NZ711762B2 - Method of producing an immunoligand/payload conjugate - Google Patents

Abstract

The present invention relates to a method of producing an immunoligand/payload conjugate which method encompasses conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof (See Figure). The method allows the conjugation of payloads to immunoligands in a site-specific and/or sequence specific manner and allows preserving characteristic features of its components. noligands in a site-specific and/or sequence specific manner and allows preserving characteristic features of its components.

Description

Method ofproducing an irnmunoligand/payload ate _____________________________________________________________________________________________________________ The present invention is related td-‘i‘fflethods ofproducing an irnmunoligand/payload conjugate Intr0duction Currently, the predominant methods to label and/or to conjugate molecules to proteins, especially, When small-molecule payloads. or labels are concerned, involve the chemical ' conjugation with specific linker molecules that ntly attach the payload to free lysine and/or cysteine amino acids of the proteins.

HOWeVer, many proteins, like e. g. antibodies that are of particular interest for nctargeting strategies, are fairly large proteins, that may contain several lysine and ne residues. Because linker—mediated, chemical conjugation is a stochastic process, linlcer~mediated chemical ligation ofpayloads leads to geneous mixtures of conjugated proteins that may difier in their therapeutic y and/or diagnostic utility. Obviously, mixtures of n~payload conjugates also represent a significant challenge in the regulatory approval process for therapeutic conjuagtes, as batch—to-batch variation and/or variations in the active pharmaceutical ingredient (API) are negatively viewed by regulatory authorities due to potential safety concerns In addition, if a defined ratio of payload to protein is desired, it is often necessary to purify the conjugate with the desired conjugation stoichiometryi This is not only tedious, but can significantly add to the cost—of~goods in the manufacturing process, as often only a on of the linker~rnediated conjugated protein represents the d ratio of payload conjugation. This is ularly true for therapeutically relevant antibody/ding conjugates (ADCs), where depending on the toxin employed, 3 to ’4 toxin molecules appear to be advantageous, but antibodies with no toxin coupled to up to 8 toxins per antibody coupled are found in typical linlcer~mediated al conjugation reactions ski ct al. (2014)).

Despite of the limitations described above, all antibody/drug ates currently in clinical trials, or approved by the health authorities for the therapy of disease, have been generated linlcer~mediated chemical conjugation of toxic small~molecule drugs to antibodies (Lambert (2012) or Mullard (2013)).

It is widely acknowledged in the industry and by scientific experts in the field, that site specific and iomettic conjugation of molecular payloads, including toxin or label molecules to immunoligands would have significant advantages in comparison to chemical, linker—mediated conjugation This is evidenced by attempts to target the chemical ation to specific amino acids inthe protein structure (PanOWSki et a1. (2014)).

StfllClIUl‘C to delete On one hand, this is ted by mutating certain positions in the protein unwanted and/or to provide desired conjugation sites (i.e. lysine and/or cysteine residues) to which the linlcer~ligation can be targeted (McDonagh et a1. (2006) or Iunutula et al. (2008)).

On the other hand, control of chemical conjugation to proteins is attempted by incorporation of unnatural amino acids at certain positions, like selenocysteine, p—azidophenylalanine, or phenylalanine (Hofer et al. (2009), Axup et a1. (2012), or Lemke (2011)).

However, all of these ches change the primary amino acid sequence of the n to be conjugated, and may result in undesired functional properties. Fultheimore, incorporation of unnatural amino acids, as described abOVe, is often low efficient, and does sites to ns. not allow for a quantitative incorporation of specific labeling Summary of the inVenticn of stochastic Therefore, there is an urgent need in the industry to overcome the known issues. conjugation methods in particular for the tion of therapeutically relevant conjugates, including, but not limited to ADCs.

It is thus one object of the present invention to provide an nt method for conjugating immunoligands and ds, e.g., drugs, toxins, cytolcines, markers, or the like, preferably full—length monoclonal antibodies to small~molecu1ar Weight toxins, for the tion of site— specifically conjugated antibody drug conjugates (ADCs).

It is another object of the t invention to create immunoligand/payload conjugates, Which have better efficacy and/or can be ed with higher reproducibility It is another object of the present invention to allow the conjugation of payloads to immunoligands in a site specific and/or sequence specific .

It is another object of the present invention to create immunoligand/payload conjugates Which preseI‘Ve the characteristic features of its components, e.g., target affinity, target specificity, target sensitivity, solubility, pharmacological function and the like These s achieved by the subject matter of the independent claims, While the dependent claims as well as the specification disclose r preferred embodiments.

Definitions As used herein, the term “immunoligand” is meant to define an , an agent or a molecule that has affinity to a given target, e.g., areceptor, a cell surface n, a cytolcine block or or the like, Such immunoligand may optionally dampen agonist—mediated interaction. Most however, the responses, or inhibit receptor~agonist importantly, ligand may seive as a shuttle to deliver apayload to a specific site, which is defined by the target recognized by said immunoligand, Thus, an immunoligand targeting, for instance, but not limited to areceptor, delivers its payload to a site which is characterized by abundance of said receptor. Immunoligands include, but are not limited to, antibodies, antibody fragments, dy~based binding proteins, antibody mimetics, receptors, e and ligands ofreceptors, decoy receptors, scaffold proteins with affinity for a giVen target odies", also synonymously called "immunoglobulins" (lg), are generally comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain, single domain antibodies (dAbs) which can be either be deriVed from aheavy or light chain) ,' including full length functional mutants, ts, or derivatives f (including, but not limited to, mmine, ic, humanized and fully human antibodies, which retain the essential epitope binding features of an lg molecule, including dual specific, bispecific, multispecific, and dual variable domain immunoglobulins,‘ lmmunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or ss (e.g., IgGl, IgG2, IgGB, IgG4, IgAl, and IgA2) and allotype. that contains An "antibody—based binding protein”, as used herein, may represent any protein context of other at least one antibody~derivad VH, VL, or CHimmunoglobulin domain in the non~immunoglcbulin, or non—antibody derived components. Such antibody—based proteins include, but are not limited to (i) Ila—fusion ns of binding ns, including receptors or receptor ents with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in Which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which innnunoglobulin VH, and/or VL, and/or CH domains are combined and/or led in a fashion not nmmally found in naturally occuring antibodies or antibody fragments.

An "antibody drug conjugate? (ADC), as used herein, relates to either an antibody, or an antibody fragment, or and antibody~based binding protein, d to a small molecular Weight active pharmaceutical ingredient (API), including, but not limited to a toxin (including cg, but not limited to, tubulin inhibitors, actin binders, RNA polymerasa inhibitors, DNA~interoalating and modifying/damaging drugs), a kinase inhibitor, or any API that interferes with a particular cellular pathway that is essential for the survival of a cell and/or essential for aparticular physiologic cellular pathvvay.

An "antibody derivatiVe or fragmen ”, as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, ing, the le not limited to (i) a Fab fiagment, which is a lent fragment ting of l (CHI) domains; light (VL), variable heavy (VH), constant light (CL) and constant heavy (ii) tWo Fab fragments linked by a a F(ab')2 fragment, which is abivalent fiagment comprising de bridge at the hinge region; (iii) aheavy chain portion of a Fab (Fa) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (FV) nt, which consists of the VL and VH domains of a single arm of an antibody, (v) in antibody (dAb) fragment, Which comprises a single variable domain; (vi) an isolated complementarity wo 40317 summon/055173 determining region (CDR); (vii) a single chain FV Fragment ; (viii) a diabody, which is ific dy in which VH and VL domains are expressed on a a bivalent, single polypeptide chain, but using a linker that is too short to allow for pairing betWeen the two domains on the same chain, y forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; and (ix) a linear antibody, which comprises a pair of tandem FV segments (VH-CHLVHCHI) which, er with complementarity light chain polypeptides, form a pair of antigen binding regions; and (x) other non-full length portions of immunoglobulin heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

The term ied antibody format”, as used herein, encompasses antibody~drug~~ ates, Polyallcylene oxide—modified scFv, Monobodies, Diabodies, Camelid Antibodies, Domain dies, bi~ or trispecific antibodies, IgA, or We IgG structures joined by a I chain and a secretory component, shark antibodies, new world primate ork + non—new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance affinity for Fc gamma receptors, dimerised constructs comprising CH3+VL+VH, and the like.

The term ”antibody mimetic", as used herein, refers to proteins not belonging to the immnnoglobulin family, and even non~proteins such as aptamers, or synthetic polymers.

Some types have an antibody—like beta~sheet structure. Potential advantages of "antibody es” better tissue or ”alternative scaffolds" over antibodies are solubility, higher penetration, higher stability towards heat and enzymes, and comparatively low tion costs.

Some antibody mimetics can be provided in large libraries, which offer specific binding candidates against every conceivable target. Just like with antibodies, target specific antibody mimetics can be developed by use of High Throughput Screening (HTS) logies as well as with established display technologies, just like phage display, ial display, yeast or mammalian display. Currently developed antibody mimetlcs encompass, for example, anlcyrin repeat proteins (called DARPins), C~type lectins, A— domain proteins of S. aureus, transfenins, lipocalins, 10th type HI domains of fibronectin, Kunitz domain protease inhibitors, ubiquitin derived binders (called affilins), gamma crystallin derived binders, ne knots or lmottins, thioredcxin A scaffold based binders, WO 40317 c acid aptamers, artificial antibodies produced by molecular imprinting of polymers, peptide libraries from bacterial genomes, SH—B domains, stradobodies, ”A s" of membrane receptors stabilised by disulfide bonds and Ca2+, CTLA4~based compounds, Fyn 8H3, and aptamers (oligonucleic acid or peptide molecules that bind to a specific target molecules) In case the ligand is not a protein or a peptide, e.g., if its an aptamer, it should preferably be provided with a peptide tag in order to provide a suitable substrate for the enzymatic conjugation disclosed further herein.

"Con'u ation“, molecule to another J g as used herein, relates to the covalent association of a molecule by formation of a covalent bond.

An ”immunotoxin", to a protein or as used herein, relates to an immunoligand conjugated ptide representing a toxin, ing, but not limited to bacterial toxins, e. g. diphteria— toxin A, Pseudornonas exotoxin, botulinum toxin, or e.g. naceous Vencms from invertebrates (e.g. but not limited spiders, scorpions, molluscs, jellyfish), or yetrebrates (e.g., but not limited to snakes), or functional fragments thereof, The term “low molecular~Weight payload" as used herein, represents a payload with a molecular Weight not exceeding 2'500 Dalton.

The term "payload”, as used herein, ents any naturally occuring or synthetically generated molecule, including small~molecular weight molecules or chemical es that or biological entities that need to be can chemically be synthesized, and larger molecules ed by fermentation of host cells and that confer a novel functionality to an ligand specific for binding to targets or ns.

The term ”small molecular Weight toxin”, as used herein, means a cytotoxic compound of small molecular Weight not eXceeding a molecular Weight of 2‘500 Dalton that is cytotoxic ' to ian cells.

A "transpeptidase”, or a as used herein, is an enzyme or a catalytic domain of an enzyme protein that is able to catalyze the breakage of peptide bonds and uently either directly, or by Way of several reaction intennediates, the formation of novel peptide bonds, such that the energy of the first peptide bond is preserved during the reaction and transfered to a new peptide bond, Preferably, said iranspeptidases preferably t the C—terminus of one e or n with the N~terminus of another peptide or protein. Due to the formation of a new e bond, these enzymes or functional domains are also refered to as in ligases", ”peptide ligases“, or med "protein or peptide staplers". Such protein ligases comprise, but are not limited to e enzymes, inteins and split~inteins.

As used herein, the term nce—specific transpepeptidase" is meant to define a transpeptidase which needs at least one substrate peptide or protein with a given peptide recognistion sequence (N—terminally and/or inally) to connect said sequence as substrate peptide or protein to another peptide or protein, or a small—molecular Weight compound containing apeptide or n component As uSed herein, the term ”site~specific transpepeptidase” is meant to define a transpeptidase Which has a specific site in at least one substrate peptide or n which it uses to conjugate to another peptide or protein, or a small~mclecu1ar weight compound containing a peptide or n component.

Background and general description of the invention The invention discloses methods that utilize site~specific transpeptidases, e.g., sortase enzymes and split~inteins, to site~specifically and selectively conjugate payloads, preferably small lar weight toxins to immunoligands, preferably antibodies, for the generation of immunoligand payloads, preferably antibody drug conjugates (ADCs). The red payloads are small molecular weight toxins modified with short, preferably less than 13' (thirteen) amino acid long synthetic amino acid sequence, which renders them as substrates for seitase enzymes or split intein mediated coValent conjugation either at the N~ or C~ terminus of the immunoligands (Figures 1 & 3). This conjugation is achieved in a Site— specific manner and with defined stoichiomeily, which is a distinguiShing feature to conventional chemical ation of payloads to immunoligands, where the conjugation a stochastic process, as disclosed further above.

The invention finther discloses site specific transpeptidase, e.g. sortase or split~intein ed conjugation of multimeric immunoligands, preferably antibodies specifically With two different toxin molecules or other labels using different modifications of the subunits ' 7 the multimeric protein, modified e. g. antibody heavy and light chains, and different payloads with different, short amino acid stretches specific for ent transpeptidases, in order to conjugate at least two different functional payloads to the multimeric immunOIigand (Fig.

The invention further discloses methods to add y purification and/or detection tags to the N or C—termini of the immunoligands, which undergo enzyme—mediated transpeptidation. such that the removal ofthe affinity purification and/or detection tag can be utilized to select for immunologands with complete (100%) conjugation of the payload to the modified g protein, by means of affinity resins that retain immunoligands that, have not been completely conjugated, and therefore still retain the additional affinity purification and/or detection tag (Fig. 4).

The invention further discloses immunoligands in which a catalytic transpeptidase domain is directly fused to the N— or C—terminus of the protein to be conjugated, such that the transpeptidation activity is integral part of immunoligand to be conjugated, and no onal soluble sortase enzyme needs to be provided in the course of the transpeptidasemediated ation reaction (Fig. 5).

All of these embodiments mentioned above allow the pecific and stoichicmetrically controlled conjugation of any payload, including small molecule toxins (chemical es), toxic proteins, or fluorescent labels, preferably small lar weight toxins to immuncligands, including preferably antibodies, which is superior to standard al conjugation oads to proteins by chemical linker chemistry methods, which cannot be controlled for conjugation ratio and site. Therefore, for the generation of antibody drug conjugates (ADCs) conjugation of toxic payloads by transpeptidaSes, preferably scrtase enzymes and split inteins to antibodies will lead to more homogeneous products with , expected improved therapeutic properties for cancer therapy (Fig. 12).

The enzymatic conjugation of ds to immuncligands by sortase enzymes and split~ intein allch site—specific and stoichiomeuic payload conjugation to proteins and ligands, chering f~gccds and providing homogeneous irnmuncligand— payload ates, especially as the selectivity of the transpeptidases allows the conjugation of payloads to immuncligands in crude cell culture supernatant, and does not PCT/.EI’2014/055173 require purified components as in traditional linker—mediated chemical ation.

Therefore, the of sequence—specific transpeptidases for ation of payloads to immunoligands could significantly IOWer the cost of goods in immunoligand—payload, and particularly ADC manufacturing.

The first type of transpeptidase disclosed herein, the sortase enzymes, has been identified in a variety of gram-positive bacteria, like Staphylococcus, Streptococcus and Pneumococcus species, and catalyse the coupling of nce s to cell wall proteoglyoans, in order to change the e signature of the bacteria for evading an efficient immune response by infected host (Mazmanian et a1. (1999)). Sortase A enzyme of the gram~positive bacterium Staphylococcus aureus has been characterized first (Ton~That et a1. (1999)) and has subsequently been characterized r as a tool for many protein modifications (Tsulciji (2009)). The attraction of sortase enzymes is that the two molecules to be ated only require to be modified or expressed on one hand with a short 5 amino~acid long peptide tag (sortase tag, LPXTG in case of Staphylococcus (litmus sortase A, X being any of the 20 naturally occuiing aminoacids), and a short, preferably 3 to 5 amino acid long glycine stretch (Antes et al, (200921)) (Fig. l), which can easily be added to each of the molecules to achieye either N—terminal or C~terrninal ation of proteins. This allOWS to utilize the for the coupling or conjugation of two proteins, but also for the system on one hand conjugation of r les to proteins.

The second type of transpeptidase resulting in peptide—bond cleavage and formation, is represented by the so~called inteins, which have originally been disaovered as protein s, that can remOVe (splice) themselves out ofprecursor proteins by cleavage ofpeptide bonds and formation of new peptide~bonds OCu et a1. (1993)) (Fig. 2a). Inteins can also occur separated into N—intein and C—intein s (so~called split~inteins) and attached to independent proteins that can subsequently catalyze the /raws~splicing of the extein domains (Fig. 2b). Split—inteins have been utilized for the nt coupling of N—extein and C~extein moieties, and also the ation and/or oircularization of proteins. (Elleuche (2010)).

However, in order to utilize split~inteins also for the conjugation of small molecule payloads, it is necessary to utilize split s that function if either the N~intein or the C- intein domain can be redused to few amino acids, that can easily be added to molecules of any size by chemical synthesis, similar to the short at preferably 3 glycine stretch required the artificial Ssp GyrB 811 for sortase—mediated transpeptidation. With the development of split~intein, in which the Caintein domain only ses six amino acids (Sun et al. (2004)), this condition has been met and this split—intein has been utilized for the C~terminal labeling of proteins with biotin ann et al. (2009)) (Fig. 3a). Likewise, the pment of a short 11 amino acid long N—intein from Ssp DnaX split~intein allows the N—terminal conjugation of proteins With any molecule, if such 11 amino acid long stretch is added by al synthesis to apayload of choice (Fig. 3b).

Therefore, 3 to 5 glycine one aspect of the invention is either to add a short, preferably ‘ acid stretch acid glycine stretch, or a short 12 amino-acid SW amino acid C~int domain (X any naturally occuring or artificial amino acids), containing a 6 amino Which is of Ssp GyrB or a 11 aminc~acid N~int domain of Ssp DnaX to apayload—molecule, sufficient to allow the respective trasnpeptidase to conjugate the modified payload to proteins and immunoligands, preferably antibodies that, respectively, contain a sortase enzyme recognition ’motif, e,g, LPXTG in case of utilization of Staphylococcus aureus Soitase A, or a 150aa N~mt domain in case of utilization of Ssp GyrB split intein, or a 139 of Ssp DnaX split intein (see Figs 1 82; 3). aa C-int domain in case of utilization acids, like e. g. 3 or 5 glycine residues to a small The addition of short stretches of amino molecular Weight toxins as required for sortaSe mediated conjugation, or 12 amino acids as required for split—intein mediated conjugation, has been found to add to the Water~solubility of certain hobic toxin molecules (data not shown), such that the amino acid—toxin adduct can be dissolved in the physiologic buffer, enSuring optimal sortase or split~intein conjugation. This prevents stress on the strutural integrity of large n molecules, and non» particularly antibodies that can easily be denatured by exposure to c solvents associated with traditional linker chemistry and conjugation. In physiologic pH often antibodies addition, conjugation of hydrophobic toxin molecules to large proteins, ular can induce certain levels of protein aggregation. Also this may be improved by using eptidases, ularly e enzymes, because further hydrophilio amino acids for aggregation of remain in the enzymatically generated conjugate, ng the propensity large protein, or antibody drug conjugates.

Sortase enzymes have been widely described in the prior art for protein~protein or protein~ peptide ligations (Mao et al, (2004), Parthasarathy et a1. (2007) or WO20ll/133704A2), et al. even including circularization of proteins (Antes (200%)). The applications of sortase protein or peptide ligation also included protein or peptide ligation using antibody fragments, like Fab» and ScFV~fragments with protein~ or peptide labels (Mohlmann et al. (2011), Madej et al. (2012), or US2010/0055761A1 and WO20l2/l42659Al), Eyen tWO prior art documents Were published, in which full—length antibodies have been e~ ligated to ns (Levary et a1. (2011), to the e. g. EGFP, albumin, gelonin were ated light chain of an antibody), or in which full—length antibodies have been e~ligated to short peptides (Swee et a1. (2013)), HoweVer, no prior art document could be identified like demonstrating the sortase-mediated conjugation of small~molecular weight toxins, e. g. auristatins or maytansins and the like, to full~length antibodies or antibody fragments. In particular no prior art documents could be identified, in which generation of ADCs with small molecular weight toxins has been been disclosed resulting in ADCs with small molecular Weight toxins homogeneously conjugated to either IgH or IgL chains to~ herein. antibody ratio 2), or to IgH and IgL chains (drug to dy ratio 4), as disclosed While the prior art also discloses the modification of non~protein substrates With glycine residues such that they could be used for e modification of simple, single-subunit proteins or peptides (Tsultiji (2009), or W0200’7/108013A3, respectively), the more molecular challenging homogeneous conjugation of non~protein substrates, ably small described before, Weight toxins, to multimeric proteins, preferrably antibodies, has not been been in the prior e the fact that sortase enzyme mediated protein or peptide ligation has art for many years.

Moreover, the conjugation of multimeric proteins, particularly full—length monoclonal antibodies with two different payloads, preferrably two different small lar Weight the fact that toxins as disclosed herein, has not been described in the prior art before, despite in the prior al't for many years sortaSe enzyme mediated protein or e ligation has been ski et a1. (2014)), It is known ficm the prior art that sortase enzymes may accept substrates that contain a minimum of 3 e amino acids (Parthasarathy et al. (2007), therefore the ion may include payloads that contain at least three (3) glycine amino acid residues added to the payload molecule of interest, although even one or two glycine residues may be sufficient, small molecular Weight and should be comprised by the method disclosed herein. In case of payloads the addition of few glycine amino acid residues .can be achieved by conventional PCT/EPZOl4/055173 synthetic peptide chemistry, as described . In case of proteins glycine residues can“ be added either by adding codons for a number of glycine residues, preferably at least three glycine residues, in~frame to the open reading frame of the protein, or by conVentional synthetic peptide chemistry such that the recombinant protein contains at least three N- terminal glycine amino acid residues.

It is known from the ture that different e enzymes, e.g. Scrtase B from Staphylococcus aureus, or Sortases from other ositive bacteria ize different pentapeptide motifs, which differ from the LPXTG sortase A recognition motif (X = any amino acid) from Staphylococcus aureus (Spirig et a1. ). Therefore, the invention shall also include the concept of adding other sortase recognition motifs to proteins and immunoligands, including preferably antibodies, that differ from the Staphylococcus oureus sortase A ition motif LPXTG, in order to prepare them for sortase conjugation with different e sortase enzyme of different gram—positive bacterial species. Therefore, proteins and immunoligands, preferably antibodies, can also be expressed with a different sortase recognition motif, a NPQTN pentapeptide motif specific for sortase B from e.g.

Staphylococcus cureus Which can then be conjugated to glycine modified payloads.

In an another aspect of the inVention, multimeric immunoligands, preferably but not limited to antibodies, which are composcd of immunoglobulin heavy and light chains, allow the utilization of said different e recognition ces added to the different ptides of such multimeric proteins (in case of antibodies adding different e ition in order to allow conjugation of different sequences to the antibody heavy and light chains), payloads to said different polypeptides by performing sequential conjugations with Glyn~ tagged payloads (11 >2) in the presence of the tiVe sortase enzyme (Fig. 6b). For this, modifications at heavy and light an antibody needs to be expressed with different C—terminal ‘ chains comprising difierent sortase recognition motifs for different Sortase enzymes. Such an antibody can then sequentially be conjugated to two different payloads containing a glycine, modification as described further above.

This format may have the advantage that ADCs specifically be loaded with two different toxins, preferably interfering with a ent cellular pathway will be more potent in cancer cell killing, because it is more difficult for a targeted cancer cell to evade the attack of two toxins comprised in the ADCs.

It is clear to aperson d in the art, that a sortase pentapeptide recognition motif, like the Staphylococcus aureus sortase A LPX-TG motif, can be added selectively to dual polypeptides of multimeric immunoligands, in order to provide desired conjugation sites.

For instance, in the case of antibodies, this allows the generation of modified antibodies, either only containing sortase recognition motifs added to the heavy chains (resulting in two payloads per antibody conjugation), or only containing sortase recognition motifs added to the light chains (resulting in two payloads per antibody conjugation), or containing sortase recognition motifs added to the heavy and the light chains (resulting infour payloads per antibody conjugation). These designed variations will allow specific conjugation ofpayloads to antibodies by sortase enzymes either to the heavy chains alone (generating ADCs with drug to antibody ratio of 2, i6. DARZ), or to the light chains alone (generating ADCs with drug to antibody ratio of 2, i.e. DARZ), or simultaneously to the heavy and the light chains (generating ADCs with drug to antibody ratio of 4, to. DAR4). This way, the conjugation sites and, stoichiometries for antibodies can be varied in a controlled fashion, either generating two payload conjugaticns per antibody heavy or light , or generating four payload conjugations per antibody by addition of the payload to the heavy and the light chains. r to the above-described variations in conjugation sites and stoichiometries using different sortase recognition motifs and sortase enzymes in multimeric proteins or immunoligands, it is to a further aspect of the ion to conjugate different payloads different tide chains of multimeric proteins combining sortase—mediated and split— intein mediated conjugation. This concept allows the simultaneous conjugation of different payloads to ent ptide chains of multimen'c proteins and immunoconjugates in are being employed (Fig, one step, because ent transpeptidases and substrates 6a).

It is to be understood that the abovamentioned conjugation of two different payloads a eric protein, ably an antibody, which is composed of each two disulphide linked heavy and lights chains, can either be accomplished by combining sortase enzyme mediated conjugation with split intein mediated conjugation, as depicted, in Fig, 6a, but that it is also possible to conjugate two different payloads to a multimeric n, preferably an antibody, by ing two different e enzymes, recognizing different sortase peptide motifs, for instance sortase A and sortase B from Staphylococcus omens, as mentioned wo 2014/140317 further above (Fig. 6b). However, this may also include sortase enzymes of other Sortase classes (e.g. seitases C, D, E, F), or sortase enzymes from other ial species, differing in their sortase motif specificity.

Sortase—mediated conjugation of payloads to proteins and immunoligands can be achieVed either by providing e recognition motif tagged proteins and at least tri~g1ycine tagged thereof as payloads and adding enzymatically active sortase enzyme or a functional fragment a Soluble enzmye. In another aspect of the invention the enzymatically active domain of C~tenninus of the ‘sortase enzyme can also be provided as a domain fused to either the N— or protein. In this variation, is is advantageous, but not ory, to add the sortase enzymatic domain either N~terminal to an N~terminal sortase recognition motif, or C~terminal to a C» reaction with a terminal sortase recognition motif. Both ilities ensm‘e that the after the glycine~tagged payload, that the enzymatic sortase domain is remQVed from the protein in the course of the on (Fig. 5). is similar in This variation of applying e~mediated conjugation of ds to ns Where the enzymatically actiVe N— t to split~intein mediated conjugation of payloads, in order to define intein domains of split inteins are tethered to the protein to be conjugated, the conjugation site in the n, Similar to the large number of different sortase transpeptidases with different substrate specificity that have been identified in the literature (Spirig et a1. (2011)), there is also a large and growing number of split~inteins known from different species and proteins with that can be retrieved ent in and C~intein sequences required for transpeptidation from the so—called InBase database (Perler (2002). ore, while the examples of Split~ diSclose the prefered intein mediated conjugation of immunoligands with payloads Ssp GyrB Sll split intein (Vollanann et al. (2009)), because the C~intein domain can be reduced mediated conjugation of ds to to a short, linear 6~mer amino acid stretch, split~intein proteins and immunoligands can also be achieved with other split inteins from the InBase database, are short enough as long as the N~intein or C~intein domains (preferably r than and addition to any payload molecule 13 amino acids) to easily allow peptide synthesis of choice. However, it is clear to a person skilled in the art that in the case of protein payloads,‘ C-intein domains of any size may be fused to the protein payload by genetic mechanistic advantage fusion to the ORF of the protein payload of interest, and there is no of using split~inteins with small (< 13 amino acids) N—intein or C~intein domains.

However, if synthetic small~molecule payloads are to be conjugated to proteins and immunoligands, then a small N~int or C~int domain of less than 13 amino acids as disclosed herin are advantageous, as in the case of the prefered C—int of the Ssp GyrB Sll split intein, of the N—int of Ssp DnaX, because such a short peptide can synthetically be added to any synthetic small le weight d by standard synthetic chemistry.

Sortase~mediated and split—intein mediated conjugation of payloads can be performed at either the N~ or the ini of proteins and immunoligands. This is only ent on how the sortaSe~mctif/glycine stretch and N—intein/C—intein domains are positioned at protein and payload (Fig. 1).

In the case of antibodies, Which are the prefeired immunoligands, it is preferred to conjugate the ds to the C~termini of the antibodies, because this positions the payloads most distally to the antigen~binding sites of the antibody. However, this preference shall not be interpreted by way of limitation, and it may be advantageous to conjugate ds to the N— terminus of other immunoligand molecules, like e.g. antibody mimetics, in which the functional binding domains are not located at the N—terminus of the le.

Another aspect of the ion is to improve the efficiency of soitase and split—intein to ns and immunoligands by adding affinity purification or ation of payloads detection tags, like e. g., but not d to small peptide tags (e. g. histidine tags, tag, MYC~tag or HA-tag) or larger n affinity purification tags (eg. maltose~binding protein (h/IBP) tag, Glutathione~S~transerase (GST) tag, or Chitin—binding tag) distal to the sortase recognition motif or the split~intein domain fused to the protein or immunoligand of interest.

With this aspect of the invention the affinity puiification tag will be d from the immunoligand to be conjugated as part of the transpeptidation reaction. This can be exploited to enrich fully payload conjugated immunoligands, as unreacted proteins and imrmmoligands, that still contain the affinity purification tag, can be remOVed by binding to and immunoligands a suitable y resin, While completely payload conjugated proteins will no longer contain the affinity pun‘fication tag, and can thus be specifically separated from the unreacted immunoligand substrates. This aspect of the invontion is particularly powerful in the context of multimeric proteins and immunoligands, like the preferred antibodies, in which seVeral payloads need to be conjugated. The use of affinity purification site ensures that one can tags located distal to the sortase or intein transpeptidase conjugation is still present due remove proteins and ligands in which the affinity ation tag to incomplete payload conjugation (Fig. 5). in the process In compaIiSOn to chemical conjugation, this provides a cant advantage to obtain homogeneous immunoligand/payload conjugates, and preferably ADCs in which small molecular weight toxins are site specifically conjugated to the Caterrnini of antibody heavy and/or light chains.

Generally, the disclosed method es a novel and efficient method to pecifically stoichiomeirically conjugate payloads, preferably small molecular weight toxins to immunoligands, preferably antibodies, by which defined immunoligand/payload conjugates, preferably ADCs are generated, that are useful for the therapy of diseaSes, preferably of ‘ . The method may also be utilized for the generation of immunoligand/payload conjugates uSeful for the diagnosis of diseases, preferably oncology diseases The nOVel utilization of method allows generation covalent irnmunoligand/payload conjugates by peptide—bond breaking and forming enzymes (transpeptidases), including sortase s and split~inteins, or catalytically active fragments thereof. Said enzymes can catalyze the covalent and site~specific conjugation of payloads containing short amino acid stretches (preferably shorter than 13 amino acids) either to the N— or C~termini of immunoligands and to form peptide Which are ly modified allowing sortaSe and split»inteins to break bonds in the coursc of the reaction. Immunoligands are ably antibodies, for the site specific conjugation of small molecular weight toxins, in order to generate antibody drug ratios. conjugates (ADCS) with defined antibody payload, or drug to antibody Embodiments of the invention conjugate is , According to the invention, a method of producing an immunoligand/payload disclosed, which method encompasses conjugating apayload to an immunoligand by means of a sequence c transpeptidase, or a tic domain thereof. the immunoligand According to apreferred embodiment of the invention, the payload and/or either a) consists, entirely, of aprotein or peptide b) comprises at least one protein or peptide domain, or 0) comprises at least one peptide chain and, r, the n or peptide or domain comprises, ably, an amino acid sequence that can be ed by the sequence—specific transpeptidase, or a catalytic domain thereof.

This means, for example, that, in case the payload and/or the immunoligand is aprotein, it means that said protein comprises, at its N or inus, an amino acid sequence which can be detected by the sequence~specific eptidase. If such amino acid sequence is lacking to the naive protein, it can be fused to the N~ or C-terminus of said protein by recombinant methods known inthe art.

In case the payload and/or the immunoligand is not a protein, such amino acid sequence which can be ed by the sequence— specific transpeptidase, is to be conjugated to the former by conVentional chemical crosslinking methods known inthe art.

Additional functionalities may be incorprated between the recognition sequence for a specific transpeptidase and the payload. This can be realized by chemical ures either being categorized by being cleavable (erg. containing hydrazone, or disulfide chemistry, or specific peptide sequences for intracellular proteases) or being non~cleavable (e.g. containing thioether chemistry) following internalization into cells.

Chemical structures containing hydrazone chemistry can selectiVely be cleaved within the intracellular compartment of lysosomes (IOWer pH compared to the systemic blood circulation).

Peptide linkers have the potential to be selectively cleaved by lysosomal proteases (cg. cathepsin~B) and have demonstrated increased serum stability and improved anti-tumor effects compared to hydrazone linkers. Valine—citruline (Val~Cit) pairs are the most ly used peptide linkers and are y suited to work With the auristatin family of drugs such as monomethyl amistatin E (WAR).

.Non—cleavable s have long been overlooked as chers were convinced the cleaving of the linker was the most reasonable way to free the drug. However, conjugates internalized and once internalized, the can, upon binding to a membrane receptor, get rapidly immunoligand can be degraded to the point where the payload, e. g,, the drug is exposed As one prominent example, thicether linkers, use the SMCC (N ~saiccinimidyl—4~(N— maleimidomethyl)— cyclchexanedwcarboxylate) linker (Fig. .

All of theses appaoraches haVe in common that there is no true site—specificty of the coupling reaction. e linker—mediated, al conjugation is a stic process, linker~mediated chemical ligation oads leads to heterogeneous mixtures of conjugated proteins that may differ in their therapeutic efficacy and/or diagnostic potential, Obviously, conjugates also represent a significant challenge in the mixtures of protein—payload for therapeutic conjuagtes, as batch~to~batch variation and/or tory approval process variations in the active pharmaceutical ingredient (AH) are negatively viewed by regulatory authorities due to potential safety concerns. have long been overlooked as chers Were convinced the Non—cleavable Linkers cleaving of the linker was the most reaSOnable way to free the drug. HOWever, conjugates can, upon binding to amernbarne or, get rapidly internalized and once internalized, the immuncligand can be degraded to the point where the payload, e.g., the drug is exp03ed.

One prominent example, thicether linkers, use the SMCC (N.succinimidyl~4 ~(N— maleimidomethyl} cycloheXane—l—cal‘boxylate) linker (See Fig. 14a, structure 2). of the coupling All of theses approaches haVe in common that there is no true site~specificty reaction. Because linker~rnediated, chemical conjugation is a stochastic s, linker~ mediated chemical ligation of ds leads to heterogeneous es of conjugated cy and/or diagnostic ial. Obviously, proteins that may differ in their therapeutic mixtures of protein~payload conjugates alSO represent a significant challenge in the approval process for therapeutic gtes, as batch-to-batch variation and/or regulatory viewed by regulatory variations in the active pharmaceutical ingredient (AM) are negatively authorities due to potential safety concerns. ing to another preferred embodiment of the inyention, the immunoligand comprised in the immunoligand/payload conjugate is at least one selected from the group consisting of or fragment, and/or an dy, modified antibody format, antibody derivative ' an antibody mimetic Preferably, in this ment, as substrate for the a small molecular payload is rendered sequence~specific transpeptidase by ng of apeptide of lees than 13 amino acids to the to the nini small molecular payload, such that it can be conjugated by a transpeptidase said of monoclonal antibody containing C~terminal modifications recognized by transpeptidases. Such Cuterminal modifications may be ned on either both heavy , or both light chains, or of heavy and light chains of a full—length antibody, thereby either drug—t0—antibcdy ratio allowing generation of a pecifically conjugated ADC with of 2 01‘ 4 (DARZ or DAR4).

According to another preferred embodiment of the invention, the immuncligand binds at least one entity selected from the group consisting of - areceptor ‘ an antigen, - apgrowth factor - a cytokine, and/0r - a hormone a cell surface As used herein, the term "receptor“ means a cell surface le, preferably molecule that (i) binds specific, or groups of specific, signalling molecules (Le, a receptor, like, e.g., the VEGF receptor), and/or (ii) has no known ligand (ie. an orphan receptor, like, of cells, are expressed on the surface of apopulation e.g, HERZ/neu). The l receptors domain of such a molecule (whether such a form or they merely represent the extracellular exists naturally or not), function in the or a soluble molecule performing natural binding , a cell or organ. Preferably, such receptor is a member of a signalling or within cascade that is involved in apaiticular pathogenic process (e. g, a or that belongs to a of a cell or particle that signalling cascade of a growth factor), or is expressed on the surface is inVclVed in apatholcgical process, e.g., a cancer cell. that has the ability to induce a ic As used herein, the term ”antigen" means a substance immune (cg, ion response, and may include surface proteins or protein complexes cancer cell. channels). Often times, antigens are associated to pathogenic entities, cg, a As used herein, the term ”cytolcine” refers to small cell— signaling protein molecules that are Secreted by numerous cells and are in a category of signaling molecules used extensively intercellular communication. Cytolcines can be fied as proteins, peptides, or glyeoproteins; the term dne" encompasses a large and diverse family of regulators - ed throughout the body by cells of diverse embryological .

As used herein, the term ”growth factor" relates to naturally occurring substances capable factor stimulating cellular growth, proliferation and cellular differentiation. Usually a growth is a protein or a steroid hormone. Growth factors are important for ting a variety of cellular processes.

As used herein, the term 'fhormone” relates to a chemical released by a cell, a gland, or an out messages that affect cells in other parts of the organ in one part of the body that sends organism. The term encompasses peptide hormones, lipid and phospholipid~derived hormones including steroid hormones, and monoamines.

In case the immunoligand binds a or or an n, the immunoligand—payload conjugate can for example be directed to a specific site, e.g., to apathogenio entity, eg, a cancer cell, Where the payload, eg. a toxin or a herapeutic agent, is delivered, Thus, the systemic toxicity or" the toxin or the chemotherapeutic agent is reduced, While the local concentration of the latter at the site of action is increased, thus provding a better efficacy while side effects are reduced, Furthermore, arespective signalling cascade can be inhibited marker the latter can thus be by the g of the immunoligand. In case the payload is a used to mark a specific site, e.g., a cancer cell charcterlzed by a given surface antigen detected by the immunoligand, for diagnosis. and!or ahormone, the In case the ligand binds a growth factor, a cytokine, innnunologand/payload conjugate can for example be directed to the site the growth factor ne or e us'uall binds to, in oder to deliver the payload in a site~Specific manner.

Further, arespective signalling cascade can be inhibited by the binding of immunoligand.

As used herein, the term “to bind“ means the well~understood interaction or other nonrandom association between ligands, e,g., antibodies, or antibody fragments, their targets. Preferably, such binding reaction is characterized by high specifity and/or ivity to the target. Preferably, the g reaction is characterized by a dissociation constant (Kd) 5 103 M, preferably 5 10'4 M, 5 10‘5 M, 5 10‘5 M, g 10‘7 M, 5 '8 M, 5 10‘9 M, and most preferred 5 10‘1“.

‘ Acccording to apreferred embodinient of the invention, it is provided that at least one catalytic domain ofthe‘ ce—specific eptidase is fused to the N—terminus or the C~ terminus of either the immunoligand orthe payload.

Such fusion may take place by recombinant engineering, or by chemical coupling, In this embodiment, the enzymatic activity leading to the site—specific oonmjugation of the immunoligand to the payload does not need to be added to the reaction as a separate recombinant enzyme, but is rather part ofprotein ate to be conjugated.

Preferably, the sequence specific eptidase is at least one selected from the group consisting of - a sortase, or one or more fragments or derivatives thereof - a spilt—intein, or one or more fragments or derivatives f. the payload, In apreferred embodiment, where the tranSpeptidase is a sortase, e.g., atoxin, is . preferably rendered as ate for sortase conjugation by addition of a small number of glycine amino acid residues, preferably 3 or 5 glycine residues.

In another preferred embodilnent, where the transpeptidase is a split intein, e. g., a Ssp GyrB split intein, the payload, e.g., a toxin is rendered as ate for split intein conjugation by addition of less than 13 amino acid residues of the sequence GVFVEN—SXD, X being any amino acid and n being an integer between 2 O and g 5. inteins for the generation of The use of transpeptidases, preferably sortase enzymes and split antibody drug conjugates, in which small lar Weight toxins are conjugated to full— length antibodies, has not yet been described in the prior art (Panowski et a1. (2014)).

Sortase enzymes have been identified in a variety of gram~positive ia, like Staphylococcus, Streptococcus and Pneumococcus s, and catalyze, in vivo, the coupling of Virulence factors to cell wall proteoglycans, in order to change the surface ure of the bacteria for evading an efficient immune reSponse by the infected host (Mazmanian et a1. (1999)).

The sortase A enzyme of the gram~pcsitive bacterium Staphylococcus aureus has been characterized first (Ton—That et a1. (1999)) and has subsequently been characterized further as atool for many protein modifications ji ).

One beneficial feature, of soitase enzymes is that the two molecules to be conjugated only require short peptide tags (“sortase tags"), which in case of lococcus aureus sortase A is for example LPXTG at the Cuterminus of one molecule (e.g., the payload), and "a short 3 to 5 amino acid glycine stretch at the N—terminus of the other molecule (cg, the immunoligand, see Fig. 1), These peptide tags can either be fused to the molecules, or utilize conjugated thereto by means of conventional crosslinldng chemistry. This allows to of small the system on one hand for the on of two proteins, but also for the conjugation In case molecular weight compounds, preferably small molecular weight toxins to proteins. is NPQTN of Staphylococcus aurcus sortase B, the respective sertase motif Inteins, which have originally been diScovered as protein s that can remOVe (splice) themselves out of precursor proteins by cleavage of peptide bonds and new peptide—bond formation (Xu et a1. (1993)) (Fig. 2a).

Naturally morning and artificial split—inteins involve that the intein coding region has been split into N~intein and in domains, which can be attached to different proteins or peptides in such Way that, subsequently the lrans~splicing of the extein domains (Fig. 2b) leads to the conjugation of the two proteins Split—inteins have thus been utilized for the covalent coupling of N~extein and C—extein moieties, and also for the purification and/or aiization of proteins (Elleuche ).

One embodiment disclosed herein is to utilize split~inteins for the conjugation of small molecular weight compounds, preferably small molecular weight toxins and other small than 13. amino acids molecule , in Which a short C—extein peptide sequence of smaller is coupled to molecules of any size, similar to the short glycine amino acid stretch ed for sortase—mediated transpeptidation. addition of a short glycine stretch (> 2 glycine residues) to a In case of soitase enzymes molecule of choice is sufficient to allow the molecule to be conjugated to irnmuncligands A of containing a penta~peptide sortase recognition motif, like e. g, LPXTG in case of sortase S. aureus. In case of split~inteins, minimally a short 12 aminc~acid GVFVHNSAGSGK from Ssp GyrB and amino acid stretch containing a short, 6 amino acid in (GVFVHN) a short C~extein (here: SAGSGK) are sufficient to modifi any payload molecule, preferably a small molecular weight toxin, for split~intein mediated conjugation to immuncligands containing the Nnintein domain of the SSp GyrB split intein (Voflmann et a1. (2009)). Other split inteins, in which functional intein domains can be reduced to small <13 amino acid long peptide stretches may be utilized as Well.

Even if, in the literature, split~enzymes are not always referred to as s, they qualify results in the breakage of apeptide bond and the as such, because the reaction they catalyze ion of a new peptide bond and this can be Viewed as transpeptidases, because the bond is ered to a new peptide bond. energy of an existing peptide Other than chemical conjugation, the transpeptidasamediated conjugation occurs under physiologic aqueous buffer conditions and logic temperatures, thereby minimally reaction, This feature ensures affecting the n or antibody ity in the conjugation optimal functionality of the resulting conjugate ding to another red embodiment of the invention, it is provided that the payload comprised in the immunoligand/ payload conjugate is at least one selected from the group consisting of amarlcer - ssing tag, and/or - adrug. molecule or The term "marker" (also called "detection tag“), as used herein, may refer to any moiety that ses one or more appropriate chemical substances or enzymes, which wo'2014/140317 2014/055173 directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction.

The term "processing tag” as used herein, may encompass affinity tags, solubilizatlcn tags, chromatography tags and epitcpe tags, Affinity tags (also used as purification tags) are appended to proteins so that they allow purifification of the tagged molecule fiom their crude biological Source using an affinity technique, These e chitin-binding protein (CBP), maltose binding protein (MBP), and glutathione~S~transferase (GST). The poly(His) tag, preferably a 6XHis tag, is a Widely~used processing tag; it binds 'to metal matrices.

Solubilizaticn tags are used, especially for recombinant proteins expressed in chaperone~ deficient species such as E. coli, to assist in the proper folding in proteins and keep them from precipitating. These include thioredoxin (TRX) and poly(NANP). Some affinity tags haVe a dual role as a solubilization agent, such as MBP, and GST.

Chromatography tags are used to alter tographic properties of the protein to afford these t of different resolution across a particular separation technique. Often, polyanionic amino acids, such as FLAG~tag antibodies e tags are short peptide sequences which are chosen because high—affinity are y d from can be reliably ed in many different species. Epitope tags the , Viral genes, which n their high immunoreactivity. Epitope tags include eg. and‘ HA~tag. These tags are particularly useful for western blotting, MYC—tag, immunofluorescence and immunoprecipitatlon experiments, although they also find use in protein purification.

Processing tags find many other , such as specific enzymatic modification (such as biotin ligase tags) and chemical modification (FLASH) tag. Often tags are combined to produce multifunctional modifications of the protein.

Preferably, said marker is at least one selected from the group consisting of or protein a radiolabel, preferably a radioacthely labelled peptide or protein, and/or a fluorescent label, preferably a fluorescent peptide - an enzyme label, preferably aperoxidaSe.

PCTIEP2014/055173 This enumeration of potential marker payloads is by no means restrictive. According to the group consisting of another preferred embodiment, said drug is at least one ed from r a. cytokine . a radioactive agent - an anti~inflammatory drug - atoxin, and/or - a chemotherapeutic agent This enumeration of potential drug payloads is by no means restrictive. As uSed herein, the term “cytokine” refers to small cell~signaling protein molecules that are secreted by in intercellular numerous cells and are a category of signaling molecules used extensively communication. Cytokines can be classified as proteins, es, or glycoproteins; the term "oytoldne" encompasses a large and diverse family of regulators produced hout body by cells of diverse logioal origin. in the present context, cytokines are for cell. e meant to impair, or even kill, pathogenic entity, e.g., a cancer " relates to an entity which has at least one atom As used herein, the term ”radioactive agen With an unstable nucleus, and which is thus prone to undergo radioactive decay, resulting beta particles, Which the emission of gamma rays and/or subatomic particles such as alpha or have a cell killing effect. In the present context, radioactive agents are meant to impair, or even kill, pathogenic entity, e.g., a cancer cell. that reduce As used , the term “anti—inflammatory drug" relates to compounds inflammation. This can be, e.g., steroids, just like specific glucooorticoids (often referred to corticosteroids), which reduce inflammation or swelling by binding to glucocorticoid nflammatory drugs S), receptors. The term further encompasses non—steroidal COX enzyme synthesizes which counteract the cyclooxygenase (COX) . On its own, prostaglandins, ng inflammation. In Whole, the NSAle prevent the prostaglandins the pain. The term r encompasses from ever being synthesized, reducing or eliminating which are a class of peptides IrnIrmne Selective Anti~lnflammatory Derivatives (lmSAle), that alter the activation and migration of inflammatory cells, which are immune cells responsible for amplifying the inflammatory response.

As used herein, the term ” relates to a molecule Which is toxic to a living cell or organism. Toxins may be peptides, or proteins or ably small lar weight that or even kill, pathogenic entity, e. g., a cancer cell. compounds, are meant to impair, , as meant herein, encompass, in particular, cellular toxins. Preferably, said toxin is a small molecular toxin, i.e., having a molecular weight of S 2500 Da.

As used herein, the term "chemotherapeutic ” agen relates to molecules that have the functional property of inhibiting a development or progression of a neoplasm, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or ia.

Inhibition of asis or angiogenesis is frequently a property of anticancer or chemotherapeutic agents. A herapeutic agent maybe a cytotoxic or chemotherapeutic agent. Preferably, said chemotherapcuic agent is a small molecular weight cytcstatio agent, which inhibits or suppreSSes growth and/or multiplication of cancer cells.

Conjugating cytoldnes, radioactive agents, toxins or chemotherapeutic agents to an immunologand to their can help to reduce side effects and risks related admmistration, because a) the immunoligand directs the conjugate to a specific site, e.g., to apathogenic , e. g., a cancer cell Where the payload effects its toxic function. Thus, the systemic toxicity of the payload is reduced, while the local concentration of the latter at the site of action is increased, thus provding ahetter efficacy while side effects are reduced, h) it can be provided that the conjugate is alized by the pathogenic entity, in such way its desired cytotoxic that after internalization, the payload is released and only then develops function, i.e., Without affecting the surrounding cells or tissue.

The following table is a non restrictive list of potential targets/antigens (1st ) and existing immunoligands targeting the former (2nd column). The 3rd column examples for shows a non restrictive list of potential toxins, cytoldnes or chemotherapeutic agents. Note that the examples from the 1st and the 3” column can be combined with one another ad libitum. while eds of further targets and ds exist. Respective /payload combinations not explicitly mentioned in the table are encompassed by the scope of the present invention. target/antigen example of an existing payload immunoligand elial Growth ' Cetuximab Maytansinoides, e.g, Mertansine, Ansamitooin' receptor (EGFR) sin, DM4, DMl CD20 Ritmdmab, Ibriturnomab, Calioheamioins, e.g.

Tosituinoinab (mAb) Ozogamioin CD44 Doxombicin Cantuzumab (mAb) bacterial Pseudornonas in PE38 Brentuximab (inAb) thyl Auristatin F (MMAF); Monomethyl Auristatin E (MMAE) inotuzumab (mAb) Pynolobenzodiazepine QED) transmembrane Glembaturnumab (mAb) Interleuldn— 10 (1110) (ant-i— glycoprotein NMCB inflammatory) (GPNMB) CD56 Lorvotuzumab (mAb) Diphtheria toxin CanAg huCZ42 (mAb) Tunior necroris factor (TNF) luteinizing honnone [DaLys(6)] LHRH RNase releasing hormone (LI-RH) receptor Pro state~ Specific membrane Yttrium;U antigen (PSMA) CD74 Milatuzumab (mAb) Iodine 131 CD70 Lutetium177 AGS— 16 poiine Integrin Methotrexate CD19 Taxanes, e.g., axel or Dooetaxel Interleuldn 2 receptor Interleukin—2 (Proieuldn) CD3 UCHTl (mAb) extra domain B of LlQ—SIP (soFV filSCd in ,fibroneotin with the constant domain SLAMF7 (CD319) Elotuzumab (ruAb) Indatmcimab (mAb) Trastuzumab (mAb) Gemtuzurnab (mAb) According to yet another embodimmit of the t invention, the immunoligand comprises at least two subunits each being conjugated to apayload.

Preferably, at least two different payloads can be conjugated to the at least two subunits.

This option provides a Versatile toolbox with which a large Vaiiety of different irnmunoligand‘payload constructs can be created. For example, a bispecific dual~dcmain immunoligand can be conjugated with tw0 different payloads, for example one marker and one toxin.

Preferably the at least two different payloads are toxic payloads interfering with one or more cellular pathways.

Such embodinmnt can be accomplished, e. g., by conjugating the two different payloads to each the 2 light chains of a full-length antibody, and to the 2 heavy chains of a filll length antibody, respectively, by utilizing two different sortase s, recognizing different different C—terminal ations sortase recognition motifs, plus an antibody that ns at heavy and light chains sing the respective recognition motifs for said different sortase enzymes. is composed of each two In such Way, an dy Drug Conjugate can be created which full~length Ig light chains and Ig heavy , ning different payloads covalently attached to said heavy and light chains. results, preferably, in the synchronous conjugation of the at least two Such embodiment subunits for the tion of nnmunoligand payloads with equal payload conjugation each of said subunits.

According to another preferred embodiment, said immunoligand with at least two subunits is being conjugated with at least 80% efficiency per conjugation site.

According to yet another preferred embodiment, said immunoligand with at least tWO subunits contains a peptide spacer sequence of at least two amino acids, preferably 2-5 amino acids, ed to the nini of at least one of the two subunits This approach results, advantageously, in synchronous conjugation of the at least th subunits for the tion of immunoligand ds with equal payload conjugation to the present inVention, the method each of said subunitsAccording to another embodimmt of allows a stoichiometn'cally defined relationship between immunoligand and payload.

According to this embodiment, a snict quantitatiVe relationship betWeen immunoligand the overall performance of payload can be provided, thus improving the ucibility and the respective immunoligand/payload conjugate particularly for clinical and/or therapeutic applications. This is accounted for by the sequence» and/or site specificity of the transpeptidase used. defined relationship According to aparticularly prefered embodiment said stoichiometrically d C~terminally between immunoligand and payload is achieved by removal ofpartially be carried out via affinity d immunoligand substrate, Such removal can, for example, purification Said approach s preferably, in a homogeneous drug to immunoligand ratio.

Preferably, said removal is carried out by affinity purification using an affinity tag methods positioned ninal to the transpeptidase recognition motif or . Standard imown to the skilled person can be used for this purpose, e.g., HIS tag, CBP tag, CYD HPC (heavy chain of (covalent yet dissociable NorpD peptide) tag, Strep II tag, FLAG tag, protein C) tag, and the GST and MBP protein fusion tags. method allows a site—specific According to another embodiment of the present invention, the it is ensured conjugation of a payload to the immunoligand. According to this embodiment, of the immunoligand, or the that the ation s does not interfere with the ty payload, itself, thus improving the reproducibility and the l performance of the respective. immunoligand/payload conjugate ularly for clinical and/or therapeutic the and/or site specificity of the ations. This is accounted for by sequence~ is not site transpeptidase used. Other than with conventional binding chemistry, which the payload is conjugated to c in most caSes, or has limited site specificity (cg, when like in Arg, Lys, Asn or Gin), the binding site can thus be exactly a free amino group, determined, of the irnmunoligand so that the characterizing features (e.g., target specificity) or the payload (e.g., toxicity) are not affected. obtained with a method The invention finther provides an immunoligand/paylcad conjugate according to the above~mentioned embodiments. the group consisting of an Preferably, said immunoligand/payload conjugate is selected from antibody/drug conjugate, and/or an antibody/marker conjugate.

The inVention further provides the use of an immunoligand/paylcad conjugate according to the above mentioned embodiments for in vitro or in vivo diagnosis of a given pathclogic condition in vitro or in viva prediction or prognosis with respect to a given pathologic condition - the treatment of a human or animal subject suffering from or being at risk developing a given pathologic condition, and/or - research and/or development puiposes Preferably, said pathclogic condition is at least one ed from the group consisting - Neoplastic disease Autoimmune e ' Neurodcgenerative diSease, and/or Infectious disease have In all theSe eaSes, the immunoligand/payload conjugate according to the invention can beneficial effects, e.g, by ing the latter to a specific site, e.g., a cancer cell, a site of neuropathology, or a site of an autoimmune on, The payload, e.g., a toxin, a chemotherapeutic agent, a cytokine or a drug is delivered at said site, eg., to deplete a cancer cell, to act anti—proliferatively on a cancer cell, to diSSOlve a plaque, to inhibit autoantibodies, and the like. the invention can have In all theSe cases, the immunoligand/payload conjugate according to beneficial effects, e.g, by directing the latter to a specific site, e.g., a cancer cell, Where the payloadJ agent, is deliveredJ e. g., to e a cancer cell, e.g. a toxin or a chemotherapeutic to act roliferatively on a cancer cell.

Thus, the systemic toxicity of the toxin or the chemotherapeutic agent is reduced, While the local of the latter at the site of action is increased, thus provding abetter efficacy while side effects are reduced. Further, a respective ling cascade can be inhibited by the binding 2014/0551’73 WO 40317 thus be used to mark a of the immunoligand. In case the payload is a marker the latter can specific site, e.g., a cancer cell charcterized by a given surface antigen ed by the immunoligand, for diagnosis. and l The site—specifity of the conjugating process ensures a high reproducibility performance of the tive immunoligand/payload conjugate particularly clinical and/or therapeutic applications.

The term astic disease", as uSed herein, refers to an abnormal state or ion of cells cell growth or sm. In a more or tissue characterized by rapidly proliferating specific meaning, the term relates to cancerous processes, e. g., tumors and/or leulcemias.

The term "neuropathological diseases” encompasses, among others, neurodegeneratiVe diseases, neuroinflammatory diseases or seizure disorders.

Neurodegenerative diseases or function of are characterized by progressive loss of structure neurons, including death of neurons. Many neurodegenerative diseases including Parkinson's, mer's, Huntington's, Amyoirophic lateral sis and Multiple Sclerosis occur as a result of neurodegeneratlve processes. There are many parallels assemblies as Well betWeen different neurodegenerative disorders including atypical protein can further be found in many different leVels as induced cell death. Neurcdegeneraticn al circuitry ranging from molecular to systemic. diseases” have apartially The terms "Neurodegenerative diseases” and "Nemoinflammatory overlapping scope. Inflammatory responses are ahallmark of neurodegenerative disease participate, or contribute, through ent mechanisms in the neuronal cell death. The these tryptophan catabolism along the Kynurcnine pathway (KP) represents. one of mechanisms. abnormal signaling between Seizure disorders are brain disorders which are characterized by brain cells. Seizure disorders can affect part of the brain (Partial seizures) or the entire brain (Generalized seizures). The most prominent Seizure disorder is epilepsy.

The term ”Autoimmune diSease", as used herein, encompasses specific autoimmune diseases, in which an autoimmune response is directed against a single , such as Crohn's diSease and ulcerative colitis, Type I es mellitus, myasthenia , vitiligo, Graves‘ disease, Hashimoto's disease, Addison' s disease and autoimmune gastritis and ‘ autoimmune hepatitis. The term also encompasses non—organ specific mune diseases, in which an autoimmune response is directed t a component t in several or many organs throughout the body.

Such autoimmune diseascs include, for example, rheumatoid arthritis, disease, systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis and deimatomyositis .

Additional autoimmune diseases include pernicious anemia including some of autoimmune gastritis, primary biliary cirrhosis, autoimmune thrombocytopenia, Sjogren's syndrome, multiple sclerosis and psoriasis. One skilled in the art understands that the methods of the invention can be applied to these or other autoimmune diseases, as desired. disease that The term “infectious disease” as used herein, includes, but is not d to any is caused by an ious organism, Infectious organisms may comprise viruses, (eg, virus single stranded RNA viruses, single stranded DNA viruses, human immunodeficiency (HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV) n~Barr virus (EBV), human oma virus (HPVDppaIasiteS (cg, protozoan and metazoan pathogens such as Plasmodia species, Leishmam'a species, Schistosoma species, osoma species), bacteria (cg, Mycobacteria, in particular, M. tuberculosis, Salmonella, Streptococci, E. coli, Staphylococci), fungi (e,g., Candida species, Aspergillus species), Pneumocystz's carinii, and prions.

The invention fithher provides a a low molecular~vveight payload modified with Glyn— modification, wherein, n>l, preferably n=3 or n=5, As used herein, the term "Gly,,~rnodificaticn” means that an oligo~ or polypepide consisting of n Glycin residues has been added to said payload. As used herein, the term “10W lar~vveight payload nd” shall ass payloads that have a molecular weight of 2500 Da or less.

Saidpayload is, ably, at least one selected from the group consisting of a marker, - aprocessing tag, and/or ~ a drug.

Said marker is at least one selected from the group consisting of - or protein a abel, preferably aradioactively labelled peptide ' or protein, and/or a fluorescent lab e15 preferably a fluorescent e - an enzyme label, preferably aperoxidase.

Said drug is at least one selected from the group consisting of a cytokine aradioactivc agent - atoxin, and/or - a chemotherapeutic agent alreadyJ said toxin is preferably a small molecular toxin, i.e., having a As disuSSed above selected firom the group molecular weight of _<_ 2500 Da, Preferably, said toxin is at least one constisting of p ' Maytansine - Monomethyl auristatin, and/or Alpha~amanitin are shown in or derivatiVes of the former. Examples for such Glyn—modified toxions structures lto 9 ofFig. 14A a 14C low molecular—Weight d The invention r provides the use of a glycine—modified for conjugation thereof to an irnmunoligand.

Preferably, and as ned above, the conjugation is a transpeptidease—mediated conjugation, preferably with a sortase and/or a split . Likewise preferably, immunologand is an antibody.

Preferably, said immunoligand is an antibody. In such way, an antibody drug ate (ADC) can be provided.

Preferably, the immunologand—payload conjugation reaction is performed in crude cell culture Supernatant. This means that, preferably, the conjugation reaction may take place With unpurified or only partially purified components.

Experiments and Figures in detail in the drawings and While the inVention has been illustrated and described foregoing description, such ration and description are to be considered illustrative ary and not restrictive; the invention is not limited to the disclOSed embodiments.

Other variations to the disclosed embodiinents can be understood and effected by those skilled in the art in cing the claimed invention, from a study of the drawings, the the word "comprising" does not exclude disclosure, and the appended claims. In the claims, other elements or steps, and the indefinite article ”a" or ”an” does not exclude a plurality. claims does The mere fact that certain measures are recited in mutually different dependent of these measures cannot be used to advantage. Any not indicate that a combination construed as limiting the scope. reference signs in the claims should notbe All amino acid sequences disclosed‘herein to C—terminus; are shown from N~terminus ‘—>3'. nucleic acid sequences disclosed herein are shown 5 of monoclonal e 1: Cloning of sion vectors and expression a CD19 antibody with C~terminal LPETG sortase tag and additional 6x—His and strepll affinity purification tags In order the C~terminal conjugation of a payload to an dy, first a to perform recombinant antibody needs to he expressed that contains C—terminal modifications, including arecognition motif, e.g. for sortase A of Staphylococcus aureus.

For this, first ORFs for heavy and light chains of an anti~human CD 19 specific antibody be gene synthesized, e.g. at contract research organizations (CROs) offering such gene synthesis services, like e. g. Genscript (WWWgenscriptcom, away, NJ, USA). As an example, the heavy and light chain Sequences of a humanized un1an CD 19 antibody hBUl2 can be found in patent US 8,242,252 B2 under Seq 53 (variant HF) and Seq 5 8 follows: (variant LG), The v H and v L regions of this anti~human CD19 antibody are as '19 antihody hBU12): SEQ ID NO 1(vH coding region cfhurnaniZed anti—human CD ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCATTGTCAGGTTCAGCTGCAAGA GTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCA CTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGG GATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTT TAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCT ACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCA This translates to the ing amino acid sequence (SEQ ID NO 2): MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWW DDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSS ' SEQ ID NO 3 19 antibody hBUlZ) (v L coding region ofhumanized anti-human CD ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGAAATTGTTCTCACCCA GTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTT ACATGGACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTEATGACACATCCAAACTGGCTTCT CCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCGAGA TGCTGTCTATTACTGTTTTCAGGGGAGTGTAIACCCATTCACTTTTGGCCAAGGGACAAAGTTGGAAA TCAAA This translates to the following amino acid ce (SEQ JD NO 4): MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEIK These sequences can be fused to human IgGi constant heavy and constant light chain realize the method disclosed regions containing additional C~tenninal tags, in order to herein.

WO 40317 In order to realize the invention, the human constant IgGl heavy chain region can be sortase synthesized with additional 3'~codons, encoding an LPETGSz‘aphylococcus aureus and a A recognition tag, ed by a 6XHis tag (HIE-IHHH), a MYC~tag (EQKLISEEDLI follows: strep II tag (WSHPQFEK) resulting in a sequence, which is as 3' extension SEQ ID NO 5 (human IgG1 heavy chain constant coding region with me encoding an LPETG sortase tag, an 6XI-Iis tag and a strepII tag): AGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCGAGCAACACCAAGGTGGACAAGAAAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGAECTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAICTCCAAAGCCAAA GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACAECGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAA GAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGA TCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAG amino acids of the tags This translates to the following amino acid sequence (SEQ ID NO 6 , are underlined): STKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKESNTKVDKKVEPKSCDKEHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SEEK QVYTLPPSRDELTKNQVSLTCLVKGFYPSD:AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGHHHHHHGEQKLI SEEDLGWSHPQFEK- Fuitheirnore, the human constant IgGl kappa light chain region can be synthesized with onal 3‘~codons, encoding an LPETG Staphylococcus auraus sortase A recognition tag, which is followed by a 6XHis tag and a strep II tag (WSHPQFEK) ing in a sequence, as follows: SEQ ID NO 7 (human IgG1 kappa light chain constant coding region with in~frame and a sirepll tag): extension encoding an LPETG sortase tag, an 6XHis tag, a Myc tag, ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA CAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCA GCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAG This translates to the ing amino acid sequence (SEQ ID NO 8, amino acids of the tags are underlined): SKDSTYSLSSTLTLS TVAAPSVFEPPSDEQLKSGTASVVCLLNNFWREAKVQWKVDNALQSGNSQESVTEQD KADYEKHKVYACEVTHQ‘GLSSPVTKSFNRGECLPETGI—lHI—IHHHGEQKL I SEEDLGWSHPQFEK )) ' The coding regions for LPETG sortase tag, 6XHis and strepll tagged heavy complete light chains of the humanized anti—human CD19 antibody hBUlZ are then as s: SEQ ID NO 9 (Complete human IgGl VH-CHheavy chain coding region for hBUlZ with C~ terminal LPETG sortase tag, 6xH1's tag, Myc tag, and a strepll tag): CTGCAAGA ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTG’I‘CCAT’I‘GTCAG GTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCA CTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATI'TGGTGG CAGTT GATGATGACAA GAGATATAA CCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTGZAAGAAC GTGCTAGAATGGAACT’I‘TGGTCCT TAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTAC'I‘ACT ACTATT'ITGACTACTGGGGCCAAGGCACCC’I’I‘GTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC TGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT CTGC CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT AACGTGAATCACAAGCCGAGCAACACCAAGGTGGACAAGAAAG’ITGAGCCCAAATCTTGTGACAAAACTCACAC ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA GAAGACCCTGAGGTCAAG CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAGGAGCAGTACAACAGCAC TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT CCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG’I‘G TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC T'I‘CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTC‘I‘CCGGGTAA ACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGA GCCACCCCCAGTTCGAGAAGTAG This translates to the ing amino acid ce (SEQ ID NO 10, amino acids of the tags are underlined): STSGMGVGWIRQHPGKGLEWIGHIWW FLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSE DDDKRYNPALKSRVTI SVDTSKNQFSLKLS TAVYYCAMLWSYYFDYWGQGTLVTvsSASTKGPSVF GLYSLS SWTVPS s SLGTQTYIC PLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSEEDPEVK FNWYVDGVEVINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAEEKT I SKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSi)GSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKLPETGHHHIHMGEQKL 1 SEEDLGWSI—[PQFEK .

SEQ D3 NO 11 (Complete human IgG1 VL—CLkappa chain coding region for hBU12 with C~terminal LPETG sortase tag, 6XHis tag, Myc tag, and a strepll tag): ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGAIGTTCTGGATTCCTGCTTCCAGCAGTGAAATTGTTCTCACCCA GTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTT ACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTATGACACATCCAAACTGGCTTCT GGTATTCCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGA GGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGGCCAAGGGACAAAGTTGGAAA TCAAAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAAQTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAA GCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAG This ates to the following amino acid ce (SEQ ID NO 12, amino acids of the tags are underlined): MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECLPETGHHHHHHGEQKLISEEDLGWSHPQFEK' CD19 specific antibody The coding regions for the heavy and light chains of the anti~human as disclosed in SEQ 1D NOS 9 and 11, respectively, can then be synthesized with flanking restriction enzyme sites (eg. Hindlll and Notl) such that they can be cloned into a standard mammalian expression vector, such standard as _pCDNA3.l~hygico (+) (Invitrogen), by molecular biology methods known in the art.

The complete DNA sequence of pCDNA3.l~hyg1‘o (+)~IgI-I chain expression vector for the tagged hBUlZ anti~hu1nan CD19 antibody Will be as follows: SEQ ID NO 13 (coding region of human IgG1 VH—CH heavy chain for hBUlZ vvith C— and Hindlll and NotI terminal LPETG e tag, 6xHis tag and a strepll tag underlined, cloning sites shaded): GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAAECTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTAETAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC TGGCCCGCCTGGCATTAEGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGGTTATCGAAATTAATACGACTCACTATAGGGAGACCC AAGCTGGCTAGCGTTTAAACTTWCCATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGC AGGTGTCCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGA CTTGTACTGTGTCTGGGGGT’I'CAATGAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAG GGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGAC AATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCT ACTACTGTGCTAGAATGGAACTTTGG’I‘CCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCC TCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCOTCCAAGAGCACCTCTGGGGGCACAGCTGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCG ACACCTTCCCGGCTGTCCTACAGTCCTGAGGACTCTACTCCC’I‘CAGCAGCGTGGTGACCGTGCCCTCC TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGA AGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAWWTCGAGTCTAGAGG GCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT’I’CCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAG CAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATC CCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCT’I‘TCCCCGTCAAGC TCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCT’I‘TACGGCACCTCGACCCCAAAAAACTTGATTAGG GTGATGGT’I‘CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGAT TTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAA TGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAAT TAGTCAGCAACCAGGTGTGGAAAGTCCCGAGGCTCCCGAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTA GTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC CCCATGGCTGACTAATTTTTTTTATITATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAG TGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGC’ITGTATATCCATTTTCGGATCTGA CGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGT’ITCTGATCGAAAAGTTCGACAGCG TCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGC!TTCGATGTAGGAGGGCGTGGATAT CGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTI‘GCATCGGCCGC GCTCCCGA’I‘TCCGGAAGTGCTTGACA’ITGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCAC AGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGAT GCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACAC TACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCG TCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTC GTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGA GGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGC AGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGC ATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATG CGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGA CCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAG CACGTGCTACGAGAT’I‘TCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGC CGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTT ATAATGGTTACAAATAAAGCAATAGCATCACAAAT’I‘TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGC'ITGGCGTAATC ATGGTCATAGCTGTTTCCTGTGTGAAATTGT’I‘ATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAA AGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA’I‘TGGGCG CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG GCGAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGAGGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC’I‘CCCTTCGGGAAGCGTGGCG CTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGA ACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT TATCGCCACTGGCAGCAGCCACTGGTAACAGGATIAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTAITTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC AGCAGAITACGCGCAGAAAAAAAGGAICTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGAETTTGGTCAIGAGATTATCAAAAAGGAECTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTLAAATCAATCTAAAGTATAIATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTG AGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAAIGATACCGCGAGAQCCACGCTCACCGGCTCCAGATTT AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG CCGCAGTGTTATCACTCATGGTTATGGGAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAIAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAAIACCGCGCCACATAGCAGAACTTIAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAIGCCGCAAAAAAGGGAATAAG GGCGACACGGAAAIGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC TCAIGAGCGGATACATATTTGAATGTAITIAGAAAAATAAACAAAIAGGGGTTCCGCGCACAITTCCCCGAAAA GTGCCACCTGACGTC The complete DNA sequence of pCDNA3.l~hyg1*o (+)~IgL chain expression vector for the tagged hBUlZ anti—human CD19 antibody will be as follows: ' for hBUl2 with C~ SEQ ID NO 14 (coding region ofhuman IgG1 VL~CL kappa light chain and Hindlll terminal LPETG sortase tag, 6XHls tag, Myo tag, and a strepll tag underlined, and Notl .ng sites ): GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACICTCAGTACAATCTGCTCTGATGCCGCATAG’ITAAGC CTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG’ITCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GAOGTCAATAATGACGTATGT‘I‘CCCATAGTAACGCCAATAGGGAC’ITTCCATTGACGTCAATGGGTGGAC’I‘ATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAG—TACGCCCCCTATTGACGTCAATGAC TGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT’I'GGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG’ITTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCA'ITGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGC'ITATCGAAATTAATACGACTCACTATAGGGAGACCC AAGCTGGCTAGCGTTTAAACTTEEEWCATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCC TGCTTCCAGCAGTGAAATTGTTCFfCAEJ—CCAGTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCC TGAGCTGCAGTGCCAGC’I‘CAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTC CTGATTTATGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGG’ITCAGTGGCAGTGGGTCTGGAACAGATTT TACACTCACAATCAGCAGCCTGGAGCGAGAGGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCAT TCACTTITGGCCAAGGGACAAAG’ITGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCTI‘CATCTTCCCGCCA TCTGATGAGCAGTI‘GAAATCTGGAACTGCCTCTGTTGTGTGCC’I‘GCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGG CCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCG AGAAGTAWWTCGAGTCTAGAGGGccCGTTTAAACcceoTeATCAGCCTCeACIGTGcCTTCTAGTTG CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT’I‘CCTTGACCCTGGAAGGTGCCACTCCCACTG’I‘CCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATI‘CTATTCTGGGGGGTGGGGTGGGGCAGGAC AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGA AAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG TTACGCGCAGCGTGACCGCTACAC’I‘TGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTC GCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGA'ITTAGTGCTTTACG GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTC GCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC GATTTAACA TCGGTCTATTCTTTTGATTTATAAGGGATT’ITGGGGATTTCGGCCTATTGGTTAAAAAATGAGCT AAAAT’ITAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCC GCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCT AACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTI‘ATGCAGAGGCCGAGGCCG CCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCG GTCTGTCGA GGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGAC GTGCTT GAAGTI‘TCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTC CGATGGTTTCTACAAAGATCGT .TCAGCTTCGATGTAGGAGGGCGTGGA’I‘A’I'GTCCTGCGGGTAAATAGCTGCGC TATG'I'I‘TATCGGCACTTTGCATCGGCCGCGCTCCCGA'I'I‘CCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAG AACCGAACTGCCCGCTG CCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCT TTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCA GCGCGATTGCTGATCCCCATGTGTA TTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATAT TCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGG CCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGC CGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATI‘CCCAATACGAGGTCGCCAACATCTTCTT CTGGAGGCCGTGGTTGGCTTGTA’I‘GGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGAT CGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAA’ITTC GCAGCT'I‘GGGCGCAGGGTCGA’I‘GCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACA AATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCC CCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGG TTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTT CGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATI‘TCACAAATA AAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCG GTTATCCGCTCACAAT TCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT GAGCTAACTCACA’I‘TAA TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGT TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT CCGCCT'ITCTCCCTTCGGGAAGCGTGGCGC’ITTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACI‘GGTAACAGGATTAGCAGAGCGA GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGT CACCGC ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC GATCTCAAGAAGATCCTTTGA TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAG CATGAGATTATCAAAA CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT ACTTG AGGATCTTCACCTAGATCC’ITTTAAA’ITAAAAATGAAGTTTTAAATCAATCTAAAGTATATAT GTCTGACAGTTACCAATGC’I‘TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA'I'ITCGTTCATCCATAGTTG CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG CAGCCGGAAGGGCCGAGCGCAGAAGTGG CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC ’I‘CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG’ITCGCCAGTTA ATAGTTTGCGCAACG’ITGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG GCATAATTCTC GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACT CATTCTGAGAATAGTGT TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA.

AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC WO 40317 like eg‘ TheSe constructs allow upon transfection into mammalian cells, - but not limited to the sion of CEO cells, that are typically used for recombinant antibody sion, the anti~human CD 19 specific humanized antibody hBUlZ with C~te1minal additions of a atboth the IgH and IgL chains. sortase Atag, a 6XHis tag, a Myc tag, and a strepll tag Example 2.: Cloning of expression Vectors for monoclonal antibody with C-terminal N~ C~terninal 6xHis and strepll intein domain of SsP GyrB 11 split~intein With additional y purification tags aureus smtase A Similar to the design of expression cassettes and vectors of Staphylococcus C~te1minal fusion of N~intein tagged IgGl heavy and light chains, the coding regions for a domain of Ssp GyrB 11 split~intein to either the IgH and IgL chain can be designed follows, in order to gene synthesize the genes by a qualified CRO (e.g. Genscn'pt away, NJ, USA), with the same elements for the anti~human (WWWgensciipteom, CD19 antibody as disclosed further above.

The 150 amino acid sequence of the N~intein domain of Ssp GyrB 11 split~intein can be found in apublication by Appleby et al. (2009), and is as follows: SEQ ID NO 15 (Naintein domain of Ssp GyrB 11 split~intein): CFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGS IGVEKIINARKTKTNAKVIKVTLDNGES IICTP RDGSYKCAMDLTLDDSLMPLHRKI STTEDSGHMEAVLNYNHRIVNIEAVSETIDVYDIEVPHTHNFAL mammalian codon usage will result in Reverse translation ofthat amino acid ce with ll split~intein as follows: the coding sequence for the N-intein domain of Ssp GyrB SEQ ID NO 16 (endocing sequence for N~intein domain of Ssp GyrB 11 Split~intein)2 TGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGA GAAGGAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACG CCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCC GACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCC CAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCG TGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTG GCCAGC With this sequence ation at hand, the complete IgGl heavy chain coding region anti~human CD19 antibody hBUlZ with C—temn‘nal extension, comprising the N~intein domain of Ssp GyrB 11 split~intein, folloWed by a Gxflis-tag and a strepll tag can be designed as sed in SEQ ID NO 17 below: ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCAGGTTCAGCTGCAAGA GTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCA CTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGG GATGAEGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTT TAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTAGTGTGCTAGAATGGAACTTTGGTCCT TTGACEACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC TGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACQAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACAC ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG .TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT CCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAICTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC TGGACTGCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAA ATGCTTCAGCGGCGACACCCTGGTGGCCCTGAGCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGG AGAAGGAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAAC GCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCC CGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGAGCCTGGACGACAGCCTGATGC ACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATC GTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCT GGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAGAAGTAG acids of the N—intein domain This translates to amino acid sequence SEQ ID NO 18 (amino are shaded): are underlined, 6xHis tag and l tag LIFLVETLKGVQCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWW DDDKRXNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGfiagFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGS IGVEKIIN‘ STTEDSGHMEAVLNYNHRI ARKTKENAKVIKVTLDNGES IICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKI yNIEAVSETIDVYDZZEVPHTHNE‘A : Lass; anti~human CD 19 antibody Likewise, a complete IgGl kappa light chain coding region for hBU12 with ina1 extension, comprising the N—intein domain of Ssp GyrB 11 split~ disclosed in SEQ ID NO intein, followed by a 6XHis~tag and a snepll tag can be designed as 19 below: PCT[EP2014/055173 ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTGACATTGTGCTGACCCA ATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATT TTGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAAGTCCTCATCTATGCTGCA TCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCA TCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATGCCAGAGAGGCCAAAGTACAGTGGAAGGT GGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCC GCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTTCAGCGGCGACACCCTGGTGGCCCTGAC CGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCA TCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATC AAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTA CAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACA GCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGAC GTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCAIGGCTGGAG CCACCCCCAGTTCGAGAAGTAG of the N~intein domain This translates to amino acid sequence SEQ ID NO 20 (amino acids are underlined, 6XHlS tag and streplI tag are shaded): MNFGLRLIFLVLTLKGVQCDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAA SNLEEGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ TKSFNRGECFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGS IGVEKIINARKTKTNAKVI LTLDDSLMPLHRKI STTEDSGHMEAVLNYNHRIVNIEAVSETID The coding regions for the N~intein modified heavy and light chains of the uman 17 and 19, respectively, can then be CD19 specific antibody as sed in SEQ ID NOs synthesized with flanking restdctlon enzyme sites (e. g. I-Iindlll and Natl) such that they can be cloned into a standard mammalian expression vector, such as pCDNA3.l-hygro (+) (Invitrogen), by standard molecular biology methods known in the art.

The complete DNA sequence of pCDNA3.l—hygro (+)~IgH chain expression vector for the follows: NHintein tagged hBU12 anti-human CD19 antibody is then as SEQ ID NO 21 g region of human IgGl VH—CH heavy chain for hBUlZ with C- terminal N—intein domain of Ssp GyrB Sll split intein, folloWed by 6xHis tag Strepll tag (underlined), and Hindlll and Notl g sites (shaded)): GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAETAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAGGACCCCCGCCCATT AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGGCAAGTACGCCCCCTATTGACGTCAATGAC 2014/055173 GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTFATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTITGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC AAGcmGCTAGCGTTTAAACTTKXEE‘TTCCATGAAHTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAA AGGCGTCCAGTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTI‘GGTTAAGCCCTCCCAGACCCTCAGTCTGA CTTGTACTGTGTCTGGGGGITCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAG GGTCTGGAGTGGATTGGACACA’ITTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGAC AATCTCTGTGGATACCTCCMGAACCAGWAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGC’I‘G’I‘CT .

ACTACTGTGCTAGAATGGAACTTTGGTCCTACTATI‘TTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCC TCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCT’I’CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT’I‘CTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC ACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAA GCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGAC GGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCT GGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCA LGEACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATG GAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACAT CGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGT TCGAGAAGTAEE?fl???EfiETCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCT’I‘CTAG CAGCCATCTGTTG’fTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCITCTGAGGC GGAAAGAACCAGCTGGGGC’I‘CTAGGGGGTATCCCCACGCGCCCTG’I‘AGCGGCGCATTAAGCGCGGCGGGTGTGG TGG’I‘TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC’ITT CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGG’ITCCGATTTAGTGCTTT ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAG’I‘GGACTCTTGTTCCAAACTGGAACAACACTCAACCCT ATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTAA’ITCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGG GAAGTA'I‘GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCA AGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCC CCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTI'TTTTI‘ATITATGCAGAGGCCGAGG CCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTC CCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGT CGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTG CTTTCAGCTTCGATGTAGGAGGGCGTGGATA’I‘GTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGAT CGTTATGTTTATCGGCACT’ITGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGA GAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCG CTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGC CCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGA’ITTCATATGCGCGATTGCTGATCCCCATGT GTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTI‘ GGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAAT GGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTT CTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAG GATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGG’I‘TGACGGCAAT TTCGATGATGCAGCTTGGGCGCAGGG’I‘CGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTAC ACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGAG GCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTI‘CGATTCCACCGCCGCCTTC'I‘ATGAA AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTT WO 40317 CCACCCCAAC’I‘TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA CATCAATGTATCTTATCATGTCTGTATA ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGG’ITTGTCCAAACT CCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT’I‘ATCCGCTCAC ACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT CGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT’I‘AATGAATCGGCCAA CGCGCGGGGAGAGGCGG’HTGCGTATTGGGCGCTCTTCCGC’I'TCCTCGCTCACTGACTCGCTGCGCTCGGTCGT TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT’I‘GCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGA'I‘ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT’I‘ACCGGATACC TGTCCGCCI‘TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTG’I‘AGGTATCTCAGTTCGGTGTAG GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA GATTAGCAGAG TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GACAGTATTT CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG’ITGGTAGCTCTTGATCCGGCAAACAAAC CGCTGGTAGCGGTGGTTITTTTG’ITTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG‘ITAAGGGATTTTGGTCATGAGATTATCA AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC CTGTCTATTTCGTTCATCCATAG TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTI‘ACCATCTGGCCCCAGTGCTGCAATGATA GCCGGAAGGGCCGAGCGCAGAAG CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAG TTAATAGTTI‘GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA GCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT’I‘ CACTGCATAATT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAG CTCT'I‘ACTGTCATGCCATCCGTAAGATGCTI‘TTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG CAGAAC’I‘TTAAA TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAG GCTGT‘I‘GAGATCCAGTTCGA AGTGCTCATCATTGGAAAACGTTC’ITCGGGGCGAAAACTCTCAAGGATCTTACC TGTAACCCACTCGTGCACCCAACTGATCT’I‘CAGCATCTTTTACTT‘I'CACCAGCGTTTCTGGGTGAGCAAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCT’I‘TTTCA ATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC The complete DNA Sequence of pCDNA3.l~hygro L chain expression vector for the Ssp GyrB Sll N—intein domain tagged hBUl2 antl~human CD19 antibody will be as follows: light chain for hBU12 with C~ SEQ ID NO 22 (coding region of human IgGl VL~CL kappa tenninal Ssp GyrB Sll N~intein domain, 6xHis tag and a strepfltag underlined, and Hindlll and Notl cloning sites shaded): GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTG'I‘TGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA CAATTACGGGGTCATTAGTTCATAGCCCA GATATACGCGTTGACATTGATTATTGACTAGTTATI‘AATAGTAAT TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GAGGTCAATAATGACGTATGTTCGCATAGTAACGCCAATAGGGACTTI‘CCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTT’ITGGCAGTACATCAATGGGCGTGGATAGCGGTI‘TGACT GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAA’I‘GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC AAGCTGGCTAGCGTTTAAACTTWCATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAA AGGCGTCCAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCA TCTCCTGCAAGGCCAGCCAAAGTGT’I‘GAITTTGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGA CAGCCACCCAAAGTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGG GTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAA GTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCA’I‘CTGTC ITCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG CTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGA AGCAGGGCAAGCAGAACTICTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCC AGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGA CCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCC TGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTG AACATCGAGGCCG’I‘GAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGC CCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAGAAGTAWfiCTCGAGTCTAGAGGG CCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCI'I‘CTAGTTGCCAGCCATCTGTI‘GTTTGCCCCTCCCCCGT GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCA’ITCTAT'I‘CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTA’I‘CC GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACITGCCA GCGCCCTAGCGCCCGCTCCITTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT CTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCITTACGGCACCTCGACCCCAAAAAACTTGATTAGGG TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTITTTCGCCCT’ITGACGTTGGAGTCCACGTTCTTTA ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTT’I‘GATTTATAAGGGATT TTGGGGATTTCGGCCTATTGG'ITAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA’I’I‘AATTCTGTGGAAT GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT AGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAG TCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA’ITCTCCGCC CTGACTAATTTTTTI‘TATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGT GAGGAGGCTTT’ITTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTITCGGATCTGAT GTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGT CTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAA’I‘CTCGTGCTTTCAGCT’I‘CGATGTAGGAGGGCGTGGATATG TCCTGCGGGTAAATAGCTGCGCCGATGGTITCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCG C'I‘CCCGATTCCGGAAGTGCTTGACA’ITGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACA GGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATG , CGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACT ACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGT CAGTGCG’I‘CCGTCGCGCAGGCTCTCGATGAGCTGATGC’I'I‘TGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCG CGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAG GCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCA ‘ GCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCAITGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGC GACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGAC CGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGC ACGTGCTACGAGAT’ITCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGC’ITCGGAATCGTTTTCCGGGACGCC GGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTA TTACAAATAAAGCAATAGCATCACAAA’ITTCACAAATAAAGCATITTFITGACTGCATI‘CTAGTTGTG GTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGAGCTCTAGCTAGAGCTTGGCGTAATCA TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGOGCGGGGAGAGGCGGTTI‘GCGTATTGGGCGC TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGITATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCG’ITGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCT CGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGAGCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA AGTGGTGGCCTAACTACGGCTACACTAGAAGGA‘CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTITGCAAGCA GCAGAT’I‘ACGCGCAGAAAAAAAGGA’I‘CTCAAGAAGATCCTT’I‘GATCTTTTCTACGGGGTCTGACGCTCAGTGGA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA GGCACCTATCTCAGCGATCTGTCTATTTCGTTCAECCATAGTTGCCTGACTCCCCGTCGTGTAGAEAACTACGA TACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTC TATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAGTGTCATGCCATCCGTAAGATGCTTTT CTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG TCAATACGGGAIAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGAECCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAG TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAEATTATTGAAGCATTTAECAGGGTTATTGTCT CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG TGCCACCTGACGTC 21 and 22 These pcDNA3.1~hygTo(+) based expression vectors disclosed in SEQ ID N03 CHO cells, that are allow upon transfection into mammalian cells, like eg. but not limited to anti~human CD19 typically used for inant antibody expression, the expression of the followed by a specific humanized antibody hBUl2 with inal N~intein domain fused, 6XI-Iis tag and a strepll tag at both the IgH and IgL chains.

Example 3 A enzyme from : Cloning and expression of recombinant Sortase Staphylococcus aureus.

The ORF of Sortase A from lococcus aureus is published in Genbank and can be found under reads is shown as SEQ ID entry: AF162687.1. The aa~sequence in that record NO 23 (amino acid ce of sortase A from Staphylococcus aureus): MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAG YIEIPDADIKEPVYPGPAIPEQLNRGVSFAEENESLDDQNI SIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGN ETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK ID NO The eonesponding nucleotide sequence inthis Genbanlc entry is provided as SEQ ATGAAAAAATGGACAAATCGATTAATGACAATCGCTGGTGTGGTACTTATCCTAGTGGCAGCATATTTGTTTGC TAAACCACATATCGATAAETATCTTCACGATAAAGATAAAGATGAAAAGATTGAACAATATGATAAAAATGTAA AAGAACAGGCGAGTAAAGATAAAAAGCAGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGC TAIATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAATAG AGGTGTAAGCTTTGCAGAAGAAAAEGAATCACTAGATGATCAAAATAETTCAATTGCAGGACACACTTTCATTG CGAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGGTGTACTTTAAAGTTGGTAAT CGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAA AGGTAAAGATAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTA AAATCTTTGTAGCTACAGAAGTCAAATAA active fragment of Technical information With respect to the expression of an enzymatically recombinant sortase A in E.coli, comprising amino acids 60~205 with 6xHis tag are disclosed inreference W02007/108013A2. The coding region for a 6XHis tagged Version of Staphylococcus s Sortase A (aa60~205) is provided below as SEQ ID NO 25: ATGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAAATTCCAGATGCTGATAT TAAAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAA’I‘AGAGGTGTAAGCTTTGCAGAAGAAAATG AATCACTAGATGATCAAAATA’ITTCAATTGCAGGACACACTTTCATTGACCGTCCGAACTATCAATTTACAAAT CTTAAAGCAGCCAAAAAAGGTAGTATGGTG‘I‘ACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAG TATAAGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAA TTACTTGTGATGATI‘ACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTI‘TGTAGCTACAGAAGTCAAA CACCATCACCATCACCATTAA This translates to amino acid sequence SEQ ID NO 26: MQAKPQII'KDKSKVAGYIE IPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNI S I AGHTFI DRPNYQFTN LKAAICKGSMVYFKVGNETRKYKMTS IRDVKPTDVGVLDEQKGKDKQLTL I TCDDYNFKTGVWEKRKIFVATEVK HHBHHH' The coding region for the 6xHis tagged sortase A fragment of Staphylococcus aureus, as provided in SEQ ID NO 25, can be cloned into a standard ial expression vector, like e.g. pET29 (Novagen), in order to transform E. coli strain BL21(DE3) en) and to scitase generate an E. coli clone that can be used for the ial production of recombinant art. In short, E. coli BL21(DE3) transformed A according to standard s known in the with pET29 expression plasmids for sortase A can be cultured at 37 °C in LB medium With IPTG can then be added to a 50 ug/mL kanamycin until until an OD 600 = 8 is reached. at 30 final concentration of 0.4 mM and protein expression can be induced for three hours °C. The cells can then be harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgC12, 2 units/mL DNAsel (NEE), 260 nM aprotinin, 1.2 uM leupeptin, and lmM PMSF). Cells can then be lysed by Sonication and clarified supernatant can then be purified on Ni~NTA agaIOSe ing cturer‘s instructions. ons that are of >90% purity, as judged by GE, can then be conSolidated and dialyzed against uffered saline (25 mM Tris pH 7 .5, 150 mM NaCl), and the enzyme concentration can be calculated from the measured A230 using the published extinction and ca. 20‘ coefficient of 17,420 M“ crn'l.The above—mentioned protocol has been followed mg of > 90% pure recombinant enzymatically active fragment (of ca. 17 kD) sortaSe A of Staphylococcus aureus has been produced and the analysis of the recombinant protein by SDS~PAGE and Western blotting is disclosed in Fig. 7.

Example 4: Expression and purification of sortase tagged or N~intein tagged recombinant antibodies in CHO cells a.) CHO cell expression: Expression of recombinant IgG1 antibodies from the expression transfection using constructs disclosed under Examples 2 and 3 can be achieved by transient c. g. comrnercially available CHO expression systems, like the FreeStyle CHO system from Invitrogen following the ctions ofthe yle CHO .

In brief, about 1 day prior to transfection, CHO cells shall be seeded at 5—6 x105 cells/ml in FreeStyle CHO medium in shaker~flasks in order to expand them at 120 rpm on an orbital shaker at 37°C in a humidified incubator at 7.5% CO The following day the 2 atmosphere. cells can be transfected, when they reach a density of 1.2—1. 5x10 5/1111. Cells then need to be added to a 125 ml diluted to 1X10 5 cells/ml. 30 ml of such a cell suspension then needs to be added to 600ul shake flask and 40 ug of 1:1 mixed IgH and IgL expression plasmid DNA is OptiPro SF—medium (Invitrogen). At the same time, 40ul of FreeStyle MAX ection both samples need to be gently reagent needs to be added to 600u1 Optil’ro SF~medium, and mixed, and incubated for 10 min at room temperature to allow DNA~transfection reagent to the 125 complexes to form. Then the DNA—transfection t mix can be added slowly ml CHO cell culture from above and the transfected cells are then grown for up to 6 days at at 7.5% CO 120 rpm on an orbital shaker at 37°C in ahunn'dified incubator 2 atmosphere. fter, cell culture supernatant can be collected and analyzed for antibody expression titer by appropriate methods known in the art , Lurninex, etc). b.) Protein A purification: Protein A purification of recombinant antibodies fiom the CH0 cell supernatant can be performed with commercially available protein A cse columns o Fisher, Pierce) according to instructions from the manufacturer.

A column of appropriate size In brief, cleared cell culture supernatant is run over a protein and capacity equilibrated with PBS. al medium is washed with PBS and eventually bound IgG can be eluted with low pH buffer, like 0.1 M citric acid—NaOH, pH 3.0. Eluted l, pH7.4. Combined IgG should be neutralized immediately with l/lOth volume of 1M 4°C. fractions ning IgG can then be dializcd against PBS over night at 2014/055173 The protocols provided in Example 4 provide the skilled perSOn in the art with the antibodies from the instruction to produce sufficient quantities of purified, recombinant constiucts disclosed in Examples 1 and2.

MJVIAE Example 5: Generation of site~specifically C~terminally toxic payload conjugated monoclonal antibodies by sortase and split~intein mediated transp eptidation and a 6 amino acid Monomethyl tin A toxin d to a 5 amino acid glycine stretch SSp GyrB Sll C~int split intein peptide according to the formulas ed below, can custom ordered from qualified chemistry CROS.

Formula 1: 5~glycine modified MlVLAE With VCPAB linker Me =methyl (~CH5) group G = glycine amino acid residue WWHasaceest Formula 2 With 6 amino acid Caintein domain : MlWAE With VCPAB linker, modified GVFVEN and 6 amino acid SAGSGK in peptide Me = methyl (CHE) group stretch GVFVHNSAGSGK = Gly~Val~Phe~Val—His~Asn~Ser~Ala~Gly~Ser~Gly-Lys a.) Toxic MlVlAE payload conjugation of LPETG eA motif tagged recombinant IgG antibodies soitase A Conjugation of 5 glycine amino acid modified MMAE toxic payload to LPETG tagged IgGl antibody (that can be produced by following Examples 1 and 4) can be achieved by mixing appropriate ratios of LPETG tagged IgGl antibody with the glycine— modified MMAE toxin disclosed in Formula 1 (e,g, at 1:1 ratio and 50 uM concentration) and with inant Soitase A ction described in Example 3) (eg. at 5 uM concentration), and using physiologic tion buffer, like e. g. ; 5 mM Tris/Cl, 15 mM NaCl, 6 mM CaClZ, pH 8.0, and incubating at 37°C to 40°C for aminimum of 2 hours. the 6xHis tag Efficiency of the conjugation can be monitored by analyzing the absence of and/or the strepII tag after stopping the reaction, e‘ g. by western~blot is or ELISA with anti~His~tag and/or anti strepll tag antibodies.

Completely conjugated product can be enriched by Niclcel—NTA columns, or streptaotin column binding, which bind to the 6xHis tag or l tag, respectively, which can only present in incompletely reacted IgGl substrate Final IgG~payload conjugate can eventually be purified using protein A purification as described above. b.) Toxic WAE payload conjugation of 88p GyrB Sll N—intein tagged recombinant IgG antibodies Conjugation of Ssp GyrB Sll C~intein amino acid d MAE toxic payload to N~ intein tagged IgGl antibody (that can be produced by following Examples 2 and 4) can be achieved by mixing appropriate ratios of N~intein tagged IgGl dy with the CHintein amino acid—modified leAE toxin disclosed inFormula 2 (cg. at 1:10 or 1:25 ratio at 5 uM concentration of the IgG antibody) using physiologic incubation buffer, like e.g.,' 20 mM Tris/C1, 250 mM NaCi, 1 mM EDTA, pH 8.5, and incubating at room temperature or 37°C aminimum of4 hours. of the 6xHis tag Efficiency of the conjugation can be monitored by analyzing the absence and/or the strepil tag after stopping the reaction, e.g. by western~blot is or ELISA with is~tag and/or anti strepll tag antibodies.

W0 40317 Completely conjugated product can be enriched by Nickel—NTA columns, or streptactin Which can only be column binding, which bind to the 6XHis tag or strepll tag, respectively, present in incompletely reacted IgGl substrate. Final IgG~payload conjugate can eventually be d using protein A purification as described above.

In summary, the Examples 1~5 disalosed above allow a person skilled in the art to ce the invention of enzymatically conjugating a toxic payload site~speeifically to the 0 terminus either using sortase A mediated or split~intein mediated transpeptidation.

Example 6: Production of Trastuzumab With C~terminal GS (glycine—serine) linkerj LPETG Sortase motif and additional 6X~His and Strep II y purification tags either heavy or light chain Antibody expression constmcts encoding monoclonal antibody Trastuzumab (Tras) heavy and light chains, either untagged (SEQ in not: 31 e 34) or C~terminally tagged with GS ID NOs: 35 ~ (glycinewerine) linker, LPETG Sortase tag, 6XHis tag, and Strep II tag (SEQ 38) were generated ially as described inExample 1, Using these expression constructs, C—GS~LHS and Tras~LC~GS~LHS (HC=heavy chain, LC=1ight chain, GS=glycine~ serine, LHS=LPETG~tag+6xlfls~tag+sirepll~tag) were produced in CHO cells by co~ is a Trastuzumab transfection of the corresponding expression constructs Tras~HC—GS~LHS light chain (SEQ ID NOS: 35 ~ 36), and variant With a heavy chain C~ an unmodified terminally tagged with GS (glycine~serine) linker, LPETG Sortase motif, 6XHis—tag, and strepll~tag (SEQ ID NOs: 33 ~ 34). Tras~LC~GS~LHS is a Trastuzumab t with an with unmodified heavy chain (SEQ ID NOs: 31 ~ 32), and a light chain C—terminally tagged GS linker, LPETG Scitase motif, 6xHis~tag, and strepll~tag (SEQ ID'NOs: 37 ~ 38). CHO transfection and affinity pmification of dies cell by nA—sepharose chromatography was done essentially as described in Example 4. chain of Trastuzumab Example 7: Sortase A—mediated ation of heavy or light With odified DMI toxin Conjugation reactions containing Glys~modified DMl toxin (ordered from Concortis, San Diego, CA, US, structure see Fig. 14 a.) and a 171d) recombinant sortase A fragment from Staphylococcus aureus (see Example 3) Were d out with 10.5mg of each monoclonal antibody (mAb) (see Example 6) in 1x Sortase buffer (25mM Tris—HCI, pH8.2,' lSOmM NaCl; 7.5mM CaCl2), as shown in Table II, below. The Tras~HC~GS~LHS ation reaction was ted at 25°C for 211; the C~GS—LHS conjugation reaction was incubated at 25°C for 18h, Each reaction mixture Was then passed over a Strep-Tactin® Sepharose columns (IBA Life—Sciences, gen, Germany). For this, 1ml of Strep~Tactin e was packed under gravity into a fritted column and equilibratcd with 2 column Volumes of equilibration buffer (100 mM Tris~HCL pH 8.0; 150 mM NaCl; lmM EDTA).

Each conjugation e was passed twice down the same column using y flow (to increase residence tune on the resin) The resin was Washed with an additional column volume of equilibration buffer to maximize conjugate yield and the pool then applied immediately to column. For this, a lml Protein A HiTrap column was a protein A equilibrated with 10 column volumes of buffer (25mM sodium phosphate pH 7.5). Each conjugation reaction was then applied to an equilibrated column and the column washed with eluted with 5 column a further 5 column volumes of buffer. Bound conjugate was volumes of elution bufier (0.1M succinic acid, pH 2.8) with 1 column volume fractions collected (into tubes containing 25% V/v M Tris Base to neutralise the acid) and analySed for n content. n containing fiactions were pooled and formulated by G25 column chromatography. For this, NAP 25 columns of an appropriate size for each scale of manufacture were used to formulate the conjugates for long term storage. The columns were equilibrated, loaded and eluted with lOmM Sodium Succinate pH 5.0, lOOmg/mL Trehalose, 0.1% % w/v Polysorbate 20 (Formulation Buffer for Kadcyla® (T~DM1), marketed by Roche/Genentech) according to the manufacturer’s instructions.

The Tras—HGGS—IHS and Tras~LC~GS~LHS DMl gate yields Were, respectively, Protein A and 8.0mg ) and 59mg (56.2%). The major process lOSSes occurred during G25 purification, most probably as a result of peak cutting to ensure maximal concentration of the product for each subsequent step or storage.

Reaction component HC LC Final concentration Tras—HCGS—LHS (5.3mg/ml) 1981u1 ~ SuM LTras—LC—GS~LHS (5.5mg/ml) — 191m SuM H20 l 7775.25u1 7714M a Glys~DMl (ImM) l 140mm l400u1 IOOuM Sortase A (0.85mg/Inl = ca, 43.75n1 175m 0.156/O.625uM 50W) 5X Sortase buffer* 2800ul 2800u1 195 __| and Tras~LC~GS~LHS: Table 2: Conjugation conditions for Tras—HC»GS~LHS and was The drug loading was assessed by Hydrophobic Interaction Chromatography (HIC), at 0.8mL/min with performed on a TOSOH Butyl~NPR 4,6rnm X 3.50131, 2.5 um column run H LSM mapzso 4, ZSmM NaPi, pH=6.95+0.05 a 12 minute linear gradient between A B IPA. The HIC profiles revealed that for both, ~ 75% ZSmM NaPi, pH=6.95+0.05, 25% Tras~HC~GS~LHS and Tras—LC~GS~LHS, there was no detectable unconjugated mAb left, and a major fraction of each mAb was loaded with 2 drugs (see Figure 8).

Trastuzumab~DMl Example 8 :In vitro toxicity assay with sortase A - mediated conjugates Cytotoxieity of DMl~sortaseA~conjugated Tras-HC~GS~LHS and DMl~sortaseA~oonjugated Tras—LOGS—LHS was igated and compared to Kadcyla® (Roche/Genentech) using SKBR3 cells, a human breast cancer cell line oyerexpressing the cognate antigen of trastuzurnab (Tras) HER—Z/neu, and T47D-5R cells, a breast cancer cell line naturally expressing low levels of HER—Z/neu, engineered to be devoid of cell surface HER~2/neu (Graus~Porta et a1. (1995)). Cells were plated on 96 well plates in IOOuI complete DMEM carefully remOVed from (100 00 cells per well). After one day incubation, dium was each well and replaced by SOul of 35—fold serial dilutions of each ADC in complete 0.25ng/ml. Each dilution DMEM, resulting in ADC concentrations ranging from 2CAg/ncl to additional days incubation duplicates cates. After 3 at 37 0C in a was done in or humidified tor at 5% CO were removed from the incubator 2 here, plates IOOuI ter—Glo® equilibrated to room temperature. After approximately 30 s, added to each Well and, after shaking scent Solution ga, Cat.No G7570) was min incubation without shaking, the plates at 450mm for 5 min followed by a time of 1 second luminescence was measured on a Tecan Infinity F200 with an integration SKBRB ADCs were highly cytotoxic for the HER-2mm overexpressing per well. All three R breast cancer cell line breast cancer cell line, but not for the HER—CZ/neu—negative ‘ PCT/EPZOl4/055173 cell line SKBR3 were: (see Fig. 9). The BCso Values for Her»2/neu positiVe breast cancer 32.4ng/1nl;‘ DM1~conjugated C~GS~LHS, 45.6ng/ml; Tras—LC—GS~LHS, Kadcyla®, cell killing 51.4ng/ml, and thus are within similar range of potency in the in vitro tumor with {the Her—2/neu experiment. Conversely, no specific cellular toxicity was detectable negative breast the functional cancer cell line T47D~5R, demonstrating equivalence ally conjugated ADC, . sortaseA, enzymatically conjugated ADC Versus traditional, toxin (DMl) When the comparison entails the same targeting antibody and the same (Fig. 9), ratio of 1.80 However, it that the lower drug~to antibody ca. (deducted fiom intergration of the DARl and DAR2 peaks in Fig. 8) for the Tras—HC~GS~LHS and Tras~ LC~GS~LHS Sortase A~conjugated ADCs, as compared to the DAR of ca. 3—4, reported for inthe in wire Kadcyla® does not translate into aproportionally different cellular cytotoxicity (Fig. 9). This cted finding may be the result of a more tumor cell killing assays A in comparison to defined and site~specific toxin~antibody conjugation mediated by e the less defined, stochastically, chemically conjugated Kadcyla®.

Example 9: Optimization of synchronizetion of sortaseA mediated antibody heavy chain and light chain payload conjugation by variation of peptide—spacer length inserted between C~tern1inal end of antibody heavy chain and light chain and the sortaseA recognition motif the C~terminus of antibody heavy The influence ide~spacer length positioned n motif was investigated. For this, antibody or light chain and LPETG sortase A recognition heavy chain and light chain expression constructs encoding ic CD30~specific mAb A010 heavy and light chains (HC sequence derived from US 2008213289Al, Seql, LC Seq9), C—terminally modified with sequences sequence derived from US 2008213289A1, comprising or not comprising a 2 amino acid GS (glycine~serine) spacer, and comprising a ID 'NOs: 39 LPETG sortaseA recognition motif, and a II purification tag (SEQ ~ 46), have been cloned essentially according to instructions disclosed in Example 1. Using these mAbs AclO~HC~GS~LHS/LC~GS~LHS and AclO~HC~LS/LOLS expression constructs, were produced in CHO cells by nsfection of the corresponding plasmids. AclO~HC~ modified GS—LHS/LC~GS~LHS at the C~ is an A0 10 t with heavy and light chains 6XHis tag, termini of each HC and LC with a GS peptide , a LPETG SoitaseA motif, a and a strep~II tag (SEQ ID NOsz39 — 42; Table 3). AciO—HCLS/LC—LS is an AclO variant LPETG Soltase motif and strep~ with heavy and light chains modified at the C—termini with II tag Without the 2~peptide GS linker (SEQ ID NOs: 43 ~ 46; Table 3). CHO cell transfection and affinity purification of dies by protein A~sepharose chromatography Was done essentially as described in Example 4.

To investigate ncy of conjugation, Serial dilutions of Sortase A Were used to ate penta—glycinemodified FITC (Glys—FITC, see Formula 3 below).

Formula 3 : penta—glycine d FITC (GlyS—FITC) - G = glycine residue For this, Glys~FITC was sortaseA conjugated to two A010 variants in lx SortaSe buffer (ZSmM Tris—HCl, pH8.2; lSOmM NaCl; 7.SmM CaClZ), as shown in Table 4. After 4h at 42°C, on products were analyzed by denaturing, reducing SDS~PAGE gel electrophoresis, and FITC was visualized by placing the gels on a UV box (Fig. 10). of the presence Conjugation to the heavy chain was found to be highly efficient irrespective and LPETG Sortase ition absence of the GS~linker betWeen heavy chain C~te1minus chain was significantly less motif Unexpectedly, sortaseA mediated conjugation to the light Furthermore, it was efficient in comparison to sortaseA mediated heavy chain conjugation. surprisingly found that coupling efficiency was ically affected by the presence (glycine-scrim) the C—terminus of absence of the 2 peptide GS spacer positioned between and the LPETG sortaseA recognition motif. Whereas in the the dy light chains to the light chain took place with about 5—10): lOWer presence of the GS~linken conjugation efficiency than to the heavy Chaim it was about 50—100); less efficient in the absence of a linker. Therefore, it was concluded that increasing the peptide spacer length betWeen the further improve conjugation light chain and the LPETG Sortase recognition motif might efficiency. between light chain Therefore, the influence of increasing the length of the e spacer and LPETG Sortase A recognition motif on conjugation efficacy was investigated next.

Expression constructs encoding mAb AclO light chains, C~terminally tagged with LPETG 2 to 5 amino acid linker (SEQ Sortase recognition motif and strep—II purification tag, with a in e 1. Using these ID N03: 47 as deScribed ~ 54), were generated essentially AclO~HC~LS/LC~GGS~LS, AclO— expression constructs, mAbs AclO—HOLS/LC—GSLS, HC—LS/LC—GGGS~LS and AclO-HC~LS/LC~GGGGS~LS Were produced in CHO cells by cowtransfection of the corresponding expression constructs. In each of these antibodies, the motif and a strep— heavy chain is inally modified with an LPETG Sortase recognition modified H purification tag (SEQ ID NOS: 43 ~ 44; Table 3). The light chain is ninally with an LPETG e tag and strepdl tag containing either a GS, GGS, GGGS, or a motif CHO GGGGS peptide spacer (SEQ ID NOS: 47 ~ 54; Table 3) in ficnt of the LPETG cell transfection and affinity purification of antibodies by protein A~sepharose chromatography was done essentially as described in Example 4. of Sortase A were uSed to conjugate To igate conjugation efficiency, serial dilutions different AclO penta~glycine~rnodified FITC (Glys~FITC, see Formula 3, above) to the four lSOmM NaCl; 7.SmM CaClZ), mAb variants in 1x Sortase bufier (ZSmM Tris~HCl, 131-182; as shown in Table 5. After 4h at 42°C, reaction products Were analyzed by denaturing, the gels on a reducing SDS~PAGE gel electrophoresis, and FITC was visualized by placing chain was equally efficient in all UVbox (Figure 11). As expected, conjugation to the heavy the light chain was ed significantly four antibody variants In contrast, conjugation to by increasing e—spacer length. Significantly, with the longest peptide—spacer analyzed chain conjugation efficiency was y efficient in comparison to ), light conjugation of the heavy chain, thereby allowing synchronous conjugation of heavy and chains of an antibody C—terrninally modified at both heavy and light chain, It is light concluded that this antibody format will facilitate Sortase iated production of homogeneous ADCs loaded with 4 dlugs per antibody (DAR4) WO 40317 .. Heavy Chain ' Light Chain Antlbody SEQ ID NOS ‘ modification modification AclOnHC~GS~ GS~LPETG~G~ GS~LPETG~G~ LHS/LC~GS~ HHHEIHILG— 39,40 . IIEIHHHII~G~ LHS WSHPQFEK WSHPQFEK ' AGIO~HC~ LPETG~G~ LPETG—G— 43 44 LS/LOLS WSHPQFEK WSHPQFEK 45, 46 AclO-HO LPETG—G~ GSMLPETG—(i 43’ 44 47 48 LS/LC—GSJJS WSI-IPQFEK WSHPQFEK AolO~HC~ LPETG~G~ , 43 44 gflS‘LPETG‘ GSuLS WSHPQFEK 49, 50 WSHPQFEK __1_ AclO~HC~ GGGS~LPETG~ LS/LC~GGGS~ TWPSEPCZSEK 43, 44 on 51, 52 LS WSHI’QFEK A010~HC~ GGGGS~ LS/LC- gggégfili 43, 44 LPETG~G~ 53, 54 LS WSHPQFEK _J Table 3: ina11y modified mono clonal dy AclO variants produced Reaction component 1~8 9-16 Final concentration I AclO-HC-GS~1HS/LC—GS~IHS (8.75mg/ml : 10 ~ 59M ZSnM) i C~LS/LC—LS (3.75mg/ml : ESHM) ~ 10 SnM H20 20 l 20 — ‘ G1y5~FITC (1mm 5 l 5 100m ‘ SortaseA (2X selial dil. of ca. 50nM) 5 l 5 5 9 0.039pM EX SOl‘tase buffer 10 t 10 1X and Ac10~ Table 4: Conjugation conditions for InAbs AclO~HCnGS—LHSILC~GS~LHS HC~LS/LC~LS Reaction component 1~7 I 8~l4 15—21 22428 i Final conc.

AclO—HC-LS/LC-GS~LS ‘ " " SHM (3.75mg/n11225nM) C—LS/LC—GGS~LS T _ # SnM (3.75mg/ml=25nM) AciO—HGLS/LOGGGS—LS T_ _ _ 10 5 M” (3.75mg/ml=25nM) _ AclO~HC~LS/LC—GGGGS~LS I _ '10 5 M” i (3.75mg/ml=25nM) H20 —i 20 20 20 2o _ Glyg—FITC (lmM) 5 ‘ 5 5 5 lOOnM i 5 l 5 1 Scrtase A (2x selial dil. of ca. ZSnM) 5 5 132590039” 5X Sortase buffer 10 j 10__] 10 10 1x J Table 5: ation conditions for mAbs AclO~HC~LS/LC~GS—LS, AclO—HOLS/LG GGS~LS, C~LSfLCwGGGS-LS and AclO~HC~LSlLC~GGGGS~LS.

Example 10: Generation of homogeneous ADC by strepll~tag affinity purification Sortase A mediated conjugation with G1ys~labeled -MMAE (see Formula 1, Example 5) was performed with anti~CD30 antibody A010 modified at the C~te1mini of either the heavy chains, or the light chains with sequences comprising an LPETG sortase A motif and a strepllw affinity ation tag as provided in Table 6 below: . Heavy Chain Light Chain dy SEQ ID NOS SEQ ID N05 modificaticn modification C~LS LPETG—G— none Ac~10~LC WSHPQFEK 43,44 29, 30 AclO~HC GS~LPETG~G— Ac10~LC—GS— none 27, 28 HHHHHH~G~ 41,42 LHS WSHPQFEK Table 6: C~terminally modified antibody AclO With either BC or LC modification 4 have The expression vectors encoding the AclO heavy or light chain sequences of Table been constructed essentially as disclosed in Example 1. CHO cell transfection and affinity purification of antibodies by protein A—sepharose chromatography was done essentially as described in Example 4.

Sortase A mediated conjugation of heavy or light chaing sortase motif tagged anti~CD30 antibodies with Glys—labeled ~MMAE (see Formula 1, Example 5) was performed essentially according to the protocol provided in Example 7.

As described further abOVe in the detailed description of the invention, ted dy will retain the C~terminal strep~ll affinity purification tag, which can be exploited to enrich fully reacted ADC with DAR2. Analysis of the heavy chain sortase A conjugation with VG— PAB~MMAE toxin via hydrophobicity interaction chromatography (BIC) (Fig. 12, panel A), shows that the majority of the sortase~motif d heavy chains have been conjugated, but a certain percentage of unreacted substrate (DARO = drug to antibody ratio = zero), or partially d substrate (DARl = drug to dy ratio = 1) was still detectable by HEC (Fig. 12, Panel A).

Therefore, the n A purified vc—PABJVJMIAE conjugate was passed 4 times times over a essentially as described . Strep’l‘actin® affinity column (IBA es, Gottingen, Germany), in Example 7 , in order to remove unreacted or partially reacted sortase A—modified antibody.

Fig. 12, Panel B shows that upon l es of the heterogeneous vc-PAB—MMAE antibody drug conjugate, completely reacted DARQ ADCs (DAR2 = drug to antibody ratio = 2) could be highly enriched. This experiment demonstrates the feasibility to utilize additional affinity purification tags added C-terminally to the sortase A LPETG recognition motif to generate homogeneous ADC with a defined drugs per antibody ratio (here DAR2).

Example 11: Synthesis of 5xg1ycine~modified maytansine and alpha~amanitin toxins In order to allow conjugation of two different payloads, preferably toxic ds to a single antibody, d with different sortase motifs at heavy and light chain C—termini, it is required to modify two different toxins with e residues, preferably toxins with different mode of actions, such that a cancer cell ed with a dual payload conjugated ADC, is attacked with Via two different, potentially synergistic routes. The synthesis of We different glycine~modified toxic payloads (here maytansine and alpha~amanitin) satisfying this requirement has been performed and is described herein. 2014/055173 11.1 Synthesis of glycine~mcdified alpha~amanitinz 30mg alpha~amanitin (Structure 1) (Sigma~Aldrich, order # A2263) Was dissolved in 1 ml added, anhydrous DMSO. To this solution 19mg NH~Boo—amino—hexylbromide were followed by potassium tert~butoxide (1M solution in THF, 35 at"). The reaction mixture was solution in THF, stirred at room temperature for 6 h and more potassium tert~butoxide (1M at) was added. The reaction was kept at room temperature for 16 h. Acetic acid (10 at") RP—HPLC directly (Sunflre C18 5p. 3 cmx was added and the crude e was purified by cm column, 50 mL/min, 560% acetonitrile/water 15 min gradient). The desired fraction 2 as a white powder was collected and lyophilized to giVe ure (15 mg), which was treated with TEA/DOM solution (1/1, WV, 11ml) for 30 minutes at room temperature. The Volatiles were removed under reduced pressure to give Structure 3 as a slightly ish without further purification. gum> which Was used in the next step Fmoc~Gly5—OH (8 mg) was dissolved in anhydrous DMF (0.5 ml). HATU —Aldrich, order # 445460) (6 mg) was added, followed by DIEA (10 ml) (Sigma~Aldrich, order and then transferred #49 6219). The mixture was agitated gently at room temperature for 30s to a solution of compound 3 in DMF (0.5 ml). After 30 mins, LC/MS analysis showed that Piperidine (30 u’f) was added and the ss of the all of compound 3 was consumed. the reaction after 1h reaction was red by LC/MS. Acetic acid was added to neutralize and the mixture was purified by RP~HPLC (Sunfire C18 5n 3 cm x 25 cm column) 50 mL/min, 240% acetonitrile/water 30 min gradient). The fractions were pooled and lyophilized to give ure 5 as a white powder (12 mg). ical data for compound provided mag. 13, panel A). 0 If?" Fmoo—GlyB‘OH HO". N O \ NH Scheme 1: Synthesis of glycine—modified alpha~amanifin 11.2. Synthesis of g1ycine~modified maytansine: Maytansinol (0.6 g, 1.1 mmol) (CleaISynth Labs, , India) was dissolved anhydrous THF (6 ml) and anhydrous DMF (3 1111) after which 1,2 ml DIEA (Sigma Zinc Aldrich, order #496219) was added. The solution was placed under argon atmosphere. triflate (1.2 g) and NMeAla NCA (0.7 g) were added in one portion. The mixture was sonioated until the solid was dissolved. The reaction mixture was stirred at room temperature for 2 days and then d with ethyl acetate (100 ml). It was washed with NaHCO3 (aq. solution, 2 X 50 ml) and brine (50 ml). The organic layer was dried (oVer MgSO 4) and concentrated to give the crude maytansinol 3—(S)-alpha—N~ methylaminopropionate which was uSed ly in the next step without r purification.

Fmoo~Gly5~OH (26 mg) was dissolved in anhydrous DMF (1 ml). HATU (Sigma~Ald1ich, mixture was agitated order #- 445460) (19 mg) was added, followed by DIEA (18 uL). The 8 in THE gently at room temperature for 30s and then transferred to a solution of compound (1 ml). After 30 mins, LC/MS analysis showed that all compound 8 was consumed.

Piperidine (40 ill) Was added and the ss of the reaction was red by LC/MS. collected and Ether (40 ml) was added to the reaction after 2 h and the precipitated solid was CIS Su 3 cm x washed with ether. The crude compound was purified by RP-HPLC (Sunfire cm column, 50 ml/min, 10-60% acetonitrile/water 20 min gradient). The fractions were pooled and lyophilized to give compound 10 as a white powder (33 mg). Analytical data for compound 10 is provided in Fig. 13, Panel B.

Fmoc—GIyS—OH —————+ Scheme 2: Synthesis of glycine-modified maytansine for sortase Importantly, it is to be noted that in principle, any toxin can be functionalized mediated enzymatic conjugation, if either 5 glycines (as shown here), or any number of the toxins 14 e residues greater or equal than one glycine, are attached to (see Fig. ).

Trastuzumab~DlVIl in Example 12: In vivo tumor inhibition of sortase A-conjugated SKOVS cvarial carcinoma xeno graft models. subcutaneously 5x10 5 SKOV3 tumor cells in ZOOulPBS/Matrigel (1:1 ratio) were ted mice. y tumor volunies Were into the left flanks of 5~6 weeks old female NMRI nude 2014/055173 monitored by calipering. After a mean tumor volume of lOQ~200mm3 was reached, tumor~ bearing s were ized into 3 Groups according to tumor sizes (10 animals per 1, 2 and 3 » group). On the day of randomization (day 0) and on day 21, animals of Groups injected intravenously with, respectively, Smlfkg PBS, 15 rug/kg Kadcyla®, or 15 were mg/lcg sortase A—conjugated Trastuzumab~DMl Tumor volumes were ed ldy by calipering (Fig. 15). The study was terminated after 39 days and animals were euthanized according to accepted animal experimentation ines.

In the couISe of the study, tumors in control animals mock~injected with PBS grew steadily to a volume of approximately 600mm3. In contrast, tumors in Kadcyla®~tteated annuals shrank and Were essentially undetectable on day 39. Anti~tumor activity of Sortase A~ conjugated Trastuzumab~DMl did not differ significantly from that of commercially available Kadcyla®, despite the fact that the sortase~conjugated T~DM1 exhibited a lOWer drug to antibody ratio of approximately 2, in comparison of a reported DAR of 3.5 of a®. In combination with the data from e 8, the results demonstrate that sortase conjugated ADCs, using identical antibody and toxin moiety, have comparable tumor killing activity in comparison to commmcially available chemically conjugated Kadcyla® in vitro and in vtvo, albeit at loWer drug to dy ratio.

Example 13: Sortase Aemediated conjugation in crude CHO cell supernatant.

Trastuzumab variant Tras—HC~LS/LC~GGGGS—LS, consisting of heavy chains C— terminally tagged with LPETG Sortase motif and Strep II purification tag (SEQ ID NOS: US$056), and light chains C~terminally tagged with a 5 amino acid Gly4~Ser spacer (GGGGS), LPETG Sortase motif and Strep II tag (SEQ ID NOS: 057~058), was produced CHO cells essentially as described in Example 4. The resulting serum—free crude cell supematant contained approximately 157mg£L Tras—HC~LS/LC~GGGGS~LS and was directly uSed for conjugation essentially as described in Example 9, by adding Sortase the supernatant. In parallel, buffer, Gly5~FITC, and serial ons of Sortase A directly to Tras~HC~LSfLC~GGGGS~LS purified by protein A affinity chromatography was also conjugated under othe1wise identical conditions. After 4 hours at 42°C, the reactions were FITC by analyzed by ning, reducing SDS—PAGE gel ophoresis. After visualizing Blue placing the gel on a UV box, protein was stained using Coomassie Brilliant (Fig. 16). of antibodies in The data shows the unexpected finding that Sortase A~mediated conjugation supernatant was efficient as that of d antibody. r, the crude cell culture as conjugation reaction was highly specific and none of the protein contaminants t in crude CHO cell supernatant Were non-specifically conjugated. Together, these data suggest that the robustness of the Sortase reaction may help facilitate ADC manufacturing by allowing to m drug conjugation directly after production in CHO cells prior to ation and downstream processing.

Figure legends Fig. 1: This figure illustrates the principle of the sortasc A mediated site—specific payload conjugation to an immunoligand (or binding protein), which can be performed at the N- terminus of a protein (a), or at the C~terminus of the protein (b). In order to achieve N» motif terminal conjugation, the payload needs to contain a sortase penta—peptide recognition there LPXTG, the ition motif of sortase A from Staphylococcus aureus (X representing any of the 20 natural amino acids), whereas the N~terminus of the immunologand/binding protein to be labeled needs to be expressed with an N—terrninal extension of minimally 3 e residues, here indicated as Gm (with n>2), that has a free N~terminal amino group (here indicated by the smaller H2N~ symbol). lly 36 Addition es are used in order to modify a substrate for sortase—mediated conjugation. then of recombinant sortas A enzyme from Staphylococcus aureus, as indicated here, catalyzes the breakage of the peptide bond betWeen the T and the C~terminal G residue in the LPXTG pentaapeptide motif and forms a new peptide bond between the N—terminal glycine of the GI, stretch (n>2) and the T residue. The C—terminal G residue of the LPXTG motif (here highlighted in boldface print) is removed in the transpepiidation reaction, (b) ConVerSely, in order to achieVe C~terminal conjugation of a payload to a protein, which is antibodies the red method for conjugation of payloads, particularly toxins, to (see Fig. needs to be added to the C~terminal 6), the LPXTG e recognition penta—peptide motif end of the irnmunoligand/binding protein (eg. by recombinant protein expression logy, as described in the Examples), and the payload needs to be modified with a short glycine stretch (Gm, with n>2, typically 3-5 glycines), As described under (a), on of the (in of sortase A fiom lococcus aureus will then catalyze the transpeptidation stretch to the LPXTG motif, whereby the terminal G residue of the LPXTG motif (in boldface) Will be removed.

Fig. 2: This figure illustrates the principle of intein (a) and split~intein (b) mediated transpeptidation. (a) Inteins can occur as led ”protein~introns” in precurs0r proteins, Where they separate N~terminal and inal parts .of a mature protein, which are generally called N~extein and C~extein. The intein "protein—intrcn" can catalyze the breakage of the peptide bond between the intein and the C~extein and the formation of a new peptide bond between the N—extein and C~extein by transfering the N~terminal amino acid of the C- extein to the C~terminal amino acid of the N—extein in a transpeptidation reaction. The result of the reaction is the removal of the intein in~intron" from the precurSOr protein and the generation of a mature protein with a newly created peptide bond betWeen the N~ extein and in s, (b) The intein activity has also been described to be ble into distinct domains, that can be ed to different proteins, for which this intein variation has been termed split-intein. The N~i11t and C~int domains of the split intein form a non~ccvalent structural complex, that can perform the same transpeptidation reaction as a contiguous intein, on the attached N—extein and C~extein domains that are then in spatial proximity and part of the complex. The result of the transpeptidation of N~int and C—int intein on is then a ”protein trans~ splicing", or essentially a protein ligation between the N-extein and in domains, by ion of anovel peptide bond.

Fig. 3: This figure illustrates how particular split inteins that are characterized by either an extremely short C~int domain or an extremely short N—int domain can be used to conjugate (or binding protein), including small molecular entities, any payload to an immunoligand because short amino acid hes can be synthesisze chemically and can easily be attached to small molecular entities by conventional chemical coupling. (a) This part of the illustration shows the use of the Ssp GyrB Sll split intein (described in Appleby et al. (2009)) for the C~tetminal conjugation of a payload to an immunoligand/binding protein.

Here the C~int domain is only 6 amino acids long and ses the amino acid sequence GVFVHN, as indicated. HoweVer, as there need to be some peptides that are the equivalent the first one needs of an C~extein domain, additional amino acids need to be added, ofwhich acid residue, Whereas the remaining amino acids can be to be a serine or cysteine amino chosen. This is indicated by the SXI, symbol, which means that a short amino acid stretch lead at the N~terminal side by serine and followed by 11 amino acids (n>2, preferably 5), which can be any of the 20 naturally cccuring amino acids (therefore indicated as X). Thus, as described in the Example, a short 12 amino acid stretch comprising a 6 amino acid mini CHint domain and 6 amino acid C—ext amino acid stretch are sufficient to allow the N~int/Ca WO 40317 bond in the int complex to catalyze the transpeptidation fiom the gine~ serine peptide GVFVHN—SXn (X any amino acid, n>2, preferably 5) to the peptide bond between the N~ extein and N~int tion. This will result in a C~te1minally conjugated amino acid nnmunologand/binding protein with the payload attached via the short C—extein intein (described stretch, (b) This part of the illustration shoas the use of the Ssp DnaX split in Song et a1. (2012)), which can be separate into a Very short, 11 amino acid Nwint domain C—int domain for N—terminal conjugation of a payload to an and a 139 aa immunologand/binding protein. As indicated here, this only requires the synthesis and coupling of a short 11 amino acid N—int domain to any payload (or the addition by. of the payload recombinant protein technology), Which then allows the specific conjugation that has a 139 amino acid long Ssp DnaX to the N—terminus of any immunoligand or n, C~int domain fused to the N—teiminus. The result of this reaction is then a N—teiminally ligand/binding protein. Therefore, like in the case of sortaSe conjugated the arrangement transpeptidation, Where the N~ oriC—terminal conjugation only depends on and d, of the LPXTG and GIl peptide motifs with regard to protein split inteins can also mediate site~specific N~ and C~terminal ation of proteins With short e modified payloads, and by t short mini Cant, or mini N—int peptide domains, like those of Ssp GyrB and Ssp DnaX split inteins, respectively. and/or Fig. 4: (a) This Figure rates the utility of adding additional affinity purification detection tags in addition to a sortase tag in the conjugation of payloads to immunoligands. added amino acids representing a 6XHiS (a) this part ofthe Figure shows how an additionally and a a Myc-detection tag (EQKLISEEDL) strepll affinity purification tag (HHHHHH), purification tag (WSHPQFEK), as described in the es are removed in the course of the C~te1minal payload conjugation via Staphylococcus aureus sortase A transpeptidase.

This allows to select for the ated product, if Ni—NTA affinity resins (for the s~ tag) or streptactin afifoity resins (for the strep ITtag) are employed to seperate non» conjugated substrate from conjugated product. This combination of tags is only provided way ofExample. is particularly useful to (b) This Figure illustrates that the use of affinity purification tags select/purifiy completely conjugated product in the case of eric proteins, like antibodies in the examples, antibodies can be modified as illustrated here, As also provided with specific conjugations sites at heavy and light chains, and if the modification is targeted be conjugated to the to the C-tennini of IgH and IgL chains, then up to four payloads may antibody. The on of (a) further ty ation tag(s), e. g. as described in Fig 4(a) allows to bind incompletely conjugated product, that may only have one, two, or three (as illustrated here) payloads conjugated to the antibody, still bind to the respectiye affinity purification resin, and can thus easily be separated from the fully payload~conjugated product. This paradigm is of course also applicable to intein~modified irnmunologands, not only to s0rtase~motif~modified logands, as depicted here. that can also be Fig. 5: This figure rates avariation of the sortase—mediated conjugation applied, in which the sortase~enzyme is not added as a te recombinant protein to the sortase tagged immunologand and glycine stretch modified payload, but where the the LPXTG sortase enzymatic sortase domain is sed as a fusion protein C-tenninal to it is not incubated with glycine~ tag. The sortase enzyme domain Will be inactive as long as substrate stretch modified payload (or substrate); As soon as glycine~stretch modified (or here payload) is added to such a construct, the fused sortase domain Will catalyze eptidation of glycine—payload substrate to the LPXTG sortase tag, by cleaving the and e~5 position of the LPXTG tag, and thereby protein between the threonine~4 that can be removing the Sortase enzyme domain with additional affinity ation tags, optionally, This procedure has the advantage that, similar to the added as depicted here. domain. can be addition of catalytically active split~intein domains, the sortase enzyme the immunoligand expressed by inant protein technology as an integral component of to be conjugated.

Fig. 6: (a) This figure illustrates the use of different transpeptidases (here sortase and split- different payloads to different subunits of a intein), in order to simultaneously conjugate multimerio protein, like e.g., the light chains of an antibody. as ed here, the heavy and In this selected e, the C~termini of the heavy chains are modified with the N—int domain of Ssp GyrB (as provided in Example 2), while the light chains are modified with the sortase A penta~peptide motif LPXTG (as provided in Example 1, the additional tags are A and With a C— omitted for simplicity). Incubation with a lglycine~ stretch modified payload int-domain modified payload B and sortase enzyme will allow the simultaneous and A to the light chains. If ive conjugation of payload B to the heavy Chains and payload payloads A and B are toxins addressing different cellular pathways, this strategy could ancer drugs, as conventional ADCs, only containing a single generate more potent toxin . (b) This figure illustrates the use of different sortase enzymes (here sortase A and sortase B from lococcus LZW‘BHS), in order to simultaneously conjugate different payloads to different subunits of a multimeric protein, like e. g., as depicted here, the heavy and the light chains of an antibody. In this selected example, the C—termini of the heavy chains for sortase B, NPQTN, While are modified With the pentapeptide recognition motif the light chains are modified with the sortase A penta—peptide motif LPXTG. Sequential and Sortase B will conjugation of glycine—stretch modified payloads A andB With sortase A allow the aneous and selective conjugation of payload B to the heavy chains and payload A to the light chains (remaining peptide Sequences from LPXTG and NPQTN are toxins addressing omitted in the conjugated structure for simplicity). prayloads A and B are different cellular ys, this gy could generate more potent anti~eancer drugs, as conventional ADCs, only containing a single toxin .

Fig. 7: GE (a) and Western~blot (13.) analysis of recombinant enzymatically active e A fragment of Staphylococcus ourcus. (a) Lane lin the SIDS—PAGE contains BSA of Genscript (Cat-N1: (ca. 66.4 kD), Lane Mi contains protein lar weight standard A of M00505), Lane 2 contains His~tag purified recombinant sortase fragment Staphylococcus aurcus. The proteins in the SDS—PAGE are stained with Commassie blue.

Cat.—Nr.: (b.) The Westem~blot was developed with an anti~His antibody (Genscript AA00186). Lane 3 contains His—tag ed recombinant sortase A fragment of standard of Genscript (Cat.~Nr.: Staphylococcus aurcus. Lane M2 contains molecular Weight Mix/£0908).

Fig. 8: Hydrophobic Interaction Chromatography (HIC) analysis of DM1 ~toxin conjugated C—GS—LHS (A) and Tras—LC—GS~LHS (B). DARl indicates drug to antibody ratio of ratio of2. 1; DAR2 indicates a drug to antibody indicated ADCs on HERZ—overexpressing Fig. 9: Dose response of cytotoxic effects of the SKBR3 (A) and HER2~negatiVe R cells (B). Cells Were incubated with serial dilutions of ADCs for 3 days, after Which cell viability Was detected by CellTiter—Glo® Luminescent Solution (Promega). LC: DlVll~SortaseA~conjugatecl Tras~LC~GS~LHS; HG: DM lasortaseA—conjugated Tras-HC~GS~LHS.

W0 2014/14031’7 conjugation of Glys—FITC to mAb AclO variants with or Fig. 10: Sortase A—mediated Without GS peptide spacer. Serial ons of SortaSe A were used to conjugate Glys—FITC tc mAb AclO~HC~GS~LHS/LC~GS~LHS (A) and rnAb AclO~HC~LS/LC~LS (B) under otherwiSe identical conditions. Reaction products were scperated by size on denaturing, reducing SDS—PAGE gels, FlTC was visualized by placing the gels on aUV box. Sortase concentrations uSed Were: lanes 1, 9: SOnM; lanes 2, 10: 25nM; lanes 3, 11: 12.5nM; lanes 4, 12: 6.25tLM,‘ lanes 5, 13: 3.13nM; lanes 6, 14: ‘l.56tLM;lanes 7, 15: ,‘ lanes 8, 16: 0.39uM.

Serial Fig. 11: Influence of peptide spacer length on light chain ation efficiency.

AclO~HC~LS/LC~GS—LS dilutions ofScrtase A were used. to conjugate ciy'srn‘c to mAbs left), AclO~HC~LS/LC~GGS~LS (A, right), AclO—HC-LS/LGGGGS—LS (B, left) and C~LS/LC~GGGGS~LS (13, right) under otherwise identical conditions. Reaction seperated by size denaturing, reducing GE gels. FITC was products were on visualized by placing the gels on aUV boX. e A concentrations used Were: lanes 1, 8, , 22: 25uM; lanes 2, 9, 16, 23: 12.5uM; lanes 3, 10, 17, 24: 6.25uM,’ lanes 4, 11, 18, 25: 12, 19, 26: ; lanes 6, 13, 20, 27: 0.78uM; lanes 7, 14, 21, 28: 3.13t1M,‘ lanes 5, 0.39aM Fig. 12: Analysis of SortaseA Vc~PAB~1VflviAE toxin heavy—chain—conjugated ADC of mAb AclO by hobicity interaction chromatography (HIC), which is able to differentiate unreacted substrate CDARO = 0 drug to antibody ratio), substrate in which one of the two heavy chains has been conjugated (DARl = l drug to dy , and substrate in which to antibody ratio), as both modified heavy chains have been conjugated (DAR2 = 2 drugs indicated Panel A shows the HIC profile after a standard e A mediated conjugation of HC modified AclO rnAb, inwhich still DARO and DARl species are detectable, next to the desired DAR2 product. Panel B showa the ETC profile after 4 passes of the ADC preparation analyzed in Panel A over a StrepTactin® affinity purification column. toxin (a) and Glys-niodified Fig. 13: Analysis of synthesized Glys~rnodified alpha~ amanitin maytansin toxin (b.). In each of the panels a.) and b.) the snthesized structure is provided on top, with the five glycines highlighted by a box, The analysis of each compound by mass spectrometry and reverse—phase HPLC is provided below. a.) The expected mass of the Glys~rnodified alpha~ amanitin toxin is 1302.07 D, the observed mass is 1325.38 D, coirespcnding to Ms + Na+. The RP~HPLC profile indicates a purity of > 95%. b.) The ed mass of the Glys—moditied maytansine toxin is 991.41 D, the observed mass is of > 95%. 957.69 D, ponding to Ms + Na+. The RP~HPLC profile indicates a purity been synthesized by Fig. 14: Structures of SXGlycine (Gly5) modified toxins that either have Concortis, San Diego, CA, US. (structures 16, and 9), or that can be synthesized (structures 7 85 mediated enzymatic 8), demonstrating that any toxin can be functionalized for e conjugation, if either 5 glycines are attached to the toxins (as shoWn here), or any number of glycine residues greater or equal than one glycine. G1ycine~modif1ed toxins can either be synthesized ning additional validated linker/spacer structures as provided in structures 1-3 in Fig. 14 a), ially adding certain additional functionality (e.g. bility in certain subcellular compartments) or Without additional s, as depicted in structures in the case of in Fig. 14 b). If several reactive groups are available at a given toxin, like eg. alpha—amanitin toxin, glycine residues can be added'to these different groups as exemplified in structures 7-9 in Fig. 14c).

Fig. 15: Tumor volumes as determined in Example 12. The results demonstrate that sortase conjugated ADCs, using identical antibody and toxin moiety, have comparable tumor killing activity in comparison to commercially available chemically conjugated Kadcyla®.

Fig. 16: Gels stained With Coomassie blue as described in example 13 The data shows the cell culture cted finding that Sortase ated conjugation of antibodies in crude supernatant was as efficient as that of purified antibody.

References Amos et al. (2009a) J.Am, Chem. Soc. 131,131). 1080040801 Amos et al. (2009b) J,B101. Chem. 284, 16028~16036 Appleby et a1. (2009) JBC 284, 619499 Axup et a1. (2012) Proc. Natl, Acad. Sci USA 109, 1610246106 Elleuche (2010) Appl. Microbiol. Biotechnol. 87, 479489 Grams—Portal et a1. (1995) M01, C611. B101. 15, p1182ff Hofer et a1. (2009) Biochemistry 48, 12047-57 Junutula et a1. (2008) Nat. hol., 26, 925~932 c (2012) British I C1111 Phalmacol 76, 248262, Lemke (2011) Methods M01. B101. 751, 3—15 Levary et a1. (2011) PLOS One 6, 018342 Madej 619.1. (2012)B1'otechn01. Bioeng. 109, 1461—1470 Mao et a1. (2004) 1.11111. Chem. Soc. 126, 2670—2671, Mamanian et a1. (1999) Science 285, 760~763 MoDonagh et a1. (2006) Prof. Engin. Design 86160121011 19, 299~307 lm et a1. (2011) Chembioohem. 12, 1774~1780, Mflliard (2013) Nature Rev. Drug Discov. 12, 329632), sarathy et a1. (2007) Bioconjugate Chem, 18, 6 Perler (2002) Nucl. Acids Res. 30, 383-3 84 Song et a1. (2012) PLOS One 7, (245355 Spirig et a1. (2011) Mo1ecular Microbiol, 82, 1044—1059 Sun et al. (2004) I.Biol, Chem. 279, 35281—35286 SW66 et a1. (2013) Proc. Natl, Acad. Sci USA 110, 14284433 at et a1. (1999) Proc. Natl. Acad. Sci USA 96, 12424-12429 Tsuldji (2009) Chembiochem. 10, 787—798) Voflcmann et a1. (2009) PLOS One 4, 68381 Xu et a1. (1993) C611 75, 13714377

Claims (20)

1. What is claimed is

   l. A method of producing an immunoligand/payload conjugate, which method encompasses

   conjugating at least one payload to an immunoligand by means of a sequence—specific

   transpeptidase, or a catalytic domain thereof,

   wherein the immunoligand sed in the immunoligand/payload conjugate is at least

   one selected from the group consisting of

   (i) an antibody,

   (ii) an antibody—based g protein being a protein containing at least one antibody—

   derived VH, VL, or CH globulin domain,

   (iii) an antibody fragment binding to a receptor, antigen, growth factor, ne and/or

   hormone, and

   (iv) an antibody mimetic selected from the group consisting ofDARPins, C-type lectins,

   in proteins of S. aureus, transferrins, lipocalins, 10th type III domains of fibronectin,

   Kunitz domain se inhibitors, affilins, gamma crystallin derived binders, ne knots or

   knottins, thioredoxin A scaffold based binders, nucleic acid aptamers, artificial antibodies

   produced by molecular ting of polymers, and stradobodies, wherein the sequence—

   specific transpeptidase is a sortase enzyme which izes a pentapeptide recognition motif,

   and n the payload is a cytotoxic compound not exceeding a molecular weight of

   2500 daltons that is cytotoxic to mammalian cells wherein either

   0 the immunoligand comprises a sortase ition motif and the cytotoxic

   compound is modified with a Glyn-modification, wherein n>1, or

   n the cytotoxic compound comprises a sortase recognition motif and the

   immunoligand is modified with a Glyn~modification, wherein n>l.
2. 2. The method according to claim 1, wherein the immunoligand binds at least one entity selected

   from the group consisting of

   v a receptor

   a an antigen

   . a growth factor,

   0 a cytokine, and

   o a hormone.
3. 3. The method according to either claim 1 or 2, wherein at least one catalytic domain of the

   sequence-specific transpeptidase is fused to the C-terminus of either the irnmunoligand or the

   payload.
4. 4. The method according to any one of claims 1 to 3, wherein said immunoligand comprises at

   least two subunits each being conjugated to a payload.
5. 5. The method according to claim 4, wherein said immunoligand with at least two subunits is

   conjugated to at least two different payloads, at least one of which is a toxin not exceeding a

   molecular weight of 2500 daltons.
6. 6. The method according to either claim 4 or 5, wherein said immunoligand with at least two

   subunits contains a peptide spacer ce of at least two amino acids, appended to the C-

   terminus of at least one of the two subunits.
7. 7. The method ing to any one of claims 4 to 5, wherein method allows a

   stoichiometrically defined relationship between immunoligand and d.
8. 8. The method ing to claim 7, in which method said stoichiometrically defined

   relationship between immunoligand and payload is achieved by removal ofpartially reacted C—

   terminally modified immunoligand substrate.
9. 9. The method according to claim 8, in which method ses the use of at least two affinity

   purification tags,

   which affinity purification tags are bound to at least two ent sites of the antibody,

   mimetic Via e

   modified antibody format, antibody derivative or nt, and/or antibody

   recognition motifs prior to the conjugation of the at least one small molecular toxin,

   which method further comprises an affinity purification step,

   in which affinity purification step conjugates which after the conjugation of at least one small

   molecular toxin to the antibody, modified antibody format, antibody derivative or fragment,

   and/or antibody c, still bear affinity purification tags, and thus qualify as incomplete

   conjugates, are separated from the complete conjugates by affinity purification.
10. 10. The method according to any one of claims 1 to 9, which method allows a pecific

    conjugation of the payload to the immunoligand.
11. 11. An immunoligand/payload conjugate obtained with the method according to any one of

    claims 1 to 10, which conjugate comprises at least one of

    (i) an antibody,

    (ii) an antibody—based binding protein being a n containing at least one antibody»

    derived VH, VL, or CH oglobulin domain,

    (iii) an antibody fragment binding to a receptor, n, growth factor, cytokine

    and/or hormone, or

    (1V) an antibody mimetic selected from the group consisting of DARPins, C—type

    lectins, A—domain proteins of S. aureus, transferrins, lipocalins, 10th type 111

    domains of fibronectin, Kunitz domain se inhibitors, affilins, gamma

    crystallin derived binders, cysteine knots or ns, thioredoxin A scaffold based

    s, nucleic acid aptamers, artificial antibodies produced by lar

    ting of polymers, and stradobodies,

    and a cytotoxic compound not exceeding a molecular weight of 2500 daltons,

    wherein the conjugate further ses at least one linker that conjugates a payload to the

    immunoligand, which linker comprises a peptide motif that is a sortase recognition motif, and

    is conjugated to the C—terminus of at least one peptide chain of the immunoligand.
12. 12. The immunoligand/payload conjugate according to claim 11, which conjugate further
13. comprises a sortase recognition motif that has undergone transpeptidation reaction.
14. 14. The irnmunoligand/payload conjugate according to any one of claim 11 to 13 for use in the

    treatment of a human or animal subject suffering from or being at risk of developing a given

    pathologic condition.
15. 15. The immunoligand/payload conjugate for use according to claim 14, wherein said

    pathologic condition is at least one selected from the group consisting of

    o neoplastic disease

    - autoimmune disease

    0 neurodegenerative disease, and

    - infectious disease.
16. 16. The method according to any one of claims 1—10, in which the immunoligand—payload

    conjugation is performed in crude cell culture supernatant.
17. 17. The method according to any one of claims 4—5, wherein said immunoligand with at least

    two ts contains a peptide spacer sequence of two—five amino acids, appended to the C—

    terminus of at least one of the two subunits.
18. 18. The immunoligand/payload conjugate of claim 11, wherein a non-cleavable linker is

    estaboished which comprises or consists of a peptidic motif resulting from specific cleavage of

    a sortase enzyme ition motif,

    wherein the peptidic motif ing from specific cleavage of the e enzyme

    recognition motif is LPET and/or

    wherein the peptidic motif resulting from specific cleavage of the sortase enzyme

    ition motif is LPQT.
19. 19. The immunoligand/payload conjugate of claim 18, n the sortase enzyme recognition

    motif is selected from the group consisting ofLPXTG, LPQTG, LPETG, NPQTN and LPLTG.
20. 20. The immunoligand/payload conjugate ofclaim 19, wherein the linker comprises or consists

    ofthe ce LPETG.

    21‘. The immunoligand/payload conjugate of claim 19, wherein the linker comprises or consists

    of the sequence LPQTG.