + CD3, EGFR EGFR + CD3, + CD3, IgE IgE + CD3, + CD3, EphA2EphA2 + CD33 + CD3, CD3, +CD33 + CD3, MCSP++CD3, CD3, MCSP CD3,PSMA PSMA + CD3, + CD3, TF TF + CD3, + CD3, CD19 CD19 + CD16, + CD16, CD19 CD19 + CD16a, + CD16a, CD30 CD30 + CD16a, CEA + CD16a, CEA ++ HSG, HSG, CD20 CD20+ +HSG, HSG,MUC1 MUC1 + HSG, + HSG, CD20 CD20 + CD22, + CD22, HLA-DR HLA-DR + + CD79, CD79, PDGFR PDGFR ++ VEGF, VEGF, IL17a IL17a ++ IL23, IL23, CD32b CD32b ++ CD25, CD25, CD20 CD20+ +CD38, CD38,HER2 HER2+ + 15 AXL,CD89 15 AXL, CD89 + HLA + HLA classII, class II, CD38+CD138, CD38+CD138,TFTF+ +cMet, cMet,Her2 Her2++EpCAM, EpCAM,HER2 HER2+ + HER2, EGFR+ +EGFR, HER2, EGFR EGFR, EGFR EGFR + c-Met, + c-Met, c-Met c-Met + non-binding + non-binding armarm and and combinations combinations of of G-protein coupledreceptors. G-protein coupled receptors.

19. A method 19. A method for for producing producing a bispecificantibody, a bispecific antibody, said said method methodcomprising comprisingthe the 20 followingsteps: 20 following steps:

a) providinga afirst a) providing firstnucleic-acid nucleic-acid construct construct encoding encoding a polypeptide a first first polypeptide comprising comprising a afirst first full-length full-length heavy chainofofananantibody heavy chain antibody comprising comprising a first a first

Fc region, said Fc region, said first first Fc region comprising Fc region comprisinga first a firstCH3 CH3 region region comprising comprising a a Phe at position Phe at position 405 405and andanan Arg Arg at at position position 409 409 (EU(EU numbering) numbering) and further and further

25 25 comprising comprising a aCys-Pro-Pro-Cys Cys-Pro-Pro-Cys sequence sequence in core in the the core hingehinge region, region,

b) providing aa second b) providing second nucleic-acid nucleic-acid construct construct encoding encoding aa second second polypeptide polypeptide comprising comprising a second full-length a second full-length heavy heavy chain chain of of an antibody an antibody comprising comprising a asecond secondFc Fc region, region, said said second second Fc region Fc region comprising comprising a second a second

30 30 CH3 regioncomprising CH3 region comprising a Leu a Leu at position at position 405405 and and a Lysa at Lysposition at position 409 (EU 409 (EU

numbering) andfurther numbering) and furthercomprising comprisinga aCys-Pro-Pro-Cys Cys-Pro-Pro-Cys sequence sequence in the in the core hinge region, core hinge region,

c) co-expressingsaid c) co-expressing said firstand first and second second nucleic-acid nucleic-acid constructs constructs in a host in a host

35 35 cell, cell, and and

d) d) obtaining said bispecific obtaining said bispecific antibody fromthe antibody from thecell cell culture; culture;

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whereinthe wherein thesequences sequences of said of said first first and and second second CH3 CH3 regions regions are different are different and and are are such that the such that the heterodimeric heterodimericinteraction interactionbetween between saidsaid first first and and second second CH3 CH3 regions regions

is is stronger stronger than eachof than each of the thehomodimeric homodimeric interactions interactions of of said said firstand first and second second CH3 CH3

regions, andwherein regions, and whereinsaid saidbispecific bispecificantibody antibody has has binding binding specificity specificity fora afirst for first and and 5 secondepitope, 5 second epitope,

whereinsaid wherein saidfirst first and secondepitope and second epitope are are located located on on a tumor a tumor cell cell and and wherein wherein the the 2022201608

bispecific bispecific antibody hasbinding antibody has binding specificityfor specificity fora target a target selected selected fromfrom the group the group

consisting consisting of: of:erbB1 erbB1(EGFR), (EGFR), erbB2 erbB2 (HER2), (HER2), erbB3, erbB3, erbB4, erbB4, MUC-1, CD19,CD20, MUC-1, CD19, CD20, 10 CD4, 10 CD4, CD38, CD38, CD138, CD138, CXCR5, CXCR5, c-Met, c-Met, HERV-envelop HERV-envelop protein, protein, periostin, Bigh3, periostin, Bigh3, SPARC, BCR,CD79, SPARC, BCR, CD79, CD37, CD37, EGFrvIII, EGFrvIII, L1-CAM, L1-CAM, AXL, AXL, Tissue Tissue Factor Factor (TF),(TF), CD74, CD74, EpCAM andMRP3; EpCAM and MRP3;oror

wherein saidfirst wherein said first and andsecond second epitope epitope are located are located on a cell on a tumor tumor andcell and wherein wherein

15 15 saidsaid bispecific bispecific antibody antibody has binding has binding specificity specificity for afor a target target combination combination selected selected

from the from the group group consisting consisting of: of: erbB1 erbB1 + erbB2, erbB2 + erbB2, erbB2+ +erbB3, erbB3,erbB1 erbB1 + erbB3, + erbB3, CD19 CD19 ++ CD20, CD20, CD38 CD38++CD34, CD34,CD4 CD4+ +CXCR5, CXCR5, CD38 CD38 + RANKL, + RANKL, CD38 CD38 + CXCR4, + CXCR4, CD20 CD20 ++ CXCR4, CXCR4, CD20 CD20++CCR7, CCR7,CD20 CD20+ + CXCR5, CXCR5, CD20 CD20 + RANKL, + RANKL, erbB2 erbB2 + AXL, + AXL, erbB1 + cMet, erbB1 + cMet, erbB2 erbB2 ++ c-Met, c-Met, erbB2 + EpCAM, erbB2 + EpCAM,c-Met c-Met++AXL, AXL,c-Met c-Met++TF, TF,CD38 CD38+ + 20 CD20, 20 CD20, CD38 CD38 + + CD138; CD138; oror

whereinsaid wherein saidfirst firstororsecond second epitope epitope is located is located on a on a cell tumor tumorandcell the and otherthe other epitope is located epitope is located on onananeffector effectorcell, cell, wherein whereinthe thebispecific bispecificantibody antibodyhashas binding binding

specificity specificityfor fora atarget targetselected selected from from the groupconsisting the group consistingof: of:FcgammaRI FcgammaRI (CD64), (CD64),

25 FcgammaRIII 25 FcgammaRIII (CD16),CD3, (CD16), CD3,CD89, CD89,CD32a, CD32a, FcRI; FcεRI; or or

whereinsaid wherein saidbispecific bispecificantibody antibodyhashas binding binding specificity specificity for for a target a target combination combination

selected selected from: from: CD3 + HER2, CD3 + HER2,CD3 CD3+ + CD20, CD20, IL-12 IL-12 + IL18, + IL18, IL-1a IL-1a + + IL-1b,VEGF IL-1b, VEGF + + EGFR, EGFR, EpCAM EpCAM ++ CD3, CD3, GD2 GD2+ +CD3, CD3,GD3 GD3+ + CD3, CD3, HER2 HER2 + CD64, + CD64, EGFR EGFR + CD64, + CD64, 30 CD30 30 CD30 + CD16, + CD16, NG2 NG2 + CD28, + CD28, HER2 HER2 + HER3, + HER3, CD20 CD20 + CD28, + CD28, HER2 HER2 + + CD16, CD16, Bcl2++ Bcl2 CD3, CD19++CD3, CD3, CD19 CD3,CEA CEA + CD3, + CD3, EGFR EGFR + CD3, + CD3, IgE IgE + CD3, + CD3, EphA2EphA2 + CD33 + CD3, CD3, +CD33 + CD3, MCSP++CD3, CD3, MCSP CD3,PSMA PSMA + CD3, + CD3, TF TF + CD3, + CD3, CD19 CD19 + CD16, + CD16, CD19 CD19 + CD16a, + CD16a, CD30 CD30 + CD16a, CEA + CD16a, CEA ++ HSG, HSG, CD20 CD20+ +HSG, HSG,MUC1 MUC1 + HSG, + HSG, CD20 CD20 + CD22, + CD22, HLA-DR HLA-DR + + CD79, CD79, PDGFR PDGFR ++ VEGF, VEGF, IL17a IL17a ++ IL23, IL23, CD32b CD32b ++ CD25, CD25, CD20 CD20+ +CD38, CD38,HER2 HER2+ + 35 AXL, 35 AXL, CD89 CD89 + HLA + HLA classII, class II, CD38+CD138, CD38+CD138,TFTF+ +cMet, cMet,Her2 Her2++EpCAM, EpCAM,HER2 HER2+ + HER2, EGFR+ +EGFR, HER2, EGFR EGFR, EGFR EGFR + c-Met, + c-Met, c-Met c-Met + non-binding + non-binding armarm and and combinations combinations of of G-protein coupledreceptors. G-protein coupled receptors.

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20. The 20. The method method according according to to claim claim 19,19, furthercomprising further comprising the the features features according to any according to anyone oneorormore more of of claims claims 2 to 2 to 13.13.

21. 21. An An expression expression vector vector comprising comprising thethe nucleic-acidconstructs nucleic-acid constructsaccording accordingto to any oneofofclaims any one claims1919toto20. 20.

5 5 22.22. A cell A host host cell comprising comprising the the nucleic-acid nucleic-acid constructs constructs accordingtotoany according anyone oneofof claims 19 to claims 19 to 20. 20. 2022201608

23. A bispecificantibody 23. A bispecific antibodyobtained obtained by by the the method methodofofany anyone oneofofthe thepreceding preceding claims claims 11 to to 17, 17, 19 19 or or 20. 20.

24. A bispecific 24. A bispecific antibody antibody comprising comprising a first a first polypeptide polypeptide comprising comprising a first afull- first full- 10 length 10 length antibody antibody heavy heavy chain chain having having binding binding specificity specificity fora a for first epitope first epitope and and comprising comprising a afirst first Fc region, said Fc region, said first first Fc Fcregion region comprising comprising aa first first CH3 region, and CH3 region, and a a second polypeptide second polypeptide comprising comprising a second a second full-length full-length antibody antibody heavyheavy chain chain havinghaving

binding specificity for binding specificity for a a second epitopeand second epitope and comprising comprising a second a second Fc region, Fc region, said said

second Fcregion second Fc regioncomprising comprising a second a second CH3 CH3 region, region, wherein wherein the sequences the sequences of said of said

15 firstand 15 first andsecond second CH3 CH3 regions regions areare differentand different andare aresuch such thatthe that theheterodimeric heterodimeric interaction interaction between saidfirst between said first and andsecond second CH3 CH3 regions regions is stronger is stronger thanthan eacheach of the of the

homodimeric interactions homodimeric interactions of of said said first and first andsecond secondCH3CH3 regions, regions, and and

whereinsaid wherein saidfirst first CH3 CH3region region comprises comprises a Phe a Phe at position at position 405anand 405 and Argan Arg at at position position 409 409 (EU (EU numbering) anda aCys-Pro-Pro-Cys numbering) and Cys-Pro-Pro-Cyssequence sequence in in thethe 20 20 core hinge region core hinge region

and and

wherein saidsecond wherein said second CH3 CH3 region region comprises comprises a Leuaat Leu at position position 405 405 and a and Lys a Lys

at at position position 409 409 (EU (EU numbering) anda aCys-Pro-Pro-Cys numbering) and Cys-Pro-Pro-Cyssequence sequence in in thethe core hingeregion core hinge regionwherein wherein said said first first andand second second epitope epitope are located are located on a on a

25 25 tumorcell tumor cell and andwherein whereinthethe bispecific bispecific antibody antibody hashas binding binding specificity specificity forfor a a target selected target selected from fromthe thegroup group consisting consisting of:of: erbB1 erbB1 (EGFR), (EGFR), erbB2 erbB2 (HER2), (HER2),

erbB3, erbB4, MUC-1, erbB3, erbB4, MUC-1,CD19, CD19, CD20, CD20, CD4, CD4, CD38, CD38, CD138, CD138, CXCR5,CXCR5, c-Met, c-Met, HERV-envelop protein, periostin, HERV-envelop protein, periostin,Bigh3, SPARC, Bigh3, SPARC,BCR, BCR, CD79, CD79, CD37, CD37, EGFrvIII, L1-CAM, EGFrvIII, L1-CAM, AXL, AXL, Tissue Tissue Factor Factor (TF), (TF), CD74, CD74, EpCAM EpCAM and or and MRP3; MRP3; or

30 30 wherein said first wherein said first and and second epitope are second epitope are located located on on aa tumor tumorcell cell and and wherein said bispecific wherein said bispecific antibody antibody has hasbinding bindingspecificity specificity for for aatarget target combination selected combination selected from from thethe group group consisting consisting of: erbB1 of: erbB1 + erbB2, + erbB2, erbB2 erbB2

+ erbB3, erbB1 + erbB3, erbB1 ++ erbB3, erbB3,CD19 CD19 ++ CD20, CD20, CD38 CD38 + CD34, CD4 + CD34, CD4 ++ CXCR5, CXCR5,

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CD38 CD38 ++ RANKL, RANKL, CD38 CD38 ++CXCR4, CXCR4,CD20 CD20+ +CXCR4, CXCR4,CD20 CD20+ + CCR7,CD20 CCR7, CD20+ + CXCR5, CD20 ++RANKL, CXCR5, CD20 RANKL,erbB2 erbB2+ +AXL, AXL,erbB1 erbB1+ + cMet,erbB2 cMet, erbB2+ + c-Met, c-Met, erbB2 + EpCAM, erbB2 + EpCAM,c-Met c-Met++AXL, AXL,c-Met c-Met++TF, TF,CD38 CD38+ + CD20, CD20, CD38 CD38 + CD138; + CD138; or or

5 5 whereinsaid wherein saidfirst first ororsecond second epitope epitope is located is located on a on a tumor tumor cell cell and theand the other epitopeis other epitope is located located on onananeffector effectorcell, cell, wherein whereinthe thebispecific bispecificantibody antibody has bindingspecificity specificity for for aa target target selected selectedfrom fromthethe group consisting of: of: 2022201608

has binding group consisting

FcgammaRI (CD64), FcgammaRI (CD64), FcgammaRIII FcgammaRIII (CD16), (CD16), CD3, CD3, CD89, CD89, CD32a, CD32a, FcεRI; FcRI; or or

wherein said wherein said bispecific bispecific antibody antibody has hasbinding bindingspecificity specificity for for aatarget target 10 10 combination selected from: combination selected from: CD3 CD3 ++ HER2, HER2,CD3 CD3+ + CD20, CD20, IL-12 IL-12 + IL18, + IL18, IL-IL- 1a 1a + IL-1b, VEGF + IL-1b, + EGFR, VEGF + EGFR,EpCAM EpCAM+ + CD3, CD3, GD2GD2 + CD3, + CD3, GD3 GD3 + CD3, + CD3, HER2 HER2 + + CD64, CD64, EGFR EGFR + + CD64, CD64, CD30 CD30 + + CD16, CD16, NG2 NG2 + + CD28, CD28, HER2 HER2 + + HER3, HER3, CD20 CD20 + CD28, HER2 + CD28, HER2 ++ CD16, CD16, Bcl2 Bcl2 + + CD3, CD3, CD19 + CD3, CD19 + CD3, CEA CEA ++ CD3, CD3, EGFR EGFR ++ CD3, IgE + CD3, IgE + CD3, CD3, EphA2 EphA2+ +CD3, CD3, CD33 CD33 + CD3, + CD3, MCSP MCSP + CD3, + CD3, PSMA PSMA + CD3,+ CD3, 15 15 TF + TF CD3, CD19 + CD3, CD19 ++ CD16, CD16, CD19 CD19++CD16a, CD16a,CD30 CD30+ +CD16a, CD16a,CEA CEA + + HSG, HSG, CD20 CD20 ++ HSG, HSG, MUC1 MUC1+ + HSG, HSG, CD20 CD20 + CD22, + CD22, HLA-DR HLA-DR + CD79, + CD79, PDGFR PDGFR + + VEGF, IL17a VEGF, IL17a ++ IL23, IL23, CD32b CD32b++CD25, CD25, CD20 CD20 + CD38, + CD38, HER2 HER2 + AXL, + AXL, CD89CD89 + + HLA class II, HLA class II,CD38+CD138, CD38+CD138, TF TF++ cMet, cMet,Her2 Her2++EpCAM, EpCAM, HER2 HER2 + HER2, + HER2, EGFR EGFR ++EGFR, EGFR,EGFR EGFR + c-Met, + c-Met, c-Met c-Met + + non-binding non-binding armarm andand combinations combinations 20 20 of of G-protein coupledreceptors. G-protein coupled receptors.

25. A bispecific 25. A bispecific antibody antibody according according to claim to claim 23 or23 orwherein 24, 24, wherein

i) thefirst i) the first and andsecond second fulllength full length antibody antibody heavy heavy chainchain comprise comprise FC FC regions as described regions as describedininclaim claim22oror3; 3;

ii) ii) the the heterodimeric interaction in heterodimeric interaction the bispecific in the bispecific antibody is as antibody is as 25 25 described in claim described in claim 44 or or 5; 5;

iii) iii)the thefirst and first andsecond second full fulllength length antibody heavychain antibody heavy chaincomprise comprise CH3CH3

regions as described regions as describedforfor respectively respectively the the first first and and second second homodimeric monospecific homodimeric monospecific antibody antibody as described as described in claim in claim 6 or 6 7;or 7;

iv) iv) the bispecific antibody, the bispecific antibody, apart apartfrom from anyany specified specified mutations, mutations, is a is a

30 30 human antibody; human antibody;

v) the v) thefirst first and secondpolypeptide and second polypeptideareare antibodies antibodies further further comprising comprising a a light light chain; chain;

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vi) vi) the the first firstand/or and/or second full length second full length antibody heavychain antibody heavy chaincomprise(s) comprise(s) a a mutation removing mutation removing thethe acceptor acceptor sitesite forfor Asn-linked Asn-linked glycosylation; glycosylation;

vii) vii)the the first firstand secondpolypeptide and second polypeptide are are antibodies antibodies that been that have have been purified priortotodimerization; purified prior dimerization;

5 5 viii) viii)the the first first polypeptide and/orsecond polypeptide and/or second polypeptide polypeptide are antibodies are antibodies

conjugated conjugated totoa adrug, drug,a a prodrug prodrug or aortoxin a toxin or contains or contains an acceptor an acceptor 2022201608

group for the group for the same; same;and/or and/or

ix) ix) the first polypeptide the first polypeptide isis ananantibody antibody binding binding to antoepitope an epitope on a on a tumorcell tumor cell and andthe the second second polypeptide polypeptide is anisirrelevant an irrelevant or inactive or inactive

10 10 antibody without any antibody without anyrelevant relevantininvivo vivobinding bindingactivity activityfor forthe the application application intended. intended.

26. A bispecific 26. A bispecific antibody antibody according according to one to any anyof one of claims claims 23 to 23 25 to 25used when whenas used as

a a medicament. medicament.

27. 27. Use Use of bispecific of the the bispecific antibody antibody according according to any to oneany of one of 23 claims claims to 25 23 in to 25 in

15 15 thethe manufacture manufacture of of a medicament a medicament forfor treatingofof cancer. treating cancer.

28. A method 28. A method of treating of treating of cancer of cancer comprising comprising administering administering the bispecific the bispecific antibody accordingtotoany antibody according any one one of of claims claims 23 23 to to 25 25 to an to an individual individual in in need need thereof. thereof.

29. A pharmaceutical 29. A pharmaceutical composition composition comprising comprising a bispecific a bispecific antibody antibody according according to any to oneof any one of claims claims23 23toto2525and and a pharmaceutically-acceptable a pharmaceutically-acceptable carrier. carrier.

20 20 30.30. A method A method of inhibiting of inhibiting growthgrowth and/or and/or proliferation proliferation and/or and/or for killing for killing of of tumorcells tumor cellscomprising comprising administration administration of aof a bispecific bispecific antibody antibody according according to any to any one of claims one of claims 23 23to to25 25totoananindividual individualinin need needthereof. thereof.

31. 31. Use Use of bispecific of the the bispecific antibody antibody according according to any to any one of one of 23 claims claims to 25 23 in to 25 in

the manufacture the manufacture of of a a medicament medicament for inhibiting for inhibiting growth growth and/or and/or proliferation proliferation and/or and/or

25 25 for for killing killing ofoftumor tumor cells. cells.

21743767_1(GHMatters) 21743767_1 (GHMatters)P36631AU05 P36631AU05

08 Mar 2022

Figure 1

5 RhlgG4-2F8 X HulgG4-7D8 OD 405 nm 4 HulgG4-2F8 X RhlgG4-7D8 2022201608

2022201608

HulgG4-2F8 X HulgG4-7D8 3 RhlgG4-2F8 X RhlgG4-7D8 2

1

0

0.1 1 10 100 1000 10000 Concentration (ng/mL)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388

2/64 08 Mar 2022 2022201608 08 Mar 2022

Figure 2

Species Isotype Amino-acid position*

(common name) Core-hinge CH3-CH3 interface 226 227 228 229 230 347 349 350 366 368 370 392 394 395 397 398 399 405 407 409 351 2022201608

Homo sapiens IgG1 CPPCP Q Y T L T L K K T P V L. D F Y K IgG2 M IgG3 R N M IgG4 S R

Macaca mulatta IgG1 (Rhesus Monkey) IgG2 I IgG3 T I IgG4 (In)* L T I IgG4 (Ch) A T L

*EU numbering; **In, Indian; Ch, Chinese

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 3

5 2022201608 08

IgG4-7D8 X IgG4-2F8

OD 405 nm 4 IgG4-7D8 X IgG1-2F8 IgG4-7D8 X IgG1-2F8-CPSC 3 2022201608

IgG4-7D8 X IgG1-2F8-ITL

2 IgG4-7D8 X IgG1-2F8-CPSC-ITL

1

0 0

0 10 20 30 Time (hours)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 4

100 Bispecific IgG (%)

lgG4-7D8 X IgG4-2F8 80 IgG4-7D8 X IgG4-2F8-CPPC* 2022201608

2022201608

60 IgG4-7D8 X IgG4-2F8-R409K* IgG4-7D8 X IgG1-2F8* 40 lgG4-7D8 X IgG1-2F8-CPSC-K409R 20

0

0 20 40 60 80 Time (hours)

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 5

2022201608 08 5 IgG4-2F8 X IgG4-7D8 OD 405 nm 4 IgG1-2F8-ITL X lgG4-7D8-CPPC 3 + 2022201608

2

1

0

0.1 1 100 10 Concentration 2-MEA (mM)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 6

A IgG1-2F8-ITL X IgG4-7D8-CPPC

146259.9

145543.7 0 mM 2-MEA 2022201608

2022201608

145000 146000 147000 148000 Da

145902.7

1,46261.6 7 mM 2-MEA

145544.8

145000 146000 147000 148000 Da

145901.0

40 mM 2-MEA

146258.3

145000 146000 147000 148000 Da

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 7/64 08 Mar 2022 Mar 2022

Figure 6 (continued)

B 2022201608 08

100 IgG4-2F8 X lgG4-7D8 2022201608

% Bispecific

80 IgG1-2F8-ITL X lgG4-7D8-CPPC

60

40

20

0

0.1 1 10 100 Concentration 2-MEA (mM)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 7

A IgG1-2F8-ITL X lgG4-7D8-CPPC control) of (% binding Bispecifc control) of (% binding Bispecifc 100 2022201608

2022201608

50

0 control +0x IgG4 x 10x ox 19G4 50x IgG4 50x no GSH IgG4 no GSH x IgG4 IgG4

B

IgG4-2F8 X IgG4-7D8 control) of (% binding Bispecific control) of (% binding Bispecific 100

50

0 50x ox IgG4 IgG4 50x no GSH IgG4 no GSH control +0x IgG4 1x19G410x IgG4

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 8

A 2022201608 08

Total IgG

10000 (ug/mL) Concentration (ug/mL) Concentration Mouse 14 Mouse 16 Bispecific Ab 2022201608

1000 Mouse 18

100 Mouse 13 Mouse 15 Bispecific Ab + 10x IgG4 10 Mouse 17

1 Mouse 19 Mouse 20 lgG1-2F8-ITL + lgG4-7D8-CPPC 0.1 Mouse 22

0 5 10 15 20 Time (days)

B

Bispecific IgG

1000 (ug/mL) Concentration (ug/mL) Concentration Mouse 14 Mouse 16 Bispecific Ab

100 Mouse 18

Mouse 13 10 Mouse 15 Bispecific Ab + 10x IgG4 Mouse 17 1 Mouse 19 Mouse 20 lgG1-2F8-ITL + lgG4-7D8-CPPC 0.1 Mouse 22

0 5 10 15 20 Time (days)

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 9A

a b Reducing Non-Reducing

M C S MCS 2022201608 08

M: Marker C: IgG1 Control S: Bispecific Sample 2022201608

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 11/64 08 Mar 2022 08 Mar 2022

Figure 9B 20.00

18.00

16.00 2022201608

2022201608

14.00

12.00 Chromatogram Auto-Scaled Chromatogram Auto-Scaled Minutes

10.00

6988 8.00

7.584

6.00

5.362 4.00

2.00

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

nv

Peak results: RT (min) Area Height % Height 1 5.362 18309 448 0.21 2 7.584 82197 2876 1.36 3 8.869 4431069 207416 98.42

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 9C

2022201608 08 IgG1-2F8-ITL 146259.5 2022201608

145000 145400 145800 146200 146600 Da

lgG4-7D8-CPPC 145542.0

145672.8

145800.2

145000 145400 145800 146200 146600 Da

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 13/64 08 Mar 2022 Mar 2022

Figure 9C (continued)

2022201608 08 lgG1-2F8-ITL X lgG4-7D8-CPPC

145901.2 2022201608

146027.7

145000 145400 145800 146200 146600 Da

Measured Measured Measured IgG1-2F8-ITL 146259.5 IgG4-7D8-CPPC 145542.0 145672.8 145800.2 IgG1-2F8-ITL X IgG4-7D8- 145901.2 146027.7

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 10 (A)

6

OD 405 nm IgG4-7D8 X IgG4-2F8 4 IgG4-7D8 X IgG1-2F8-ITL - IgG4-7D8 X IgG1-2F8-IT 2022201608

2022201608

IgG4-7D8 X IgG1-2F8-IL 2 -O- IgG4-7D8 X IgG1-2F8-TL

0

10 100 1000 10000 100000 Concentration Ab (ng/mL)

(B)

6 IgG4-7D8 X IgG4-2F8 OD 405 nm

lgG4-7D8-CPPC X IgG4-2F8 4 lgG4-7D8-CPPC X IgG1-2F8-ITL . lgG4-7D8-CPPC X IgG1-2F8-IT 2 lgG4-7D8-CPPC X IgG1-2F8-IL lgG4-7D8-CPPC X IgG1-2F8-TL 0

10 100 1000 10000 100000

Concentration Ab (ng/mL)

(c)

6

OD 405 nm

4 IgG1-2F8-F405L+IgG4-7D8 IgG1-2F8-F405L+IgG4-7D8-CPPC 2

0 G

10 100 1000 10000 100000 Concentration Ab (ng/mL)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 15/64 08 Mar 2022 08 Mar 2022

Figure 11

6

5 OD 405 nm

4 lgG1-2F8-ITL X lgG4-7D8-CPPC (37°C) 2022201608

2022201608 IgG1-2F8-ITL X lgG4-7D8-CPPC (20°C) 3 lgG1-2F8-ITL X lgG4-7D8-CPPC (0°C) 2 1

0

0 50 100 150 200 250 Time (min)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 16/64 08 Mar 2022 Mar 2022

Figure 12

IgG1-2F8-ITL X lgG4-7D8-CPPC 2022201608 08

5

OD 405 nm 4 DTT 2022201608

3 2-MEA GSH* 2 1

0

0.01 0.1 1 100 10

Concentration reductant (mM)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 17/64 08 Mar 2022 Mar 2022

Figure 13

A Positive Control

2022201608 08

lgG1-2F8-ITLxlgG4-7D8-CPPC

lgG1-2F8-ITLxlgG1-7D8-K409A lgG1-2F8-ITLxlgG1-7D8-K409D 2022201608

lgG1-2F8-ITLxlgG1-7D8-K409E 6 lgG1-2F8-ITLxlgG1-7D8-K409F lgG1-2F8-TLxlgG1-7D8-K409G

OD 405 nm lgG1-2F8-ITLxlgG1-7D8-K409H lgG1-2F8-ITLxlgG1-7D8-K409I 4 lgG1-2F8-ITLxlgG1-7D8-K409Q lgG1-2F8-ITLxlgG1-7D8-K409R lgG1-2F8-ITLxlgG1-7D88-K409S, 2 lgG1-2F8-ITLxlgG1-7D8-K409T lgG1-2F8-ITLxlgG1-7D8-K409V lgG1-2F8-ITLxlgG1-7D8-K409W

0 lgG1-2F8-ITLxlgG1-7D8-K409Y lgG1-2F8-TLxlgG1-7D8-K409N

10 100 1000 10000 100000 lgG4-2F8xlgG4-7D8 Log conc Ab in ug/ml lgG1-2F8-ITLxlgG1-7D8-K409L lgG1-2F8-ITLxlgG1-7D8-K409M

gG1-2F8-ITLxlgG1-7D8

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 Positive lgG1. IgG1- Control IgG1. IgG1- 7D8 lgG1. IgG1. /////// IgG1. lgG1. lgG1. lgG1. IgG1. lgG1. lgG1. ////// ////// lgG1. IgG1- IgG1. IgG1- 708-K409Y

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 14

6 IgG4-2F8+IgG4-7D8 OD 405 nm -0- IgG4-2F8+IgG4-7D8 (deglyc) 4 2022201608

2022201608

IgG1-2F8-ITL+IgG4-7D8-CPPC * IgG1-2F8-ITL+IgG4-7D8-CPPC (deglyc)

2

0 8

10 100 1000 10000 100000 Concentration Ab (ng/mL)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 19/64 08 Mar 2022 Mar 2022

Figure 15

A 125 (%) binding Bispecific (%) binding Bispecific 2022201608 08

100 lgG1-2F8-CPSC X IgG1-7D8-CPSC 75 IgG1-2F8-CPSC-K370T X IgG1-7D8-CPSC-K370T 2022201608

IgG1-2F8-CPSC-T3501 X IgG1-7D8-CPSC-T3501 50 IgG1-2F8-CPSC-ITL X IgG1-7D8-CPSC-ITL

25 X IgG1-2F8-CPSC-K409R X IgG1-7D8-CPSC-K409R

0

0 5 10 15 20 25 Time (hrs)

B

125 (%) binding Bispecific (%) binding Bispecific IgG4-2F8 X IgG4-7D8 100 IgG4-2F8-R409K X lgG4-7D8-R409K IgG4-2F8-R409A X IgG4-7D8 R409A 75 IgG4-2F8-R409L X lgG4-7D8-R409L IgG4-2F8-R409M X lgG4-7D8-R409M 50 lgG4-2F8-R409T X lgG4-7D8-R409T 25 lgG4-2F8-R409W X lgG4-7D8-R409W lgG4-2F8-F405A X lgG4-7D8-F405A 0 IgG4-2F8-F405L X IgG4-7D8-F405L

0 5 10 15 20 25 Time (hrs)

c

125 (%) binding Bispecific (%) binding Bispecific 100 IgG4-2F8 X IgG4-7D8 IgG4-2F8-Y349D X lgG4-7D8-Y349D 75 lgG4-2F8-L351K X IgG4-7D8-L351K IgG4-2F8-E357T X IgG4-7D8-E357T 50 lgG4-2F8-S364D X lgG4-7D8-S364D 25 lgG4-2F8-K370Q X lgG4-7D8-K370Q lgG4-2F8-K370E X lgG4-7D8-K370E 0

0 5 10 15 20 25 Time (hrs)

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 15 (continued)

D IgG1-based molecules 2022201608 08 (%) binding Bispecific (%) binding Bispecific 100 2022201608

50

0

10- 10-4 10 10 10 10 Apparent KD (M)

E

IgG4-based molecules (%) binding Bispecific (%) binding Bispecific 100

50

0

10-9 10- 10- 10 10 10 Apparent KD (M)

SUBSTITUTE SHEET (RULE 26)

2022201608 08 Mar 2022

EU S 9

I E

2 L

* 8 6 T0 12 WVROAPGKGLEWVAVIWD WVROAPGKGLEWVAVIWD 2F8-51 OVQLVESGGVVOPGRSLRLSCAASGFTFSTYGMH--WRQAPGKGLEWVAVWD-DGSYKYYGDSKGRFTISRNSKNTLYLOMSLRAEDTAVYCARDGITVGMDYFDY 2F8-G4 OVQLVESGGVVOPGRSLRLSCAASGFTFSTYGMH--WRQAPGKGLEWVAVWD-DGSYKYYGDSKGRFTISRNSKNTLYLOMSLRAEDTAVYCARDGITVGMDYFDY 2F8-G4 KABAT Figure 16

I 0

2

t S L

9

E 8

D 6

ABC ABC

AB ABCDEFGH WO 2011/131746

EU TL

12 IE T9

T5

10 LI I6

T8

IV N0 90123456 7890123456 00684 9 7890 90123456 7890123456 00684 9 7890 068 TYIC NSGALTSG-VHTFPAVLOS-SGLYSLSSVVTVPSSSLGT-- TYIC NSGALTSG-VHTFPAVLOS-SGLYSLSSVVTVPSSSLGT-- WGQGTLVTVSS WGQGTLVTVSS 2F8-G1 - 84 MS WGQGTLVTVSS WGQGTLVTVSS 258-54 TYTC

- KABAT IL

L0 TT It I9

[ N [S

IE I8 20

I 6 8295be2T0682994e21068295fe2106829548210682954e21068095be2106895fe210681956e21068195be2068299beeeee2068942 8295be2T0682994e21068295fe2106829548210682954e21068095be2106895fe210681956e21068195be2068299beeeee2068942 ABCDE 2

EU 2I

20 2D

22 2 S

E 234 12345678901 234 12345678901 345678

06 12

78 56 1 0689506

KTHTCPP VEPKSC KTHTCPP VEPKSC DKR NVNHKPSNTKV DKR NVNHKPSNTKV 2F8-G1 ELLGGR

CP

2FB-G3 SELECT

AP AA EXG-AXLNSHXHOAN EXG-AXLNSHXHOAN SVFLFPPKPKDT EFLGGP AP SVFLFPPKPKDT EFLGGP AP PPCPS

2FB-G4 VESKYG CP

KABAT 2A

22 TO 23

2N 25 29 21/64

ABCDEFGHI ABCDEFGHIJKLMNOPQRSTUVWXYZABCDEFGHIJKLMNOPORS ABCDEFGHI ABCDEFGHIJKLMNOPQRSTUVWXYZABCDEFGHIJKLMNOPORS ABC ABC V AB

EU 2L

29 2B 26

zS 30

106 068 VEVHNAKTKPREEQYN VEVHNAKTKPREEQYN 2F8-G1 STY

TO DG STY VEVHNAKTKPREEOFN DG LMISRTPEVTCVVVDVSQEDPEVQFNWYV STY VEVHNAKTKPREEOFN DG LMISRTPEVTCVVVDVSQEDPEVQFNWYV SUBSTITUTE SHEET (RULE 26) 258-54

KABAT 30

2L I

28 26

N9

na E5

EC 33 E9 EL D 0

38 6

E 0 E I Et 0684950684 0684950684 78901 01234 95

3456 PENNYKTTPPVLDS PENNYKTTPPVLDS QPREPQVYTLPPSRDE QPREPQVYTLPPSRDE SKAKG EWESN

2F8-G1 EKTI RVVSVLTVLHODWLNGKEYKCKVSNKGLPSSI-EKTI-SKAKG-QPREPOVTLPPSQE--MTKNQVSLTCLVKGFYPSDIAV--EWESN-GO--PENNYKTTPPVADS RVVSVLTVLHODWLNGKEYKCKVSNKGLPSSI-EKTI-SKAKG-QPREPOVTLPPSQE--MTKNQVSLTCLVKGFYPSDIAV--EWESN-GO--PENNYKTTPPVADS 2F8-G4

KABAT ES 3 6

E9 L E8 D 2

E3 40

E2 D T

Et V

EU 3 V

V1 N

D01 2F8-G1 288-G4 PCT/EP2011/056388

KABAT tL

VS

D3 4 9

t t

08 Mar 2022

Figure 17

IgG1-2F8-F405L X lgG1-7D8-K409R control) of (% Ab Bispecific control) of (% Ab Bispecific 100

TCEP 2022201608

2022201608

DTT 50 2-MEA GSH *

0 0.1 1 10 100 Concentration reductant (mM) -50

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 18

A

Bispecific ELISA

5

lgG1-2F8-F405L X lgG1-7D8-K409R 4 2022201608

2022201608

OD 405 nm

lgG4-2F8 X lgG4-7D8 3

2

1

0

0.1 1 10 100 Concentration 2-MEA (mM)

B

MS-data

100 lgG1-2F8-F405L X lgG1-7D8-K409R % Bispecific

lgG4-2F8 X lgG4-7D8 75

50

25

0

0.1 1 10 100 Concentration 2-MEA (mM)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 19

146290.9 lgG1-2F8-F405L 2022201608

2022201608

145000 145500 146000 146500 147000 147500 148000 Da

146030.5 gG1-7D8-K409R

145000 145500 146000 146500 147000 147500 148000 Da

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 25/64 08 Mar 2022 08 Mar 2022

Figure 19 (continued)

IgG1-2F8-F405L 146160.7

X lgG1-7D8-K409R 2022201608

2022201608

147027.6

145000 145500 146000 146500 147000 147500 148000 Da

Measured MW IgG1-2F8-F405L 146,290.9 IgG1-7D8-K409R 146,030.5 IgG1-2F8-F405L X IgG1-7D8-K409R 146,160.7

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 20

A

Total IgG

1000 (µg/mL) Concentration (µg/mL) Concentration Mouse 26 2022201608

2022201608

Mouse 28 Bispecific Ab 100 Mouse 29

10 Mouse 25 Mouse 27 Bispecific Ab + 10x lgG4

Mouse 31 1

0.1

0 5 10 15 20 25 30 Time (days)

B

Bispecific IgG

1000 (µg/mL) Concentration (µg/mL) Concentration 100 Mouse 26 I Bispecific Ab Mouse 28 Mouse 29 10

Mouse 25 1 I Bispecific Ab + 10x IgG4 Mouse 27 Mouse 31

0.1

0 5 10 15 20 25 30 Time (days)

SUBSTITUTE SHEET (RULE 26)

Figure 21 2022201608 08 Mar

A CDC on Daudi cells

100 cells Pl-positive % cells Pl-positive % lgG1-7D8 2022201608

80 lgG1-7D8-K409R 60 lgG1-2F8

40 lgG1-2F8-F405L

Bispecific antibody 20

0

0.01 0.1 1 10 100 Concentration Ab (µg/mL)

B

CDC on Raji cells

100 cells Pl-positive % cells Pl-positive % 80 lgG1-7D8 lgG1-7D8-K409R 60 lgG1-2F8 40 lgG1-2F8-F405L

20 Bispecific antibody

0

0.01 0.1 1 10 100 1000 Concentration Ab (µg/mL)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 22

ADCC on A431 cells 30 lgG1-2F8 2022201608

2022201608

20 lgG1-2F8-F405L % lysis

8 lgG1-7D8 10 lgG1-7D8-K409R

Bispecific antibody 8 0 0.1 1 10 100 Concentration Ab (µg/mL)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 29/64 08 Mar 2022 Mar 2022

Figure 23

A Positive Control

2022201608 08

lgG1-2F8-F405AxlgG1-7D8-K409R

lgG1-2F8-F405DxlgG1-7D8-K409R igG1-2F8-F405ExlgG1-7D8-K409R lgG1-2F8-F405HxlgG1-7D8-K409R 2022201608

lgG1-2F8-F405xlgG1-7D8-K409R 5 lgG1-2F8-F405KxlgG1-7D8-K409R

lgG1-2F8-F405LxlgG1-7D8-K409R OD 405 nm 4 lgG1-2F8-F405MxlgG1-7D8-K409R

3 lgG1-2F8-F405NxlgG1-7D8-K409R IgG1-2F8-F405QxlgG1-7D8-K409R

2 IgG1-2F8-F405SxlgG1-7D8-K409R lgG1-2F8-F405TxlgG1-7D8-K409R 1 lgG1-2F8-F405VxlgG1-7D8-K409R lgG1-2F8-F405WxlgG1-7D8-K409R 0 lgG1-2F8-F405YxlgG1-7D8-K409R

lgG4-2F8xlgG4-7D8 10 100 1000 10000 100000 Log conc Ab in ug/ml IgG1-2F8-F405GxlgG1-7D8-K409R lgG1-2F8-F405RxlgG1-7D8-K409R

lgG1-2F8xlgG1-7D8-K409R

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 IgG1. IgG1 Positive IgG4-2 Control 2F8x19G1. IgG1. IgG1- IgG1- K409R IgG1. IgG1- IgG1. K409R lgG1. K409R 708-K4009R lgG1. IgG1- IgG1. IgG1- IgG1- K409R K409R K409R

B

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 30/64 08 Mar 2022 Mar 2022

Figure 24

A Positive Control 2022201608 08

lgG1-2F8-Y407AxlgG1-7D8-K409R lgG1-2F8-Y407GxlgG1-7D8-K409R lgG1-2F8-Y407LxlgG1-7D8-K409R 5 lgG1-2F8-Y407MxlgG1-7D8-K409R 2022201608

lgG1-2F8-Y407NxlgG1-7D8-K409R OD 405 nm 4 lgG1-2F8-Y407WxlgG1-7D8-K409R

lgG1-2F8-Y407HxlgG1-7D8-K409R 3 lgG1-2F8-Y407lxlgG1-7D8-K409R lgG1-2F8-Y407VxlgG1-7D8-K409R 2 lgG4-2F8xIgG4-7D8

1 lgG1-2F8-Y407DxlgG1-7D8-K409R lgG1-2F8-Y407Ex lgG1-7D8-K409 lgG1-2F8-Y407FxlgG1-7D8-K409R 0 lgG1-2F8-Y407KxlgG1-7D8-K409R lgG1-2F8-Y407QxlgG1-7D8-K409R 10 100 1000 10000 100000 lgG1-2F8-Y407RxlgG1-7D8-K409R

Log conc Ab in ug/ml lgG1-2F8-Y407TxlgG1-7D8-K409R lgG1-2F8-Y407SxlgG1-7D8-K409R

lgG1-2F8xlgG1-7D8-K409R

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 IgG1- IgG1. Positive 2F8x19G1. Control IgG1. IgG1. 19G1-2F8-1 Y407E IgG1. + 708-K409 lgG1. lgG1. 708-K409R 1961-2F8-1 708-K409R 708-K409R /////// IgG1. IgG1.

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 25

A Non-reduced B Reduced

M 1 2 3 4 5 1 2 3 4 5 M 2022201608

2022201608

- - 1) IgG1 control B12

2) IgG1-7D8-K409R

3) gG1-2F8-F405L

4)IgG1-7D8-K409R + IgG1-2F8-F405L (1:1)

5) IgG1-7D8-K409R X igG1-2F8-F405L (Exchanged)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 26

8.614

8.927

A B 2022201608

2022201608

7.433 7.508 9.462

A AA 4.00 6.00 8.00 10.00 12.00 4.00 6.00 8.00 10.00 12.00 minutes 8.900 minutes 8.631

8.767

C D

10.598 10.826 7.497 9.483 7.677

4.00 6.00 8.00 10.00 12.00 4.00 6.00 8.00 10.00 12.00 minutes minutes

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 27

146028.0 146289.0

2022201608 08 A B 2022201608

*147732.5 *147473.6

144000 145000 146000 147000 148000 Da 144000 145000 146000 147000 148000 Da

C 146288.7 D 146159.7

146027.4

* 147472.2 * 147609.7

143500 144500 145500 146500 147500 Da 143500 144500 145500 146500 147500 Da

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 34/64 08 Mar 2022 Mar 2022

Figure 28

A

0.4 0.4 2022201608 08

A b 0.3 0.3 b S S Or or 0.2 0.2 2022201608

b b a a 0.1 0.1 n C C e e 0.0 0.0

7.0 7.5 8.0 8.5 9.0 9.5 10.0 pl

B

0.4 0.4

A A b b 0.3 0.3 S S O r O r 0.2 * 0.2 b * b a a n 0.1 0.1 n C c e e 0.0 0.0

7.0 7.5 8.0 8.5 9.0 9.5 10.0 pl

c

0.4 0.4 r S n C a b b e A r S n C a b b e A A 0.3 0.3 b S

r 0.2 * 0.2 # b a 0.1 0.1 n C 0.0 e 0.0

7.0 7.5 8.0 8.5 9.0 9.5 10.0 pl

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 28 (continued)

D - 0.4 0.4

A b 0.3 0.3 S 2022201608

2022201608

r 0.2 * 0.2 * b a 0.1 0.1 C e 0.0 0.0

7.0 7.5 8.0 8.5 9.0 9.5 10.0 pl

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 36/64 08 Mar 2022

2022

Figure 29 08 Mar

A 2022201608

2022201608

B C D

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00

Minutes

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 30

146159 .3

*

146344. .5

* 2022201608

2022201608

t 146026.7 145972.4 + t * 146 284.2 t 146477.0 1458 45.8 t t 14 6664.9

145660.6

145400 145600 145800 146000 146200 146400 146600 146800 147000

Mass (Da)

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 31

A t=0

t = 45 B 2022201608

2022201608

t = 90 C

D t = 135

t = 180 E

F t = 225

G t = 270

H t = 450

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 Minutes

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 32

A 2022201608

2022201608

B

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 Minutes

SUBSTITUTE SHEET (RULE 26)

Figure 33 2022201608 08 Mar

A

20 mg/mL IgG, 90 and 300 min 4 10 mM 2MEA 15C 90 min

10 mM 2MEA 15C 5 hr OD 405 nm

3 10 mM 2MEA 25C 90 min 2022201608

10 mM 2MEA 25C 5 hr 10 mM 2MEA 37C 90 min 2 10 mM 2MEA 37C 5 hr 25 mM 2MEA 15C 90 min 1 25 mM 2MEA 15C 5 hr 25 mM 2MEA 25C 90 min 25 mM 2MEA 25C 5 hr 0 25 mM 2MEA 37C 90 min 25 mM 2MEA 37C 5hr 100 1000 10000 100000 50 mM 2MEA 15C 90 min Concentration Ab (ng/ml) 50 mM 2MEA 15C 5 hr control

B

20 mg/mL IgG 90 and 300 min 4 50 mM 2MEA 25C 90 min 50 mM 2MEA 25C 5hr OD 405 nm

3 50 mM 2MEA 37C 90 min 50 mM 2MEA 37C 5hr 100 mM 2MEA 15C 90 min 2 100 mM 2MEA 15C 5hr 100 mM 2MEA 25C 90 min 1 100 mM 2MEA 25C 5hr 100 mM 2MEA 37C 90 min

0 100 mM 2MEA 37C 5hr 0 mM 2MEA 4C

0 mM 2MEA 37C 5hr 100 1000 10000 100000 control Concentration Ab (ng/ml)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 41/64 08 Mar 2022

2022

Figure 33 (continued) 2022201608 08 Mar

C

10 mg/mL IgG 90 and 300 min 4 10 mM 2MEA 15C 90 min

10 mM 2MEA 15C 5 hr OD 405 nm

3 10 mM 2MEA 25C 90 min 2022201608

10 mM 2MEA 25C 5 hr 10 mM 2MEA 37C 90 min 2 10 mM 2MEA 37C 5 hr 25 mM 2MEA 15C 90 min 1 25 mM 2MEA 15C 5 hr 25 mM 2MEA 25C 90 min 25 mM 2MEA 25C 5 hr 0 25 mM 2MEA 37C 90 min 25 mM 2MEA 37C 5hr 100 1000 10000 100000 50 mM 2MEA 15C 90 min Concentration Ab (ng/ml) 50 mM 2MEA 15C 5 hr control

D

10 mg/mL IgG 90 and 300 min 5 50 mM 2MEA 25C 90 min 50 mM 2MEA 25C 5hr 4 50 mM 2MEA 37C 90 min OD 405 nm

50 mM 2MEA 37C 5hr 3 100 mM 2MEA 15C 90 min 100 mM 2MEA 15C 5hr 2 100 mM 2MEA 25C 90 mn

1 100 mM 2MEA 25C 5hr 100 mM 2MEA 37C 90 min 100 mM 2MEA 37C 5hr 0 0 mM 2MEA 4C 0 mM 2MEA 37C 5hr 100 1000 10000 100000 control Concentration Ab (ng/ml)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 42/64 08 Mar 2022 Mar 2022

Figure 33 (continued)

E

20 mg/mL IgG o/n 2022201608 08

6 10 mM 2MEA 15C o/n

10 mM 2MEA 25C o/n OD 405 nm

10 mM 2MEA 37C o/n 2022201608

4 25 mM 2MEA 15C o/n

25 mM 2MEA 25C o/n 25 mM 2MEA 37C o/n 2 50 mM 2MEA 15C o/n

50 mM 2MEA 25C o/n 50 mM 2MEA 37C o/n

0 100 mM 2MEA 15C o/n 100 mM 2MEA 25C o/n 100 mM 2MEA 37C o/n 10 100 1000 10000 100000 0 mM 2MEA 4C Concentration Ab (ng/ml) 0 mM 2MEA 37C o/n control

F

10 mg/mL IgG o/n 5 10 mM 2MEA 15C o/n 10 mM 2MEA 25C o/n 4 10 mM 2MEA 37C o/n OD 405 nm

25 mM 2MEA 15C o/n 3 25 mM 2MEA 25C o/n 25 mM 2MEA 37C o/n 2 50 mM 2MEA 15C o/n

1 50 mM 2MEA 25C o/n 50 mM 2MEA 37C o/n

0 100 mM 2MEA 15C o/n 100 mM 2MEA 25C o/n 100 mM 2MEA 37C o/n 10 100 1000 10000 100000 0 mM 2MEA 4C Concentration Ab (ng/ml) 0 mM 2MEA 37C o/n control

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 34

A

10 mM 2MEA: % cFAE 120 control to compared % control to compared % 100 2022201608

2022201608

80

60

40

20

0

5hr 14th 1442 14th $45 control 3,6 3/8 90min 5hr25C 15C 15C 37C 3>C5hr 3>C 90min 5hr 2ahr5hr 24hr 15C 15C 25C 15C 15C B

25 mM 2MEA: % cFAE 120 control to compared % control to compared % 100

80

60

40

20

0

90min 15C 15C 37C 15C 37C 5hr 5hr 5hr 290min 5hr 24hrmin Shrahrmin 37C 5hr 24hr control

15C 25C 15C 2537C 25C 37C 15C

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 34 (continued)

C

50 mM 2MEA: % cFAE 120 control to compared % control to compared % 2022201608

2022201608 100

80

60

40

20

0

37C 3715C 5hr 24hr 90min gomin 5hr 24hr gomin 5hr 37C control 5hr 5hr 5hr 24hr 24hr Milk 25C 15C 25c 290min 15C 15C 25C 15C 15C 37C

D

100 mM 2MEA: % cFAE 120 control to compared % control to compared % 100

80

60

40

20

0

15C gomin 15C 15C 5hr 25C 24hr gomin 25C 25C 5hr 37C 24hr gomin which 14th 2442 14th gooding $hr $he $he 5hr control TYE

25C 15C 37C 3>C 25C 15C 15C 25C 37C

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 45/64 08 Mar 2022 2022201608 08 Mar 2022

Figure 35

A A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0 mM 2-MEA 4°C control: 0.10 -0.10

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + gG1-7D8-K409R 0.20 2022201608

10 mM 2-MEA 15°C 5 hr 0.10 0.10

0.00 0.00

A280 (AU)

020 IgG1-2F8-F405L + gG1-7D8-K409R 0.20 10 mM 2-MEA 15°C 24 hr 0.10 0.10

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + lgG1-7D8-K409R 0.20 10 mM 2-MEA 25°C 5 hr 010 0.10

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R -0.20 10 mM 2-MEA 25°C 24 hr 0.10 -0.10

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 10 mM 2-MEA 37°C 5 hr 010 0.10

0.00 0.00

IgG1-2F8-F405L + IgG1-7D8-K409R A280 (AU)

0.20 -0.20 10 mM 2-MEA 37°C 24 hr 010 -0.10

0.00 0.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time (minutes)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 46/64 08 Mar 2022 Mar 2022

B A280 (AU)

0.20 gG1-2F8-F405L + IgG1-7D8-K409R 0.20 0 mM 2-MEA 4°C control 0.10 0.10 2022201608 08

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + igG1-7D8-K409R 0.20 25 mM 2-MEA 15°C 5 hr 2022201608

0.10 0.10

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 25 mM 2-MEA 15°C 24 hr 0.10 0.10

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 25 mM 2-MEA 25°C 5 hr 0.10 0.10

0.00 0.00 A280 (AU)

IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0.20 25 mM 2-MEA 25°C 24 hr

0.00 0.00

A280 (AU)

IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0.20 25 mM 2-MEA 37°C 5 hr

0.00 0.00

A280 (AU)

IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0.20 25 mM 2-MEA 37°C 24 hr

0.00 0.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time (minutes)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 47/64 08 Mar 2022 08 Mar 2022 c A280 (AU)

0.20 igG1-2F8-F405L + IgG1-7D8-K409R 0.20 0 mM 2-MEA 4°C control 0.10 0.10

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 50 mM 2-MEA 15°C 5 hr 2022201608

2022201608

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 50 mM 2-MEA 15°C 24 hr

0.00 0.00 A280 (AU)

IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0.20 50 mM 2-MEA 25°C 5 hr

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 50 mM 2-MEA 25°C 24 hr

0.00 0.00 A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 50 mM 2-MEA 37°C 5 hr

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 50 mM 2-MEA 37°C 24 hr 0.10 0.10

0.00 0.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time (minutes)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 48/64 08 Mar 2022 2022201608 08 Mar 2022

D A280 (AU)

0.20 igG1-2F8-F405L + IgG1-7D8-K409R 0.20 0 mM 2-MEA 4°C control 0.10 0.10

0.00 0.00

A280 (AU)

IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 0.20 100 mM 2-MEA 15°C 5 hr 2022201608

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 100 mM 2-MEA 15°C 24 hr

0.00 0.00

IgG1-2F8-F405L + IgG1-7D8-K409R A280 (AU)

0.20 0.20 100 mM 2-MEA 25°C 5 hr

0.00 0.00

IgG1-2F8-F405L + IgG1-7D8-K409R A280 (AU)

0.20 0.20 100 mM 2-MEA 25°C 24 hr

0.00 0.00

IgG1-2F8-F405L + IgG1-7D8-K409R A280 (AU)

0.20 0.20 100 mM 2-MEA 37°C 5 hr

0.00 0.00

A280 (AU)

0.20 IgG1-2F8-F405L + IgG1-7D8-K409R 0.20 100 mM 2-MEA 37°C 24 hr 0.10 0.10

0.00 0.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time (minutes)

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 49/64 08 Mar 2022 Mar 2022

Figure 36

2022201608 08

A Positive Control

lgG1-2F8-L368AxlgG1-7D8-K409R 2022201608

lgG1-2F8-L368DxlgG1-7D8-K409R lgG1-2F8-L368ExlgG1-7D8-K409R

3 lgG1-2F8-L368GxlgG1-7D8-K409R lgG1-2F8-L368HxlgG1-7D8-K409R

lgG1-2F8-L368xlgG1-7D8-K409R OD 405 nm

lgG1-2F8-L368NxlgG1-7D8-K409R 2 lgG1-2F8-L368QxlgG1-7D8-K409R

lgG1-2F8-L368RxlgG1-7D8-K409R lgG1-2F8-L368SxlgG1-7D8-K409R 1 lgG1-2F8-L368TxlgG1-7D8-K409R lgG1-2F8-L368VxlgG1-7D8-K409R lgG1-2F8-L368WxlgG1-7D8-K409R 0 lgG1-2F8-L368KxlgG1-7D8-K409R

10 100 1000 10000 100000 lgG4-2F8xIgG4-7D8

Log conc Ab in ug/ml lgG1-2F8-L368FxlgG1-7D8-K409R lgG1-2F8-L368MxlgG1-7D8-K409R lgG1-2F8-L368YxlgG1-7D8-K409R

lgG1-2F8xlgG1-7D8-K409R

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 19G1. 19G1 19G1. Positive 7D8-K439R 08-K409R 108-K409R D8-K409R 8-K409R B-K409R IgG1. 708-K409R IgG1. //////// ////// /////// lgG1. Control

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 37

A 2022201608 08

Positive Control

lgG4-2F8xlgG4-7D8

lgG1-2F8-K370WxlgG1-7D8-K409R 2022201608

lgG1-2F8-K370AxlgG1-7D8-K409R 3 lgG1-2F8-K370DxlgG1-7D8-K409R lgG1-2F8-K370ExlgG1-7D8-K409R

OD 405 nm gG1-2F8-K370FxlgG1-7D8-K409R

2 gG1-2F8-K370GxlgG1-7D8-K409R lgG1-2F8-K370HxlgG1-7D8-K409R gG1-2F8-K370xlgG1-7D8-K409R

1 lgG1-2F8-K370LxlgG1-7D8-K409R lgG1-2F8-K370MxlgG1-7D8-K409R lgG1-2F8-K370NxlgG1-7D8-K409R lgG1-2F8-K370QxlgG1-7D8-K409R 0 lgG1-2F8-K370RxlgG1-7D8-K409R lgG1-2F8-K370SxlgG1-7D8-K409R 10 100 1000 10000 100000 lgG1-2F8-K370TxlgG1-7D8-K409R Log conc Ab in ug/ml lgG1-2F8-K370VxlgG1-7D8-K409R lgG1-2F8-K370YxlgG1-7D8-K409R

gG1-2F8xlgG1-7D8-K409R

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 Positive IgG1. ////// 8-K409R lgG1. 708-K409R 708-K409R 708-K409R ////// 708-K409R 708-K409R Control

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 51/64 08 Mar 2022 Mar 2022

Figure 38

2022201608 08

A Positive Control

lgG1-2F8-D399AxlgG1-7D8-K409R 2022201608

lgG1-2F8-D399FxlgG1-7D8-K409R lgG1-2F8-D399HxlgG1-7D8-K409R lgG1-2F8-D399xlgG1-7D8-K409R lgG1-2F8-D399KxlgG1-7D8-K409R 4 lgG1-2F8-D399LxlgG1-7D8-K409R lgG1-2F8-D399MxlgG1-7D8-K409R OD 405 nm

3 lgG1-2F8-D399NxlgG1-7D8-K409R lgG1-2F8-D399RxlgG1-7D8-K409R lgG1-2F8-D399SxlgG1-7D8-K409R 2 IgG1-2F8-D399TxlgG1-7D8-K409R IgG1-2F8-D399GxlgG1-7D8-K409R 1 lgG1-2F8-D399WxlgG1-7D8-K409R lgG1-2F8-D399YxlgG1-7D8-K409R

0 lgG4-2F8xIgG4-7D8

lgG1-2F8-D399ExlgG1-7D8-K409R 10 100 1000 10000 100000 -V-- lgG1-2F8-D399QxlgG1-7D8-K409R Log conc Ab in ug/ml lgG1-2F8-D399VxlgG1-7D8-K409R

lgG1-2F8xlgG1-7D8-K409R

B

100 (%) binding Bispecific (%) binding Bispecific 80

60

40

20

0 19G1- Control Positive IgG1. 19G1. 19G1- 19G1. 19G1- 19G1- 19G1 19G1 19G1 19G1 19G1- 2F8xigG1. 7D8-K409R TD8-K409R IIII IIIIIIII IIIIIII

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 52/64 08 Mar 2022 Mar 2022

Figure 39

t=0 t=30 2022201608 08

4 4

OD 405 nm 3 OD 405 nm 3 2022201608

2 2

1 1

8 0 0

0.01 0.1 1 0.01 0.1 1 10 100 10 100 Conc Ab in ug/ml Conc Ab in ug/ml

t=60 t=105 4 4

OD 405 nm 3 OD 405 nm

3

2 2

1 1

0 0 d

0.01 0.1 1 0.01 0.1 1 10 100 10 100 Conc Ab in ug/ml Conc Ab in ug/ml

t=200

4 Postive control

gG1-2F8-F405SxlgG1-7D8-K409A OD 405 nm 3 <> gG1-2F8-D399RxlgG1-7D6-K409G

lgG1-2F8-L368RxlgG1-7D8-K409H 2 gG1-2F8-F405LxlgG1-7D8-K409R

1

0

0.01 0.1 1 10 100 Conc Ab in ug/ml

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 40

100 lgG1-7D8-K409A (%) binding Bispecific (%) binding Bispecific lgG1-7D8-K409G 80 igG1-7D8-K409R I 60 lgG1-7D8-K409H 2022201608

2022201608

40

20

0 IgG 10G1-2F8-FA0M

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 54/64 08 Mar 2022 Mar 2022

Figure 41

2022201608 08

Phospho c-Met (PY1234/1235)

I 2022201608

Total c-Met

ß-actin

- b12 / 069 IgG1 Bispecific b12 / 069 IgG1 Bispecific b12 / 058 IgG1 Bispecific b12 / 058 IgG1 Bispecific HGF 50 ng/ml medium UniBody-5D5 IgG1-058 IgG1-069

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 42

2022201608 08 NCI-H441 Proliferation Assay 7 day incubation [mAb ] 10 µg/ml

200 2022201608

Proliferation (%)

150

100

50

0 IgG1 Bispecific IgG1 069 069/b12-b12 none

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 43

A 2022201608 08

CDC on Daudi cells

100 2022201608

EN cells PI-positive % cells PI-positive % 80 No Antibody

lgG1-7D8 60 lgG1-7D8-K409RxlgG1-7D8-F405L

lgG1-7D8-K409RxlgG1-2F8-F405L 40 Negative Control

20

0

0.001 0.01 0.1 1 10 100 Concentration Ab in ug/mL

B

CDC on Raji cells

100 cells PI-positive % cells PI-positive % 80 No Antibody

lgG1-7D8 60 lgG1-7D8-K409RxlgG1-7D8-F405L

40 H lgG1-7D8-K409RxlgG1-2F8-F405L

Negative Control

20 +

0

0.001 0.01 0.1 1 10 100 Concentration Ab in ug/mL

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 44

IgG1-005-ITL X IgG1-169-K409R 3000000 IgG1-025-ITL X IgG1-005-K409R intensity Fluorescence intensity Fluorescence IgG1-025-ITL X IgG1-153-K409R IgG1-025-ITL X IgG1-169-K409R 2000000 IgG1-153-ITL X IgG1-005-K409R 2022201608

2022201608

IgG1-153-ITL X IgG1-169-K409R IgG1-005-ITL 1000000 IgG1-005-K409R IgG1-025-ITL IgG1-153-ITL 0 10 100 IgG1-153-K409R IgG1-169-K409R mAb concentration staurosporin (ng/mL) Herceptin isotype control

SUBSTITUTE SHEET (RULE 26)

08 Mar 2022

Figure 45 cells) untreated to (compared cells) untreated to (compared 100

80 % HER2 2022201608

2022201608

60

40 T 20

0 Herceptin. 19G1-1- -005- ITL 1gG1- -K409R ITL TTL K409R K409R -K409K409R -005- -K409R K409R -K409R K409R 15otype

19G1 19G1 19G1. 19G1 19G1- + + + 19G1- 19G1- 19G1- 19G1- + IgG1- + IgG1- + IgG1-

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 59/64 08 Mar 2022 Mar 2022

Figure 46

A 2022201608 08 lysosomes in intensity pixel FITC lysosomes in intensity pixel FITC 150000 2022201608

100000

50000

0 19G1. 19G1. 19G1. 19G1. 19G1. 19G1- 18G1-153-ITL -153-ITL COOS-ITL -025-ITL + + + + + + 19G1- 19G1- 19G1- 19G1- 19G1- 19G1- -ITL 169-K4D9R K409R -K409R K409R K409BR KAOGOR 19G1 Herr -ITL -ITL Herceptin

B lysosomes in intensity pixel FITC lysosomes in intensity pixel FITC 150000 IgG1-153-ITL X gG1-169-K409R IgG1-025-ITL X lgG1-153-K409R IgG1-005-ITL X lgG1-169-K409R 100000 gG1-153-ITL X IgG1-005-K409R gG1-025-ITL X IgG1-169-K409R IgG1-025-ITL X IgG1-005-K409R IgG1-025-ITL Herceptin 50000 lgG1-169-K409R

IgG1-153-ITL IgG1-005-ITL

0 bispecific monospecific

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 60/64 08 Mar 2022 08 Mar 2022

Figure 47

120

% viable cells 100

80 * 2022201608

2022201608

60

40

20

0 Herceptin 1gG1- 19G1- ITL 1gG1- K409R -K409R K409R 169-005R K409R K409R 169-005-K K409R K409R 1sotype -K409Rontrol

19G1 19G1- 19G1 19G1- 19G1- 18G1-005-ITL + + + + + + 19G1- 19G1- 19G1- 19G1- 19G1- 19G1- 19-1-153

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 61/64 08 Mar 2022 Mar 2022

Figure 48

A pH 7.4, IgG1 pH 7.4, hinge-deleted-lgG1

2022201608 08 1.5 1.5

OD 405 nm OD 405 nm

1.0 1.0 2022201608

0.5 0.5

0.0 0.0 0 10 100 1000 10000 100000 10 100 1000 10000 100000 Concentration Ab (ng/ml) Concentration Ab (ng/ml)

pH 6.0, IgG1 pH 6.0, hinge-deleted IgG1

1.5 1.5

OD 405 nm OD 405 nm

1.0 1.0

0.5 0.5 X

0.0 0.0

10 100 1000 10000 100000 10 100 1000 10000 100000 Concentration Ab (ng/ml) Concentration Ab (ng/ml)

IgG1-2F8 IgG1-2F8 lgG1-2F8-ITL/lgG1-7D8-K409R Uni-G1-2F8-ITL/Uni-G1-7D8-K409R lgG1-2F8-ITL/lgG1-7D8-K409R-H435A Uni-G1-2F8-ITL/Uni-G1-7D8-K409R-H435A lgG1-2F8-ITL Uni-G1-2F8-ITL gG1-7D8-K409R Uni-G1-7D8-K409R IgG1-7D8-K409R-H435A Uni-G1-7D8-K409R-H435A

SUBSTITUTE SHEET (RULE 26) wo 2011/131746 PCT/EP2011/056388 62/64 08 Mar 2022 Mar 2022

Figure 48 (continued)

B 2022201608 08 pH 7.4, IgG1 pH 7.4, hinge-deleted-lgG1 1.5 1.5

OD 405 nm OD 405 nm

1.0 1.0 2022201608

0.5 0.5

0.0 0.0

10 100 1000 10000 100000 10 100 1000 10000 100000 Concentration Ab (ng/ml) Concentration Ab (ng/ml)

pH 6.0, IgG1 pH 6.0, hinge-deleted-IgG1

4 4

OD 405 nm 3 OD 405 nm 3

2 2

1 1

0 0

10 100 1000 10000 100000 10 100 1000 10000 100000

Concentration Ab (ng/ml) Concentration Ab (ng/ml)

lgG1-2F8 lgG1-2F8 lgG1-2F8-ITL/lgG1-7D8-K409R Uni-G1-2F8-ITL/Uni-G1-7D8-K409R lgG1-2F8-ITL/IgG1-7D8-K409R-H435A Uni-G1-2F8-ITL/Uni-G1-7D8-K409R-H435A lgG1-2F8-ITL Uni-G1-2F8-ITL lgG1-7D8-K409R Uni-G1-7D8-K409R lgG1-7D8-K409R-H435A Uni-G1-7D8-K409R-H435A

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 49

2022201608 08 YTH12.5 4.0x10

3.0x10 Herceptin 2022201608

Duo YTH12.5/153 MFI 2.0x10 Duo YTH12.5/169 Duo YTH12.5/B12 YTH12.5 1000000 B12

0

0.01 0.1 1 10 100 1000 10000 Concentration Ab (ng/ml)

huCLB-T3/4 4.0x10

3.0x10 Herceptin Duo huCLB/153 MFI 2.0x10° 6 Duo huCLB/169 Duo huCLB/B12 huCLB 1000000 B12

0

0.01 0.1 1 10 100 1000 10000 Concentration Ab (ng/ml)

SUBSTITUTE SHEET (RULE 26)

Mar 2022

Figure 49 (continued)

2022201608 08 YTH12.5-N297Q 4.0x10

3.0x10 2022201608

MFI Herceptin 2.0x10 Duo YTH12.5-Q/153-Q Duo YTH12.5-Q/B12-Q 1000000 YTH12.5-Q B12-Q

0

0.01 0.1 1 10 100 1000 10000 Concentration Ab (ng/ml)

huCLB-T3/4-N297Q 4.0x10

3.0x10 V Herceptin MFI Duo huCLB-Q/153-Q 2.0x10 Duo huCLB-Q/B12-Q huCLB-Q 1000000 B12-Q

0

0.01 0.1 1 10 100 1000 10000 Concentration Ab (ng/ml)

SUBSTITUTE SHEET (RULE 26)

SEQUENCE LISTING 08 Mar 2022

<110> Genmab A/S

<120> Heterodimeric antibody Fc-containing proteins and methods for production thereof

<130> P/61.WO

<160> 3 2022201608

<170> PatentIn version 3.5

<210> 1 <211> 107 <212> PRT <213> homo sapiens

<400> 1

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105 08 Mar 2022

<210> 2 <211> 107 <212> PRT <213> homo sapiens

<400> 2

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 2022201608

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105

<210> 3 <211> 107 <212> PRT <213> homo sapiens

<400> 3

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 08 Mar 2022

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 2022201608

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr 85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105