AU2015202283B2 - Anti-GCC antibody molecules and related compositions and methods - Google Patents

Abstract

Antibodies and antigen-binding fragments of antibodies that bind GCC are disclosed. The antibodies bind an extracellular domain of GCC and can be internalized. In some embodiments, the antibodies are humanized, chimeric or human. Nucleic acids and vectors encoding the antibodies or portions thereof, recombinant cells that contain the nucleic acids, and compositions comprising the antibodies or antigen-binding fragments are also disclosed. The invention also provides therapeutic and diagnostic methods utilizing the antibodies and antigen-binding fragments provided herein.

Description

ΑΝΉ-GCC ANTIBODY MOLECULES AMD RELATED COMPOSITIONS

AND METHODS

This application is a divisional of Australian Application No, 2010310545, the entire contents of which are incorporated herein by reference.

RELATED APPLICATIONS !§M J The present application claims the benefit of CIS. Proviskmai Application

Serial No< €1/254,474, lied October 23,2009, The entire content of US. IhxmsionsI Application Serial No, 61/254,474 ts irteorpomted. hereto by flag reteace.

FIELD OF INVENTION im The invention relates to antibody molecules which bind GCC, as well, as to related molecules, &amp;g>5 nucleic adds which encode such antibody molecules, coffipositiims, and related methods, e.g\, therapeutic and diagnostic methods, BACKGROUND . {D03J Guaaylyi cyclase C (GCC) is a tausmembrme cell surface receptor that

Factions is the maintenance of intestinal fluid, electrolyte homeostasis and cell prollferarien, see, e.g., Carriihers &amp; at, Proc. Mail Acad. Set USA 100:3018-3020 (2003). , GCC is expressed at the mucosal cells lining the small intestine, large intestine sad rectum (Canitheis et at, Dk C&amp;km Rectum 39:171*481 (1996)). GCC expression, to mamtatoed upon neoplastic transformation of Intestinal epithelial cells, witli expression to all primary and metastatic edoreetal teas (Camttas et at,.Uto Coion Rectum 39:171-181 (1996);

Biic et at. Bur J Cancer 41:1618462? (2005); Camthem et at. Gastramterofogy 107; 1653-1661(.1994)).

SUMMARY (164] ’Hie investors have discovered numerous anibGCC antibodies, iueludiog· both human and murine antibodies, Accordingly, in one aspect, fee invention features an anti~GCC antibody molecule, as disclosed herein. The aati-GCC antibody molecules are useful as naked antibody molecules and as component of imniunoconjugates. Accordingly, in another aspect, tbe invention features fenuunoeonjugaies comprising m anti~GCC antibody molecule and a therapeutic agent or label. The aivention also features pharmaceutical compositions comprising the anti-GCC antibody molecules and imnumoconjugates described herein, 1¾¾ invention also featimss methods of using the aatt-GCC antibody molecules and tmmunoconiugaies described herein for detection of GCC and. of ceils or tissues feat express GCC; for diagnosis, prognosis, imaging, or staging of a GCC-mediated disease; fer modulating an activity or function of a GCC protein; and for treatment of a GCX-mediated disease, as described herein. In another aspect, the invention also features isolated and/or mcombmaatnuelde acids encoding anti-GCC antibody molecule amino acid sequences, as well as vectors and host cells comprising such nucleic acids, and methods fer producing auti~GCC antibody molecules. I'tidSJ AH publications, patent applications, patents and other references mentioned herein are incorporated by inferences in their entirety. mi Other features, objects, and advantages of the invention(s) disclosed herein will be- apparent from the description aid drawings, and from tire deans.

DESCRIPTION OF THE FIGURES (667] Figure 1 depicts tumor growth in 293~GC€#2 bearing SOD mice heated with 5F9vc-MMAF, -DM1, and ~DM4 on a q!4d schedule. (688] Figure 2 depicts lung weight of mice treated with 0.9%Na€l; 269 antibody at 48mg/kg; or 5F9 antibody at 16 or 40mg/kg on day 41 p.i.

[869J Figure 3 depicts the survival curve of CT26-hG€C tumor-bearing mice treated wife 5F9 antibody. 1«! 6] Figure 4 depicts ELISA binding assays to test antibody cross-reactivity of GCC orthologs,

DETAILED DESCRIPTION

[Mil Gusayiyl cyclase € (GCC) (also known as STAR, ST Receptor, GUC2C, and GCJCY2C5 is &amp; tsmmmxtimm cell surface receptor that ftroctkms in the maintenance of fetesHaa! Said, electrolyte homeostasis asd cell proliferation (Carrifhers et aL, Proc Mail Acad Set U S A 100: 3018-3020 (2003); Mass et al, Moekem M&amp;pfys Res Commun 239:463*466 (1997); Pitad et ύ.,Ρκχ)NatlAemiSciUSA 100: 2695-2699 (2003)); GesSank Accession No . NMJMM963, each of which is meorporated harem by reference). This ftmctifM is mediated, through binding of geanylk (Wfcsgand et al, FEES Led. 311:150-154 (1992)}. GCC also is a receptor for heat-stable enierofoxk ( ST, e.g,, having an amis» add sequence of NTPYCCELCCNPACAGCY, SEQ ID NOD 16) which. is &amp; peptide produced by K coli, as well as other iafeettous organisms (R.ao, M,C< Clba Found. Symp, 112:74-93 (1985); Knoop F.C. and Owens, M. J. Phanmml Toxicol. Meifctds 28:67-72 (1992)). Binding of ST to GCC activates a signal cascade that results in enteric disease, e,g.s diarrhea. |M2] Nucleotide sequence for human GCC (GanBank Accession No. NM0&amp;4963): 1 atgaaga<it tgotgttgga etiggctttg tggtcactgc tettccagcc egggtggetg 61 teettlagtt eee&amp;ggfgag tcagaactgc eaeaatggca gctatgaaat eagcgtcctg 121 aigaigggaa aeteageett tgeagsgcee ctgaaasaet fggaagatge ggigaatgag 241 aelttcaigt atteggatgg tcigatfcat aaeteaggeg actgccggag tagcaectgt 361 gggceeteat gtaeataete eaecttccag atgtaeefig acacagnatt gagciaeece 421 atgatctcag etggasgttt tggattgto tgtgactsta aagaaacctt aaccaggctg 481 atgtetecag eiag&amp;aagtt gatgtaette ttggttaaet tttggaaaac eaacgatctg 541 cccttcaaaa cltaticctg gageaetteg tatgittaea agaatggtae agaaaetgag 601 gactgtUct ggtaccttaa tgetciggag getagegttt eetadtete ceacgsacfc 661 ggefitaagg tggtgitaag acaagataag gagtitesgg atatettaat ggaccaeaac 721 aggasMgea atgtgMiat tatgigtggi ggi«cagagt teciclaeaa gefgaagggt 78 1 gaccgagcag tggcigaaga eattgt eatt astcfaglgg atctttteaa tgaccagtae 841 fltgaggaca atgtcacagc ccctgactat algaaaaaig tccitgttet gacgcigtot 901 cci^gaati cecitcf&amp;aa tagcieftte tccaggaatc taieaecaac aaaacgagaa 961 tttgctettg cetatttgaa tggaatecig e&amp;fttggae ataigcigaa gat&amp;6tolt 1021 gaaaatggag aaaatatiac eaecceeaaa tttgctcafg ctScaggaa tete&amp;etttt 1081 gaagggtatg as^glceagt gaediggat gaetgggggg atgttgacag taecatggig ! 141 cttctglata eefciglgga caeeaagaaa tacaaggtlc ttttgaccta tgaiacccac 1201 gtasaiaaga ccfaiccigt ggaiatgage cccacattca etiggaagaa eislaaaett 1261 cetaatgaia l&amp;caggccg gggeccteag atccigatga ttgcagtctt caccetcact 1321 ggagefgtgg tgcigcicct pjfsgtegct ctcctgatgc toagaaaata tagaa&amp;agai 138.1 tatgaaeite gteagaaaaa atggtcccac atteetectg aaa&amp;fatctt fcaieiggag 1441 accaatgaga ccaatcatgl lagcetcaag atcgatgatg aeaaaagaeg agataeaate 1501 cagagaciac gacagtgcaa staogseaaa aagcgagtga ttctcaaaga tctcasgeac 1561 aalgatggia aittcsctga aaaacagaag aiagaattga acaagttgci toagatlgae 1621 iaftacaace fgaccaagtt ctacggeaea gtgaaaettg ataee&amp;tg&amp;t cttcggggtg 1681 afagaataet gigagagagg afceeteegg gaagitltaa atgacacaal Itcctacccf 1741 gatggp&amp;cat fcatggattg ggagtttaag afcietgiet Igtaigacai. tgdaaggga 1801 atgteatatc igcactccag taagacagaa giee&amp;tggtc gietgaaate laecaact ge 1861 giagiggaea gtagaatggt ggtgaagalc a&amp;gaifltg gctgcaattc eattttacct 1921 ecaaaaaagg acetgiggae agctccagag cacetocgcc aagccaacat etaicagaaa 1981 ggagatgtg! acagctatgg galeategca c&amp;ggagatea tcctgcggaa agaaaccttc 2041 tacactttga getgteggga ccggaatgag aagatMca gagtggaaaa liccaalgga 2101 atgaaacect tcegceeaga iitattetig gaaacagcag aggaaaaaga gedga&amp;gig 2161 iacctacitg taaaaaactg tigggaggaa gatecapaa agagae«aga fasasaaaa 2221 attgagacta caefigccaa gaiafttgga etttttcalg aceaaaaaaa tgaaagclal 2281 atggatacci igatecgacg ictacagcta taitctcgaa acciggaaca tctggiagag .2341 gaaaggaoac agctge&amp;caa ggcagagagg gacagggctg acagacifm cttlaqjttg 2401 cttccaaggc tagtgglaaa gtcixagaag gagaaaggct ttgiggagcc ggaaciatat 2461 gaggaagtta caafcctacii cagigaeatl glaggfttca etaetatcig eaaatacago 2521 acececaigg aagtgglgga catgctfaat gacaiclata agagitttga ceaeattgff 2581 gakate&amp;tg atgfctacaa ggtggaaacc- aleggtgalg egtaeaiggt ggetagtggt 2641 ttgcataaga paaiggcaa tcggcatgca atagacsttg eoaagatgge cftggaaatc 2701 de&amp;gcttca iggggaectt tgagctggag catctlcctg geetcecasl aiggattcgc 2761 attggagtte actctggtcc ctgtgctgci ggagttgtgg gaatcaagai geetegttai 2821 tgtciattfcg gagatacggt caacacagcc tetaggatgg aaiccactgg cetcccttyg 2881 agaaticacg tgagiggeie oacaatagac afcctgaaga gaaotgagig ccagttcctt 2941 tatgaagtga gaggagaaao atacttaaag ggaagaggaa aigagaciae claelggetg 3 001 actgggatga aggaccagaa attcaaeeig ccaaccecte ciaetgtgga gaaicaacag 3061 cgtdgcaag cagaaltttc agacatgatt gecaactett: iaeagaaaag acaggeagca 3121 ggg?ia.agaa gccaaaaacc eagacgggia gceagetata aaaaaggcac- tdggaaiae 3181 tigcagetga ataccacaga eaaggagage acciattitt aa (SEQ ID 140:227) {013] Amko add aeqwaace for humaa GCC (GeaPept Accession No, NPJ194954): 1 laktlMdiaf wsli%?gwl s&amp;sqvsqnc hagsyeisyl mmpaafaep Ikakdsvae 61 gldvirgriq aa^bvtvna ifmysdgEh Mgdcrssie egidllxMs naqrmgovli 121 gpsoiysifq myldtdsyp misagsfgls cdykedtrl msjwdkimyf ivnfwktodi 181 pfklpw'^s y?yknglete defwylnaie asvsyfehel g&amp;wlrqclk efqdilmdki 241 ifeaviiffieg gpefiyklkg dravaecSM ilYdlfedqy Mtxvtapdy jnknvhrltls 301 pgnsltassf smisptkrd falaytogll ligtalMCI engemtipk feha&amp;nlif 361 egydgpvtid dwgdvdstmv Ilytsvdtkk ykvlltydth vmkiypvdms ptfrwinsfcl 421 pnditgrgpq limiayfrl.t gawSHva HmlrkyrM yelrq.klr.wsh ippemfple 481 metahvslk idddkrrdti qdrqckydk kmlMlkh ndgnftekqk ielaldlqid 541 yyaltktygt vkMiaiilgv feycergslr evindbsyp dgtfindwetk isvlydiakg 60.1 msylhsskte vhgrffcstno yydsrmvvirii td%cnsilp pkkdtwiape Mrqanisqfc 661 gdvysygila qeiilifcetf yitodme k^v&amp;mg mkpftpdlil et aeekelev 72! yllvkacwee tipekrpdidt iettiaM% libdqknesy mdtlirrfql ysmlehive 781 ertqiykaer dradrluiml Iprfwkslk ekgfvepely eevdyfsdi vgftlickys 84 ί qtmewdmin diyksfdhiv dhhdvyfcvet igdaymvasg Ipkmgnrha idiakm&amp;lei 901 Isfingt&amp;le Mpglpiwir IgyhsgpcM gwgikaipry dfgdtvata snnestgi.pl 961 nhvsgsda ilkrtesqll yevrgetylk g^etiyw! tgmkdqMhl pippiYenqq 1021 rfqaefcdrai amlqfcmaa girsqkprrv asykkgtlej' Iqlnttdkes tyf (SEQ ID NO:228) [614] The GCC protein has some generally accepted domains each of which contributes a separable fimction ίο the OCC molecule. Hie portions of GCC include a : signal sequence (Sir directing the protein to the ceil surface) Som amino acid residue I to about residue 23» or residue 1 to about residue 21 of SEQ ID SI0:228 (excised for maturation to yield functional mature peotssm. from about amino acid residues 22 or 24 to 1073 of SBQ ID NO:228), an extracellular domain for ligand, e.g„, guanylin or ST, binding from about amino acid residue 24 to about residue 420, or about residue 54 to about residue 384 of SBQ .13 NO:228, a hansmemferane domain from about amino scad residue 431 to about residue 454, or about residue 436 to about residue 452 of SEQ ID N0:228, a kinase homology domain, predicted to have tyrosine kinase activity from about amino acid

residue 489 to about residue 749, or about residue 508 to about residue 745 of SBQ ID NO;228 and a gnanylyi cyclase catalytic domain from about residue 750 to about residue 1007, or about residue 816 to about residue 1602 of SEQ ID NO:228, filSJ In normal human tissues, GCC is expressed at the mucosa! cells, e,g.s at the apical bnish bolder membranes, lining the small intestine, large intestine and rectum (Csmthets et ai, Dis Calm Rectum 39; .171-181 {1996)). GCC expression is maintained upon neoplastic traagfostm^io» of intestinal epithelial cells, with expression is all primary and metastatic colorectal honors (Camthers et al, Dis Colon Rectum 39:171481 (1996);

Bucetat EurJCmcer 41:1618-1627(2905); CurTithers et al, Gas&amp;wnieroiogy 107: 1653-1661(1994)). Neoplastic cells from the stomach, esophagus and the gastroesophageal janctios also express GCC (see, fcg., U,S. Patent No. 6,767,704;

Debruync etal Gastroenterology 130:1.191-1206 (2006)), The'feme-specific expression and association with cancer, c.g,, of gastrointestinal origin, (e.g,, colon cancer, stomach cancer, or esophageal cancer), can be exploited tor the use of GCC m a diagnostic marker tor this disease (Carathers et at, Dis ColmMectm 39; 171-181 (1996); Sue et al. EurJ Cancer 4J; 1618-1627 ¢2005)), f0!6jf As a cell surface protds, GCC can also serve as a therapeutic target for receptor binding proteins such as antibodies or ligands, la normal intestinal tissue, GCC is expressed on the apical, side of epithelial cell tight junctions that form, an impermeable barrier between the luminal environment and vascular compartment (Almmoff et al, Mol

Microbiol 8: 865-873); Guarino ct al, BigBisSci 32: 1017-1026 ¢1987)). As such, sy&amp;emic intravenous administration of a GOC-Mnding protsin therapeutic will have minimal effect on intestinal GCC receptors, while having access to neoplastic cells of the gastrointestinal system, including invasive or metastatic colon carreer cells, extramiesllna! or metastatic colon tumors, esophageal tumors or stomach tumors, adenocarcinoma at the ’ gastroesophageal junction. Additionally, GCC tetoaliaes through receptor mediated endocytosis upon ligand binding (Sue ct al EurJ Cancer 41:1618462? (2005); Urbtmski ct. at, Btochem Biophys Ada 1245:29-36 (1995)).

[017] Polyclonal antibodies raised against the extracellul ar domain of GCC (Nandi et 4, Protein Expr, Purif, 8:.151-159 (1996)) were able to inhibit the ST peptide binding to human and rat GCC and inhibit ST-mediated eGMP production by human GCC.

[018J GCC has been ehamtottzed as a protein involved is cancers, iaciudmg colon cancers. See also, Carriihers et al, Dis Colon Rectum 39:171481 (1996); Bsc et al.

EurJCmcer 41:161.8-1627 (2005); Carriihers etal., Gastroenterology 107; 1653-1661(1994); Ofbaaski cl al., Biochem Siophys Acta .1245:29-36 (1995), Antibody molecule therapeutics directed to GCC can be used alone in ^conjugated form to thereby inhibit the GCC-eapressing cancerous cell s. Anti.-GCC antibody molecules of the invention can Uni huaian GCC, In some entoodiraenis, an anfi-GCC antibody molecule of the jtoveutio» can inhibit the binding of a ligsad, ag., guanyito or heat-stable entorotoxin to GCC, la other embodiments, an anti-GC€ antibody molecule of the invention does aot inhibit the binding of a ligand, e,g., guanylin or host-stable enterotoxia to GCC, {019f Monoclonal antibodies specific for GCC include GCC;BI0 (Nandi et ah, J.

Cell. Bmckem, 66:500-511(1997)), GCC :4D? (\tijayachandra et al Biochemistry 39:160754 6083 (2000)) and GCCC8 (Bskre et al Bur. J. Biochem, 267:179-187 (2000», GCC:B10 has a kappa light chain and an IgG2a isofypc and cross-resets to rat, pig and monkey GCC, OCC;Bl 0 binds to the fust 63 ammo acids of the intracellular domain of GCC, specifically to residues 470-480 of SBQ ID NO;22S (Nandi et si. Protein Sci 7:2175-2183 (1998)). GC€:4D? binds to die ktoase homology domain, withto. residues 491-568 of GCC (Bfeandari et at Biochemistry 40:9196-9206 (2001». G€C:C8 binds to the protein Mmse-like domain to the cytoplasmic potto» of GCC. f020| Unless otherwise defined herd», meatifk aid technical tons used is connection with the present invention have the meanings that are commonly understood by those of ordinary skill to the art. Generally, nomenclature utilized in connection 'with, and techniques of, cell and tissue culture, molecular biology, and protein and align- or polynucleotide diemistiy and hybridization described herein are those known in the art. OenBank or GenPept accession numbers and useful nucleic acid and peptide sequences can fee found at the website maintained by the National Center tor Biotechnolopcal information, Bethesda MD, Standard techniques am used tor recombinant DNA, oligonucleotide synthesis, and tissue culture ami transformafion and transfection (e.g., electroporation, lipofechon). Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished ia the art or as described hereto. The foregoing techniques and procedures are generally performed according to methods known to. the art, c,g,, as described in various general and more specific references that am cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory e&amp;, Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, NX ¢2000)) or see generally, Harlow, B. and Ime, B, (I$8B) Antibodies: A Laboratory Manned, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The sommdatares utilized in cotmeetiba with, and the laboratory prmtedures and tedtaiqaes of, analytical dbesraMry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry describedherein are 1310¾¾ in the art. Standard techniques are used for chemical syntheses, chemical analyses, pfearmacenttcal preparation, formulation, and delivery, and treatment of patients. Furthermore, unless otherwise inquired by coatesO, singular terms shall include pluralities and plural terms shall include the singular, im As used herein, the term '‘antibody molecule” refers to an antibody, antibody peptkteCs) or immunoglobulin, or an antigen binding fragment of any of tire foregoing, e,g.s of m antibody. Antibody molecules include single chain antibody molecules, e,g,, scFv, see. e«g., Bird et al, (1988) Science 242:423-426; and Huston et al (1980) Pmc. Nail Acad, ScL USA 85:5879-5883), and single tfomatn antibody molecules, see, e,g., WO9404678. Although not within the term “antibody molecules/’ the invention also includes “antibody analog^}/’ other non-antibody molecule protein-based scaffolds, e,g., fusion proteins and/or tmmunocmjugates that use CDRs to provide specific antigen binding, 1022] As “anti-GCC antibody molecule” refers to an antibody molecule (ie., an antibody, antigen-binding fragment of an aadibody or antibody analog) which interacts with or recognizes, e,g,, binds (e.g., binds spedfically) to GCC, e.g,„ human GCC. Exemplary antx-GCC antibody molecules are such as those summarised in Tables 1 and 2. 1923} As used herein, the term “antibody/’ “antibody peptide(s)w or iiimmtmoglobuim,‘ refers to singl e chain, two-chain, and multi-chain proteins and glycoproteins. The term antibody includes ftofydoaaf, .monoclonal, chimeric, CDR-grafted and human or humanized antibodies, all of which are d&amp;enssedin more detail elsewhere herein. Also included within the term are eamelid antibodies, see, e.g,, U82O05/003742I, and mnobodies, e.g.s igNARts (shark antibodies), see, e.g., W003/0.14161, The term “antibody” also includes synthetic and genetically engineered variants, |'#24J As used herein, the term “antibody fragment” or “antigen binding fragment" of an antibody refers, e.g., to Fab, Fab’, F(ab% and Fv fragments, single chain antibodies, fonctional heavy chain antibodies (nanobodies), as well as any portion of an antibody having specificity toward at least one desired epitope, that competes with the intact aafflbody for specific Muding (e.g., a fragment having sufficient CD&amp; sequences and having sufficient framework sequences so as to bind tpedficafiy to m epitope), E.g., an antigen binding fragnient can compete for binding to an epitope which binds the antibody from which the fragment was derived. Derived, as used in this and similar contexts,, does not imply any particular method or process of derivation, but can refer merely to sequence similarity. Antigen binding fragments can he produced by recombinant techniques, or by enzymatic or chemical cleavage of an intact antibody. The term, antigen binding fragment, when, used with a stogie chain, e.g., a heavy chain, of an antibody having a light and heavy chain means that the fragment of the chain is sufficient such that when paired with a complete·variable region of the other chain, e.g., the light chain, it will allow binding of at least 25,50,75, 85 or 90% of that seen with the whole heavy and light variable region,) [¢251 The term, %rttgea binding constellation of CDRs” or “a number of CDRs sufficient to allow binding* (and similar language), as used herein, refers to sufficient CDRs of a chain, e.g., the heavy chain, such that when placed in a framework end paired with a complete variable region of the other chain, or with a portion of the other chain's variable region of similar length and having the same number of ODEs, e,g„ the light chain, will allow binding, e,g., of at least 25,50,75,85 or 90% of that seen with the whole heavy and light variable region. |1M) As used herein, the term “human antibody” includes an antibody that possesses a sequence that is derived from a human gera^line .immunogtolrniin sequence, such as an antibody derived from transgenic mice having human immunoglobulin genes (e,g., XENOMOUSEmi genetically engineered mice (Abgemx, Fremont, €A)}, human phage display libraries, human myeloma ceils, or human B cells. P27J As used herein, the term “humanized, antibody” refers to an antibody that is derived from a.non~husmn antibody e.g,, rodent (e.g., murine) that retains or substantially Mains the audgen-binding properties of the parent antibody but is less immunogenic in humans. Humanized as used hereto, is intended to include dehnmunlzed antibodies. Typically humanized antibodies include non. -human CDRs and human or human derived framework and constant regions, [028| The term “modified” antibody, as used herein, refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e<g,, a mouse, sheep or goat) that is transgenic- for-human irnmnno^obalin genes or antibodies propped, expressed, created or isolated by Say other means that involves splicing of human immunoglobulin gene sequences to other DMA sequences, Such modified antibodies include hnnianksed, COR grafted (&amp;&amp;, a» antibody haring CDRs ftom a first antibody and a feaaieworfe region from a different source, e.g,, a second antibody or a consensus framework), chimeric, in vitro generated (e,g., by phage display) antibodies, and may optionally include variable or constant regions derived from human germline ittummoglobuim sequences or human immtmogioMm genes or antibodies which have been prepared, expressed, created or isolated by any means that involves splicing of human irnmnnoglofewlin gene sequences to alternative inmtunogiobuMn sequences. In embodiments a modified antibody molecule Includes mi antibody molecule having a sequence change fern a reference antibody, [§S9| The term "monospecific antibody " refers to an antibody or antibody preparation that displays a single binding specificity and affinity for a particular epitope-This term includes a "monoclonal antibody” or "monoclonal antibody composition,” (93$! The term "bispecific antdmdy” or "bifimetiona! antibody” refers to an antibody that displays dual binding specificity for two epitopes, where each binding site differs and recognises a different epitope.

The terms %on-conjugated antibody" and "naked antibody" are used interchangeably to refer to an antibody moleeole feat is not conjugated to a non-antibody moiety, e.g., a therapeutic agent or a label im j The tern® "Imnumoconjugate” "antibody conflate”, "antibody drug conjugate”, and "ADC” are used interehangeabiy and refer to m antibody that is conjugated to a non-antibody moiety, e,g., a therapeutic agent or a label f$?3 J The term "agent” is used herein to denote a dnumcai compound, a mixture of chemical compounds, a biological macmmoleoule, or an extract made from biological materials. The term “therapeutic agent” refers to an agent that has biological activity.

[834] The term "anti-cancer agent” or "chemotherapeutic agent” is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neopl asm in. a human* particularly a malignant (canceious) lesion., sigh as a caroawna, sarcoma, lymphoma, or leukemia, toMbition of metastasis or angiogenesis is frequently a property ofaatitoaaoer or chemotherapeutic agents. A chemotherapeutic agent may be a cytotoxic or cytostatic agent The term “cytostatic agent* refers to an agent which inhibits or «oppresses cell growfe and/or multiplication of ceils.

[03S] “Cytotoxic agents* refer to compounds which cause ceil death primarily by arierferiag directly with the cell's fenctioning, metudmg, kit not limited to, alkylating agents, tumor necrosis facte inMbttofs, totecalatots, microtubule khltotors, kinase Inhibitors, proteasome inhibitors and topoisomemse inhibitors, A ‘"toxic payload” as used herein refers to a sufficient amount of cytotoxic agent which, when, delivered to a cell results m cell death. Deliway of a toxic payload may be accomplished by aknlsistration of a sufficient amount of inanunoeoujugate comprising an antibody or antigen binding fragment of the invention md a cytotoxic agent. Delivery of a toxic payload may also be accomplished by atomnistration of a sufficient amount of an immunocortjugaie con^risirtg a cytotoxic agent, whereat toe irnmnnoconjugate comprises &amp; secottdaty antibody or antigen binding fragment thereof which «cognizes and binds an antibody or antigen binding fragment of toe invention. I&amp;M1 As used herein the phrase, a sequence ’"derived from” or “specific for a designated sequence” refers to a sequence that comprises a contiguous sequence of approximately at least 6 nucleotides or at least 2 amino acids, at least about 9 nucleotides or at least 3 amino acids, at least about 10-12 nucleotides or 4 ammo acids, or at least about 15-21 nucleotides or 5-7 amino acids comtspondiag, he., identical or eomplemeataty to, e.g., a contiguous region of the designated sequence, to certain embodiments, toe sequence comprises all of a designated nucleotide or amino acid sequence. The sequence may be comptementory (in the ease of a polymioieotide sequence) or identical to a sequence region that is unique to a particular sequence as determined by techniques known in the art. Regions from which sequences may be derived, include but are not limited to, regions encoding specific epitopes, regions encoding CDRs, regions encoding framework sequences, regions encoding constant domain regions, regions encoding variable domain regions, as well as aon-franslated andtornon-traascribed regions. The derived sequence will not necessarily be derived physically from the sequence of interest under study, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or tran.scrlpf.ion, that is based on the information provided by the sequence of bases k the reglonCs) fiom which the polymicleofide » derived. As such, it may represent either a sense· or a» antisense orientation of the original polynucleotide. la addition, combinations of regions corresponding to that of the destp&amp;ted sequence may be modified or combined k ways known m the art to be consistent with the intended use. For example, a sequence may comprise two or more contiguous sequences which each comprise part of a designated sequence, and are interrupted with a region which is not identical to the designated sequence but is intended to represent a sequence derived from the.desigsated sequence. With regard to antibody molecules, “derived therefrom* includes an antibody molecule which is feuctionally or structurally related to a comparison antibody, e.g„ “derived iherefionf* includes an antibody molecule having similar or substantially the same sequence or structure, e.g„ having the same or similar CDRs, framework or variable regions. “Derived therefrem” for an antibody also includes residues, e.g., one or more, e.g,, 2,3,4, 5,6 or more residues, which may or may not be contiguous, but are defined or identified according to a numbering scheme or homology to general antibody structure or three-dkmrional proximity, Le., within a C.DR or a framework region, of a comparison sequence. The term “derived therefiom’* is not limited to physically derived therefieia but includes g<meraribn by any maimer, e.g., by use of sequence reformation item a comparison antibody to design another antibody, ft37J As used herein, the phrase “encoded by” refers to a nucleic acid sequence that codes for a polypeptide sequence, wherek the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, at least 8 to 10 amino adds, or at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. |#3S] Calculations of “homology’’ between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e,gM gap can bo introduced is one or both of a first and a second amino add or nucleic add sequence for optimal alignment and nen-horeoiogous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned, for comparison purposes is at least 30%, 40%, or 50%, at least 60%, or at least 70%, 80%, 90%, 95%, 100% of the length of the reference sequence. The ammo acid residues or nucleotides at corresponding amino add positions or audeoride positions are then compared. When a position in the first sequence Is occupied by the same amino add residue or nucleotide as the corresponding position ία the second sequence, fees the Molecules are identical at that position (as used herein amino acid or nucleic acid ‘%icntity,s k equivalent to amino acid or nucleic add “homology”). The percent identity between the two sequences is s function of the number of identical positions shared by the sequences, taking into account the number of gap®, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. mn The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid sequences can be determined using any method known la the art. For example, the Needleman and Wunsck, J. Mol Biol 48:444-453 (1970), algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossom 62 matrix or a PAM250 matrix, aud a gap weight of 16, 14, !2,10,8,6, or 4 and a length weight of 1,2,3,4,5, or 6. The percent homologs? between two nucleotide sequences can also be determined using toe GAP program in fee GCG software package (Aceelerys, Inc. San Biego, CA), using an KWSgapdna.CMP matrix and a gap weight of40,50,60,70, or 80 and a length weight of 1,2,3,4,5, or 6,

An exemplary set of parameters tor drtemtioation of homology are a Blossom 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a framesfaift gap penalty? of 5, [040J As need hernia, fee tens ‘hybridizes under stringent conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found i n Current Protocols in Molecular Biologyt hha Wiley &amp; Sons, NX (.1989), 6.3,1-6.3.6, Aqueous and nooaqneous methods am described in feat reference and either can be used. Specific hybridisation conditions refereed to herein are as follows: 1) low stringency hybridization conditions in 6X sodium cMoride/sodiura citrate (SSC) at about 45eC, followed by two washes in 0.2X SSC, 0,1% SDS at least at 5ft°C (the temperature of fee washes can be increased to 55°C for low stringency conditions); 2) medium stringency hykifezatiem conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0J% SDS at 60°C; 3) high stiingeaoy hytmdizatk» conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0,1% SDS at 65°C; and 4) wry high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0,2X SSC, 1% 80S at 65°C, Very high stringency conditions (4) are often the preferred conditions and the ones that should be used, unless otherwise specified. |041i It is understood that the antibodies and antigen binding fiagmenf thereof of the invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide fimsfioos. Whether or not a particular substitution will be tolerated, f e., will not adversely «fleet desired biological properties, such as binding activity, can be determined as described in Bowie, JU et al. Science 247:.13064310 (1990) or Padlaa et at FASKB J, 9:133439 (1995). Λ “conservative amino acid substitution” is one in which the amino add residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains hav e been defined in the ait These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g,, aspartic add, gbitemie acid), uncharged polar side chains (e.g„ asparagine, glutamine, serine, tbxeomne, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleudne, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleacme) mid aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). 1042) A “non-essetrtkr amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or, without substantially altering a biological activity, whereas an “essential** amino acid residue results In such a change. In an antibody, an essential amino acid residue can be a specificity determining residue (SDR).

[043] As used herein, the teem “isolated5* refers to material that is removed from its original environment (e.g., the natural environment If it is naturally occurring). For example, a naturally occmrmg polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DMA or polypeptide, separated from some or «II of the coexisting materials in the natural system, is Isolated. Such polynucleotide could foe part of a vector and/or such polynucleotide or polypeptide could be pan of a composition, e.g., a mixture, solution of suspension, or comprising an isolated cell or a cultured cell which comprises the polynucleotide or polypeptide, and still he isolated in that tire vector or composition is not pari of its natural environment.

[M4i As used herein the term “repiicon” refers to any genetic element, such m a plasmid, a chromosome or a virus, that behaves as m autonomous tunt of polynucleotide replication within a cell

As used 1-1¾¾¾ the term “operably KakedP refers to a situation wherein the components described are in a relationship permitting them to function in their intended meaner. Thus, for example, a control sapience “opembly linked” to a coding seqasace is ligated in such a manner that expression of the coding sequence is achieved under conditions eonqiatible with the control sequence. fiHti] As used herein, the term “vector refers to a teplioon I» which another polynucleotide segment is attached, such m to bring shout the replication and/or expression of the attached segment. (©47| As used herein, the tom “control sequence?5 iste to a polynucleotide sequence that is necessary to effect the oppression of a coding sequence to which it is ligated. The nature of such control sequences differs depending upon the host organism.

In prokaryotes, such control sequences generally include a promoter, a ribosomal binding site and tenmnatots and, in some instances* enhancers. The term “control sequence* thus is intended to include at a minimum, all components whose presence is necessary for oppression, and also may Include additional components whose presence is advantageous, for example, leader sequences. im As used herein. the term “purified product” refers to a preparation of fee product which has bee» isolated from the cellular constituents with which the product is normally associated and./ or from other types of cells that nay he present in fee sample of interest |04§} As used hernia, fee ten» “epitope” refers to a protein determinate capable of binding specifically to an antibody. Epitopic determinants usually consist, of chemically active surface groupings of molecules such as amino adds or sugar side chains and. usually have specific three dimensional structural chanctotistics, as well as specific charge characteristics. Some epitopes are linear epitopes while others are conformational epitopes, A linear epitope is an epitope whereat a contiguous amino acid primary sequence comprises fee epitope recognized. A linear epitope typically includes at least 3, and more usually, at least 5, for example, about 8 to about 10 contipous amino acids. A conformational epitope can result from at least two situations, such as; a) a linear sequence which is only exposed to antibody binding in cataea protein combinations, e.g., dependent on ligand binding, or dependent os modification (e.g., phosphmylatioa) by signaling molecule; or fe) a eornMnation of structural features from more than cate part of the protein, of in multebunit proteins, from more ter one subunit, wfeein the features are in sufficiently dose proximity in 3«dhnens«mal space to participate in binding. |βδ»| As need herein, “isoiype” refers to the antibody class (ag., IgM or IgGl) that is encoded, by heayy chain constant region genes, [0S1J As used herein, the terms “detectable agent,15 “labef or “labeled” are used to refer to incoiporaiion of a detectable marker <a* a polypeptide or g!yco|sihek. Various methods of labeling polypeptides and'glycoproteins are known in the art and may be used. Examples of labels ibr polypeptides include, but are sot limited to, the following: radioisotopes or radionuclides (e,g., indium (UIIa), iodine (° lI or mI), yttrium f*Y% lutetiatn {mLu), actinium <335Ae), bismuth (3i3Bi or SBBi), snlfitr (3¾ carbon (i4C), tritium (3H), rhodium (m&amp;fe)} technetium (S9mTe), praseodymium, or phosphorous (^P) or apoatiron-emiMng radionuclide, e.g., carbon-11 (aC), potaasi.um-40 ^SK), nitrogen-13 (dN)s oxygm-15 (xS0% fluorine-18 (iSF), and iodine-121 (R'I)), fluoreseeoi labels (eg., FITC, rhodamiae, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, heta-galactosidixse, Inciierase, &amp;lkrdme phosphatase), ehmtihunineseeai, bfotiayl groups (which can be detected by a marked avidin, e.g,, a molecule containing a streptavidm tnoiety and a fluorescent marker or art enzymatic activity that can be detected by optical or calorimetric methods), and predetemiined polypeptide epitopes recognized by a secondary reporter (e.g,, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential sterie hindrance, ί§52| As used herein, “specific binding,” “hindfs) specifically" or “binding specificity” meatss, tor an asti-GCC antibody molecule, that the antibody molecule binds to GCC, e,g,, tinman GCC protein, with greater affinity than it does to a non-GCC protein, e.g., BSA, Typically an antriOCC molecule will have a K4 for the tm-GCC protein, e.g.* BSA, which is greater than 2, grater than 10, greater than 100, greater than 1,000 times, greater than 104, greater than 10s, or greater than. 1# times its K4 for GCC, e.g., human GCC protein. In determination of K&amp; the 1¾ for GCC and the nat-GCC protein, e,g., BSA, should be done under the same conditions. |®S3J As used herein, the term “treat” or “treatment” is defeed as the administrate of an ant?«OCC antibody molecule to a subject, e.g., a patient, or administration* e.g,? by application, to an isolated tissue or ceil from a subject which is returned to the subject. The a&amp;ti-G€€ antibody molecule cm be administered alone or 1« combination. with a second agent. The tmatmeut can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder, e.g., a cancer. While not wishing to be bound by theory, treating is believed to cause the inhibition, ablation, or killing of a cell in vitro or in vivo, or otherwise reducing capacity of a ceil, e.g., an aberrant cell, to mediate a disorder, e.g., a disorder as described hemic (e.g., a cancer).

[§541 As used hereto, the term "subject** is intended to include mammals, primates, humans and non»fcmnan animals. For «temple, a subject can be a patient (e.g., a human patient or a veterinary patient), having a cancer, e.g., of gastrointestinal origin (e.g., cobs cancer), a symptom of a cancer, e.g,, of gastamtestinal origin (e.g,, colon cancer), m which at least some of the cells express GCC, or a predisposition toward a cancer, e.g., of gastrointestinal origin (e.g„ colon cancer), m which at least some of the cells express GCC. 'The term teon-human animals** of the invention iaeludes all nonrimman vertebrates, e.g., non-human mammals arte non-mammals, such as aon»feuman primates, sheep, dog, cow, chickens, amphibians, reptiles, etc, nates otherwise noted. In an embodiment subject excludes one or more or all of a mouse, rat, rabbit or goat. 1855} As used herein, an amount of an anti-GCC antibody molecule “effective” or “sufficient” to treat a disorder, or a “therapeutically effective amount55 or “therapeutically sufficient, amount* refers to an amount of the antibody molecule which is effective, upon single or multiple dose admtestration to a subject, in treating a cell, e.g., cancer cell (e.g., a GCC-expressing tenor cell), or in prolonging caring, alleviating, relieving or improving a subject with a disorder as described herein beyond that expected m the absence of such, treatment. As mod herein, “inMhitkg the growth” of the tumor or cancer reiess to slowing, interrupting, arresting or stopping its growth and/or mefastases and does not necess arily indicate a total elimination of the tumor gr owth, 18561 As used herein, “GCC ” alsoknown as “STAR” “GCJC2C”, **GUCY2C** or “ST receptor*5 protein, refers to mammalian GCC, preferably human GCC protein. Human GCC refers to die protein shown hi SEQ ID N0:228 and naturally occurring allelic protein variants thcraofr The allele in SEQ ID NO; 228 can be encoded by the nucleic add sequence ofGCC shown in SEQ ID NO:22X Other variants are known k the art. See, eg., accession number EnspOOO026117e, Basembl Database European Bioinfomnatics institute and Wellcome Trust Sanger Institute, Which has a lencke at residue 281; SEQ ID NO: 14 of published OS patent application number US 2OD6O035852; ctGeoBank accession number AAB19934, Ty$catty, a satem% occurring allelic variant baa an amino add sequence at least 95%, 97% or 99% identical to the OCC sequence of SEQ IP NO:228. The transcript encodes a protein product of1073 amino adds, and is described in GenBank accession no,: NMJJ04963. GCC protein is characterized as s ixausmembrane cell surface receptor protein, and is believed to play a critical role in the maintenance of intestinal fluid, electrolyte homeostasis and cell proliferation, 18571 Unless otherwise noted, the term “alkyl” rote to a saturated straight or branched hydrocarbon having ten about 1 to about 20 carbon atoms (and all combmations and subcombmations of ranges and specific numbers of carbon atoms therein), with fern about 1 to about 8 carbon atoms being preferred. Examples of alkyl groups are methyl ethyl, «-propyl, feo-propyi, «-butyl, .iro-buiyl, <ree-buiyl, mri-butyl, #* pentyl, 2-p entyl, 3-pentyi 2-methyI-2-butyl, «-hexyl, n-heptyl, n-octyl «Haouyl, «-decyl, 3*ftteihyi~2~buiyl, 3-methyl- 1-butyl, 2~methyl~l-butyl, I-hexyl, 2-hexyl, 3-hexyl, 2-methyi-2-pentyI, 3~meihy!~2~pentyi, 4-metoyl“2-pentyh S-methyl-S-pentyl, 2-raelhyl~3~ pmtyl, 2,3-dimethyl-2~huiyl, and 3,3-dimethy t~2~butyl. f®SS] Alkyl groups, whether done or as part of another group, may be referred to as "substituted,” A substituted alky l group is an alkyl group that is substituted with oae or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to, -halogen, ~0-(€rC* slfeyd), -0-{€2-C8 alkenyl), -<HCrCs alkynyl), -aryl, -€(0)RI -0€(O)R*, ~C<0)ORS, -C{0)NH2, -€{0}NMR\ -C<Q)N(R%;NH€(Q)R% -SR.', -S03R% -S(0)2r, -S{0)R', -OH, -O, -N3, -NH2, -NH(R')} -19(115)¾ aud -CM, where each R' Is independently selected from -H, -Ct-Cg alkyl, -CrC8 alkenyl, -C?.>Ca alkynyl, or -aryl, and wherein said -0-(0-08 aUsy!)» *<ΜΟΚ* alkenyl), -0-(02-08 alkynyl), -aryl, -Cj-Cg alkyl, -CrCg alkenyl, and -02-Cs alkynyl groups can be optionally further substituted with one or more groups including, but not limited to, «Cj-C* alkyl, -CrCs alkenyl, ~CrCs alkynyl -halogen, -0-{€rCa alkyl), -O-(Q-Cs alkenyl), -0-(€rCs alkynyl), -aryl, -€(0)R" -00(O)R'S, -0(0)0R55( -C(0)NH2, ~C(0)MHR", -C(0)N(R”>, -NHC(0)R»5 -SR", -SO#”, -S(0)aR”-S(0)R", -OH, ~N3 , -NEg, “NH(R5 f% ~N(M”h md -CN, where each R? 5 is mdepetidenfly selected from -¾ -CrCs alkyl, ~C2-C8 alkenyl, -CVCg alkynyl, or «aryl f0$fJ Unless otherwise noted, the tern “dlkenyf:5’ and “alkynyf5 refer to straight and branched carbon chafes having from about 2 to about 20 carbon atoms (and all combinations arid s^BomUestkm of ranges aad specific numbers of carbon atoms therein,), with from about 2 to about § carbon atoms being preferred. An alkenyl chain has at least one double bond in the chafe and an alkynyl chafe has at least one triple bond fe the chain. Examples of alkenyl groups include, bat are not limited to, ethylene or vinyl, ailyl, -I-butenyl, “2-butenyl, -isobfeylenyl, 4-peatenyl, -2-p cntenyl, «3“toethyl“l“hBieay!s ~2-methy1~2~hutenyL and -2»3-dimcdtyl«2-bufeayf. Examples of alkyayl groups include, but arc not limited to, acetylenic, propatgyl, aeefylenyl, propynyi, -d-bntyny!, -2-butynyI, -l-pentyoyl, -2~peniyayl, and -S-methyl-l batynyl

|0601 As with. alkyl groups, alkenyl and alkynyl groups, can be sobstitnkxL A “substituted” alkenyl or alkynyl group Is one that is substituted with me or more groups, preferably 1 to 3 groups (and any additional snbsdtuents selected from halogen), including but not limited to, -halogen, -0-(0-0¾ alkyl), ~0~(CrC* alkenyl), ~0~(QrCg alkynyl), -aryl, -C(0)Ts <XXO)R\ -0(0)011% -0(0)«%, ~0(0}ΜΙ®% -€(0)N(R% -NHC(OW> -SR\ ~SGaR\ "Sip)2r, -S(0)R\ -OH, -O, -N3, -NH2, -NH(R')S ~®{R% and «CN, where each R? is independently selected from -if, -€i~Cs alkyl, -Cg-Q affeyenl, ~ CfeCi alkynyl, or -aryl and wherein said -0-(Cj,-C* alkyl), -OCQrCe alkenyl), -0-(0g-Os alkyayl}, -aryl, «C*-€s alkyl, -CfeCg alkenyl, and -C2-G5 alkynyl groups can be optionally further substituted with me or more sebstitmns fedtidmg, hfe not Ihnited to, -C j«Cs alkyl, -Cj-Cg alkenyl, -Cg-C* alkynyl, -halogen, -0-(0-0¾ alkyl), -0«<C*-Ca alkenyl), -©-(CgCs alkynyl), -aryl, -C(0)Rr» ~0C(0)R”, ~C(0)Gr\ -C{0)M;i2, -€{0)NBR5% -NHC(0)Rs\ -SR”, -SO3R”, -S(0)3R’% -S(0)R’% -OHf -N3, -NHg, -NHCR”), -N0f”)t and -CN, where each R” is fedepeadeatly sdectedfiom -H, -Ci-C*alkyl, -Q-C$ alkenyl, -€2-(¾ alkynyl, or -aryl.

[061] Unless otherwise noted, the term ’’aikykne” refers to a saturated branched or straight chain hydrocarbon radical having fern «boot 1 to about 20 carbon atoms (and all combinations and subcofebinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 8 carbon atoms being praferred and haviag two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two difimart carbon atoms of a parent alkane. Typical *&amp;y$enes include, but m sot limited to* methylene, ethylene-, propylene, butylene, peaiyiene* hexylene, heptyleae, ocytylem, nmyleae, decalene, 1,4-cyclohexylene, and the Eke. Alkytene groups, whether alone or as part of another group, cm be optionally substituted with one or mom groups, Inferably 1 to 3 group (and any additional sufesdtoents selected from halogen)* moluding, but not limited to, -halogen, -0-(0^-¾ alkyl), ~Q~(Cr€k alkenyl), -©-(Cj-Cg alkyuyi), -C(G)R’, -0C(0)r, -€{0)0R’, ~C(0)Ne2, <0)NHT, -qO)K0t% » mC(QW, -sir, ~SOsR!5 -8(0)^, -S{0)r, -OH, *0, -1%, -Mfe -MH(r), -Wfc aad -ON, •«tee each IT isindependently selected from -H, -CrCs alkyl, -Ch-Cs alkenyl, -Cr €$ alkynyl, or -ary! and wherein said -CHCrCs alkyl), ~0-(C2~Ca alkenyl), -CHCrCs alkynyl), -aryl, -Ci-Q alkyl, -Cj«C$ alkenyl, and -CVCS alkynyl groups can be further optionally substituted with one or more subshtuenteincludiag, but not limited to, -Cj-C? alkyl, -CVCs alkenyl, «C2~Cg alkynyl, -halogen, -0-{CrCs alkyl), »0-{C2~€s alkenyl), -0-(QrCa alkynyl), -aryl, -€(0)R” ~OC(OW\ ~C(0)QR” -C(0)NH2, ~€(0}ΗΗΙΙ”, - c(ojn(r*% ~mc(ow\ -sr*» ~$o3r \ -s(0)2rs, -s<p)R”, -oh, -Ns, ~nh2, - NH(R”), -NiR”)2 and ~CN, where each R” is independently selected from -I-l, -C\-C% alkyl, ~Gr€s alkenyl, -Cb-Cg alkynyl, or -aryl (062j Unless otherwise noted, the term, “aikenylme5’ refers to an optionally substituted alkylene group oonMining at least one carbon-carbon double bond. Exemplary aikesylsne groins include, for example, etheoyleue (-€H==CH-) and propenylene (-CHCHOfc-), fW3] Unless otherwise noted, the term “alkynyiese” refers to an. optionally suhstiteiedalkylcne group containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, acetylene (~C®C~), propatgyl (~€H20a€-), and 4~ pentynyl (-CHZCH2€H2CMTT). 10641 Unless otherwise noted, the term “aryF refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (and all combinations and suhoomfckaiions of ranges and specific numbers of carbon atoms therein) derived by the remo val of oae hydrogen atom, from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented k the exemplary structures as “Af*. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like. |06S| An aryl group, whether alone or as part of another group, can be optionally substituted with one or more, preferably I to 5, or even 1 to 2 groups including, bat not limited to, -halogen, »€i-C$ alkyl, -Q-Cg alkenyl, ~Cz-C$ alkynyl, -<>-(€ r€s alkyl), -O-(CrC* alkenyl), -CKCrCg alkyayl), -aryl, -€(0}ΚΛ ·0€{0’β% -€(0)0ΚΛ -C(0}NH2, -€<0)Ν1®\ -C(0)M(R% ~MHC{0)R% ~SR!,«-S(0},R\-S{0)R\ -0¾ -N02, -Ns , -1¾. ~NB(R*), »NpU)3 and -CM, where each R; is indcpendoHiy selected from -¾ -€r Cs alkyl, -CrQt alkenyl, -C*-Cs alkynyl, or -«tyl sad wherein said «Cj-C* alkyl, -CrCg alkenyl, -C2-C&amp; alkynyl, 0-{Ci“C§ alkyl), -0-(02-¾ alkenyl), -0~(CrCg alkynyl), and -aryl groups can be farther optionally substituted with one or more sabstitnents including, but not limited to, -Cj-Cg alkyl, -CrC8 alkenyl, -Qj-Q alkynyl, -halogen, -0~{Ci-€&amp; alkyl), ~ <MCrC* alkenyl), -0-<€3-€s alkynyl), -aryl, -C<0)R’\ -QC(0)R!\ ~C(0)0R”, -C(0)», ~€<0)NHR% -0(0)^(10¾ ~ΝΗ€(0)!Τ, -SR”, -S03R”, -8(0^2% -S(0JR.”, -OH, -N3, -NH3, ~NH<R.ss), -Ν(Ε’% and -CM, where each R” i&amp; independently selected from ~H, -Cj-Cs alkyl, -€2-C8 alkenyl, -Cj-Cg alkynyl, or -aryl.

Iftbd] Unless otherwise noted, the term “aryleno” refers to an optionally substituted aryl group which is divalent (In... derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent aromatic ring system) and can be in the ortho» raeta, or para configurations as shown in the Mowing structures with phenyl as tire exemplary aryl group:

Typical “-(CrC3 alkyime)aryl,>5 “-(€rC3 aikmy1sne)aryr, "and-fCrCa alkynylene)aryr groups include, but arc not limited to, benzyl, 2-phenyIetfaan4-yl, 2-phenyleihen-l -yl, naphthylmethyl, 2»nsghthyietfaan-i»yl, 2-naphihyIethm-l»yh naphthobenzyl, 2-ttaphthop.henyIeihan-:l -yl and the like, im Unless otherwise noted, the term f,heteriteyde,fi refers to a monocyclic, bieyclic, or polycyclic ring system having from 3 to 14 ring atoms (also referred to as ring members) wherein at least one ring atom in at least one ring is a heteieatom selected from N, 0. P, or S (and all combinations and subeombmations of ranges and specific numbers of carbon atoms aad heteroatoms therein), The keierocyele can have from 1 to 4 ring heteroatoms independently seleeted from N, 0, P, orS. One or mom N, €, or S atoms in a faetemcycle can fee oxidized, A monoeylie heteroeyde preferably has 3 to 7 ring members (e,g,s 2 to 6 carbon atoms and I to 3 heteroatoms kdepeadesuly selected from N, Ο, P*or S), and a Mcyelie hetetocyele preferably has 5 to IQ ting members (e,g.f 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from Ν» Ο, P, or S). The ring that includes the heteioatam cm fee aromatic or non-aromatic. Unless otherwise noted, the hetetocyele is attached to its pendant group at any hetematom or carbon atom that remits m a stable structure.

[868] Heterocyeies are described in Paquette, "Principles of Modem. Hotorocyebe

Chemistry" (WA Benjamin, New York, 1968), pmticuiarfy Chapters 1,3,4,6,7, and 9; 'The Chemistry of Heterocyclic Compotmds, A series of Monogrsqths" (Mm Wiley &amp; Sons, New York, 1950 to present), m particular Vetoes 13,14,16,19, and 28; and/

Am* Chem, Sac, 82:5566 (I960), [169] Unless otherwise noted, the term “heterocyelo” refers to an optionally substituted hetecoqycle |px>np as deSaed above that is divalent; (in., derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent heterocyclic ring system), [070] Examples of “heteroeyele” groups include by way of example and not imiitetion pyridyl, dihydropyridjd/tofrahydropyridyl (piperidy!), thiazofyl, pyrimldkyl, furanyl, thienyl, pyrrolyl, pytazolyl, midazolyl, tehazolyl, beumfutaayi, thianaphthalenyb iadolyl, indblenyl, quinolmyl, isoqoinolinyi, benriniid&amp;zolyl, jnperidroyi, 4-piperidcmyb pynolidinyl, 2~p ynroildonyi, pyrrolmyl, tetrahydrofuranyh feis-tefrahydrefuranyl, iefrahydit^yraayl, bis-tetrahydmpymnyl, tetmhydroquinolinylstetrahydroisoqmnoMnyi, decahydmqnmolinyl, o ctaitydroisoqumolinyl, azodayt, ttmriuyi, 6H~ 1,2,5-thiadiaziny 1, 2H,6H~l,5,2-^isia3inyl, tMenyl, thiantbrenyl, pyranyl, isobenzoftiranyl, ck'amenyl, xassthenyl, phenoxatoinyl, 2H-p)nxolyi, isotMazolyl, isoxaaolyl, pymzinyl, pyridtd&amp;yh mdolkkyl Isoiadolyl, 3H-kdoiyl, IH-mdaxolyi, porinyl, dH-qumolizinyl, phthalaamyls naphthyridiayl, qnfeoxalinyl, qumaxoliayl, cmnolkyf pieridloyl, 4H-earbazolyl, carbazolyl, p-carboiinyi, phenanthridinyl, acridinyl pyrimidiavl, phenantkniinyl phena:rinyi,phenoMaknyi, fnrazaayi, phenoxamnyl, ssochmmanyl, dmorasnyl, mddazolidkyt, ioadmlmyl, pyrarolidtoyl, pyraeolinyi, piperazinyl, mdoliayl, isomdolitiyl, quinuelidinyi, morpfeolinyi, oxazolidinyl, henzotriaaolyl, benzisoxazolyl, oxkdolyl* feenzoxazolinyi, and isatkoyl Preferred iteterocyde® groups include, but are not limited to, betmafuranyl beaaofetapheuyf, indoM, bena»p>nmolyl coumaiiayL isofraaolmyi, pynolyl, iMophenyt, furaayl, thiasoiyl iaadeaotyl, pyraaolyl triassc^!» qafeolinyi, pyrnnidmyl, pyridiayl* pyridonyl, pymkyl, pyridaaayl, Isofoianolyf isoxaasoiyl and tetsmlyi (171! A hetetocyele group, whether alone or as part of another group, can be optionally substituted wife one or more groups, preferably 1 to 2 groups, including but sot Untried to, ~CS~C&amp; alkyl, ·<#€&amp; alkenyl, -C2«C$ alkynyl, -halogen, -0-(Ci -€$ alkyl), -0-(Qt~C&amp; alkenyl), -CKGrCs eftyny!}, -aryl, ~C{0)tl% -00(0)¾% -C(Q)0R\ -€{0)KH2, -CXO)HHR% -C(Q)N<r}2* «M1C(0}R% -SR\-SOsR% -8(0½¾% <S(0)R%-OH, -N3,-Nlfc, -Md(R5), -NCR.^ and -CM, where each R* is independently selected from -H, -€i-€* alkyl, -CrQ alkenyl, -Cz-Ce alkynyl, or -aryl and wherein said -0<CrC* alkyl), -0-(02-Cs alkenyl), -OnfOa-C* alkynyl), -Cj-Cs alkyl -C2-Cg alkenyl, -CyC* alkynyl, and -aiyi groups can bo further optionally substituted wife one or more su bsiituents including, but not Mmited to, ♦€*-€* alkyl, ~C2-C$ alkenyl -Cr€« alkynyl, -halogen, -0-(Ci-Cg alkyl), -0-(¾-Cg alkenyl), -0~<C2-Cs alkynyl), -aryl, -0(0)¾51 ~OCfO}RR, -C(0)<>R5\ -C(0)MH2, -€(0)ΜΗ1,!, ~C(0:iMCRs% ~MHC(0)R” -SR”, -S03r\ -$(0^*% -S(0)R*% -OH, -Nj, -M% -NH(R”), -NCR”! and -CM, whm each R” is independently selected from -H, -Cj-Og alkyl, *€rQ alkenyl, -Cs-Cg alkynyl, or aryl (072j By way of example and not limitation, carbon-bonded lieteroeycles can be bonded at fee following positions: position 2,3,4,5, or 6 of a pyridine; position 3,4,5, or 6 of a pyridszine; position 2,4,5, or 6 of a pyrimidine; position 2,3,5, or 6 of a pyraame; position 2, 3,4» or 5 of a fiaaa, tetrabydro&amp;ran, iMoforan, fekqsheac, pyrrole or teirshydropymde; position 2,4, or 5 of m oxazole, imidazole orthiazole; position 3,4, or 5 of aa isoxazole, pyrazole, or iaofeiaxole; position 2 or 3 of an azlridine; position 2,3, or 4 of an aaetidine; position 2,3,4,5,6,7, or 8 of attmnolkie; or position 13,4,5,6,7, or 8 of an isotjusnoliae. Still «tote typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pytidyl, 6-pyridyl, 3-pyridaziny!, 4-pyiidaaiityl, 5-pyridazkyl, 6-pyridafonyl 2-pyrimidinyl 4-pyrimidinyl, 5-pyrimidinyi, 6-pyramdisyl, l-pyrarmyi, 3-pyrasdnyl, 5-pyxazmyl b-pyrazinyl, 2-fejazolyI, 4-thiazolyl orS-feiszolyl, (#731 By way of example and sot limitation, nitrogen bonded heterocycles can be bonded at position 1 of an aritidine, areddine, pyrrole, pyrrolidine, 2~pyrroline, 3-pyrtolins, imidazole, iaiidazoUdme, 2~imidazolioe, 3-kudazoline, pyrazole, pyraaolins, 2-pymzolms, 3~pyra®oli.ne} piperMIne, pipamdoe, indole, indoiroe, or IfJ-indazole; position 2 sfa isoiudolc, or isotsdoline; po stem 4 of a moipholise; and position 9 of a catbszoie, or β-caifelme, Still more typically, nitrogen bonded heietoeycles include 1-szMdyl, b azetedyl, 1-pytrolyl* l-femdazolyl, I-pyrazolyl, sad l-piperidinyl.

[074] IMess otherwise noted, the term “caiboeycle/* refers to a saturated or unsaturatod non-aromatic monocyclic, McycHc, or polycyclic ring system having from 3 to 14 ring atoms (and all combinations and subcombmafcns of ranges and specific numbers of carbon atoms therein) wherein all of the ring atoms are carbon atoms. Monocyclic c&amp;rboeycles preferably have 3 to 6 ring atoms, still more preferably 5 or 6 ring atoms,

Bieyeife ctoisocycfes preferably haw 7 to 12 ring atoms, e.g,s arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 16 ring atoms arranged as a bicycle [5,6] or [6,6] system. The term, “carlxtoycle” includes, tor example, a monocyclic carbocycle ring fused to an aryl ring {e.g„ a monocyclic cafeoeyde ring fused to a benzene ring), Cartsocyles preferably have 3 to 8 carbon ring atoms, [¢75] Carbocycfe groups, whether atone or as part of another group, can be optionally substituted with, for example, one or mom groups, preferably I or 2 groups (and My additional substituents selected .from halogen), including, but not limited to, -halogen, -CrCg alkyl, -CrC$ alkenyl, -C*-C8 alkynyl, -D4€5-Cs alkyl), -0-(€rCg alkenyl), -G- (CrC% alkynyl), -tnyl, -C(G)T, -0C(O)r, -C(0)0r f -C(0)NH2, ^OJNHr, - ! C<0)N(Rs)a, -NHC(0)R\ -SEN -S03R5, S{0}zR\ -S(0)R\ -OH, «0, -bfe, -N%, - NH(RS), ~Ν(Ε’)2 and -CM, where each &amp;* is Independently selected, from -H, -Ci-C* alkyl, -CrCg alkenyl, -Qj-€$ alkynyl, or-aryl and wherein said -Cj-Cs alkyl, -CrCs alkenyl, -CrCs alkynyl, -0-(€j-€s alkyl), ^KCrCs alkenyl), -G~{CrCs alkynyl), and -aryl groups can be further optionally substituted wife one or more substituents including, but not limited to, ~CrCs alkyl, *€*-€* alkenyl, -<VCa alkynyl, -halogen, dHCrCs alkyl), -0-(CrCs alkenyl), -CKCrCg alkynyl), -atyl, -€{0)R*% -OC<0)R*\ C(0)0r5, ~C(0)NB2, -C(0)NHR”, -€(0)N(R”)2, -NHC(0)R;\ -SR”-W, ~S(0)2R.,?, ~S(0)R” -00, ~Nj, -NH2s "NH(R5i), ~Ν(Κ/% and -CM, where each R” is independently selected fas -B, -Cj-C* alkyl, -C2-Cs alkenyl, -Cj-Cs alkynyi, or -aryl

[076] Examples of monoeyclie oarbocyhc substituents include -cyclopropyL -cyclobutyi, ~<yciopcntyk ~1. -cyclopent-1 -enyl, -l-cyelopent-2-enyi, -l-cyclopent-3-enyl, cyelohexyl, -l-cyckfeex-l -eayl, -1 ~cydohex~2~cnyl, -l-eyctohcx-S-enyl, -eycloheptyl, -eyciooetyi. 4,3-c^tehesuHdieayl5 4,4-cyciohexadienyl, -U^ydo^tadieaijil» -1S3S5~ oyciaheptofeiesyl, aid -cydoociadienyl.

[0?7J A ‘tonrboeyclo,” better used alone or as pad of another group, refers to an optionally substituted caifeocycle group as defined above dial is divalent (ie., derived by the removal of two hydrogen stems from the same or two different carbon atoms of a parent csrhocycKc ring system). I®5*l Unless otherwise indicated by context, a hyphen (-) designates toe point of attachment to the pendant moleatie, Accordingly, the term “-(Cr-€s alkyfcoekryP or “«C j-Cs atoylenefaryl)” refers to a Q-Cg alfcyiemt radical, as defined herein wherein ihe alkylenc radical is attached to the pendant molecule at any of the carbon atoms of toe alkylene radical and one of toe hydrogen atoms hooded to a carbon atom of the alkylene radical is replaced with an aryl radical as defined herein, fflfy When a partiafiar gtonp is ^bstitofed**, that group may have one or more substifeenti, preferably from one to five substituents, more preferably horn one to three substituents, most preferably from one to two substituents, independently selected from the list of substituents. The group can, however, generally have any number of sabstitoents selected from halogen. Groups that are substituted are so indicated. im It is intended that toe definition of any substituent or variable at a particular location in a molecule be independent ©fits definitions elsewhere in that molecule. It is under stood that substituents and substitution patterns on the compounds offers invention can be selected by one of ordinary skill in toe art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in toe art as well as those methods set forth herein. IΘ811 Protective groups as used herein refer to groups which selectively block, either tempsrarily or permanentiy, one reactive site in a mulfi&amp;ncfiosm! compound Suitable hydroxy-protecting groups for use m toe present invention are pharmaceutically acceptable and may or may not need to he cleaved item toe parent compound after administration to a subject in order for the compound to be active, Cleavage is through normal metabolic processes within the body. Hydroxy protecting groups are well known in toe set, see, Pr.OTBCX1VbOrotJPS IK ORCtoMCSYKiHESIS by T. W. Ornern and P, G. M.

Wuts (John Wiley &amp; sens, 3* Edition) incorporated heroin by reference in its entirely and for all purposes and include* tor example, «tier (e.g,, alkyl ethers and silyt ethers including, &amp;r example, dia&amp;ylsiiyle&amp;er, trialkyMyiether, dialkylalkoxysilylether), ester, carbonate, carbamates, sulfonate, and phosphate protecting groups. Examples ofhydroxy protecting gmups include, bat are not limited to, methyl ether, methoxymethyl ether, TnediyltMomethyi ether, Cphe»y1dimethy!sllyi)mdhoxymethy! ether, benzykntymethyl ether, p-methoxybenxyloxyinetbyl ether, p-niteobeasyloxymethyl ether, o-mteobemyloxymethyl ether, C4mie!box>phenoxy)methyi ether, gnaiarxrfmetbyl ether, t-hirtoxymethyi ether, 4-peuienyloxymetbyl ether, slioxymeibyl ether, 2-meteoxyethoxymethyl ether, 2*2,2-McMeroetoxym8ihyi ether, bis(2^tifemeth0'xy)methyl ether, 2~(irimethy!s.iIyI)ethoxyniethyl ether, menihoxymethy! ether, tetrahydropyranyl ether, I-rnethexycyla>hexyl ether, 4~n'ie£hox>i:etrahydrothiopyninyl ether, 4» methoxydetrahydrcdhiopyTanyl ether S,S~I>i0xlde, 1 «[(2~ehom-4’'m.eth.yl)phenyll~4~ meihoxypipernMn-d-yl ether, l-(2~Sffim>pfcteyIM~methoxypiperidin*4“yI ether* .1,4-dbxaa-2~yi ether, f etehydrofisrany! ether, tetrahydrothtefinanyi ether, substituted ethyl ethers sack as l-ethoxyethyl ether, l-(2-cMoroefhoxy)ediyl ether, 1«[2« (mrnethyLsilyOethoxylethyl. ether, hanethyi-l-methoxyethyi ether, I-methyb-i-henzyloxyelhyl ether, l~me%'!~l~beaxs>'loxy»2»Suoroethyl ether, l-m^yi-lphenoxyeihyl ether, 24«methylsilyl ether, t-hutyl ether, ally! ether, propargyl ethers, p-ehkaophenyt ether, p-methexyphenyl. ether, beaxyl e&amp;er, p-meihoxybesxyl ether 3.4-dtmeteoxybeaayl ether, Iritneihylsiiyl ether, MethyMIyl ether, tripiopylsilyiether, dimethyllsopropylsilyl ether, diethylisopmpyMyl ether, dimethylhexyMlyl ether, t-bthyfdimetbylsilyl ether, dipheaylmetbykilyl ether, henzoyifoimate ester, acetate e$er* cMoroacetate ester, diehloroaeetate ester, trlchloimcetate ester, ttffimroaeeiate ester, metlioxyacetate ester, triphiieyhnethoxyacetate ester, phenylacetate ester, benxoafe ester, alley! methyl carbonate, alky! 9-t]i»teny!raefhyi carbonate, alky! ethyl carbonate, alkyl 2,2,2,-tdchloroethyl carbonate, hlj-diatethyl-S^sS-iricMoroethyl carbonate, alkylsttlibnate,mcdhsaesidfanate, henaylsul&amp;aate, tosylate, methylene acetal, ethyHdeae acetal, and t-butyimdhylidene ketal. Preferred protecting groups are represented by the formulas -R* »Si(li)CR)(R), -C{0)R, ~C(0)GR* ~C(0)NH(R)s ~S(0}2R, -8(0)/)¾ P(0)(0H>., and ~P(OXOH>OR, wherein R is Ci«€a) alkyl, CrCaa aUceayi Ca-Qas aikynyi, -CrCao a%lene(caibcscyde)s -CrCze alkenyleneteurho^ude), -C2-C2&amp; aLkynyleae(earbocyde), -Ce-Qo aryt, -Cj-C» aikylene(aryl), /¾¾¾ alkenylenetaryi), -C2-C2&amp; MkynylendCaryl), «Cj-G^s alkyleue(heterocyele), -Cj-Qso aJkenyie«e{heierocyde), or -CrCgj alkynylene(heferocyde) wherein &amp;mi alkyl, alkenyl, al&amp;ynyl, alkylene, alkenylene, alkynylene, atyl, caobocyd^ md heteracyole radicals Aether alone or as pari of another group are optionally substituted, £082} The abbreviation “AFP* refers to femethylualme-valioe-doiaisolenme» doIaprom^idwoyMaame-p^imyleoediaiiiiae (m'P&amp;tm&amp;Bt (XVJII) infm), mi The abbreviation “MMAE” refers to nrmcsnethyl aaristatin E (see Formula (Mil) infra),

10841 Ifce abbreviation “AEB” refers to am ester produced by reacting aurist&amp;titi E with, parascetyl benzoic acid (see Formula (ΧΣΙί) infra), . t*l The abbreviation “AEVB* refers to an ester produced by reacting auristatio E wish benzoylvaleric add (me Formula (X%J££) infra), [I8f i The abbreviation “MMAF® refers to monometbyl axtristatm F (see Formula (Mi) infra). ASlMigi mi la certain sheets, the invention relates to anti-GCC antibody molecules with features such, m those summarized in Tables 1 and 2. In other aspects, the invention relates to aati-GC€ antibody molecules with features such as those summarized in Tables 3,4, 5 andtor 6, [0881 I»· «a embodiment, the anti-0€€ antibody molecule is a human hybridotna antibody and is one of antibody 5P9,5H3S 6H8,8C2,10C10,16D3 and 103. la an embodiment, the anti-GCC antibody molecule m derived ftom antibody 5P9,5113,6118, 8¾ IOC10,1003, or 103. In an embodiment, the artd-GCC antibody molecule is produced hybybridoma SF9 (ΡΪΑ-8132), [089| In an embodiment the anti-GCC antibody molecule is a selected lymphocyte antibody and is one of antibody Ate-12, Abx-020, Ate-106, Abx-198, Abx-221, Ate-229, Abx-338, and Ahx-393. In an embodiment, the anti-GCC antibody molecule is derived from antibody Ate-12, Ate-020, Ate-106, Abx-198, Ate-221, Ate-229, Ate-338, and Ate-393, [090J In an embodiment the anti-GCC antibody molecule is a. murine antibody and is one of antibody mAb 301, mAb 8E12, mAb 1088, and mAb 8F1, In an erabodimsaf, fee aatbGCC antibody molecule is derived ftom aaiifcody mAb 3GI, mAh 8E12,andmAh8Fl. IW1] In an embodiment an aaih-GCC antibody molecule will have an affinity for GCC, e.g., as measured by direct binding or competition binding assays, in. a range described herein, in an embodiment die anii-GCC antibody molecule has a IQ of less tram UlO'^M, less than IxlO"7 M, less than lx 10 s M, less time Ixl0"s M, less than lxlΟ40 M, leas than lxlff11 M, less than lxl042 M, or less than Μβ43 M. In an embodiment the antibody molecule is an IgG, or am%en~binding fr^nwt thereof, and has a IQ. of less than IxKT^M, less than !x!04 M, less'than Ιχΐθ4 M, or leas than lx Iff* M. In an embodiment, an anthGCC antibody molecule, e.g., a 5F9 antibody or antibody derived therefrom has a IQ of about 80 to about 200 pM, preferably about 100 to about 1 §0 pM or about 120 pM, In m mbodimeat, an anti»G€€ antibody molecule, e.g., a 5F0 antibody or antibody drived therefeom has a ka of about 0.9 to about 1.25 xl 05 M4»4, preferably about 1.1 xl05 M4s4. In an embodiment the antibody molecule is m ScFvand has a 1¾ of less than IxiCT^M, less than M04 M, less than lx Iff8 M, less than lxl O'9 M, less than lxl04Q M, less than ΙχΙΟ4* M, less than lxl0'si M, or less than lxiO**3 M. 10¾] In embodiments, the antibody molecules are not inminnoeonjugates, Le.„ are “naked” and in embodiments cause a cellular reaction upon binding to GCC, la related en&amp;odSmeats, the cellular reaction is performed by the GCC-expresang cell to which the antibody binds. Such a cellular reaction can fee signal transduction mediated fey GCC, eg., if the antibody molecule is an agonist of GCC (see, e.g., US Patent Application publication no. 1320040258687. In other embodiments* the cellular reaction is performed by a second cell, eg, an immune effector cell (e.g., a natural killer cell) which recognizes the antibody molecule bound to GCC on the first cell. In some embodiments, surveillance molecules, e.g., complement molecules, contact the GCC-feound antibody molecule prior to fee cellular reaction. The cellular reactions in these embodiments can cause death of the GC€~expreasing cell. 1093] In further embodiments, antibody molecules which are immunoconjugates can both cause a cellular reaction upon bindi ng to GCC and. i nternalize to deliver an agent to the GCC-expressmg cell to which it hinds. | 0MJ In some embodiments, an anti-GCC antibody molecule of the invention can block ligand binding to GCC. |$9SJ Is as embodiment, tiie asti-GCC antibody molecule fails to stew substantial cross reaction with one or both of tat GCC and mouse GCC. fiMI Is as embodiment, the antibody molecule is not GCCSIO, QCC'ADl or GCC: €8, In another embodiment, as anti-GCC antibody molecule does not hind an iafraceliukr domain of GCC, about atniso add residue 455 to 1073 ofSEQ ID Mfc22R For example, in this embodiment, as asti-GCC astibody molecule does not Wad the kinase homology domain or the guaaylyi cyclase domain of GCC, f #?| The naturally occurring mammalian antibody structural unit is typified by a tetramer, Bach trimmer is imposed of two pairs of polypeptide chains, each pair having ose “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa), The ammo-tmainal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, The carboxy-tmniiMi portion of each chain defines a cons tant region primarily responsible for effector function, Burma light chains can be classified as kappa and lambda light chains, Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG» IgA, and IgB, respectively. Witiriu light and heavy chains, the variable and constant regions are j oined, by a WJT region of about 12 or mom amino acids, with the heavy chain also including a *T>* region of about 10 more amino acids. See generally, Fundamental Immumbgy Ch. 7 (Paul, W., edL, 2n,d ed, Raven Press, N,Y. (1989)). The variable regions of each ligfh/beavy chain pair form the antibody binding she, Preferred isotypes for the anti-GCC antibody molecules are IgG immunoglobulins, which can be classified into four subclasses, IgGl, IgG2, lgG3 and lgG4, having different gamma heavy chains. Most therapeofic antibodies are human, chimeric, or humanized antibodies of the IgGl type. In a particular embodiment, the aati~GCC antibody molecule has the IgG! isotype, |§9hf The variable regions of each heavy and light chain pair form the antigen binding site. Thus, an intact IgG antibody has two binding sites which are the same. However, hifimefional or bispecific antibodies am artificial hybrid constructs which have two different heavy/light chain pairs, resulting in two different binding sites, f§99| The chains all exhibit the same general structure of relatlvety conserved framework regions (PR) joined by three hypetwariafeie regions, also called complementarity determining regions or CDRs, Tbs CDRs from the two chains of each pair are aligned by the fismewsik regions, enabling binding to a specific epitope. From N* terminal to Cdemnnal, both light and heavy chains comprise the domafes Fill, CDR1, FR2, CDR2, FR3* CDR3 and FRA The of ammo adds to each domain is in acccndance with the definitions of Rabat Sequences ofPtoteiBS of Immunological Interest (National Institutes of Health, Bethesda, Md (1987 and 1991)), or ChotMa &amp; Lesk J, Mol Biol 196:901-917 (1987); Chofhiaet al Nature 342:878-883 (1989). As used herein, CDRs ate referred to for each of the heavy (BCDRI, HCDR2, HCDR3) and light (LCDRI, LCDR2, LCDR3) chafes.

[01.WJ An &amp;nti~GC€ antibody molecule cm. apprise all, or an antigen binding subset of the CDRs, of one or both, the heavy and light chain, of one of the above-referenced human hybridoma, selected lymphocyte, or murine antibodies. Amino acid, sequences of human hyMdoma, selected lymphocyte, and murine antibody portions, iaelndmg variable regions and CDRs, can. be found in Table 3 and Table 5, [01 il| Thus, in an embodiment fee aalihody molecule Includes one or both of: (a) one, two, three, or an antigen binding number of, light chain CDRs (LCDRI, LCDR2 and/or LCDR3) of one of the above-referenced human hybridoma, selected lymphocyte, or murine antibodies. In embo&amp;nenis the €DR($) may comprise an amino acid sequence of one or more or all of LCDRI-3 as fellows: LCDRI, or modified LCDRI whereat one to seven amko acids are conservatively substituted) LCDR2, or modified LCDR2 wherein one or two ammo acids am «stservaitvely substituted); or LCDR3, or modified LCDR3 whereat one or two amino acids are conservatively substituted; and (b) one, two, three, or an antigen binding number of, heavy chain CDRs (HCDER HCDR2 and/or HCDR3) of one of the above-referen.ced human hybridoma, selected lymphocyte, or murine antibodies. In embodiments the CDR(s) may comprise an amino acid sequence of one or more or all of HCDR1-3 as follows: HCDR1, or modified HCDR1 wherein one or two amino acids are consemiively substituted; HCDR2, or modified HCDR2 wherein one to four anrino acids are conservatively substituted; or HCDR3, or modified HCDR3 whereas one or two amino acids are conservatively substituted.

f 01®2[ Useful immunogens tor production of anti-GCC antibodies include GCC e.g,, human GCC-expressing cells (e,g., a tumor cell Ike, e.g,, T84 cells, or fiesh or itoaasa colon tenor cells, recombinant cells expressing GCC); membrane fractions of-GCC-expressing cells (e.g,, &amp; colon tunior ceil line, e.g„ TS4 cells, or ftesh or fiwsea colonic tumor cells, recombinant cells expressing GCC, e.g., HT~29~GCC#2 cells, which express full-length GCC, ora portion thereof, e.g., CHO GCC #2? cells which excess a portion comprising the GCC extracellular domain, e,g., SEQ E> NG:3!S); isoktedorpunfied GCC, e.g„ human GCG protein (e.g., hiochemicaiiy isolated GCC, e.g., isolated from gastrointestinal tumor cells or recombinant cells expressing GCC or a variant thereof), or a portion thereof (eg., the extracellular domain, of GCC, the kinase homology domain of GCC or the gnaaylyi cyclase catalytic domain of GCC or peptide corresponding to a portion thereof, eg., comprising at least about 8,10,12,14,16,20,24,28 or 32 amino acid residues of SEQ ID NG:228); or an immunogen comprising SEQ ID M>;229 or compmipg a mature portion thereof without the signal sequence (i.e., without amino add residues 1 to about 21 or 23 of SEQ ID NO:229), e.g,, the mature TGK107-hIgG protein, SEQ ID NOG 17. fdli3| Immunogens can be fused to heterologous sequences to aid in. bfoohenuoal manipulation, purification, immunization or antibody titer measurement. Such immunogens can comprise a portion of GCC, e.g, the extracellular domain, and a portion comprising a non-GCC polypeptide. Many options exist fer coMtrudmg a fusion protein for ease of puriieatioa or immobilization onto a solid support, e.g, an affinity column or a microliter plate or other suitable assay subsirata^chip. For example, a fusion moiety can add a domain, e.g.»^utaihione-S-tmnsferasdldnase (GST), which can bind gl ukthione; an Fc region of an immimoglobnHn, which cm bind to proto» A or protein G; amino add residues, e.g, two, throe, four, five, preferably six histrdme residues which can bind nickel or cobalt on an affinity column; an epitope tag, e.g, a portion of e-myo oncogene (myc~ tog), a FLAG tag (U.S. Patent No. 4,703,004), a bsmagglutmto (HA) tag, a T? gene J 0 tag, a VS tag, an HSV tag, or a VSV-G tag which can bind a tog-specific antibody; at a eofactor, e.g., biotin, which can bind steptavidtn. 101041 Immxmgem which comprise the Fc portion of an imniunogiobnlin can hold the GCC, either in solution or attached to a cell, in a configuration which allows structural access to GCC epitopes by the host immune surveillance compouenk for efficient antibody generation. Because unmnaogbbulin heavy chains comprising the Fc regions associate Into dimers through interchain disulfide bonds, immunogens resulting from fusion with Ec regions are dimers. Valency of fusion proteins can reflect the type of immunoglobulin contributing an Fc region. For example, fusions with IgG proteins cent be dimers.» IgA fimons dan. make tefeameric femmogens, and IgM fusions can make decaraeric immunogens, the latter two is facilitated with eo-taasfection of fee 3 chain. An exesqslsry immimogiobulm for an Pc fusion pmtefe Is IgOl. The portion used typically lias the IgGl hinge, CH2 and CB3 domains encoded by a single exon. Because this exon also has a portion of the CHI region, which has a cysteine oriented to disulfide bond with a cysteine from the light chain, a useful modification is to mutate fee CHI cysteine, e.g., to a seriae, to ensure there is no impaired cysteine in the foston protein. Such a mutation also increases flexibility of the hinge. |WM| An Fc portion derived horn a non-host species, e.g.. human Ig Pe region, for fusing to an immunogen for immtmfeatton in a host species, e.g,, mouse, rah rabbit, goat, acts as an adjuvant. This adjuvant function can trigger specific antibodies against both Fc and GCC epitopes, Pe-reactive antibodies cm be identified and discarded during screening. The Fc portion can have a wild type sequence or a sequence which Is mutated to modify effector function. Bor example, a mutated constant regfon (variant) can be feocapm^ed into a fusion protein to minimize binding to Fc teceptas and/or ability to fix congjlement (see e.g. Winter et ai,GB 2,209,757 B; Morrison et at, WO 89/0714¾ Morgan et to., WO 94/29351), In a preferred example, lysine 235 and glycine 237, numbered according to Fc region standards, are mutated, e.g., to alanine. An irnmnnogen/fusion protein with Fc-tnuteted IgG can have reduced interaction wife Fc receptors in the host. A preferred soluble immunogen fusion protein (after maturation to cleave fee signal peptide and secretion) is TGK.407~hIgG (alt. name hGCC-ECD/MgGl Fc), which consists of amino add residues 24 to 430 of SEQ ID NO;228 fused to mutated human IgGl immunoglobulin Fc (SEQ JD NO:317). P106| To prepare a cdh&amp;xpmteed immunogen, the inmiimogtebuliu portion can be structured to mimic an immunoglobulin portion of the B cell receptor. For example, the immunoglobulin Fc region can be further fused to a polypeptide comprising a transmembmne region from an immune receptor, such as Fey receptors, Foot receptors, Pca/μ recep tor or Fee receptors. Proper orientation, of such an Fc receptor cell-bound immunogen wife adequate exposure on fee cell surface may be improved if the cell" expressing the immunogen fktion pmfein further comprises additional components of the antigen receptor complex, e.g., B cell IgM receptor or IgB receptor. Suitable components of the complex include immunoglobulin (Ig) sheafe proteins, such m MB-.I and B29 (CD79A and CD79B; Hbe&amp;acfe et al Em J. Immm&amp;l 20:2795-2799 (1990) for IgM receptor), which form a heterodimer, The Ig sheath pmtems cm he provided efitihgeaoesly by fee tmnsfectcdeeil, e.g.} if transfecting a B cell lymphoma cell line* or by co-transfeefiou of fee immunogen wife feeath ptoteins, e.g.. in a separate vector or in fee « vector. deferred IgG sheafe pmteitts for femnnisafem in moose are mouse €D79a and CD79fe (GenBank Accession Nos. NM_007655 and NM_(X>8339, respectively)- A preferred ced-bound immunogen feion protein (after maturation to clwe the signal peptide md translocation to fee eel! surface) is fee TOO11 product, consisting of fee TOK-iOftlgO (hGCC-ECMgGI Fc) feed to fee.mouse IgG2a (e.g., GeaPept Accession No, AAB59661) tranMiembraae and intracellular domains (SEQ ID MO:3iB). {01 07] Useful epitopes, e.g., reference epitopes, horn the GCC molecule, to which fee anti-CCC antibody molecules, e,g.s monoclonal antibodies, human antibodies or bumarired antibodies, as described herein, can bind, can be fend on fee extracellular portion, of GCC. Such GCC epitopes can bind antibody molecules on fee surface of cells, e,g,s on fee cell exterior, fOUSJ For example, an epitope for an anti-OCC antibody molecule can reside within, or include a .residee(s) fem, residues 1-50 of SEQ ID NO:228, or a fragment thereof that binds an aad-GCC antibody molecule of the invention, e.g., a 5F9-hmding fiugment thereof Such fragments cat? comprise residues 1-25,5-30,10-35,15-40,20-45, 25-50,5-45,10-40,15-35,20-30 or 33-50 of SEQ ID NO;228. is some embodiments, an epitope for an anti-GCC antibody molecule, e.g., a 5F9 antibody, is a conformational epitope further comprising one or more additional amino acid residues in fee GCC amino acid sequence beyond residue 50, ie„ selected from about residue 30 to 1073 of SEQ ID NO :228. (01091 Ϊ» Mother example, an epitope for «η anti-GCC antibody molecule can reside-wifeln, or include a resklne(s) from, SEQ ID NO :225, or residues 271.-300 of SEQ ID NO:22gf or a fragment thereof feat binds an anti-GCC antibody molecule of fee invention, e.g.* an ABX-19S-, a 3G1 -, an 8F1-, or a 10B8-binding fragment thereof. Such fragments can comprise residues 281-290 of SEQ ID NO:22S, or residues 281-290 of SEQ ID 190:228 wherein residue 281 is leucine, or residues 281-300 or residues 2?I -290 of SEQ ID NO:228. In some embodiments, an epitope for an anti-OCX antibody molecule, e.g., an ABX-198-, a 301», an 8F1-, or a 10B8 antibody, is a conformational epitope farther comprising one or more additional amino acid residues, i.e,, non-SF.Q ID $0:225 residues in. the GCC amiuo acid sequence e,g,, selected fens about residue i to 270 aad/or about 301 to 1073 ofSEQ ID NO *228, iOlltJ In another example, an epitope for the aoti-GCC antibody molecule can reside within, or include a residne(s) fas SEQ ID $0:226* or residues 351-375 ofSEQ H> $0:228» or a .fragment thereof that binds an anti-GCC antibody molecule of the invention, e.g,, a® ΑΒΧ*012~» ABX-338-, or ABX-I 06~hmdmg fragment thereof Such ffagmsnts can comprise 356-370 ofSEQ ID $0:228, or residues 351-370 ofSEQ ID $0:228, or residues 356-375 of SEQ ID $0:228, In some embodiment^ an epitope for m anti-OCC antibody molecule, e.g.» an ABX-0S2-, m ABX-338-, or an ABX-106 antibody, is a conformational epitope further comprising one or more additional amino add residues, Le., non»SEQ ID $0:226 residues in the- GCC amino acid sequence e.g., selected from about residue 1 to 350 and/or about 376 to 1073 of SEQ ID $0:228. pit 1 If Antibodies raised against such epitopes or the extracellular domain, e.g., epitopes that reside within, or include a residue(s) from amino acid residues 24 to 420 of SEQ ID $0:228, or a reference portion thereof, e.g,f matte 24 to 75,75 to 150,150 to 225,225 to 300,300 to 375 or 375 to 420 of GCC, or antibody molecules derived ihersibom, can be useful as therapeutic or diagnostic antibodies, as described herein, |0I12| In an embodiment, the anti-OCC antibody molecule has one or more of the Mbwing properties: a) it competes tor binding, e.g., binding to cell surface GCC or purified GCC, with one of the abov e-referenced astt-GCC antibody molecules summatmsd in Tables 1 and 2 e.g., human liybridoma antibodies (e.g., 5F9), selected lymphocyte antibodies (e.g., Abx-229), or marine antibodies (e.g., 3G1); b) it binds to the same, or substantially the same, epitope on GCC as one of the above-referenced antr-GCC antibody’·molecules summarized in Tables 1 and 2, e.g., human hytedoma antibodies (e.g., 5F9), selected lymphocyte antibodies {e.g,, Abx-229), or murine antibodies (e.g., 3G1), In an embodiment, the antibody binds the same epitope, as determined by on e or more of a peptide array assay or by binding to truncation mutants , chimeras or point mutants expressed on the cell surface or membrane preparations, e.g., as those assays am described herein; c) it Muds to m epitope which. Ms at least 1,2,3,4,5, 8, ID, 15 or 2D contiguous amino acid residues is common with the epitope of one of the above* mfereneed anti~GC€ iraritedy molecules smmnarized ia Tables ! aad % c.g.> human hj&amp;ridoma antibodies <e.g.} 5F9), selected lymphocyte antibodies (e.g., Abx-229) or morale antibodies (e.g.s 3G1); d) it binds a region of human GCC that Is bound % an anh-GCC antibody of.the invention, wherein the region e.g., an exiraeelMar or cytoplasmic region, is 10-15, 10-20,20*30, or 20*40 residues In length, and binding is determined, e.g,, by binding to truncation mutants; In an embodiment the anti-GCC antibody molecule binds the exiraediuter region of human GCC, In an embodiment an anti-GCC antibody molecule can bind die human GCC portion of the extracellular domain defined by amino acid residues 24 to 420 efSEQ IDNO:228. hi an embodiment an aati-GCC antibody molecule can bind the guanylate cyclase sgnatiue site at amino acid residues 931 to 954 of SEQ SO MO:22S; or e) it binds to a reference epitope described herein,

|0i 13J la a» embodimaii the and-GCC antibody molecule binds the GCC sequence ILVDLFWQYI^DNVTAPDVMKNVLVLTX,S (SEQ ID NO:225), ί#114| In an embodiment the anii-GCC antibody molecule binds the GCC sequence I^VHAFRNL'G'EG'YDGPVIXDDWGDV (SEQ ID NO:226). 101151 in an. embodiment the antibody molecule binds a conformational epitope.

In other embodiments an antibody molecule binds a linear epitope, [§ 1 IhJ The anti-GCC antibody molecules can be polyclonal antibodies, monoclonal antibodies, monospecific antibodies, chimeric antibodies (See U,S. Pat No, 6,020,153) or human or Imraanmed antibodies or antibody fragments or derivatives thereof. Synthetic and genetically engineered variants (See 1J,S. Pat. No. 6,331,4.15) of any of the foregoing are also contemplated by the present invention. Monoclonal antibodies can be produced by a wdy of techniques, including eom'entionsl murine monoclonal antibody methodology e.g., the standard somatic celt hybridisation tedmisiue of Kohler aadMilstem, Nature 256:495 (1975). See generally, Harlow, E. aad Lane, D. (1988) Antibodies: Λ Laboratory Mmmi, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY. |M1?| Inmuurizafion wife protein, e,g,, GCC or a soluble potion» or fusion protein, comprising a portion of GGC (e.g., TOitie?~feXgX or cells or membrane fractions therefrom, e.g,, cells oxpressing surface-exposed GCC or a porfio«feeteo£(e,g,, fee pLICT0&amp;4 product, or fee pUpFOKl Ϊ1 product), can bo performed with fee immunogen prepared for injection in a manner Pa induce a response, e.g., with adjuvant, eg., complete Freund’s adjuvant. Other suitable adjuvants include, T1TERMAX GOLD® adjuvant (CY'TRX Corporation, Los Angeles, CA) and alum, Small peptide immunogens «so be finked to a larger molecule, such as keyhole limpet hemoeyamn, Mice can be injected in a «amber of manners, e.g,, subcutaneous, intravenous or intramuscular at a number of sites, e.g.f in fee peritoneum (Ip.), base of fee tail, or foot pad, or a combination of sites, e.g., IP and base of tail (BXP). Booster injections can include fee same or a different immunogen and cm additionally include adjuvant, e.g., incomplete Freund’s adjuvant Immunization wife DNA, e.g., DNA encoding GCC or a portion thereof or feskm protein comprising GCC or a portion thereof (e.g,, encoding TOK1107»Mg) can be injected «sing gene gan teebsoiogy. For example, DMA is loaded onto microscopic gold particles and injected into mice at frequent intervals over a brief period |iI18j Generally, where a mmtoeional antibody is desired, a hybridous» is produced by fusing a suitable cell from aa Immortal, cell Hue (eg., a myeloma cell line such as S ¥2/0, P3X63Ag8.653 or a heiemmyefoma) with miibody-paodudng cells. Atitibody-pioducing cells can be otained from the peripheral blood or, preferably the spleen or lymph «odes, of humans, human-antibody transgenic aaimals or other suitable animals immunized wit h the antigen of interest Cells that produce antibodies of human origin (e.g,, a human antibody) can be produced using suitable methods, for example, fusion of a human aniihody-pmducing cell and a hetmsmyeloma or trioma, or immortalization of an activated human δ cell via infection with Epstein Barr virus, (See, &amp;$., U.S. Patent No. 6,197,582 (Trakht); Niedbala et al, ffyhridoma, 17:299-304 (1998); Zandte etal, JImmunol Methods, 156:205-215 (1992); Gustafsson te al, Htm Antibodies Hybridomas, 2:26-32 (1991),) The fused or immortalized antibody-producing cells (hybridoams) can be isolated using selective culture conditions, and cloned by limiting dilution, Cells which produce antibodies wife fee desired specificity can be identified using a suitable assay (s.g,, ELISA (e.g., wife immunogen, e.g„ TOi<.107-feIgG, munobilhsed on the microtiter well) or by FACS on a cell expressing GCC or a portion thereof, e.g., a cell expressing the pLKTOKl 11 piodnct), For example, if the GCC-inunuaogen ««metises a fusion moiety that is an affinity reagent, this moiety cm show the fusion protein comprising GC€ or a portion thereof to be bound te a matrix, e.g„ protein G-eoated, stmptevidm-vxmted, glteadxtone-dmvatiised or aatibody-coated Merotifec plates or assay chips, which am then combined with the immune serum or conditioned medium fern a hybridoma or antibody-expressiag recombinant cell, and the mixture incubated under conditions conducive to complex formation (&amp;,g,, at physiological conditions ibr salt and pH). Following incubation, the microtitre plate wells or chip cells are washed to remove any unbound components and binding by anri-GCC antibody is measured. fftl Iff in embodiments, for therapeutic applications, the antibodies of the present invention are human or humanized antibodies. The advantage of human or humanized antibodies is that they potentially decrease or eliminate the immunogenicity of the antibody in a host recipients thereby permitting an increase in the Unavailability and a reduction in the possibility of adverse immune reaction, thus potentially enabling multiple antibody admiaistratiom [0120] Modified antibodies include humanized, chimeric or CDR-gafted antibodies. Hum® anti-mouse antibody {HAMA)m^xmses have led to development of chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a murine variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will fce observed, particularly in domic or mufti-dose utilizations of the antibody. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient In order to avoid the utilization of murine or rat derived antibodies, humanized antibodies where sequences are introduced to an antibody sequence to make it closer to human antibody sequence, or fully turn»» antibodies generated by the miroduetion of human antibody function into a rodent have been developed so that the rodent would produce antibodies having fully human sequences. Human antibodies avoid certain of the problems associated with, antibodies that possess murine, rabbit, or rat variable and/or constant regions. liman Antibodies [112,1] Fully human antibody molecules can minimize the immunogenic and allergic responses intrinsic to mouse or orouseMermtized mAbs and thus to increase the efficacy *nd safety of the administered antibodies. He use of felly human antiborfy molecules can provide &amp; substantial advantage in the treatment of chronic and recurring inanrn diseases, such m inflammation, .aafesmajaaity, and cancer which require repealed antibody administrations, Also, human antibody molecules can be produced using genetically engineered strains of animals k which the antibody gens expression of the animal &amp; «oppressed and functionally replaced with human antibody molecule gene expression, [61221 Methods -for making human antibodies me known in the art. One method lor making human antibodies employs the use of hmsgeme ammals» such as a transgenic mouse. These transgenic animals «(attain a substantial portion of the human antibody producing genome, e.g., a human immunoglobulin locus that can undergo functional rearoangnment, inserted into their own genome arid the animaTs own endogenous antibody production is rendered deficient in the production of antibodies. Methods for making such transgenic animals are known in the art. Such transgenic animals can be made -using XENGMOIJSE m technology or by using a i4mmiloc«s·5 approach. Methods for making XSNOMICB™ are described m U.S. Pat. Nos, 6,162,963, 6450,584,6,114,598 and 6,075,181» which are incorporated herein by reference. Methods for making transgenic animals using the “mmitocus” approach are described in U.S. Pat. Nos, 5,545,807, 5,545,806 and 5,625,825; also see International Publication No, WG93/12227, which me each incorporated herein by reference, Other transgenic human antibody- producing mice include HUMAB-MOUSB Φ» KIRIN TO MOUSE*** tianschromosome mice, KM-MOIISE® (MEDAREX, Princeton, Nl). |#l23f Using the human antibody transgenic animal technology, e.g., XENOMOUSE ™ technology, human antibodies can he obtained by immaniaing a XBNOMOUSB m mouse {Abgemx, Fremont, Calif) with an antigen of interest, The fyasfoatio cells (such as B-cefis) are recovered (e.g„, isolated from, spleen tissue) from the mice that express antibodies. These recovered cells can he fused with a myeloid-type cell line to prepare immortal hybridoma cell lines, using standard methodology. These bybridoma cell lines can he screened and selected to identity hybridoma cell lines that produce antibodies specific to the antigen of interest [0124] Himiaa-antibody transgenic animals provide a source of nucleic acids that can be enriched in nucleic acids that encode antibodies having desired properties, such as specificity and affinity. For example, nucleic acids encoding atkbodies or antibody variable regions can be isolated from hmmn-anfibody transgenic mice that have been immunized wife a GCC protein or variant or pmtfcm thereof. The iaolated m<Mc adds or portions thereof (e.g,, portions encoding variable mgkris, CDRs, Mmework regions) can be expressed using any suitable method (e.g., phage display) to produce a library of antibodies or antigen-binding fragments of antibodies (e.g,, single chain antigen -binding fragments, doable chain autigea-binding fragments) that is enriched tor antibodies or anfigen-bkdfog fragments that bind a GCC protein, Such a library can exhibit enhanced diversity (e.g.f combinatorial diversity through pairing of heavy chain variable regions and light chain variable regions) relative to the repertoire of antibodies produced in the immunized hum&amp;n-antihody transgenic animal. The library can be screened using any suitable assay (e.g,, a GCC protein blading assay) to identify antibodies or anfigen-binding fegmenis having desired properties (e.g., ^edfieiiy, affinity). The nucleic acids encoding antibody or m%en~biadlng fragment having desired properties can be recovered using my suitable method, (See, e.g., U.S. Patent No, 5,871,90? (Winter et si) and U.S. Patent No, 6,057,098 (Buechler et ai.,)<} ¢1125] Alternatively, the antibodies can be expressed in cell Ikes other than hybridoraa cell lines. More specifically, sequences encoding particular antibodies can be cloned from cells producing toe antibodies and used tor transformation of a suitable mammalian host ceil. In a preferred method, spleen and/or lymph node lymphocytes from immunised mice are isolated from toe mice and plated in plaque assays as described previously in Babcook et al, Proc Nat! Acad Sci USA, 93; 7843-8 (1996), which is kcorpmatod herein by reference. Briefly, cells are plated in agar with sheep red blood cells, coated with GCC antigen and cells secreting mAh against the GCC antigen would fix complement and lyse toe red blood cells immediately stirrounding the mAh producing cells. Cells within the cleared plaques are lifted for sequencing of the immunoglobulin sequences and suhdoning into expression vectors. Supernatants front transiently transfected cells containing GCC specific mAh are subsequently screened by ELISA and fo binding to cells by flow cytometry, the variable sequences, or a portion thereof of the produced human antibodies comprising CBKs which bind particular epitopes may be utilized for production of modified antibodies, For example, the variable regions of the produced antibodies may be spliced into an expression cassette for ease of transfer of constructs, increased expression of constructs, and/or incorporation of constructs into vectors capable of expression of fell length antibodies, see, e.g., 0820660147445. In a particular embodiment, the expression, cassette comprises the heavy chain constant region of the fg<31 isotype. 1#126| The Selected Lymphocyte Antibody Method (SLAM, see O.S. Pal No. 5,627,652, Babeook et si. Proc. Natl Aead Scl UJSLA 93:7843-7848 (1996)) can also be used to identify cells which caa provide fee aaiftfedy of lateral In SLAM, B-cel!s are cultured directly, feus bypassing hybridoma technology, which typically captures only small percentage of the antibodies originally generated by a mouse. Using microplata-based assays, the B-eella are rapidly assayed over a period of several days. Typically, thousands of aniigen-reaeiive cell-clones are identified, represenring thousands of individual antigen-specific, e.g., GCC-specific, monoclonal antibodies. The number of different aatigm“reactive monoclonal antibodies identified in a single experiment is typically Increased many-fold, After applying additional rapid mcreplate-based assays to measure and rank antibodies by affinity and inaction, individual B-eell clones producing extremely high quality antibodies can he selected, In addition, by bypassing the hybridoma generation step, production can move rapidly into a recombinant manufacturing cell line. Individuals cells selected using the technology ere isolated and the antibody genes can. be directly introduced into a manufacturing cell line. The resulting cel! line then can be developed lor crimes! trial testing over essentially tire same timeline as that required for hybridoma cell line development (01271 Human mAh 5F9 <%G2, kappa) can he produced by hybridoma 5F9, also referred to as hybridoma 46.5F9.8.2, which was deposited on January 10,2007, on behalf of Mill<mniurn.Fharmaceuticals fee., 40 Landsdowne Street, Cambridge, MA, 02139, USA, at the American Type Culture Collection, 10861 University Boulevard, Manassas, Virginia 20110, 0.S.A., mider Accession No, PTA4132. (The deposit was made pmmoi to, and in satisfaction of, the requirements of the Budapest Treaty on the Mcmational Recognition of fee Deposit of Microorganisms for the Purposes of Patent Procedure.) The invention relates to hybridoma 5F9, to the antibody it produces, antigen-binding fragments thereof, mi to uudde adds encoding the mtibody and portions thereof (e.g,, heavy chain, heavy chain variable region, light chain, fight chafe variable region, CDRs), As described herein, hybridoma SF9 produces an Ig02s kappa antibody.

Btman&amp;atmn and Display? Technologies mdModlfice&amp;m to Antibodies {§128] As discussed above, there are advantages ίο producing antibodies wife reduced immmrogemdty, This can be accomplished m connection wife techniques of humanization and display techniques using appropriate Mbmri.es, It will be appreciated that murine antibodies or -aotibcsdies from other species can be humanfeed or primatized usiag techniques known in the art. See tjg.t Winter and Harris Immunol Today 14:43-46 (1993) and Wright et al Cril Reviews in ImnmtoL 12125-168 (1992), The antibody of interest may be engineered by recombinant DNA techniques to substitute die CHI, CH2, CM3, binge domains, and/or the framework domain with fee concluding human Sequence (see WO 92/02190 and U,S. Pat Nos. 5,530,101,5,585,089,5,693,761,5,693,792,5,714,350, and 5,777,085). Also, fee use of Ig cDNA for construction of chimeric kmumogiobalm genes is knows iu the art (Liu et at Pmc Natl Acad Sei USd. 84:3439 (198?) and J. Immmol. 139:3521 (1987)). «ιΒΝΑ is isolated from a hybridoma or other ceil producing fee antibody and used to produce cDNA: The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).

[0129] Alternatively, phage display technology (see, e.g., MeCafferty et al, Nature* 348:552“ 553 (1990)) cm be used to produce human antibodies and antibody fragments in vitto, from immunoglobulin variable (V) domain genes, e, g., from repertoires from mihnsiunfred donors. According to tins techuiqne, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or id, and displayed as functional antibody fragments on fee surface of fee phage particle. Because fee filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on fee functional properties of fee antibody also result in selection of the gene encoding fee antibody exhibiting those properties. Thus, the phage mimics some of fee properties of fee B cell Phage display' can be performed in a variety of formats; tor their review sea, e,g<, Johnson and QmwcII, Current Ofmion in Strueiuml Biology , 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxaeolone antibodies from a small random combinatorial library of V gases deri ved from the spleens of imniuufred mice. A repertoire of V genes from imimmunfeed human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can fee Isolated essentially following the techniques described by Masts at at, / Mol Mot., 222:581- 59? (1991), or Griffith et al,EMBOJ.> 12:725-734 (1993). See.

Patettf Nos. 5,565*332 and 5,573,995. Display libraries can. contain antibodies or antigen-binding &amp;agraeats of antibodies that contain artificial amino acid sequences. For example, the library can contain Fab fragments which contain artificial CDRs (e,g,, random amino acid sequences) and human Immework regions. (See* for example, US, Patent No. 6,300,064 (Knapplk, et ai).) [1139) Butman antibodies may also be generated by m vita* activated B cells (see US, Patent Nos. 5,567,610 sad 5,229,275).

[0131] The sequences of human constant region genes may be found k Kabai et al. (1991) Sequences of Proteins ofMmumhgical Merest* Ν.Ι,Η. publication no. 91-3242, Human C region genes are readily available from know» clones. The choice of isotype will be guided by the desired effector functions, sack as complement fixation, or activity in antibody-dependent cellular ototoxicity. Isotypes can be IgGl, IgG2, IgG3 or IgG4, In particular embodiments, antibody molecules of the invention are IgGl and IgCKL Biker of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is feat expressed by conventional methods, [9132] Is some embodiments, an anti-GCC antibody molecule of the invention cm draw antibody-dependmh cellular cytotoxicity (ADCC) to a sell expressing GCC, e.g., a tumor celt Antibodies with the lg<31 and IgG3 isotypes are useful for eliciting effector function in an antibody-dependent cytotoxfe capacity, tine to their ability to bind the Pc receptor. Antibodies with the IgG2 and IgG4 isotypes are useful to minimize m ADCC response because of their low ability to bind the Fc receptor, hr related embodiments substitutions in fee Fc region or changes in fee glycosylation composition of m antibody, e.g,* by growth in a modified eukaryotic cell line, can bo made to enhance the ability of Fc receptors to recognize, bind, aud/or mediate cytotoxicity of cells to which anti-GCC antibodies bind (see* e.g,, U.S. Pat No. 7,317,991,5,624,821 and publications including WO 90/42972, Shields, et at,/. Biol Chem. 276:6591-6694 (2001),, Lazar et at Ph>c. Natl. Acad. Sd. USA, 193:4005-4910 (2906), Satob etat Expert Opin Biol Then 6:1161-1173 (2996)), 1» certain embodimeafs, the antibody or antigen-binding Segment (e.g., antibody of h uman origin , human antibody) can include amino acid substitutions or tenements that alter or tailor function (e.g., effector function), For example, a. constant region of human origin {«.&amp;> yl constant region, y2 constant region) can be designed to reduce complement activation and/or Be receptor binding- (See, for example, U.S, Patent No&amp; 5,648,260 (Winter et a!.), 5,624,821 (Winter et at) and 5,834,597 (Fao et al), the cadre teachings oi which ore incorporated herein by reisrenee,) Preferably, fee Ammo acid sequence of a constant region of human origin that contains sadi amino add substitutions m replacements is at least about 95% Identical over the MS length to fee amino add sequence of the unaltered constant region of human origin, more preferably at least abo ut 99% identical over fee fed length to fee amino acid sequence of fee unaltered constant region of human origi n, C$1331 In still another embodiment, effector functions can also be altered by modulating the giycosyfalkm pattern of fee antibody, By altering is meant deleting one or mere carbohydrate moieties found in fee antibody, and/or adding one or more glyeosylatioa sites that are not present in fee antibody. For example, antibodies wife enhanced ADCC activities wife a mature caibohydtate structure feat lacks fueose attached to an Fc region of the antibody are described in US, Patent Application Publication Mo, 2003/0157108 (Presfa). See also US, Patent Application Publication No, 2004/0093621 (Kyowa Hakko Kogyo Co,, Ltd), Giycofi has also developed yeast cell lines capable of producing specific glyeoforms of antibodies.

[01341 Additionally or alternatively» an antibody can be made feat has an. altered type of giycosylatiou, such as a hypofueosylated antibody having reduced amounts of fneosyl residues or m antibody having increased bisecting GIcNac structures, Such altered glycosylatioa patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for «cample, expressing the antibody in a host cel! wife altered giycosyiation machinery. Cells wife altered glyeosyMon machinery have bean described in the at and can be used as host cells in which are engineered to express recombinant antibodies of the invention to thereby produce m antibody wife altered giycosyiation.. For example, BP 1,176,195 by Hang at al describes a cel! line wife a functionally disrupted PUTS gene, which encodes a fucosyl transferase, such feat antibodies expressed in such a cell tine exhibit hypofucosyisfem. PCX Publication WO 03/035835 by Presta describes a variant CEO ceil line, Lee!3 colls, wife reduced ability to attach fecose to Asn(2$7)4inked carbohychates, ala? resulting in hypofneosylatkm of antibodies expressed in feat host cell (see also Shields, R. L. et aL, 20O2 J, Biol Chem. 277:26733-26740). PCX Publication WO 99/54342 by Umana et ai describes cel! lines eagfeessredto express glyeopioiem-modtfying glycpsyl transferases (ag,s hets(I;4>N ac^ylgiucosamkylimssfease ΙΠ (GnTIH)} such that antibodies expressed in the raipnaered cell ikes exhibit increased bisecting GlcNac structures which «Salts in increased ADCC activity of the antibodies (see also Oman® et a!., 1999 Nat Biotech, 17:176*180). mm Humanized antibodies can also be made using a CDR-gmtod approach.

Techniques of generation of such humanized antibodies are known in the art. Generally, humanized antibodies are produced by obtaining sueleto add sequences that encode the variable heavy and variable light sequences of an antibody that binds to GCC, identifying the complemrataiy determining region or {i€DR" in the variable heavy and variable light sequences and grafting the CDR nucleic acid sequences on to human framework nucleic ack sequences (See, tor example, U.S. Pat Nos. 4,816,567 and 5,225,539). The location ofthe CDRs and framework residues cm he determined (see, Rabat, F.A., etai. (1991)

Sequences of Proteins of Immunological Interest, Fifth &amp;&amp;&amp;οη> US. Department of Health and Human Services, N3H Rxtblication No, 91-3242, and Chothi®, C. et at. J, Mol, Biol 196:981 »917 (1987)). AntNGCC antibody molecules described herein have the COR amir» add sequences and nucleic acid sequences encoding CDRs listed in Tables 5 and 6.

In some embodiments sequences from Tables 5 and 6 can be incorporated into molecules which recognize GCC for use in the therapeutic or diagnostic methods described herein.

The human, framework that is selected is one that is suitable for in vivo administration, ; meaning that it does not exhibit im®uoogmdty. For example, such a determination can be made by prior experience with in vivo usage of such antibodies and studies of ammo acid similarities, A suitable framework region era be selected from an antibody of human origin having at least about 65% amino acid sequence identity, rad preferably at least about 70%, 80%, 90% or 95% amino acid sequence identity ova· the length of the framework region within the amino acid sequence of the equivalent portion {e.g,, framework region) of the donor antibody, e.g., raatdTGCC antibody molecule {e.g., 301),

Amino acid sequence identity era he determined using a suitable amino add sequence alignment algorithm, such as CLUSTAL W, using the default pammeters. (Thompson AD. et al5 Nucieic 4cids Ref. 22:4673*4680 (1994).) ($136] Once the CDRs rad FRs of the cloned antibody that are to be humanized a® identified, the amino acid sequences encoding the CDRs are identified and the contending nucleic add sequences grafted on to selected human FRs. This era be done «sdog known primes end linkers, the selection of which are known m the art All of the CDRs of a particular human antibody may be replaced with at tost a portion of a non-humaa CBR or only some of the CD.Es may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanised antibody to a predetermined antigen, After the CDRs are grafted onto selected human Ms, die reselling ”ktmaaizedM variable heavy and viable light sequences are expressed to produce a humanized Fv or humanized antibody tot binds to OCC, Preferably, the CDR-grafted (e,g,, humanized) antibody binds a G€€ protein with an affinity similar to, substantially the same as, or better than, that of the donor antibody. Typically, the humanized variable heavy and light sequences are expressed as a fusion pmiek with human constant domain sequences so an intact antibody that hinds to GCC is obtained. However, a humanized Fv antibody can be produced that does not contain the constant sequences.

[91371 Also wittan the scope of the invention am humanized antibodies in which specific amino acids have been substituted, deleted, or added. In particular, humanized antibodies can have amino acid substitutions in to framework region, such as to improve binding to the antigen, For example, a selected, small number of acceptor ffcamewoik residues of the humanized immmoglohuiia chain cm be replaced by the corresponding donor amino acids. Locations of the substitutions include amino acid residues adjacent to the COR, or which are capable of interacting with a €DR (see e,g.s 1LS, Patent Nos, 5,585,089 or 5,859,205), The acceptor fraraeworii can be a mature human antibody hamewoifc sequence or a consensus sequence. As used herein, the term '‘consensus sequence” refers to to sequence feund most fosquently. or devised front the most common residues at each position in a sequence k a regjon among related family members, A number of human antibody consensus sequences are available, including consensus sequences for the different subgroups of human, variable regions (see, Rabat, E.A,, et ai, Sequences of Proteins oflmmumbgicai Merest, Fifth Edition, US. Department of Health and Human Services, U.S, Government Printing Office (1991)). The Rabat database and its applications arc freely available on line, e,g. vie IgBLAST at the National Center for Biotechnology Information, Bethesda, MD (also see, Johnson, G. and Wu, T.T., Nudeic Adds Research 29:205-206 (2001)), f§I38f Other techniques for humanizing antibodies are described in Padlau et a!, BP 519596 Al, published on December· 23,1992, [Θ139| m mti-GCC «tribady molecule includes other humanized antibodies which may also be modified by specific deletion ofbuman T cell epitopes or ‘Vldnrniumzatwrf* by the methods disclosed in PCT Publication Nos. WO 98/52976 and WO 00/34317, the contents of which are incorporated herein by reference. Briefly, the murine heavy and light chain variable regions of as antMSCC antibody can be analyzed &amp;r peptides that bind to MHC Class II; these peptides represent potential T~cdEl epitopes. For detection of potential T-cell epitopes, a computer modeling approach termed s<pepride threading* can be applied, and in addition a database of tern MHC class II binding peptides can be searched for motifs present in the marine VH and VL sequences, as described k. PCT PuMleatton'Nos. WO 98/52976 and WO 00/34317, these motifs bind to any of toe IS major MHC class 0 DR allotypes, and thus constitute potential T cell epitopes. Potential T-cel) epitopes detected can be eliminated by substituting small numbers of amine add residues in the variable regions, or preferably, by single amino acid siifesfsturions, As &amp;r as possible, conservative substitutions ate made, often but not exclusively, an amino add common at this position in human germline antibody sequences may be used. Human germline sequences ere disclosed in Tomlinson, LA. et at., J. Mol Biol 227:776-798(1992); Cook, G. P. et aL, Immunol Today Vol. 16 ($): 237-242(1995); CholMa, D. et at, X Mol Sfo. 227:799-817(1992). The V BASE directory provides a comprehensive directory of human ixmnunogtebuHn variable region sequences (compiled by Tomlinson, LA. et at MRC Centre for Protein Engineering, Cambridge, UK.). After tiro deimmmriaed VH and VL of an anti-GCC antibody are contracted by mutagenesis of the murine VH and VL genes, toe mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human. IgGI or κ constant regions.

[0148] la other embodiments, reduction of an immunogenic* response by a CDR~ grafted antibody can be achieved by changes, e.g., deletions, substitntioas, of amino acid residues in CDKs (Kaslnmri et aL Methods 36:25-34 (2005), U.S.. Fat No. 6,818,749, Tan. et aL J. Immunol 169:1119-.1125 ¢2006)). For example, residues at positions involved ia contact with toe antigen preferably would not be changed Typically, such residues, the SDKs, are in positions which display high levels of variability among antibodies.

Consensus sequences (e.g., SEQ ID NOs:3G2~307, Table 5) deri ved, e.g., by the Closlal method (Higgins D. G. et al, Metk Bmsymol 266:383-402 (1996)), ire® anti-GCC antibody molecules, e.g., from antibodies described herein, aid in identifying SDRs. Ia the human anti-GCC antibody molecules described herein, the SDRs are the following, at least tiie first residue or m some embodiments, the first four residues ofiheavychaia GD&amp;l; at least the N-tesnmaal portion, e.g,, the first semi, tea or 13 residues of heavy chain CDR2; nearly all of heavy chain CDR3; the C-terminal portion, e,g„ after residue six, eight, or nine of light chain CDR1; about the first, middle and/or last residue of fight chain CDR2; and most of fight chafe CDR3, or at least after residue two or three, Accototogiy, to maintain binding to GCC prefer» after hnmankahon or modification of m aafi-GCC anti'itody molecule, such SDR residues in CD&amp;s of the a»ti*GCC antibody molecules are less amenable to changes, e.g,, from murine residues to human consensus residues than are residues in other residues of the CDEs or the framework regions. Conversely, it can fee beneficial to change residues in aon-hnman, e.g,, nmrme CDRs to residues identified as consensus to human CDRs, e.g., CDRs of a nti-GCC antibody molecules described herein (e.g., the sequences fisted to. Table 5). For example, a serine can represent a human residue for toe C*fea»au*s of heavy sham CDR1, and/or a tyrostos can represent a human residue tor the second and/or third residues of heavy chain CDRI; heavy chain CDR2 can end to 8~{Τ/ν)~Κ~(8/Ό) (SEQ ID NO:3I2) to represent a human CDR; to represent a human CDR3, there can be a glycine after four to six residues and/or an aspartate six to tone residues in heavy chain €DR3; light chain CDS! can begin with (K/S)~(A/S)~SQS-(WL)~(S/L) (SEQ ID N0;313) to represent a human CDR; light ehato CDR2 can have a seriae to the third residue and/or an arginine to the fifth residue rqsreseat a human CDR; and/or light chain CDR3 can have a glutamine in the second residue and/or a tyrosine or serine in the third residue represent a human CDR.

[CM 41 ] Anii-GCC antibodies that are not totaet antibodies are ate useful to this tovetoion. Such antibodies may be derived from my of the antibodies described above. UseSil antibody molecnles of this type include (i) a Fab fragment, a monovalent fragment consisting of toe VL, VH» CL and CHI domains; (n) a Ε{βΚ)2 fragment, a bivalent fragment comprising two Fab fragments finked by a disulfide bridge at the hinge region; (Si) a Fd fragment, oonsisttog of toe VH and CHI domains; (Iv) a Fv fragment consisting of the VL and VH domains of a single arm of m antibody, (v) a dAb fragment (Ward et at, Nature 341:544-546 (1389)), which consists of a VH domain ; (vii) a single domain fimctional heavy chain antibody, which consists of a VIIH domain (known as a naaobody) see e.g„ Corteg-Retamoao, et aL, Cancer Res. 64:2853-2857(2034), and references cited thereto; and (vl) an isolated CDR, e.g., one or more isolated CDRs together with sufficient framework to provide an antigen binding fragment. Furthermore, although the two domains of the FV ihtgmeui, VL and VHS are coded for by separate genes, they can be joined, using reoomiramit methods, by a synthetic linker that enable# then* 1» be made as a single protein ehaio is which the VL and VH regions pah' to form moeovaknt molecules (knows as single chain-Fv (scFv); see e.g.„ Bird et ai Scumce 242:423-426 (1988); and Huston et al Prvc. Nail Acad\ Set USA 85:5879-5883 (1988). Such single chain antibodies are also intended to be aaecanpassed within, the term wantige®-hmdmg fragment* of an antibody. These antibody Imprests are obtained using conventional techniques known to those with skill-in the art, and the fragments are screwed for utility in. the same manner as are intact antibodies. Antibody fragments, such as Fv, Έ(&amp;% and Fab may he prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage. (0142] In embodiments, some or ail of the CDRs sequences, of one or both the heavy and light chain, can be used m another antibody molecule, e.g., m a CDR-grafled, humanized, or chimmo antibody molecule. |0143] Embodiments include m antibody molecule that comprises sulSdeut CDRs, e.g., all six CDRs from one of the abore-referenced human fcyferidoma, selected lymphocyte, or murine antibodies to allow binding to cell surface OCC. (01441 In an embodiment the CDRs, e.g., all of the HCDRs, or all of the LCDRs, or all sk, are embedded in human or banian derived homework region(s). Examples of human If amework regions include human germline framework sequences, human germline sequences that have been affinity matured (either in viva or in vUm% or synthetic human sequences, e.g., consensus sequences. In an embodiment the heavy chain ffamewoik is an IgOI or IgG2 framework. in an embodiment the light chain framework is a kappa framework. (0.1.451 In aa embodiment the asti-GCC imtibody molecule, e.g,, a €DR-graffed or humanisred antibody molecule, comprises sufficient CDRs* e.g., all six CDRs from one of the antibodies described herein, e.g„ sequences listed in Table 5, to allow binding to GCC. (Exemplary nucleic acid sequences which can encode the CDS. amino acid sequences listed In Table 5, are provided, in Table 6 herein). In particular embodiments, aa anti-GCC antibody molecule can comprise CDRs from $F9or.A.bx~229, (0146| Antibody fragments for in vivo therapeutic or diagnostic use can benefit from modincations which improve their semis half lives. Suitable organic moieties intended to increase the in vim serum half-life of the an tibody can include one, two or m>m linear or broached moiety selected imm. a hydrophilic pofymcric groop (&amp;g., a linear or a branched polymer <e.g., a polyalksoe glycol suck as polyethylene glycol, moaomeltey-polyethyleoo glycol and the like), a carbohydrate (e.g., a dexiran, a cehnlose, apolysaechari.de and the like), a polymer of a hydrophilic amino add pofylysine, polyaspartate and the like), a polyalkaae oxide sod polyvinyl pyrrolidone), a feity acid group (e.g,t a mono-carboxylic add or a di-carboxyllc acid), a My add ester groap, a lipid group (e.g, dlacylglycero! group, sphfegolipid group {e.g., ceramidyi)) or a phospholipid group (e.g,, phosphatidyl ethaoolamiue group). Preferably, fee organic moiety is baond to a pmdetmned site where fee organic moiety does not impair fee function (e.g„ decrease the antigen Wading affinity) of fee resulting immimoeonjugale compared to fee «oiHtonjugated antibody taoiefy. The organic moiety can have a molecular wight of about 500 Da to about 50,000 Da, prefembly about 2000,5000, 10,000 or 20,000 Da. Examples md methods for modifying polypeptides, e,g,, antibodies* wife organic moieties can he found, tor «rumple, in ITS. Patent Mos. 4,179,337 md 5,612,460, PCI Publication Nos. WO 95/06058 md WO 00/26256, and U.S. Patent Application Publication No. 20030026805.

[0147] An anti-GCC antibody molecule can comprise aft or an antigen binding fragment of the variable region, of one or both, fee heavy and light chafe, of one of tire ahove-refeaced human hybridoma, selected lymphocyte, or murine antibodies.

[0148J In an embodiment the light chain amino acid sequence of (a) can differ from, cate of fee reference amino acid sequences) referred to in (a)(i-ii) by as many as 1,2, 3,4,5,10, or 15 residues, in embodiments fee differences are conservative substitutions.

In embodiments, the differences are In the fmmewofe regions. In an embodiment the heavy chain amino acid sequence of (b) can differ from one of fee reference amino acid sequencers) referred to in (b)(i~ii) by as many as 1,2,3,4,5,10, or 15 residues, la embodiments fee differences are conservative sitostitutions. In embodiments the differences arc in the framework regions.

[01491 In an embodiment the anti-GCC antibody molecule comprises one or both of:

(a) a light chain amino acid sequence of all, oral antigen binding fragment of, either, (i) a light chain variable region amino acid sequence from Table 3, e.g., SEQ ID NO;20, or (ii) a light chain variable region ammo acid encoded by a nucleotide sequence from. Table 4, e.g, SEQ ID NO: 19; and (b) a heavy chain amino add sequence of all, or an antigen blading fragment of, either (1) a heavy chain variable region amino acid sequence from Table 3, e.g., SEQ ID NO:l 8» or (ii) a heavy chain ammo add sequence encoded by a nucleotide sequence font Table 4, e.g., SEQ ID M):l?. 1,9159 J In an embodiment the aoti~G€€ antibody molecule comprises one ear both of: ft) ft light chain variable region, or an antigen binding fragment thereof, having at least 85,90» 95,97 or 99 % homology with the light chain variable region of an anti-OCC antibody molecule of the invention, e.g, one of the above-referenced human hybridoma, selected lymphocyte, or murine antibodies; and (b) a heavy chain variable region, or as antigen binding fr agment thereof, having at least 85,90,95,97 or 99 % homolo gy with the heavy chain variable region of an anti-GCC antibody molecule of the invention» e.g., one of the above-referenced human hybridoraa, selected lymphocyte, or murine antibodies. 10151] Amino acid sequences of the variable regions of human hyhridoma, selected lymphocyte, and murine antibodies can be found in Table 3.

[MS2f In an embodiment, the anti-GCC antibody molecule is a SF9 antibody molecule and. includes one or both of: a) al l or a fragment of die heavy chain constant region from SEQ ID NO: 231; and b) all or a fragment of the fight chain constant region from SEQ ID NO: 233.

[0153| I» another embodiment» the anti-GCC antibody molecule is an Ahx-229 antibody molecule and includes one or both of: a) all or a G€C~frmding fragment of the heavy chain variable region from SEQ B> NO: 46; and b) all or a GCC-friralmg fragment of the light chain variable region from SEQ ID NO: 48.

[9154| In one approach, consensus sequences encoding the heavy and light chain 3 regions may be used to design oligonucleotides ionise as primers to introduce userid restriction sites ado the I region for subsequent linkage of V region segments to human € region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

{0155| Expression vectors include plasmids, retroviruses, co&amp;mids, YACs, EBV derived episomes, and the like. A eonvenieht vector Is one that encodes a fiinctionally complete human CH or CL rnmooigtotmlm sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted sad expressed. .6* such vectors, splicing usually ©corns between the splice donor site in the inserted $ region and the splice acceptor site preceding the human C region, and also at the splice regions Hud occur within the human CH ««ms. Suitable exjamion vectom can contain a number of components, for example, an origin of replication* a selectable master gene, one or more expression control elements, such as a trarrsenpdon control element (e.g., promoter, enhancer, terminator) and/or one or more translation signals, a signal sequence or leader sequence, and the like, Polyadenylation and transcription terasmadoa occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to my strong promoter. Examples of suitable vectors that can be used include those that are suitable tor oasnrmalias bests and based on viral replication systems, such as simian vims 40 (SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papilloma virus (BFV), papovaviius BK mutant (BKV), or mouse and human cytomegalovirus (CMV% and moioney murine leukemia virus (MMLV), native ig promoters, etc; A variety of suitable vectors are known in the art, including vectors which are mainlined In single copy or multiple copies, or which, become integrated into the host cell chromosome e.g,, via LTRs, or via artificial chromosomes engineered with multiple integration sites (Lmdeubaaut et al. Nucleic Acids Res, 32;ei72 (2004), Kaunard et al. Bimechnol Biomg. Online May 20,2009). Additional examples of suitable vectors are hated in a later section. 101561 Thus, the invention provides an expression vector comprising a nucleic aeid encodkg m antibody, antigen-binding fragment of an antibody (e.g,, a human, humanised* chimeric antibody or anti gen-binding fragment of any of the foregoing), antibody chain (e.g,, heavy chain, light chain) or anti.gen-bin.ding portion of an antibody chain that binds a GC€ protein, 101571 Express»» k eukaryotic host cells is useful because such cells are tscae likely than prokaryotic cells to assemble and secrete a properly folded mid immaaoiogieally active antibody. However, any antibody produced that is inactive due to improper folding may be renaturable according to known methods (Kim and Baldwin, "Specific Intermediates in the Folding Reactions of Small Proteins and the Mechanism of

Protein Folding", Amu Rev. Mochem, 51* pp. 459-89 (1982)), 1 is possible that the best ceils will produce portions of Intact antibodies, sack as light chain dimers or heavy cbam. dimers, which also are antibody homologs avoiding to the present fcweation. filSSJ Further, as described elsewhere herein, human antibodies or antibodies from other species can be gmemied through display-type tedmologies, including, without lintitation, phage display, reumdral display, rfbosamal display, and other tedmiques, te&amp;ug teclmiques well known % the art and thq resulting molecules can be subjected.*» additional maturation, such as affinity maturation, as such techniques are known in the art. Winter and Hamslmmwnoi Today 14:43-46 (1993) and Wright et ai. Crit Review in Immmol .12.125468 (1992), Hanes and PfaethauFNAS USA 94:4937-4942 (1997) (ribosomal di^slay), Pamtiey and Smith Gene 73:305-318 (1988) (phage display), Scott TIBS 17:241-245 (1992), Cwiria et al. Proc Noil Acad Sci USA 87:6378-6382 (1990), Eassel et al HucL Adds Research 21:1081-1085 (1993), Hoganhootn et al, Immunol Reviews 130:43-68 (1992), CMswell and MeCafferty TIBTECM 10:80-84 (1992), and US. Pat. No. 5,733,743. If display technologies are utilized to produce antibodies that ate not human, such antibodies can be humanized as described above. 18159] It will he appreciated that antibodies that are generated need sot initial iy possess apartieuter desired isoiype but, mther, the antibody as generated can possess any isotype. For example, the antibody produced by the 5F9 hybridoma (ATCC deposit no. PTA-8132) has the IgG2 isoiype. The isotype of the antibody can be switched thseafter, e.g., to IgGl or IgG3 to elicit as ADCC response when the antibody binds GCC os a cell, tisiag oonventiosa! techniques that are known is the art. Such techniques include the use of direct recombinant techniques (see e.g., U.S. Pat. No, 4,816,397), cell-cell fusios techniques (see e.g., U.S. Pat No 5,916,771), among others. In the cell-cell fusion, technique, a myeloma or other cel! line is prepared that possesses a heavy chain with any desired isoiype and another myeloma or other cell line » prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated.

[0166] In certain embodiments, the GCC antibody molecule is a human anti-GCC

IgGl antibody. Since such antibodies possess desired binding to the GCC molecule, any? one efsach antibodies can be readily isotype-wwiiched to generate &amp; human IgG4 isotype, ha*example, while still possessing the same variable region (which defees the antibody's specificity and affinity, to a certain extern). Acoefemgiy, as antibody candidate are generated that meet desired "strocftaaT attributes as discussed above, they can generally be provided with at least eertaiu additional "f&amp;acibaal” attributes that are desired through isotype switching. idldlf In an embodiment the variable region or antigen binding fragment thereof can be coupled to a constant region (or fragment thereof) other than fee constant region it was generated with, e.g., a constant region (or fragment thereof) Son* another antibody or to a synthetic constant region (or fragment thereof). In embodiments the constant region is an. IgGl or lgG2 constant region (or fragment thereof!. Sequence change cm be made in the variable or constant regions to modify effector activity of the antibody molecule. |§M2f The antibodies that are produced and characterized herein wife respect to GCC provide for the design of other therapeutic modalities inc luding other antibodies, other antagonists, or chemical moieties other than antibodies is fadlitated. Such modalities include, without limitation, antibodies having similar binding activity or functionality, advanced antibody therapeutics, such as bispecilc antibodies, immnntwpigafes, and radiolabeled therapeutics, generation of peptide therapeutics, particularly intrahodies, and small molecules, fferfeermore, as discussed above, the effector fimetien of the antibodies of the invention may he changed by isotype switching to an IgGl, IgG2, IgG3, lgG4, IgD, IgAI, %A2, IgE, or IgM for various therapeutic uses.

In connection with bispeeifie antibodies, bispeeifie antibodies can be generated that comprise (i) two antibodies, one with a specificity to C1CC and another to a second molecule feat are conjugated together, <«) a single antibody that has one chain specific to GCC and a second chain specific to a second molecule, or (hi) a single chain antibody that has ^edfidty to GCC and fee other molecule. Such bispedSc antibodies can be generated using techniques that are known. For example, hispetiilc antibodies may be produced by crosslinking two or more antibodies (of fee same type or of different types). Suitable cmsslinkera include those feat are heterobifimetional, having two distinctly reactive groups separated hy an appropriate spacer (e.g., m-maiemtidohenzoyl-N»hydrox.ysttcctntei<fe ester) orhomobifimctional (eg,, disuednimidyl suberate). Such linkers are available from Pierce Chemical Company, Rookferd, IL, See also, e.g.t Fanger et al immmomethods 4:72-81 (1994) and Winter and Hams Immunol Today 14:43-46 (1993) and Wright et si Oil Reviews in Immm&amp;l. 12125468 (1992) and in connection with (ill) see e.g,s Tr&amp;unecker et at fni / Comer (Suppl.) 7:51-52 (1992), Soagsivilai4 Laehmarm Clin, Bxp< fmmmol. 79:315-321 (1990), Kosielny et si. J. Immunol* 148:1347-1553 (1992), [#1441 In addition, ‘ICappabodies51011. et al. “Design aid eonslriiction of a hybrid iianmoglobilk domain with properties of both heavy and light chain variable regions” Protein Bag 10:949-57 ¢1997)), ^Mimbodies” (Martin et al. EMBOJ 13:5303-9 (1994), US Patent No. 5,83 7,821), “Diahodfas” (Holligsr et al Proc KM Acad Set USA 90:6444-6448 (1993)), or “Jasasins” (Tteneoker et al M/?/10:3655-3639 (1991) and Traoaeeker et al, MJ Corner Suppi 7:51-52 (1992)) may also be prepared, 10.1651 1» «tote embodiment, the present kveatioa relates to polymideksde and polypeptide s&amp;pgBces that encode for or represent the antibody molecules described herein. Such polynucleotides encode for both the variable and constant regions of each of the heavy and light chains, although other combinations are also contemplated bv the present invention k accordance with foe compositions described herein. The preseat invention also contemplates oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid, sequences complementary to these polynucleotides.

[01661 The polynucleotides can be k the form of RNA or DMA. Poiymicleotides in the form of DNA, cDNA, geatoroic DNA, nuelek acid analogs and synthetic DNA are within tire scope of foe present invention, the DNA. may be double-stranded or skgle-sttaaded* and if single stranded, may be the coding (sense) strand or non-coding (anti-sense) strand. The coding sequence that encodes the polypeptide may be identical to foe coding sequai.ee provided, herok or may be a different coding seqneace which coding sequence, as a residt of the rechmdaacy or degeneracy of foe genetic code, encodes the same polypeptide as foe DNA provided herein, [0167f In embodiments provided, polynucleotides encode at least one heavy chain, variable region and at least one light chain variable region of the present invention, e.g., as summarised k Table 4, 101681 The present invention aim includes variant polynucleotides containing modifications such as polynucleotide deletions, substitutions or additions, and any polypeptide modification msulting from the variant polynucleotide sequence. A polyniadeotide of the present myenrion may also haves coding sequence that Is a wfeal of the coding sequence provided herein. For example, a variant polynucleotide cm have at least 50%, 00%, 70%, 75%, 80%, 85¾¾ 90^.95%.«* 9^100*% wiAh'fi^auifec^^i^k TaMe 4. la embodiments, fee mnaatpo%uecleoiMeMeed® !bf sa anti-GCC antibody moleade. 10169} The present fevenrio® tether relates to polypeptides that ap®t |e antibodies of the p'eseMki?eatiaa as well as tegmenta, analogs and derivatives of such pol^eptfdes. The jteypeprides of trie present invention may fee tecomHaafe polypeptides, ttermily induced polypeptides or s^titek polypeptides, $be tegmeat, derivative or «salons of tfcepolyp^tidos of fee present Invention may be one m which one or more of the amko add residues is substituted with a conserved or ttoomaoserved amino add residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded fey fee pseiie cadet or it may be cue in which one or a^.ofAO:ip^©.amd.rssifee«kefedl^ a substituent group; or it may be one m which the polypeptide is fused with another compound, life of the polypeptide (for example, polyteylene glycol); or it maybe one in which the additional amino adds are fused to the polypeptide, such as a leader or secretory sequence or a sequence that is .ia^ioyed.^:iprifi<^^a'.«if!Ao polypeptide or a pmpmidn sequeooe*

Such tegmenta, derivatives and analogs ax® within fee scope of the present invention, In varions aspects, tie polypeptides of fee invmtfeu xMy fee pa*rialiy pariled. Or purified product. :

IfeiTOj A polypeptide of fee present invention can have an amino acid sequence that is identical to that of the antibodies described feetem, e.gi;, spnmarfeed In Tafefes 2 car 3, Or that Is different by minor variations due to one or more amino acid sufestihxtions, ’The variation may fee a “eoasenative change” typically in the range of afeo«t 1 to $ amino adds, wherein fee sntetitofed ammo add has stellar «teetotal or chemical properties, eg,* toplacemeat of louane with isoteidae of feroonme wife serine; replacement of lysine with arginine or Msiidine. In contrite, variations may include nonoonsercate® changes, e.gxf replacemeut of a glycine with a t-yptophaa, Similar minor variations may also Include amino acid, ddntions or mssriioas orhoth, Guidance In dstenteting which and how many amino acid residues may fee stestifutefe Inserted, or deleted without changing biological or lirimtmoldgicai activity may fee found using rornputet programs imown In the art, for etefetleBMASTm softer Inc., Madison, Wis.). £0171} In aaofoeraspect, tto invention features, minted aiid^rTecoxnbaiaat nucleic acids encoding anii-GCC antibody molecules. In embodiments, foe nnoleic acids encode one or mote of as antibody molecule, a heavy chain, a light chain, a light chain variable region, a heavy chain variable region, portions of fee heavy chains and light chains of the antibody molecules described herein (e,g., a light chain, variable region fragment which when paired with a Hill, length heavy chain variable region is antigen binding, or a heavy chain variable region fragment which when paired with a foil length light chain variable region is antigen binding)» and CDRs, Embodiments include such nucleic acids disposer! m vectors, e.g,» expression vectors, to specific embodiments foe invention includes plasmids pTOK58D«5F93X and pT0K5§B-SF9H€, Still further, the invention encompasses antibody molecules produced by tost ceils, e,g., expressing the antibody molecules encoded by plasmids pTOK5SD~5F9t€ and pTDK580-5F9IiC. £017¾ to an embodiment, is provided a vector, e,g,, an expression vector, comprising one or both of; £01731 sequences encoding a light chain variable region, eg., sequences listed in Table 4, an antigen binding fragment thereof, or one, two or three CDRs from a light chain (and optionally a framework region), described herd®, e.g., to Table 6; and £0174} sequences encoding a heavy chain variable region, e.g., sequences listed in

Table 4, an antigen binding fragment thereof, or one, torn or tore© GDIs 0am a heavy chain (and optionally a framework region), described hereto, e.g., m Table 6. |017S| in embodiments provided, polysucleotifo» encode at least one heavy chain variable region or at least one light chain variable region of the antibodies of the present toven.ti.oa to embodiments provided, polypeptides can encode at least one heavy chain variable region and one light chain variable region of foe antibodies of the present invention. £01T6]| In an embodiment the anti-GCC antibody molecule comprises one or both of; (a) a light chain variable region, or an antigen binding fragment thereof, encoded by a nucleic acid that hybridises under selected stringency conditions with, (i) the complement of an anti-GCC antibody molecule-encoding-nudeic acid sequence described herein, e.g., in Table 4, or (ii) any nucleic acid sequence that encodes a light chain of an $Mi-QCC antibody molecule of the invention, e.g.» one of ilteEbove-refrreneed fem» fcyferidoaa* selected lymphocyte, or murine antibodies summarized in Tables1 sod 2; and (b) a heavy chain variable region, or an. antigen binding fragment thereof, tended by a nucleic acid that hybridizes under selected stringency conditions with* φ the complement of an aaii~GCC antibody mofecule-eiieading-nnoieie acid sequence described heraa, e. g., in Table 4, or (2) any nucleic add sequence that encodes a heavy chain of an anth-GCC antibody molecule of the invention, e,g„ one of the above-referenced human hybridoma, elected lymrphocyte, or murine antibodies summarized in Tables 1 and 2, $1?7J Is an. embodiment selected stringency conditions are high stringency or very high stringency conditions, e.g,, as those conditions are described herein. $17¾ In additional aspects, the antibody or the antigen binding fragment comprises m ammo acid sequence of the light chain variable re gion, amino acid sequence of the antibod}·· encoded by the DMA having ATCC Accession Number PTA-8132, hi other additional aspects, the antibody or the antigen binding fragmeat comprises m amino acid sequence of the heavy chain variable region sequence of the antibody encoded by the DMA having ATCC Accession Number PTA-8132, $179 j The present invention also provides vectors that include the poly&amp;ucleorides of the present invention, host cells which are genetically engineered with vectors of the present invention and the production of the antibodies of the present invention by recombinant techniques, $1801 The appropriate DMA sequence may be inserted into the vector by a variety of procedures. In general, the DMA sequence is inserted into appropriate restriction endonuclease sites by procedures known in the art. The polynucleotide sequence in the exfuession vector is operatively linked to an appropriate expression control sequence (Le, promoter) to direct mRNA synthesis. Examples of such promoters include, hut are not hunted to, the Rous sarcoma virus LIU or the early or late SV40 promoter, the E, coll lac or trp, the phage lambda Ft promoter and other promoters known to control expression of genes in prokaryotic (eg., fee, T3, T7 promoters fa £ coii) or eukaryotic (e,g., cytomegalovirus promoter, adenovirus late promoter, EF-I a promoter) ceils or their viruses. The expression vector also contains a ribosome binding site for 'translation initiation and a. temaaription tmnmator. The vector may also include appropriate eeqpmom foramplliylng exfU'esski». f or example, the vector cm contain enhancers, which aretraiise^titm'.&amp;limaiatkg DMA sequences of krai origin, such as those derived iotm. simian virus Mcba»SV40, polyoma vims, cytomegalovirus, bovine papilloma vta or Moloney sarcoma virus, or genomic, origin. The vector preferably also contains m origui of replication. The vector ess be constructed to contain an exo genous origin of replication or, suets an origin of replication can be derived from SV40 or another viral source, or by the host cell chromosotnal replication. mechanism, itlStf ife addition, the valors c^tioaaily contain a marker gene ibr selection of tianxfected host cells such m dtoydrofohde reductase marker genes to permit selection with methotrexate in .a variety of boats, or antibiotics, such as jf-lactamase gene (arnpieilliu resistance), Tei gene (fbr tetracycline resi.siu.acc) used in prokaryotic cells or neomycin, GA4I8 (gsneticin, a nemnyeln-derivarive} gpt (mycopheuoHc add), ampicdlm, or .hygromydn resistance genes, or genes which complement a genetic lesion of the host cells sock ns the absence of thymidine kinase, hypoxanthine pkosphonfcosyi tiansierase, dihydrolokte reductase, etc. Genes encoding the gene product of auxotrophic markers of the host (&amp;g.s LEU2, USAS·, MSS) are often used at» selectable markers In yeast. 11182-1 &amp; order to obtain the antibodies of the present kveBtkm, one or more polynucleotide sequences that ateotic lor the li ght and heavy chain variable regions and light and heavy drain consists* regions of ilm antibodies of the present invention should be incorporated into a vector. Polyimeleotide sequences encoding the light and heavy chains of the antibodies of the present invention can be inarrporated into one or multiple vectors and the» incorporated into toe host cells. |tl83! Suitable expression, vectors for expression In mammalkn cells include, for example, jCDMS, pCDNALItomp, pcDNASX pRadLSV, pBF-I Cfevitrngeo life Technologies, Carlsbad» CA)S pCMV-SCRIKR pFS, pSG5, pXTI (Sfrstagene, La Mia, CA), pCDEFS (Goldman, L.A„ a# at, Bimeekmiques, 21:101.3-1015 (.1996)), pSVSPQRT (GIBC0 division, of Invitrogea Lite Technologies, Carlsbad, €A)S pEF-Bos (Misrahima, $., etui, Nuckk Acids Res,, 18:5322 (1990)), Mmtomic GPEX# Reriovcetor (Gala Biotech, Middleton, Wl) and the like. Exprestioo vectors which are suitable &amp;r use in various exfxession hosts, such asproksyotic cells {£, cod), insect cells (DmmpMki Schmeder S2 cells, Si9) nnd yeast (P. mMumoUce, P, pm&amp;ris, S, cerensiae) are also available. Exemplary vectors are pLKTOK58 (mid type IgG 1 Fc sequence) and pLKTOK59 (mutated IgGl Fc sequence) {see U.S. Patent Application publication no, 20060147445). mm As mil fee appreciated, antibodies m accordance with the present invention can fee expressed in cell Ikes other than hybridoma cell lines. Sequences encoding the cDNAs or genomic clones for the particular antibodies can be used for a suitable mammalian or nonmammalian host cells, Trmsformation can be fey any known method for mteodactfig polynucleotides into a host cell, including, for example packaging the polynucleotide in a vims (or into a viral vector) and hmtsditcing a host cel! with the virus (or vector) or by transfection procedures knows in the art, tor introducing heterologous polysudeotides info mammalian cells, e.g., dextrmonMiafedtrattsfeetion, calcium phosphate precipitation, polybrone mediated transfection. protoplast fesiao.» electroporation, encapsulation of toe poiynudeodde(s) into liposomes and direct mieroinfection of the DMA molecule. The tronsferm&amp;tion procedure used depends upon toe host to be transformed. Methods for introduction of heterologous polynucleotides Into mammalian cells are known in toe art and include, hut arc not limited to, dextmn-mediated transfection, calcium phosphate precipitation, polybrone mediated transfection, protoplast tosioa, dectroporatksn, particle hombatommi, eitoapulation of the polyaucleotidefs) in liposomes, peptide empgates, dendrimers, and direct mieromjection of the DMA into nuclei. i§!S5J in another aspect, the invention features, a host cell compiising a nucleic add described herein. fa embodiments toe cell expresses an antibody molecule, or compontmt thereof, described hereto. Still further embodiment provides a method of producing an antibody molecule, e.g., an mtoGCC antibody molecule described herein, e.g, a human or humanized antibody molecule comprisingmaintmning the host cell under conditions appropriate for expression, whereby imsniaoglobnlia chains) are expressed aad an antibody molecule ia produced. An additional embodiment provides a host cell misprising may of the foregoing expresshm vectors encoding heavy and light chain antibody sequences» The host cell can bo a eukaryotic cell, e,g.s a mammalian cell, an bisect cell, a yeast cell, or a prokaryotic ceil, e.g., E. colt. For example, toe raammaltaa ceS can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic roll tinea (e.g., NS0), Chinese hamster ovary ails (OHO), COS cells in a particular embodiment, the cultured host cell is a COS cell comprising nucleic acid sequences encoding a 5F9 antibody molecule, in another emk>dimexd, the host ceil isHybridoma 519 p'TA-iBS), cells, and cells from a tonsgenie animal, For example, nod etc adds encoding &amp;&amp; antibody

molcctde described heidn can. be expressed M P 1SI| Mammalian eeO lines avdlaMg as hosts for expression are known in the art pd include mmy mnniriaikeri ceil lines available bom the American Type CMture touted ΐ» Chinese hamster ovary (OK>> pto, NSf> cells, BeLa cells, baby hamster kiduey (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e,g,, Hep <32), aadaaumber of other ceil toetopp~ mamnmliao cells including hut am limited to tocterM, yeast, insect, and plants can also be medto express repaxbkptantfcdies. Site dircctcdmtotageaesis of the antibody GH2 domain to etotoate giycosyktiou may be preferred m order to prevent cbPges k either ke kmmaogemPity, pharmaeokins^ie, and/or effector fundioas resulting from noa-kmmn ' glycosylaton, The expresska mdheds are selected by detetmirsiog which system gmetoes the highest exppssktt. levels and prtouce mtoodas with oonsbtirims GCC binding properties,

|MS7| A m fmfher pkodmaent pmvides a method of produckg an ato43QC antibodymolecule, e.g., &amp; toman or humanized aaibody molecule, con^risisg niaktainmgkehostceficomprMngnttcleic aeysdperibedherek, e,g,? ojtemrmom nucleic acid sequence toted in Table 4 or b, under pndnioos appropriate lor expression of art tomimoglobolto whereby immuaoglobalm etoum are exposed and an antibody molecule, e„g,5 ahuman or humanized anybody molecule that binds GCC, or a liagmem or variant thereof, is produced. For example, methods ofegression of autltedy molecules include the use of host cells wherein a first mcomhkaaf nncieic add moiecaie eacoding an antibody molecule, e.g,s a human or huomrized antibody light chain, and a second rccomMnaat mtcleic add molecule eueodisg an aattlxMF mol<mrde, e,g.s &amp; human or hxmiMisedpflbody henvy etom, are comprised kasingle expmsriou vector. In^oiher (msbodiments, key are in sepamte vectors, The method <m further eompriMvthestgp of isolating w recovering the antibody, antigcn-bmdkg togment of an antibody* antibody chain or Pdgeariunding ftapnek of an anribody ckda, if desired. 191¾¾ Bor example, a nucleic add molecule f£«.* one or more nucleic acid

molecules) encoding the heavy mid light chains of ahuman antibody that binds a G€C protein, or m expression construct {£e„ one or more constructs) comprising such nucleic add molecule^), can be introduced into a suitable host ceil to create s mcombmant host cell using any method appropriate to the host cell selected (e.g,, Uasiriormation, transfection, electroporation, infection), such that the nucleic acid moleculefs) ate opctaMy linked to one or more expression control elements (e.gt, m a vector, in a construct created by processes in the cell, integrated into the host cell genome). The resulting recombinant host cell can be maintained under conditions suitable fix: expression (&amp;g,> in the presence of m inducer, in a suitable non-human animal, in suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nnaittonal supplements, efe.), whereby the encoded poiypeptide(s) ere produced. If desired, fee encoded protein. can be isolated or recovered (eg,, from the animal, the host cell medium, milk), 'This process encompasses expression in a host cell of a transgenic non-human animal (see, e,g., WO 92/03918, Oenfhann httematienal) or plant f01S9J Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known technique®. For example, the glutamine synthetase and DHFRgeae expression systems am common approaches for euhaacmg expression under certain confeiions. High expressing cell clones can be identified using conventional techniques, such as limited dilution cloning. Microdrop technology, or any other method® known In the art:. The GS system is discussed in whole or part in connection with Eurcpean Patent Nos. 0 216 846,0 256 055, and 0 323 99? and European Patent Application No, 89303964,4, 101.901 la an exemplary system for recombinant expression of a modified antibody, or antigen-binding portion thereof, of the invention, a recontbiuant expression vector eacodiag both the antibody heavy chain and the antibody light chain is introduced into dhfr- OHO cells by calcium phosphate-mediated transfection. Within the recombinant expression rector, the antibody heavy and light chain genes are each operatively linked to enhmeer/pmmoter regulatory elements (e,g„ derived from SV40, CMV, adenovirus and the like, such as a CMV etihanctWAdMLPpmmotermgulatory element or &amp;» SV40 enhanc^/AdMLF promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also catties a DHFR gene, which allows for selection ofCHO cells feat have been transfected wife fee vector using methotrexate seleetioj^amphEcation. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the suites medium. Standard molecular biology techniques are used to prepare toe mcmnbtoant esptessaoa vector, transfect the host pells, select for ttaesfosaaitoi, culture the bast cells am! recover the antibody frcm toe culture medium. f §19.!.] Antibodies of toe mventxon can also be produced iraasgenieally through toe generation. of a mammal or plant tout to transgenic for toe toummoglobulm heavy and light chain sequences of interest and production of toe antibody in a reeovemble form toere&amp;om. In connection with the rtansgeafo production in mammals, antibodies can be produced to, and recovered bom, toe milk of goats, cows, or other mammals, See, e.g., US. Fat Nos, 5,827,(590,5,756,687,5,750,172, and 5,741,957. {0.1.92] The antibodies, antigea-btodtog fia^nents, antibody chains and antigen-binding pardons thereof described hereto also can be produced is a suitable to wow expression system, by chemical synthesis or by any other suitable method.

Fusion Proteins and Imrunnoconl unates (01931 The anti-OCX! antibodies described herein can be fuactomslly linked by any suitable method (e.g., chemical coupling, genetic fusion, noneovalent association or otherwise) to one or more non-antibody molecular entities. (0194] Fusion proteins can be produced to which an auti-OCC antibody molecule as described hereto and a non-antibody moiety are components of a single continuous polypeptide chain. The non-antibody moiety can be located N-tenntoaUy, Oteramially, »r totewlly, with respect to the antibody moiety. For example, some embodiments can be produced by toe insertion of a nucleic acid encoding immunoglohulto sequences into a suitable expression vector, such as a pBT vector (e.g„ pET-lSb, Movagen), a phage vector (e.g„ pCNATAB 5 E, Pharmacia), or other vector, e.g., pRIT2T Protein A fusion vector, Phunnada), The rmilttog constract can be expressed to produce antibody chains that comprise anon-antibody moiety (e,g,, Histidine tag, £ tag, or Protein A IgG binding domain). Fusion, proteins can. be isolated or recowed using any suitable technique, such m chromatography using a suitable affinity matrix (see, e,g.s Current Protocol* in MolecularBiafogy (Ausubel, F.M et at, eds,, Vol. 2, Suppl. 26, pp. 16,4.1-16.7,8 (1991)). mn\ The invention provides anti-OCX antibody molecules which are directed to and, in embodiments, are foteraalfeed into cells. They are capable of delivering therapeutic agents or detectable agents to or into cells expressing OCX, but not to or into cells where the target is act expressed. Thug* Use mvehtioa also provides and-G€C immirooconjug&amp;fes ccorpasag m anti-GCC antibody molecule as described herein, which is conjugated to a ihcrepeniie agent or a detectable agent la emkHliments, the affMiy fer GCC of a» anti-'GCC imaJHtrocoaja^te is at least IQ, 25,50,75,80,90, or 95% of tfiat for the unconj ugated antibody. TMs can be detemiined using cell stalks GCC or isolated GCC. In m embodiment the anti-GCC antibody -molecule, e.g,s m immtinoconf agate, has an LD50, as detennined by m assay described herein, of less 0m 1.,090,500,250,10¾ or 50 pM. (0196( The anti-GCC antibody molecule can be modified to act as an immunity «.gate uiillxmg techniques that are known in the art See e.g„ Viteita immmol Today 14:252 (1993), See also US. Pat No. 5,194,594, The preparation of radiolabeled antibodies can also be readily prepared ufilkhrg techniques that are known in the art. See e.g,, Juughans ei at m Cancer Ck&amp;mtkempy andBtotiwopy 655-586 (2d edition,

Chafeer and Long», eds,, Lippmeoft Kaven (1996)). See also US. Pat, Nos. 4,681,581, 4,735,210,5,101,827,5,192,999 <U,S, Re. Pat. No. 35,500), 5,648,471, and 5,697,902. (0197J In some embodiments, the antibody molecule and non-antibody moiety are connected by means of a linker, &amp;t such embodiments, the immnaomnpgate is represented by formula (7):

00 wherein,

Ab is an anti-GCC antibody molecule described herein; X is a moiety which' connects Ab sad Ζ» e.g., the residue of a linker described herein after covalent linkage to one or both of Ab and Z; Z is a therapeutic agent or label; and m ranges from about 1 to about 15.

[9198] The variable m represents Sis number of ~<X~Z ntoieties per antibody molecule is m imntonoconjugate of foastula (I). Is various embodiments, m ranges bom 1 to 15,1 to 10s 1 to 9,1 to % I to 7,I to 6, 1 to 5» 1 to 4,1 to 3* or 1 to 2. I» tome effittodimenfs,, m ranges Sum 2 to 10, 2 to 9,2 to 8,2 to 7,2 to 4 2 to S; 2 to 4 or 2 to 3. In other ernkfoiments,, m is I, 2,3, 4,5 or 6, Is compositions comprising a pimmlity of Itomunoconjugistos of formula 0,mk Sts avenge fis&amp;afeesr of ~X~Z moieiie s per Ah, also» referred to as the average drag loading Average drug loading may mage feia 1 to about 15 -X“Z moieties per Ab< I». some embodiment «ires m represents die average drag loading, m is about 1, about 2, abeto 3, aboiS 4, about 5, about 6» about 7, or about 8. In eK^spIary emhodimests, m is from about 2 to shoot 8. In one embodiment, ® is about 8. la another emltofemeat, m m about 4 la another embodiment, m k about 2, |1!»| The average number of -X«Z moieties per Ab may be characterised by eot«feationai mesas such m mass gpsobxseopy, SUSA assay, and HPLC. The qaaatitative distribution of immnnoeonjugates in terms Of λ may also be determined. Is some instances, separation, purification, sad chamctemation of homogessoas imnumoermjug&amp;ies where m k a certain value, a$ distiagmshed from, immnnoconjugates with other drag loadings, may be achieved by means snch as reverse phase HPLC or eJechnpboresk [9299] The iramunocenfugates of formula 0 may exist as mixtures, wherein each component of the mixture has a different m value, For example, m imarisKtoonj ugate of formula (ϊ) may exist m a mixture of two separate innattooconjogate components: oae iassattoconjugate coinponent whereto m m 7, and the other immttnoconjugafe component wherein m is 8, [9201] In one embodiment, the immnaoeostugafe of formula (1) exists as a mixture of three separate hnimmoconf ugates wherein m for the three separate inrntuntKtopJugates is 1., 2, and 3, raspecdyely, [92921 Is one embodiment, the immunocosjugate of formula (J) exists as a mixture offeree separate InKnunoeonjugales wherein m for the three separate ummuaceonjugates is 3,4, and 5, respectively, [1203] is one embodiment, fee humunoconjugate of foranda (J) exists as a mixture of ferae separate immnnoconiugates wherein m for the three separate losasaoeonjugatea is S, 6, and ?, respectively.

[0204) In aae embodiment, the tmmnaacoipgg^ of formula (i) exists ns a mistee of three separate nnnimmeonjtigstes whereat m for the three sepamte iffimunoconjugates is 7,8, and 9» respectively, [020Sf In one emtedimant, the kumimocopragate of formula (i) exists as a mixture of thme separate ifemmoconjugates wherein m-ifer the three separate immimaconjugates is % 10»·and Hi tespeefeely, f 1206! la one embodiment, the immtmoeoajugaie of formula (J) exists as a mixture of three s^asate imnamocoajugafes wherein m for the three separate immynoeoajugatts is 11» 12» and 1.3, respectively. 102071 ί» one- embodiment» the hnrmmooonjugate of formula φ exists m a mix ture of three separate mtmnnoeoqngates wherein m for the three separate knmmtoeonjngales is 13,14» aad 15, respectively. fS20§) A vmsty of suitable linkers (e.g,, heterobiSmetioaal msgeafx for coimectteg m antibody molecule' to a therapeutic agent or label) and methods for preparing imniuuoconiegates are know in 1½ art (See, for example, Chari et at. Cancer Research 52; 127-131 (1092).) The linker caa be eleavsble, e.g., under physiological conditions,» e.g., under iutracelMar conditions., such that cleavage of the linker release the drug (tbempeutic agent or label) In the intracellular envlminnent In other emlx>diments3 the Maker is not deavaMe, aad the drug is released, far example, by antibody degradation. 102001 The Maker can be bonded to g chemically reactive group on the antibody moiety, e.g.» to a tree amino, iraino, hydroxyl thiol or carboxyl group (e.g,, to the N~ or €~ terminus, to the epsilon. amino group of one or mom lysine residues, the Res earboxylfc add group of one or more glutamic acid or aspartic acid residues, or to the sni&amp;ydry! group of one or more cysteinyl residues). The site to which the Maker is bound cm be a natural residue is the amino acid sequence of the antibody moiety or It can be infeduced mto the antibody moiety, e.g.» by DMA meombinant technology (e.g,s by introducing a cysteine or protease cleavage site in the amino acid sequence) or by prxjtein bioeheniistr/ (e.g,, reduction, pH adjustment or proteolysis).

[0310] One of the most commonly used oon-speciSc methods of covalent attachment is the eerbodlimide reaction to link a carhoxy (or ammo) group of a compound to amino (or carboxyl groups of the antibody molecule. Additionally, blfimctional agents suck as dialdehydes or amdoestera haw beea used to link foe amino granp of a composed to amino poups of an antibody molecule, Also «vailafeie for sttedhmcat of drugs to antibody molecules is the SchxH: base reaction. This method Involves foe periodate oxidation of a drug that contains glycol or hydroxy g&amp;ag®, thus tmoring aa. aldehyde which is risen reacted with foe antibody molecule. Atiadhmcst: occurs via formation of a SeMif base with, amino groups of trie antibody molecule. Jsofoioeyanates can also be used as coupling agents for covalently atfeeliing drugs to antibody molecule. Other techniques are known to the skilled artisan and within trie scope of foe present kventlon. 10211) In certain embodiments, an mtormediate, wrileh is foe precursor of foe Maker (X), is reacted with foe drug (Z) under appropriate conditions, la certain eatbodimeois, reactive groups are used on foe drag and/or the intermediate, Trie product of foe reaction between trie drug and trie intermediate, or trie dsrmtiznd drag. Is subsequently reacted wifo foe antibody molecule under appropriate cwdltlons. : i#212| The manunocosjugate can be pariried -ftom reactants by employing methodologies well known to those of skill in trie art, e.g., column dnonmtograpriy (e,g., affinity clmaiatography, ion exchange cfoiomntpgi-aphy, gel filtratfon/riydmpboble interaction chromatography), dialysis, diafitofoat mpredpitatton, Trie irammioconfugate can be evaluated by employing metriodologles wdflitowa to those skilled in foe art, e.g,, SDS-PAGE, mass spectroscopy, or c&amp;pillaty dectmphoresis, [i£B] Is some embodiments, the linker Is deavable by s oleavmg agent that is present in the intracellular environment (nf,9 within a lysosome or endosome of cavsolea).

The linker can be, e.g., a peptidyl linker that is deswt by an iatraeeLlukr peptidase or protease enzyme, mefudiag, bat not limited to, a lysosomal or endosoma! protease, fa some eanbodlments, trie peptidy! linker is at least two ammo acids long or at least three amino acids long. Cleaving agents can include cafoegsfos B and D and plasmltu all of which are known to hydrolyzedipepiade drug derivatiyos resulting in foe release of .active drag .inside target cells (met e,g.t Duhowehik and Walker, 1999» Pham. Therapmttes §3:67423), Most typical are pepridyf linkers that are cleavable by enzymes trial are present in GCC-expreasmg cells. For example, a peptidyl linker trial Is cleavafek by foe foiol-dependeni protease catriepsln»B, which is highly expressed in cancerous tissue, cm. be used (eg,, a Phe-Lea or &amp; Gly-Pbefocu~01y linker (SEQ ID N0:3I9)>, Other examples of such linkers are described, e.g.»in US, Patent No, 6,214,345, foeotpomted herein by inference its. its entirely and for all purposes, la a specific cofeodiment, the peptidyl linker cleavaMe by an intracellular piotease is a Vat*C&amp; linker or a Phe~Lys Suker {see, e.g., U.S. patent 6,214,345, which describes the synthesis of doxoinbick with fee vul-cli linker).

One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high., 102:1.4) In other embodiments, the eleavable linker is pH~smMve, i e,,sensitive to hydrolysis at certain pH vainer Typically, the pH-ssssitive linker is hydrolyzable under acidic conditions. For example, an add-labile linker that is hydrolyzable k fee lysosomc {kg,, a hydiazone, semicawbaaone, thlosemicarbazoae, ds-aconitk amide, orfeoester, acetal, ketal, or fee like) can he used, (See, e.g., 11$. Patent Nos, 5,122,368; 5,824,805; 5,622,929; DubowcMk and Walker, 1999, Pham. 'Therapeutics 83:67-123; Neville et al, .1989, Bhl Chem, 264; 14653-14661.} Such linkers arc relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5-0, the approximate pH of the lysosome, k certain embodiments, the hydrolyzable linker is a tMoether linker (such as, e,g„, a thioeiher attached to the therapeutic agen t via an acyfeydrazose bond (see. eg., U.S, Patent No, 5,622,929). |I215) In yet other embodiments, fee linker is deavable under reducing conditions (e.g„ a disulfide linker), A variety of disulfide linkers are known in fee art including, for example, those feat can be formed using SATA (N~aicda^dyi-S~«;etyifeioacetate)s SPDP (N-snccMmidyl~3-(2-pyridyl.ditMo)pfopionate), 3PDB (NLsuccinimidyl-3"(2-pyridyldhhio)butymte) and SMPT (N~,mceinhnidyi-oxycarboayl”alph.a“methyl--alpha»(2-pyridyhdithio)toiucne), SPDB and SMPT (See, e.g„ Thorpe etal, 1987, Corner Res. 47:5924-5931; Wawrzynczak et al, k Immmoemfitgates: Antibody Conjugates in Radtotmagety and Therapy of Corner (C. W. Vogel ed, Oxford U. Press, 1987, See aim U.S, Patent No, 4,880,935.) 10216) In yet other specific embodiments, the linker is a malonate tinker (Johnson eiaLf 1995, AnlieaneerRes. 15:1387-93), a malcimidobenzoy! linker (Lau etal, 1995, BioorgMed Chem. 3(10):1299-1304), ora 3-N-amido analog (Lau etal, 1995, Bimrg-Med-Chem. 3(10):1305-12). f®2 t 7| Is yet other embodiflisais* the linker «nit is oof eteavable and the teg is

Messed by antibody defp^atiom (See for example OS. Pnbiie^on Mo, 20050238649 incorporated by reference herein is Its entirety md for all praposes)- PBISI Typically,. the linker is not sa&amp;staatiaSy sensitive to the mdfaoellulsr envtonnient As used herein, fetof Mmtanferily sensitive to tbs extraceilnbr erndmiaaeat,” In the content of a Ifeker, mem that no more than about 20%, typically no more than about. 15%, more typically no more than abou t 1-0%» and even mom typically so. more than about 5%, so more than about 3%, or no mor e than about 1% of fee bakers» is a samgile of immunocoajugate, are cleaved when, the inmnmoamjngat e presents in an emraeelbtlar envimoment (e,g.»In plasma). Whether a linker is not substantially' sensitive to the extracellular environment can be determined, for example, by incubating with plasms the Immmtoaiittjugste for a predetemtiaed time period (rig,, 2,4, S, lb, or 24 tours) mi then qnantitomg the amount of fine drug present in the plasma, [§2I9) in other, toa-mafeally excksiye embodiment, the linker promotes eelhdsr felemalkarion, In certain embodiments, the linker promotes cellular fetemalkation when conjugated to fee therapeutic agent or label (2), la yet other embodiments, fee linker pmmtm cellular Infomafeation when conjugated to both fee Z moiety' and fee anthCiGC antibody molecule. (022§) A variety of exemplary linkers feat cm be need with Re present mmposidone and mefeods are described in WO 2004-010957, IIS. Publication No. 20060074008»U.S. Publication Ho, 2095023864¾ audXi.S. PiibheationHo. 20060024317 (each of which is Incorporated..by reference herein is its entirety and for all purposes). |0321| Examples of bakers capable of being used to couple an antibody mol earls to a therapeutic agent or label include, for example, nMlemritocapmyl (me); mEleintidocapmyl-jjmminotoii^icarbstnateymaleimidoe^foybpeptide» ammobenayicaibamate linkers, e.g,» maieimldocapmybL-phenyiabinine-L-lysme-p-etnnmbmmylcafeaaiMe aad ma!einhdoe8pmyi4,"VaEae4u“Cifod).fne»p" ammobemyteafeamnie (vo); N-succlntmidyi lH2“pyrIdyidlfeio)pi»prionate (also Mown, as N-saecmmidyl 4--(2 "pyridyldithIo)pntmioate or SPP); 4~sucekttOiMyl^xycarbonyb'2~ iueihyl~2^2myridyldifeioHoteae (S^IPT): N-aaccimmidyl 3-'(2>p}ridyldifel0)propioirale iSPDP); N-suecinmudyl 4~(2"pyrldyldit.hio)buiy,Ta.tc (SPDB); 2-immofeiotans; S» acmylsuednfc anhydride; disuffide benny! carbamate; carbonate; hydrs?.on.e linkers; KT~(a-

Maleirkdoaeetoxy) succminsde ester; i¥“[4»|^AMd.osdicy1aiaidc}}butyi]“3''"(2'-' (BMPS); |N“8~MateImidoc8pmy!0xyjs^cdamiide ester (EMCS); N~[y~

Mdediddome%13Q’ct0hexaae~l-c&amp;rtx>xji-i6"SmideeaproMe3 (LC~SMCC); Saedassid^ hydroxysucdnbdde ester (MBS); Ar~SuedBimldyIi4-bdotce^d]aadaol)eK2ioate (SIAB); SiicemiiftidyI4~(A?~maIeimidc}metliyl]cyelah.exaae~l'-earboxykie (SMCC); iV^Socemteddyi 3~C2“l>>^#4^diio|“pK>pit>iwmldo (SPDP); [N^Maicimidocapi»yIoxy]saIi%sacemijmde ester (Sdfb-EMCS); N-[y'Ma!eimtdobirly^doxy]sx3!&amp;stteekimide ester {Snlio-GMBS}; 4~Sidfastteeiaimidyi-6-mefbyl“a-(2»pyTi^ddiddo)tefuafflido]liexaB0ate) (Suifo-LC-SMFT); Snliosueekiimdy! 6»(3H2“p}Mdy!diteioj«propteftairiido)hex8EOste (Stdfo-LC-SPDP); ^MaIejffiid0beRKoyI“N“&amp;ydroxysalfosscciatmide ester (Skfo-MBS); A-Sdfixsecdakaldyl(4“l0doacetyijamia0feeazoa.te (S«H»-SIAB); Suifosueckimidy! 4-fA-maleimMcimedryljeyOtodexane-! -carboxyl ate (Siilfe»SMCC); Sulfosacdnimidy! Φ[ρ* aiaieimidophaiyljbufyrate (Stdfo-SMPB); ethylene g!yml4>is{suce£nic acid N-liydroxysaeckimide ester) (EGS); dtsueckimidyl tartrate (DST); 1,4,7,16* teti'aazacyciQdodecane-l^syjlO-ietmaeetic acid (DOTA); didhyiendriamke-peatMeatic add (DTPA); and thiourea Rakers. P222-1 In some embodiments, the linker “X*· has the formula ~A*»W«rYr, and the imraimoconjugate of formula (J) is characterized by formula (Si):

wherein,

Ab is an antROCC aatiboify molecule described task; Ά- ts a Stretcher unit; a is 0 or i; sack —W~ independently is an -Amino Acid unit; w is an integer ranging from 0 to 12; -Υ» is a seli-miffiQ&amp;tive spacer unit; y'isO, 1,οε2; Z is a therapeutic agent or label; md m ranges from about 1 to about 15> f tlSJ The Stretcher unit { A )s when present, is capable of liakin g an. Ab wit to ait

Amino Acid «sis (~W~)5 if present* to a Spacer unit (»¥--), if present; or to a therapeutic agent or label ( Z }. Useful fimefioaal groups that can be present oa as auti-CICC an tibody molecule, either naturally or via chemical manipulation include, but are net limited to, nililydtyL ammo, hydroxyl, ike anomerie hydroxyl group of a carbohydrate, mid carboxyl Suitable fuucdonal. groups are sulfhydryl and amiao. In one example,

Mkhydryi groups can be generated by reduction of the kirsmoleenlar disulfide bonds of mi atei-GCC antibody molecule. In another embodiment, adfhydryl gmups can be generated fey reaction of m amino group of a lyske poieiy of an. anfi~GO€ antibody molecule with 2-hninotMolaue (Traufs· reagent) or other sidfhydryl generating reagents.

In certain embodiments, the anb-OCC antibody molecule is a recombinant antibody and is engkeered to cany one or more lysines, li certain other embedments, the rgcooibinaat ami-GCC antibody molecule Is engineered to cany? additionai suliliydryl groups, e.g,> additional cysteines, 1%$M\ In one embodiment* the Stretcher unit forms a bond with a sulfur atom of the Ab unit Hie sulfur stem, can fee derived from a suiffiydryi group of an Ak RepresenMke Sfeddbermtlts of ^is embodim^ are depicted within the sguam brackets of Formulas ίβΜ} and (MM), whereinAb-, -W~, -Ϋ-, -Z, w and y are as defined above, and R* is selected fern -Ci-Cw alkylene-·, -Cr€>e alkenylene~, <ferCi« alky&amp;yleoe-» -earboeyck-, -0-(€rCs alkylene)-, 0-(CrCs alfcenyleneK "0-(Ck~Cs alkynyleneK ~ arylene-, ~€ι-€» alkyleoe-arylene-, Au-Cho allmnylene-arylene, ~Ca.~€ia a&amp;ynytene-atydeue, -asytetioCi-Cjo alkylene-, -aiylene-CrCiG alkenyleoe-, *arylene~CrCis alkynylene*, «Cj-Cw ^lsyl^te<caitocydo>, -CrCm alk^jyletae^caiboc^doK ^CrC» ulky’tiylene-Ccarboeyelo)", -(earlmcycioKlrCac alkylcue-, »(eatbseyeto}Cr€iG alkenylene-·, -{carbooytdol-C'rCYo alkynylenc, heietocyelo-, «Ci-Cto alkylened keietocyclo)-, ~€VCu> alkenyleiie‘-{heierocyeto)~, -Ca-Cte alkynyleae“(hetetoC3tolo>f ~ (keterocydo>-€rCKi alkyieBC-, -(hetotocyc!o)-€a-Cie alkenyleBe-, '-(hetercioycto^Ca-Cn) alkynylene*, <CH2GH20)r:! or *<CH2€lfeO>€Hr* swdrk a» integer ranging- bom. I-10, wherein md alkyl, alkenyl, alkynyl, aJkyime, ajfaytae, 8¾¾}½¾¾ aryl, carbocyie, carboeyclo, heteroeycio, and sryleae radicals, whether alone or as part of another group, are optionally substituted. In some emhodimeiifs, said alkyl* alkenyl, alkynyi, alkylane, aikenyiene, alkynyieae* aryl, casboeyle, carbocycio, beterocyelo* and aryieae radicals, whether alone or as part of mother group, are ««substituted. In some embodiments, B8 is selected horn -Ci-Cio alkylene-, - eaifcocycfo», ~&amp;{Ct~Cg AyleueK -aryleao-, -Cj-Cjio alkyleue-orylena-, -aryleae-Ci-Cjo dkylme-» -Cj-Cie al^aie^carbcwycb)», -( carhocyelo)-CrCio alkyleae-, -CrCg lieterocyelo-, -Ci-Cjo alky1ene~(hefeTO<yda)-} -( heterocyclo)“€i-€H} aikyiene~, -(QlaCHaO),--, and *(CH^HaO)rCH2S and r is an integer ranging from 1-10» wherein said alkyiene groups m «substituted and the remainder of the groups am optionally substituted. (122S| It is to be understood from ail the exemplary embodiments that even where not denoted expressly, from 1 to 15 drag moieties can be linked to an Ah ( m = I~ id),

ί 92281 An illustrative Stretcher unit is that of Formula (Ms) wherein Ra is - iC%)3~:

[022f 1 Another illustrative Stretcher unit is time ofForanls (Mid) where® R* is -(CH2CaO)r(Mr; and r is 2;

{0230] Another illustrative Stretcher unit is that of Formula (life) wherein R* is - arytene- or aryleBe-Ci-Cio al&amp;ylene-. la some embodiments, the aryl group is an anssibstitatesd. phenyl group, [0231] Still another illustrative Stretcher unit Is that of Formula (Mb) wherein R* is~(CH2)r:

[0232] la certain embodiments, the Stretcher ysmt i$ linked to the Ab «nit via a disulfide bond between a sulfur alum of the Ab unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is depicted within the squats brackets of Formula (IF), wherein R*, Ab*, -W~, -Y-, -Z, w and y are as defined above, [0233]

[0234] It should be noted that throughout this application, the S moiety in the formula below refers to a suite atom, of tbe Ab «sit, unless otherwise indicated by context,

[0235.1 In yet other embodiments, the Stretcher, prior to attachment to Ab, contains a reactive sits that can form a bond with a primary or secondary amino group of the Ab. Examples of these reactive sites include, but are not limited to, activated esters such as auccMmide esters, 4 mnophcayl esters, pentafiuorapheayl esters, tetratluorophmyl esters, anhydrides, add chlorides, sulfonyl chlorides, isocyanates and isotfaioeyanafes. Representative Stretcher units of this embodiment are depicted withm the square brackets of Formulas (Ve) and iyb\ wherein ~K\ Ab-, -W~, «Υ-» ~Z, w and y areas defined above;

[I238J la some embodtmeais, the Stefcher contains &amp; reactive site that Is reactive to a modified etrltohydrare’p f CHO) group feat eaa be present m as Ah, For example,a carbohydrate eas be mildly oxidized using a reagent such as sodium periodate sad the resoMog fCHO) suit of the oxidized cafix%dMe cm be eoadeased with a Stretcher to contents s fimctksaaMty such as a hydtod^ as oxime, a pnmasy or seeosdaty amine, a hydrazine, a tMeuemlearbazese, a hphaame omboxylate, end as arRliydraaMe such, as those described by Kauako ei at, WM. Mocmjugate Chew., 2:133-41. Representative Stretcher units of this emboduiaesi are depicted within the square brackets ofFermaias (¥$&amp;}, (fffl), and (Fie), wherein-~RS~S Ab~s -W~, Ύ-* ~Zt %v and y ais-as defined as above.

|I239] The Amino Acid taut (- W~}3 when present, links the Stretcher unit to tbs

Spacer rent if the Spacer unit la present, links t he Stmicher unit to the Drug mo iety if the

Spacer lost is absent, mid Maks the Ab aait to toe therapeutic agent or label moiety ffih© Stretcher met aad Spacer unit are absent

Ww- can Me, for example, a moaopepti.de, dipeptide, irtpeptide, tetrapepiide, penlapeptido, hexapeptide, keptapeptide, octapeptide, aouapeptlde, decapeptide, imdt^apepfido or dodeeapepfefe aait Bach. -W- tmft md«fseade«% has toe formula denoted below at too square brackets, aad w is aa «sieger raogiag Ufom. 0 to 12:

where!» Rb is hydrogen, methyl, isopropyl, isotmtyl, reetoatjd, benzyl pdtydroxyhenxyl - cifcoii, «cscoffices, -eaayscHj, -¾^¾ -CH^eooH, -cHrce^ccMffc jmMsOzm, -{ce^MHcocH,, <€η2>νηο{-νη)Κχ% <οη^μ*2, "(CH^NHCOCHi, -(CH^NHCHO, -{€%3KHCOm&amp; ^CHj&amp;NHOONBh, -CB-sCBsGl^OI-QCHjNHa, 2«pyridytoethy!>, 3 -pyridyimeihyl·, 4-pyridylmethyl-, phenyl cydohexyi,.

|.8240j la some embodimeais, the Amino Add trnii caa be eszyimiically cleaved by one or mom enzymes, taefodmg a cancer or tamor-assrodated protease, to liberate the therapeutic agent of label moiety {-7), which ra o»e embodiment Is potonated in vim upo® release to provide a therapeutic agent or label (Z), [8241] M certain embodimeats, the Amino Add unit east comprise natural amino adds. In other embodiments, the Ammo Acid unit am comprise non-natural amino adds. Illustrative Ww units are represented by formulas (ΨΉ)~(£%}ι (8242]

where*» Re and Rd am as follows;

wherein R°. Rij and R6 am as follows;

m wherein R5, R“\ R® a&amp;d R* are as follows:

f§243J Exemplary Amino Acid unite include, but are not limited to, unite of formula ( m) where: Re is benzyl and R* is R* is isopropyl and is -(0¾)^¾ or R* is isopropyl and Rd is -(CHaJjNHCONHa, Another exemplary Amino

Add unit is a unit of formula (VM) wherein Re is benzyl, R** is feeazyi, and R* is - (CH^NHb. ' 1(12441 Usefo! -Ww~ units can be designed and optimized is their sdeetmty for ; enzymatic cleavage % a particular enzyme, for example, a tamor-assoctafed protease, in one embodiment, a »Ww - mat is that whose cleavage is catalysed by cathepsm B, C and D, or a plasmin protease. 102451 In one embodiment, ~%V is a dipeptide, tripeptide, tetrapeptide or pentapepti.de. When R* R®, Rd, R* or Rf is other than hydrogen, the caribou atom to which Rb, R®, Rd, Re or Rf is attached is chiral. 1024^1 Each carbon atom to which R\ Re, R* or Rf is attached is independmily m the (S) or (R) configuration. 102471 In one aspect of the Amino Add unit, the Amino Acid unit is valise- citrulfine (vc or val-cit). In another aspect, the Amino Acid unit is pheny1alanine~iyshi.e (i,e., fk). In yet another aspect of the Amino Acid unit, the Amino Acid unit is N-niitbylv&amp;ifeo-dtnfeine. In yet another aspect, the Amino Acid «aft is S-aminovderic acid» homo phenylalanine lysine» teifaisc^mooHoecafboxykte lysine, q/elohexyMamse lysine, isonepeeotic acid lysine, bda-alanine lysine, glycine serine valine glutamine and isoaepecclic acid i®248j The Spacer unit (~Y~), when present, links an Ammo Add unit to fee therapeutic agent or label moiety (-Z-) when m Amino Acid unit is present. Alternatively, the Spacer «nit links the Stretcher nnit to the therapeutic agent or label moiety when the Amino Acid unit is absent. The Spacer unit also links the therapeutic agent or label moiety to the Ab unit when both fee Amino Acid unit and Stretcher unit are absent 1β249] Spacer units are of two general types: nos seli-tenoiative or seif-

Immoladve. A non self-lmmolaiive Spacer suit is one in which part or all of the Spacer unit remains bound to the therapeutic agent or label moiety ailer cleavage, particularly enzymatic, of an Amino Add unit form the antibody» drug co.n]ugate. Examples of a non. selfdmmdative Spacer unit isolode, but are not limited to a (gly«ne»g[yeiae} Spacer «nit and a glycine Speer unit (both dpicted In Scheme 1) {infra). When a conjugate amtaimng a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via m enzyme (&amp;gy, a tumor-cell assoeiated-ptoiease, a egncer-eeli-associated. protease or a lymphoeyteassocisted protease), a glpine-glycme-Z moiety or a glycme-Z moiety is cleaved hom Ab-Aa«Ww-. hi one etobodimeni, m independent hydrolysis reaction takes place within fee target cell, cleaning fee glyciue-Z moiety bond and liberating the therapeutic agent or label.

ft25$] la some embodiment^ &amp; nos. self-mnaoiative Speer unit (~Y~) is ~GIy~v in some embodiments, a non setMmmolalive Spacer unit (-Y-) is -Gly-Gly-. 1^2511 la one embodiment, the invention provides an immtmeeonjngste of iormnla (JB) wherein the Spacer unit is absent (y - ϋ% or &amp; phamiaceutiealiy acceptable salt thereof. (9252) Alternatively, a conjugate containing a seif-mimokiive Spacer unit can release ~Z. As used hersm, the term “selfdramolative Spacer-1’ refers to a bihmcdonal chemical moiety that is capable of covalently linking together two spaced chemical moMes into a stable tripartite molecule, ft will spontaneously separate fern, the second chemical moiety if its bond to the first moiety is clea ved. 10253J la some emhodancais, -Yy~ is a p-^imaobeBayi alcohol (FAB) unit (see

Schemes 2 and 3) whose phenylene portion is suMtmted with Qs wherein Q is ~€? ~€§ alkyl, -CrCg afeny!, ~CrC* alkynyl, -O-CCrCg alkyl), AKCrCs alkmyl), -0-<C2-Cg aifcynyl), dmlogen,~nite or -cyan»; and n is an integer ranging ftom (M, The alkyl, alkeayl and alkynyl groups, whether alone or as part of another group, can be optionall y substituted 102541 la some embodiments, -Y« is a FAB group that is linked to ~ Ww - via the amino nitrogen atom of the FAB group, and connected directly to «2 via a carbonate, carbamate or ether group. Without being bound hy any particular theory or mechanism,

Seta»© % depicts a possible mechanism of release of a therapeutic agent or label (»Z) front a PAB group that is attached directly to ~Z via a carbamate or carbonate group m described by Told ete!., 2002, /. Org. Ckm. 67:1866-1872.

|i2SS| la Schema 2, Q is -CrCs alkyl, -Cr€$ alkenyl, -C^-Cg alkynyl, -O^Cj-Cg alkyl), -0~C€rCg alkenyl), -0-(€r€g alkynyl), «halogen, -mteo or -cyano; m is an integer ranging from 0-4; and m ranges feas 1 to about 20. The alkyl, alkenyl and aStyuyl groups, whether alone or as pat of another group, can be optionally substituted. 10256) Without being bound by any particular theory or mechanism, Scheme 3 depicts a passible meahanism of release of a therapeutic agent or label moiety (~Z) horn a PAB group which is attached directly to »Z via an ether or amine linkage, wherein -Z includes the oxygen or nitrogen group that is part of the therapeutic agent or label moiety.

[§2S7| In Scheme 3,Qis -Cj-C* alky!, ~CrQ alkenyl, -CrCs alkynyf -0-(¾ ~Cg alkyl), -0-(CrCa alkenyl), -0-(CrCs alkynyt), -halogen, -ntes or -cysao; a Is an integer ranging from (M; and. w rangas fiem 1 to about 20. Tim alkyl, alkenyl and alkynyl groups, whether alone or as part of another group, can be optionally substituted. (0258] Other examples of selfrimmoiarive spacers include, but are not limited to, aromatic compounds that are eteetronienlly similar to the PAB group such m 2~ aniinoi»iida>mk5“jsethanol derivatives (Hey ef aLt I99%3tw?g< Med Ckem. Lett 9:2237) and ortho or pam-amkobenaylacetais. Spacers can be used that undergo cycfeation upon amide bond hydrolysis, such as substituted and nnsubstitided 4> inmnobidyrie acid amides (Rodrigues ei &amp;!., 1995, Chemistry Biology 2:223), appropriately substituted bicycIo[2.2J] and bkyclo|2.2.2| ring systems (Storm ei at, 1972, J, Amer. Ckem. Soe, 94:5815) and 2-aminopheaylpsx>pionic acid amides (Amsbeny etal, 1990, J. Qrg: Ckem, 55:5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury et al„ 1984, J, Med, Ckem, 27:144?) are also examples of self-imrnotative spacers, f!25f j Is one embodiment, the Spacer unit is,a branched Ms{hyd®xymethyl}~

Create (BHMS) unit as depicted m. Sefeenre 4, which can be used to incorporate md release multiple drugs,

\mm\ la Scheme 4, Q is ~Ct-€$ alkyl, *€rCs alkenyl, ~CrC8 aikynyl, «0<Ci-C8 alkyl), -0»(C;rCs alkenyl), ~G~{€rCs elkyayl), -halogen, -nitro or «cyaao; n is an latter ranging from 0-4; &amp; is 0 or 1; and m is an integer of from 1 to about 20. The alkyl, alkenyl, and aikynyl groups, whether alone or as part of another group, can be optionally substituted.

[#2611 In some embedments, the «Ζ moieties are the same, in yet another emhodhoent, the *Z moieties are different mm ia cam aspect, Spam* units {»Yr} are represented by Formulae (X>(30l)r

<20 wherein Q is -CVCs alkyl, -Ca-Cg alkenyl, -Cr€$ aikynyl, -O^CrCg alkyl), -0-(CrQ alkenyl), -G-fCrCs aikynyl), • halogen, -slim or ~eya.no; and m is an integer ranging ton 0*4. The alkyl, alkenyl and alkysyl gamps, whether alone or as part of another group, can he optionally substituted,

[0263] In a group of selected embodimeafc, the conjugates of Formula (i) and (M) are:

wherein w and y are each. 0,1 or 2:

wherein w and y are each 0;

wherein A* WWi Yy* Z and Ab have the meanings provided above.

The variable Z in femmla (I) is a therapeutic agent or label The therapeutic agent can be any agent capable of exerting a desired biological effect. In. some embodiments, the therapeutic agent sensitizes the eel! to a second therapeutic modality, e.g., a ehemoifeeropmtle agent, radiation therapy, ismrunoiher&amp;py.

[§2hSJ In some embodiments, the therapeutic agent is a cytostatic or cytotoxic? agent Examples include, without limitation, anbmetaholites (e,g,, azatiuojxine* 6* mereaptepmne, bdfeioguamne, fludarafeine, peotostahn, ekuinhioe, 5-iteouracal (5FU), fbxuridmo (FXJDR), cytosiae assbiaoside (cy&amp;rabiae), nieihotfsxaie, trimethoprim, pyrimetbamme, pemetrexed); alkylating ageats (o.g., cyclophosphamide, mecMoiefhammo, uramestine, meiphaimi, chlorambucil, thiotepa/cMorambacii, ifeslamide, cmxmsiim, imnusime, strepiozeein, feuseifan, dibronmniannitol, eisptetio., carboplatin, nedaplalin., oxaKplatm, satmplatin, triplstiu ietrsoitmfe, procarbazine, altretamiae, dacsrbazme, miteiforaide, tamozoloimde); ffiihmeydines (e.g, danuorublcin, doxorubicin, epirabicm, idsrabidn, tuirabiem); antibiotics (e.g,s dacdaomyem, bleomycin, mifemnyein, sathramyem, srieptozotocin, graaitcidin D* mitomycins (e.g., mitomycin €), dmwarmydns {e„g.s €0-1065), cnlieiieamieins); antimitotic agents (mduding, e.g.5 mayteashtoids, aiaistatam, dcdasMins, cryptophyclns, vinca alkaloids (e.g., vincristine, vinblastine, vmdesine, vinoreiMae), taxanes (e.g., paclitaxel, doeetaxel, or a novel teas® (see, ag., ktemationa! Patent.PublicationNo. WO 01/38318, published May 31,2001)), and coldddnes; topolsomerase iaMbitors (e.g.s irinoteean, topotecan, amsscrine, etoposide, teaipoad®» mitoxasttorte); and proteasome inhibitors (e.g.5 peptidyi bororuc adds). M&amp;y&amp;msmo'id mmrnnocoHjugmes [02¼) la some embodiments, fee therapeutic agent is a maytamiaoid.

Maytansiamd compounds and methods for their conjugation to antibodies are described, for example, in Chari etui, CmcerRes^ 52:127-131 (1992); Widdison etal.,/. Med, Chem. 49:4392-4408 (2006)' aud U.S. Patent Nos. 5,208,020 and 6,333,410. Examples of maytassmoids include mayiaasine analogues having a modified ammaife ring (e.g., €~19~ deebioroj €~20~demethoxy> C-20-acyloxy) and those having rmxllfieaiions at other positions (e.g., C-9-CH, C-l 4-aIkoxymethyl, C~14-hydmxymethyl or acyloxymethyi, ΟΙ 5*hydi»xy/aey!oxy, €-15-mettioxy, €-18~N-demetbyi, 4,5-deoxy), In certain embodiments, the maytansmoid is N2’-deacety'hN3'44-'atereapto4maopeatyl)mayi8nsine (DM3), N^-d«ac^tyl“N^34am»piO“l“Oxopiopyl)“irmyt8nsinLe (DM1), or N"~deaa4yl-N^~(4*m.eTcupto^~metbyi*l-oxopen.ty!.)mayiaiisine(DM4).

[0267) Maytansinoid eomponads that comprise a sulfhydryl group can be coupled to antibodies using a hsterofei&amp;actional linker feat is connected to the maytansinoid compound by way of a thiodher or disulfide linkage. In some such embodiments, the linker is coupled to att amino group on the antibody (e.g., a terminal amino gmup or the epsilon amino group of a lysine residue. In some embodiments, the hetembifeuetbnal

Hate that is used, to couple a aiaytaas&amp;oid compounds to m antibody is N-sucdnirnMyl 3"C2“|?jrMyMithio)pR>pnosate (also known as N-sucehmmdyi 4-(2-pyridyidithio)peotas0at»i or SPP). 4~suedmt»ii^©^ai!myl~2~iteb.yl~2«^-pyrid>4ditli.io)«lo!u«ae (SMPT), N~$ueciaimidyl. 4-(.¥”nMofmld0iseihyi]cyclohes.ane-I - succMmMyi 4-(2~pyTidyldit!iio)butyrate (SPDB), 2-mmoiMolane., iff S-aeelyismeame |Θ2681 In certain embodiments, the iramunocenjugate of formula (I) is (formula ¢1-4¾ or Ab-(SPDB-DM4).w (formula (/-¾ wherein Ab is an anti-OCC antibody molecule m described herein,, and m has foe values and preferred values described above for formula (I).

(0260} In some embodnsmts, the variable Ab in formula (1¾ (1¾ or (/-3) is aa antibody molecule with features summarised in Tables i to 6, In certain embodiments, the variable Ab is a $F9 antibody molecule or an Abx-229 antibody molecule, (1270] In some embodiments, {fee variable m in formula (/-1), (/¾ or (/-3) ranges : from about 1 to about 10, from abou t 3 to about 7, or from about 3 to about 5. I®271] la certain particular embodtiKnis, the invention relates to an immursnconjugate of formula (/-/), (/-/), or (1¾ wherein Ab is a 5F9 antibody raolecufe and m is about 4,

Dofastatin and aunsiatin immunoconjugates (0272] In some other embodiments the therapeutic agent is a Mastatm. Is some : embodiments, the therapeutic agent is an aurisiatiu, such as auristaim E (also blown i». the art. as a derivative of dolastatia-10) or a derivative thereof In some embodiments, the therapeutic agent is a compound selected from compounds of formulae p/ii)-(,¥H^), or a pharmaceutically acceptable salt form thereof:

102731 Auristatm compounds and methods for their conjugation to antibodies are described, for example, m Doionka et ai„ Natur* Bioteek, 21:778-784 (2.003); Hambldt el al, Clin, Cancer Res,t 10; 7063-7070 (2004); Carter and Sender, Cancer J,, 14 154-169 (2008); US. Patent Nrn 7,498,298,7,091,186,6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414; U.S, Patent Publication Nos, 20890010945, 20060074008, 20080300192,20058009751,20050238649, and 20030083236; and International Patent

Publication 04/0105)57 and. WO 02/088172, each ofwhieh is mcorpofated by tej^ease here®» in fts. entirety msd for all f ixzpmm. 10274] Ths S sad a kete acid, Pot example, aaristltm E can be reacted with pamacetyl beaaole acid or bffisoylv&amp;lme acid to produce AEB and AEYB, respectively. Other typical aurisfifoas iuefode pheaydeaediaatme (AFP; monofitfithyl aifostatin E (MM A!; (MM)% and tBoarwefoyl anristatk P (M&amp;4AFj: (MM}% [0275] Aoristatifis have been shown to interfere with microtubule dynamics &amp;nd nuefcar aad cellular division aad have antleancer activity, Ainfotatma lor use in die prc-sem iaveatiefi bifid ifibalin asd r»u eserta eyteteic or cytotfode effort os a GCC~ expressing cell line, Methods ^tusd^-;a cofflpouod triads tubulin are knowm la the art. 8¾¾ for 20¾ '78,4200-4397; Ilafoel fit al, Moiecular Phtimaeofagg, 1995 47; 065-976; sad Haoiei fit si* TheJmmai gf Βίό$®0€&amp;ί<$&amp;Μίφγ, 1990265:2¾. 17141-17149, Porparpos^ ofthep^oatiavendofi, the relative affinity of s oompotmd to tabula cw.be determified, Some preferred aunstadfis c^^'jase^iifiyakwbtftd-dibfiiM with as affinity ranging from. 10-fold lower f weaker affinity} than the binding sffifojty of MMAE fo fohoiia fb 10-fold, 20-fold or dvea 100-foldhigher (higher affinity} burn the binding affinity of MMA.E to tubulin. 102761 the^am a.-»utate:of diffeaMsssep, kahwn foal ca»%:ase$ for determining whhfoer' aa fifiiisfoda or resultant foifin«<>coa|tig8fo exerts a cytostatic or eytotoMc effect os a desired cell lias, Per example* the cytotoxic or eyfostaik artifoty of M:'*ppia8c»»jagke. can be measured by: exposing msunmaliaa cells expressmg a target ^o^:of^'fosasW3«x*|fi^fo ifi a cell efilfore tsedium; culturing fo.fi cells for a period from about 6 loam to about 5 days; and measafog cell vkMlily. Ceilfoased in ϊ0ρ assays can he fised to measure viability (proHfemrion), apoptosis (caspase activation.} of the iramfiaocofil^ate, |0277| for drtermimng whether an immnnoconjagate exerts a cytostatic effect;, a t%foidinc iacorporaMoa assay maybe used. For sptnple, cancer cells expressing a target sptigesMa density of 5,000celWwellofa 96-well plated can be oultoted for a ?2~hour period and exposed to 0,5 gCi of fehymidiae during the find 0 horns of the 72-hour period. The IficorpOiarioa 0f%»&amp;yfiridine into cells of the culture is measured in the presence and absence of the itamoaoeofijfigate. can be measured, Necrosis is typify accompanied by Increased permeability of the typically characterised by membrane blehbiug, condensation of cytoplasm, and the activation of endogenous endonucleases, Detemrimtion of any of these effects on cancer cells indicates that m hnnamoeOTj agate is useful in the treatment of cancers, ff 279J Cd! viability can be .measured by determining in a cell the uptake of a dye such as neutral ted, trypan Mue, or ALAMAH™ bine (see, e.g,, Page ei al.} 1993, Mil J,

Omxibgy 3:473-476). In such aa assay, the cells are incubated in media containing the dye, die cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spcctephommehicaliy. The protdn-binding dye sttiforhodaniine B (SEB) can also be used to measure cytotoxicity (Skehan d,t 1999, X Nuti Concur-Inst 82:1107« 12). f#288J Alternatively, a teimnoHnta salt, such m MIT or WST, is used in a quantitative colorimetric assay for mammalian cell survival and ptxdifemfioa by detecting living, but not dead, ceils (sees e.g., Mosmaan, 1983, J. Immunol. Methods 65:55-63).

|0281 J Apoptosis can be quantitated by measuring, for example, SNA fragmentation. Commercial pliofemetric methods lor the quantitative in vitro deimninationof DMA fragmentation are available, Examples of such assays,, including :; TIJMEL (which detects inootpomiron of labeled nucleotides m fragmented DMA) and BLSSA-based assays, are described to Bioch&amp;mica, f999, no, 2,. pp. 34-37 (Roche Molecular Bioehemieals), {«282] Apoptosis can also be determined by measuring morphological changes in a cell For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (eg,, a .%omsoeni dye such as, for example, acridine orange or etMdium bromide). A method fbr measuring apoptoiic cell number 1ms been described by Duke and Cohen, CasmafcProtocols in Immunology (Collgan et al eds., 1992, pp. 3.17,.1-3.!7.16). Cells also cm be labeled with a DNA dye (c.g,, acridine orange, ethidium bromide, or propidiuoi iodide) and the cells observed for eln-omatla condensation. and marginatioa along the inner nuclear membrane. Other morphologieal changes that csa.be measured to detmarne apoptosis include, eg,, cytoplasmic condensation, increased membrane blobbing, and cellular shrinkage. 10283] The presence of apoptotic cells cm be measured m both the attached and “floating1* compartments of the cultures. For example. Both compartments can be collected by removing die supernatant, tryp&amp;midsg the attached cells, combkmg the preparations following a centrifugation wash step (kg,, 18 minutes at 2000 rpm), arid detectiug apoptosis (e.g., by measuring DMA flagmen! ah on). (See, eg., fi&amp;aza et d., 1995 , Cancer Research 55:3110-16), C$2841 The effects of tsmtmroeonjugates can fee tested or validated m animal models, A number of established animal models of carreers are known fo tire skilled artisan, say of which can fee used to assay the efficacy of an immunocengigate, Non-limiting examples of soeb. models are deserifced iffm. Moreover, small animal models fo examine the m vivo efficacies of immunoconfugates can fee created fey implanting human tumor cell fines into appropriate immuimdeficiiaittodcnt strains, «.gv, athymic nude mice or SCID mice. 10285] la some mhodiments, the variable -Z in formula (i) is an anristatia moiety of the formula (AvI) or formula (3T-R):

wherein, independently at each location: the wavy line indicates a bond; R* is -C{ -(¾)) alkyl, -Ck-C® alkenyl, or -Cj-Cao alkyny I; R3 is -H, -Ct-Caa alhyl, -Cr€» alkenyl, -Cj-Cjo alkynyl, carhoeychs, -Cj-Cbs alkyleae (carbocycle), -QrCjs alkenylene(catb©eycle>, -CrC® alkynyleitefcarfmcycle), -aryl, -Ci-Csti aikylcn.e(aryl)s -C2-Cb alkmyieue(&amp;ryl), -Cj-Cjc alkynykoe(aryi), -hetemcycle, -Cj-Cao alkylene(hetet»cycle), -Ca-C® alkcaybneiheterocyde},, or -C2-C20 alkyrrylene(hetemcyele): R4 is «Η, “CrCas alkyl, «0*«€κί alkenyl, *€&amp;€&amp; alfcynyi, carbocyde, -R^-Qc d&amp;ytene (earboeyde), -Cy-Ca) aikmy!ene(caritfiK^cle), -C2-C20 aikyB^eaeCearbocycIc), -atyl, * C1-C20 alkylen^atyl), -Cr-C^ aikenyteae(aiyl)s «Cr€® dkynylme(&amp;ryl), -heterocyde, "Ci-Cse alkylme(hetmoyci®)? «CrC» aifeenyimeitoetocyele), or -Ca~€ae alkyTsyleae(lieraxiCycle);

Rs is -H or *Cj-Ce alkyl; or R4 asd &amp;5 joisfly fom? a carbocyoKc ring am! have the formula ~{CRaRb)s" whereia R* md. Ra are independently ~H, -Ci-C® alkyl, -Cr€® alkenyl, -CrCjo aikyayl, or -earboeycle and &amp; is 2, 3,4,5 or 6, R4 is -H, -OrC® alkyl, -CrC® alkenyl, or -Cs-C® alfcyayl; R7 is »H, -Cj-C® alkyl, -Ca-Q® alkenyl, ~C2-Cb alkynyl, -earbecyde, -€j-C® alky lose (carbocyde), -CrC® alkCTyka^caxbocyote), -Cs-C® alkyiiylai^arboeycle), -aryl» -Ci-Cae &amp;lkylme(aryl), -CrC® aikeayleaeCaryl), ~Cr€® aIS^yie»e(ajyl)> keterocycla, -Ci-C® idkylme(heteocyole), -CrC® aikeayleaellieteocyde), or -CrC® d kynyl enc(h etsrocy cle); each R$ is independently ~H, -OH, -Cj-C® alkyl, -CrC® alkenyl, -Ca-C® aikyayl, -0»(Cr C® alky!), ~G~(CrC® alkenyl), ~0-{CrC® aikyayl), or -carbocyde;

Is is -H, -Ci-C® alkyl, -CrC® alkenyl, or ~€h«C® dkynyl; R19 is -aryl, -fecterocycie, or -earboeycle; R29 is "H, -CrC® alkyl, -€a*C® alkenyl, -Ca-C® alkynyl, -earboeycle, -0-(CrC® alkyl), -0"(Cr€® alkenyl), -0»(CrC® aikyayl), or QRW wherein R1* is -0, a hydroxyl protecting group, or a direct bond where OR1® represents *-0; R1S is ~I1 ~GrC;«> alkyl, -CrC® alkenyl, or -CrC® alkynyl, -aryl, -hetcrocyele, or -carboeyde; R18 is -aryl or -heierocydc; G is -0-, -S-, -NR-, or wherein Rn is -Cj-C® alkyl, -CrC® alkenyl, -C3-C20 aikyayl;

Ru is -H, -Cj-C® alkyl ~CrC® alkenyl, -CrC® aikyayl -aryl, -heterocycle, -(R^Oj^R14, or -(RB0)s-CH(Rls)a; s is an integer ranging from 0-1880; R13 is -€j«C® alkyleae, -CrC® alkenytene, or -Ca~C® a&amp;yayleae; &amp;14 is -H, -CrC® alkyl, -CrC® alkenyl, or -CrC® aikyayl; each occurrence ofRls is independently -Η, «€00Η, H£H2>N(R.le)2, -{Cifc)rSOA "'{CHijrSOi-Oi -C20 alkyl, —(O'lsVSOs-Ci-Ciesi^sylj or alkyuyi; each ocettnres.ce of M5® Is indepetdeaily ~H, -CrO® alkyl, -CrC» alkeiQd» -QrCus alkynyl or~(CB?}r€OGH; and t is as integer ranging from 0 io 6; wherein said alkyl, alkenyl, alkynyl, aikylcns, alkraylene, aikynyklcne, aryl, carboeyie, and hetereeyele radicals, whether alone or ^ part of another group, ate optionally substituted, |0286j Auristatins of the formula Q&amp;A) include those wherein said alkyl, alkeuyls alkynyl, alkyieue, alkenyleae, alkynyklene, aryl, carboeyle, and heteroeycle isdicals are uusubstiruied. P2S?1 Auristatins of the fomiula (X~J) mclude those wherein the groups of R, &amp; » R4, Rs, R\ R7, R® and R5' are uusubstteied and the groups of Rl?, R2eand R21 am optionally substituted as described herein.

[02SSJ Auristatins of the formula {X~A) include those wherein R2 is -€r€« alkyl; I*, R4 and &amp;7 are independently selected from -H, -Ci-Cso alkyl, -Ca-Qo alkenyl? -Ca-Qjc alkynyl, rnonoeycHc CrQ earbocycle, ·€»-β» dkyienefmonocydlc CrQ carbocyde), -tVQo &amp;lkeuyl.ene(mtmocyo!ie Cs-Q carbocycie), -QrC» alkynylme(moiiocydic Q-Q carbocyde), -CtrQo and, -Cj-C;® dkyleueCCrQo »yl), ~ CrC» alkenyiene(CrCjf) aryl), -CrC® alkynylene(CV€ie aiyl)» -heterocyde, -01-(¾ afkyiene(kelerocyde), -CrCao dkeuyleneCheiew&amp;rycIe}, or -QrCso ajOkyuyIene(hcierGcyde); wherein said alkyl, alkenyl, aikynyi, alkylcae, alkeny lene, alkynylme, carbocyde, aryl, aud heterocyde radicals are optionally substituted; R® is -hydrogen; R*is-Q~Cs alkyl; each K* is Independently selected from -OH, -0-(CrCao alkyl), -0<CaC» alkenyl), or -0-(QrQ» alkyayl) wherein said alkyl, alkenyl, ami alkynyl radicals ape optionally substituted; R* Is -hydrogen, or -Ci~€s alkyl; R*9 is optionally substituted, phenyl; R39 $$ OR18; wherem R*8 is H, a hydroxyl protecting gronp, or a direct bond where 0RiS represents K); &amp;** k selected from -Β» -ChC» alkyl, *G?€® alkenyl, -QrOo alkyayl, ar -earboeyeie; wherein said alkyl, altatyl, aikynyl, and carboeydo meticals are optionally sabstsfeted; ar &amp; plwrmaeeotieally acceptable saltibrol thereof, fiBif | AuriMaims of the formtik <JMt) kchide those wherein R2 Is methyl; R* is -¾ -Cr-Cg alkyl, -Cg-Cs alkenyl, or -OrCg atkyayl, vYherein safe! alkyl, aikeny! and aikynyl radicals are optionally substituted; R4 is ~B, -CVCs alkyl, ~Cg~€g alkenyl -CrCg afkynyl, monocyclic CrCe earbocyclc, Alg-Ciui atyl, -CrCg alkyleneCQ'CA aryl), ~€-r€s alkeayksasCQ-Cja aryl), ~ €rC$ alkyrtylen^CrCio aryl), -€i-€&amp; sikylene (mottoeyelie CyCs cerboeyclc), ~Ch-€s alhenyiene {monocyclic CrQ cathocyde), ~€h~€g alfejmyleneiraoaoeydic Cs-Q eartacycle); wherein said alkyl, alkenyl, aikynyl, alkyfene, ahmnykme, alkynyiene, aryl, and earhoeycbmdic&amp;ls whether alone or as part of another gronp are optionally substituted; R5 Is Η; 114 is methyl;

Mv is «Cj-Cg alkyl, -Cg-Cg alkenyl or -Cs-Cg aikynyl; each Rs is melhoxy; R is -hydrogen or -CrC§ alkyl; ft*® is phenyl; jy!4 _ a «v R4 is OR ; wherein R · is *H* a hydroxyl potecting group, or a direct bond where OR1* represents ”0; K2* Is methyl; or a phamsaeeulkally acceptable salt fbmt thereof.

Autistatitis of the fonanta (2M) inelnde those wherein R2 is methyl; R3 Is H or Q-Ch alkyl; R4 m Cj-Ch alkyl; Rs is H; M® is methyl; R7 is isopropyl or see-butyl; R* k methoxy; R® is hydrogenor €1.4¾ alkyl; R*® is phenyl; R3® m OR1*; wherein Rts k H, a hydroxyl pmtectmg group, or a direct bond where OR18 represents »0; and R** Is methyl; or aphammceiiiically acceptable salt form thereof, |i2SI| Anristatlas of the formula (JM) include those wherein R2 is methyl Or Ci«C3 alkyl; Is is H or CrC3 alkyl; R4 is C}-Cs. alkyl; Us is H; R* is CrCs alkyl; R? is Cj-Cj -alkyl; R* is Ci«Cj alkoxy; R® is hydrogen or Cj-Cg alkyl; R** is phenyk R28 is OR18; wherein R*8 is Η» a frydmxyi protecting group, or a direst bond where GRiS represents =0; sad R21 is C1-C3 alkyl; or a phamiaceutiealiy acceptable salt form, thereof m%] Auristatim of the formula (X~8) include those wherein R2 is methyl;

Rf, R4, and R' are independently selected from -Η, -Ci-Cm alkyl* -Ca-Ca» alkenyl, *Q»C» alkynyl monocyclic Cr^g e&amp;rbocyele, -Cj-Qo alkyIeae(tmmocycHc· C3-C&amp; carhoeyde), -QrCao alkenyleaeCmonoeycEe C3-A carbocycie)* «C-t-Cga MkynykmeCmonoeyelie Cs-Cg carbecyde), -Cg-Cfo aryl, ««Cj-Cai alkyleneCGrCja aryt>„ -(4-C20 alkeaylsae-(Cs"C;o aryl), -CrCj© alkynyi®ie(Cg~Cio aryl), -heieKxyde, ~Ci~Cn alkylcne(hderaeyele)} -Cg-Css aikcnyieneCheterocyde), or -C2-C20 atkyn}dene{hetemeycle); wherein said alkyl, alkenyl, alkynyl, alkylenc, aikeuyte&amp;e, alkyaylene, earbocyle, aryl, and heterocyclic radicals whether alone or as part of another group am optionally substitu ted; R5 Is -H; R* is methyl; each Rs is methoxy; R* is ~H, -Ci-Cks alkyl, -CrCn alkenyl, or -CrCjo alkynyl; wherein said alkyl, alkenyl and alkynyl radical are optionally substituled;

Rw is optionally substituted aryl or optionally substituted hetemeye-ie; G is-CK -S-* -NH-* or *m\ wherein R18 is -C3~CKt alkyl, -CrC2s alkenyl or -CrC?,» alkynyl each of which is optionally substituted;

RiJ is “Hs -Cj-Chg alkyl, «C^-C^ alkenyl, -OrCso alkynyl, -aryl -heieroeyeie, -(R^OVR14, or"(R?3OX-~CH(Ri5}2s wliemin said alkyl, alkenyl, alkynyl aryl, and hdnroeyele radicals are optionally substituted; # is an integer ranging from. 04060;

Rb is -CrCao alkylme, -C2-C29 alkeaylene, or -Q-Cj© alkyaykne* each of which is optionally substituted; R“ is ~H, -Ci-C» alkyl ~€rCn alkenyl, or -Cj-Cjo alkynyl wherein sard alkyl, alkenyl and alkynyl radicals are optionally substituted; each occurrence of R3S is independently -H, -COCO, Η£Η;ξ)τΝ(Κ.Ι6)&amp; “{CHjV SO3H, ~CCHa)rSO;rCrC:20 alkyl ^O^Os-CrC» alkenyl, or ~(CH2)rSOr€rCM alkynyl wherein said alkyl, alkenyl and alkynyl radicals are optionally substituted; each occemsaea of R** is ladepeadeMy ~H? ^-¾ alkyl* ~€rCm alkenyl* ~Cr C» ahcyayl or ^Ofi$)rCOOH wherein said alkyl, alkenyl and alkynyl radicals ai§ optionally substitute^ # is as integer ranging .Item 0 te 6; or a phanmoeatteally acceptable salt Sam thereof f0293f la certain of these embodiments* Rm is optlojiaily sabsliiaied phenyl; : |0294j Auristati&amp;s of the formula (X~M)' include those whemln the groups of Ra, Rs* R* Rs, R* %?, R*,. and Rs are unsuhstiMed as! the groups of Ri0 and R.u are as described herein. f§29 SJ Aoristatins of the formula 0~B) include those wherein said alkyl, alkenyl, alkyayl* slkytae* alkeayleae, alkynyklens, aryi* eebocyle, and keteroeycle radicals sic

Msnbstiiuied. , !0290| Aurisfatms ofthe formula (E~M) include those wherein. , R3 is CV-C3 alkyl; R* is H or C{-C3. alkyl; I4 is C5C5 alkyl; U$ h B; Is is Ci~t3 alkyl; R7 is Cj-Cj alkyl; R*is Ci-Ca aikoxy; R7 Is hydrogen or €ι«€* alkyl; %» is optionally substitutedphenyl; G is G*-S* or NH; aadRn k as defined herein; or a pharmaceutical 1y acceptable salt form thereof, Ι929Π Auristafins of the formula 0f«J) include those wherein . R* is methyl; R3 is H or CrCs alkyl; R4 is Ci-Qs alkyi; R8 is H; 1*is methyl; R? is isopropyl or sec-butyl; is methoxy; R8 is hydrogen or Ci~Ca alkyl;

Rs® is optionally substitute phenyl; G is Os % or MH; and Ra is as defined herein; or a pharmaceutically acceptable salt form thereof 102981 Aurisiatinsof the formula {X~E} include those wherek R3 is methyl; R3 is H or Ci-Cj alkyi; R4 is Cj-€s alkyl; R8 is H; R* is methyl; R’’ is isopropyl or sec-butyl; R8 ismethoxy; R8 is hydrogen or CrCs alkyl; R*8 is phenyl; md G Is 0 or Ml and Rp Is .as defined herein, preferably hydrogen; or a pharmaceutically acceptable salt form, thereof. 102091 Anrlstafins of the formula (X~%) Include those wherein R* is CrC3 alkyl; R* Is H or e5~Cs alkyl; R4 is €-¾ alkyl; Is is H; R* is CrC3 alkyl; R7 is C1-C5 alkyl; R8 is Ci-Cj alkoxy; R* is hydrogen or CrCs alkyl; R*® is phenyl; and G is 0 or NH sad R11 is as dsfissd kerem, preferably hydrogen; or a phamiaceiaicatly acceptable salt fbtm thereof [0300] Auristafias of the formula Q&amp;A) or (X-B) include those wherein R'\ R4 and R7 are mdepmfestly isopropyl or sec-butyl and R5 is 41 la aa exemplary embodiment, R" and R are each isopropyl, R" Is H, and BJ Is sec4>t^L lire remainder of the sobstitnenis are as defined herein, [ill ] Audsfetms of the formula (X~A) or (X~M) include those wherein R2 mid R6 are each methyl, and R9 is H. The remainder of the substituents are as defined herein.

[0302J Anristatins of fee tormina (X~A) or (X~B) include those wherein each occurrence of R1 is ~OC%. The remainder of the substituents are as defined herein.

[#303| Anristatins of file formula (Χ-Λ) or (X’-S) include those wherein RJ and R4 are each isopropyl, R2 and R4 are each methyl, Rsis H, R7 is sec-bntyl, each occurrence of R8 is -OCHs, Md R* is H. The remainder of fixe substituents are as defined herein.

[0304] Auristatms of the formula (XB) Include those wherein G is -G- or -NHk

The remainder of the snbstteats are as defined herein. fH305j Anristatins of the formula (X«Jfr) indude those wherein Ri0 is aryi. The remainder of fee substituents are as defined herein.

[030fij Anristaiins of fee fomaila (X-B) include fees© where Riif is «phenyl. The remainder of the substituents are as defined herein, · [0307] Anristatins of fee fonsuta (A«l?3inci«de those wherein G is -O»* and Ra is B, methyl or t-butyi The remainder of the substituents are as defined hems.

[030$] Aurisiailns of fee formula (X~B) include those wherem, when G is ~MH, Ru is “(Ri30).rCB(Rls)2, wherein RiS is «(CH^bp.18}*» and Ri4 is -Ct-C* alkyl or ~(CH2)r COOH, The remainder of the substituents are as defined herein.

[0300j Anristatins of the formula (X~B) include those wherein when G is ~NH, R5 * is “iRi30)rCHCR.35)2? wherein R15 is H or “(CH^rSOrfeL The remainder of the snbstiteents are as defined herein. {9310J In preferred embodiments of the irsnumoconjugatus of formula (11), when Z is aa auristatia molecnie of'&amp;mula QC~A% w is an integer ranging from 1 to 12, preferably 2 to 12, y is 1 or 2, and a is preferably 1, |0511! la some embodiments of the imm»iso<XHy «gates of firamk (1% wkaa % is as surisiatia molecule of formate (3^8), a is 1 andw and y are 0. f t31.2f lliastmtive therafjeode sgeate (-¾) iadade those haying the Mlowing structures;

1031.3} In one aspect, bydtopMEc groups, such as hut sot ligated to toethyiene glycol esters (TEG) can be attached te the auristatm molecule of fermuia ζ&amp;Μ) at Ra. Without being bound by theory, the hydrophilic groups assist hi the internalization and non-agglomeration of the therapeutic agent.

[03143 some embodiments, the foarapeuiic agent is sot ΤΖΓ-4027. la some «ikxfiments, the therapeutic agent is not amistatm B* dolastatiu 10, or aaristatin PE, [0315J In. some emliodimeats, the auristatin molecule is linked to a cysteine moiety «1 the antibody molecule by way of a linker comaiaisg a maldmide moiety, e,g.f a malemddoeaproyl moiety.

[03X63 In some embodiments, the auristatinmolecule is coaled to die antibody molecule using a hetorobifsneifouai linker that is connected to a hydroxyl group on the auristatio molecule, In some such embodiments, the linker comprises a hydraxone, In some embodmm% hie linker is a hydrazone compound formed by reaction of maldmidocaproylbydrazMe and a ketnoarfeoxylic acid, e.g., S-benroylvaleric add. In particular embodimmts, the tinker is (2)--6^2-(6-(2,5410^0-2,5-^1^1110-111^0111-1-)4)-aex;moyi)hydmKono)"6'phetiylhexanoic add, |§31?1 I». some other msbodrmcsts the atmstadn molecule is coupled to the antibody using a heterobifuuctioaal linker that is cottaeded to a monomethy! amino group oa the auristatin molecule. In some cmfcodimeut^ the linker comprises a cleavable moiety, e.g., a peptide moiety, and aself~mmoMive/?~aimaob«U2;yli«rbamate spacer, Eserupiary tinkers include maleimidocapmyl (me), mddrmdo^»yl-L-pbmiylatarmie*-L~ !yske»|>aminoberizyicatbmnate, fuuimaleimidocaproyl~I,~va1iue”L-dtmlliaeTp-am too bonjy tear hamate (ve), [03I8J In certain emliodimmts, the imffiuaoeonjugate of formula (i) Is characterised by die formula AX^vc-MMAF)* (formula (f~4)); Ab-(vo-MMAE)*, (formula (1-5)3; AMme-MMAE)* (formula (M>); or Ab-(me-MMAFU (formula (1-% wherein Ah is an autI-G€€ antibody molecule as desmbed herein, S is a sulfur atom of the antibody, and m has the values and preferred values described above for formula (I). In certain emhodinrents, m is an integer fern .1 to about 5.

[¢301 in some embodiments, the variable Ab is femiula (JMX (1-5),, {/»$), or (/- 7) is m antibody molecule with features sutnmffoKed is Tables 1 to 6, In certain embodiments, the variable Ab is a SF9 antibody imlmtle or m Ahx~229 antibody molecule. f®3201 I» some embodiments, the variable w in formula (jM}, (M), (1-3% or (/-7) mages from about 2 to about 10, from about 6 to stent 8, or Item about 4 to about 6, pS21] In certain particular embodiments, the invention .relates to as immtmooonpgate of formula (/-#}, {!-$% (/~d), or (/-?), wherein Ab is a 5F9 antibody molecule and m is about 4. p322| The immuttoeonjugaies dkelosed torn can be i&amp;ed for 100¾¾.¾¾ a given biological response. The thempeutio agoutis vat to be construed as limited to classical chemical therapeutic agents. For example, tbs therapeutic agent may be a nucleic acid, pmtein, or polypeptide possessing a desired biological activity. For example, the antibody molecule can be conjugated to m antisense molecule, mi siANA molecule, sfaKMA molecule or mi&amp;NA molecule that css interfere with. exjwessioa of a gene, thereby |aod«ciaga desired biological effect 18323) Proteins mid j&amp;ilypep&amp;tes that can be conjugated. to the anybody molecules of the iaveatkm iadu.d e, for example» toxins and oompmenrs thereof such as akin, abrin A chain, «da, riels A chain, naodeecin, modeccm A chain, alpha~saretn, exoioxin A (from Pseudammas mnigm>sa% PE38 (truncated pseudomonas exoioxin), geionk, diphtheria toxin, diphtheria toxin A fragment, certain Aleurites fimin proteins, certain BUmtktts aeyaphpgus proteins (e,g.s diaotlnn 30 and diaalhin 32), certain Phytolacca Americana pmtefris (e.g., PAP, ΡΑΡΙΪ, and FAP-S), certain Sap&amp;nmiaqfflcmlm proteins (e.g., saporin 6), Momordica dxtrmim inhibitor, ourem, ctotttt» ntifogillin, restrietoein, phenoniycin, and enomycm; proteins to engage the immune system at the tumor or induce an effector function at the tumor, such as tumor necrosis factor, interferon, nerve growth factor, platelet derived growth factor, and tissue plasminogen activator; and biological response modifiers such as, for example, cytokines and lymphokiaes (e.g., feterieu.km-1 (“It-l>f), luteriettkin~2 (“IL-i**}» interleukin-ti (‘IL-fF), granulocyte macropbase colony stimulating factor (**GM«CSF*X and granulocyte colony stinndatmg factor {“G-CSF”», and other growth factors, |1324j The antibodies of the* invention can also be conjugated or fused to viral surface proteins present on viral particles. For example, a single-chain aidi-GCC antibody of tine present, invention could be fused (e.g., to form a fusion protein) to a viral surface protein. Alternatively, a whole aati-GCC antibody of the present invention, or a fragment thereof could be chemically conjugated (e.g., via a chemical linker) to a viral surface protein. Preferably, the virus is one that feses with eadocytie membranes, e.g., an infiuestxa virus, such that the vims is intemallxed along with die anti-GCC antibody and thereby infects GCC-expxessiog cells. The vims can be genetically engineered as a cellular toxin. For example, the vims could express or induce the expression of genes that are toxic to cells, e.g., cell death promoting genes. Preferably, such viruses would be incapable of viral replication. (0325) An anti-GCC antibody molecule described herein can also be conjugated to a ptodmg or prodtug activator, in a method to kill or suppress tumor cells, a first anti-GCC antibody molecule of the invention is conjugated, with a prodrug that is activated only when la close proximity with, a prodrug activator, The prodrag activator is coafugated with a second antibody molecule, preferably one that binds to a nomcompeting she eo the GCC molecule. Whether two antibodies bind. to competing or non-competing binding sites can be determined by conventional competitive binding assays.

Drug-predrug pahs suitable for use in the practice of the preseat invention are described k Blakely el at, "ZB2767, an Improved System for Antibody- directed Bmyrae Prodrug Therapy That Results in Tumor Regressions m Colorectal Tumor Xenografts,* (1996) CmcerRmmrch 56:3287-3292.

[β326| Therapeutically active radioisotopes can also be coupled to aati-GCC antibodies, or antigen binding fragments, or derivatives thereof. Radioactive isotopes can be used in diagnostic or therapeutic applications. Radioactive isotopes that can be coupled to the anti»GC€ antibodies include, but are not limited to α«, β», or y-emiiters, or β- and y* emiitem. See, e,g,, SJE, Order, "Analysis, Results, and .Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” Mmmkmal Antibodies for Cancer Deteetim and Thew$$% R.W. Baldwin et ai (eds.), pp 303-316 (Academic Press 1985. Such radioactive isotopes include, bin are not limited to copper (**Cu}, iodine (?3i| 0r yttrium <**¥), lutetmra (mLa), actinium (^Ac), praseodymium, astatine {2iiAi), rhenium (i$6Re), bismnth (mBi or 2%i), indium (ΪΠΙη), technetium fsmTc), phosphorus (3¥), medium (mRh), suffer (35S), carbon (?4C), tritium (”H)S chromium (5iCr), chlorine (¾). cobalt f 'Co or ssCo), iron fsFe)s selenium (75Se), or gallium f7Oa). Radioisotopes useful as therapeutic agents include yttrium (i?iLu), actinium (^Ac), praseodymium, astatine (211 At), rhenium f%)s bismuth imBi or 2liRi}, and rhodium (*%h), Radioisotopes useful as labels, e.g., for me m diagnostics, include iodine (UII or mI), indium {ml»}, technetium f®mTc)5 phosphorus (3¾ carbon (uQt and tritium fli), or one or more of the therapeutic isolopes listed above.

[Θ3271 Ifediokmumotherapy (ΕΠ’) using antibodies labeled with. *3JI, ®Y, and i7iLu is under intense clinical investigation. Them are significant differences in the physical characteristics of these three nuclides and as a result, the choice ofradiosradidc can be important in order to deliver maximum radiation dose to the femur. The higher beta, energy particles of*°Y may be good for bulky tumors, but It may not be necessary for small tumors and especially bone metastases, The relatively low energy beta particles of ml are ideal, but in vivo dehalcgenaiioa of radioiodinaled molecules is am^or disadvantage kr internalizing antibody. In contrast, l' 'Lu has low·' energy beta particle with only 0,2-03 mm range m&amp; delivers much lower radiation dose is bone msmm compared to 98Y, la addition„ dee to longer physical half-life (eoo^paeed is mY% the tumor residence times are higher. As a result, higher activities (moremCi amounts) of ;mLu labeled agents eats be administered with comparatively less radiation dose to marrow. There have bee® several clinical studies immsiigafesg the use of mLu labeled antibodies in &amp;efreatmsiit of vanotts cancers. (Mulligan T et at, Clin CancerMe&amp;\. 1: 1447-1454(1995); Meredith SF, et si JNudMed 37:1491-1496(199% Alvares; ED, et al Gynecologic Oncology 65:94-101(1997». 1032SJ Useful detectable agents with which art antibody or an antibody jrortion of the invention may be derivatixed (or labeled) include fluorescent compounds, various eaaymes, prosthetic pongs, inmhieromi materials, Hotersinescari materials, fluorescent emitting metal atoms, e.g., europium (Eu), and other anihamdes, and radioactive materials (described above). Exemplary fluorescent detectable agents include fluorescein, fluoresce® isothioeyanais, rhodaroxne, S-dimethylamme-l-napthabaesulfonyl chloride, phycoeiythrin and the like. An. antibody may also be dmvafized with detectable eircymes, such as alkaline phosphatase, horseradish peroxidase, p-galactosldase, acetylcholmesterase, glucose oxidase and the like. When an antibody is derivatised with a detectable enzyme, it is detected by adding additional reagents that die eazyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and. di aminobenridine leads to a colored reaction product, which m detectable. An antibody may also be derivatmed with a prosthetic group (e.g., steptevidm/biotia and avidin/biotia), For example, an antibody may be derivatked with biotin, and detected through indirect measurement of avidin or sfeeptevidin Hading, Exarnpl.es of sui table fluorescent materials include umbelliferone;, fluorescein, fluorescein imithxocyaaate, Aodamiae, dieMoroniazlnyiamine fluorescent, danay! chloride or pfeycoeryJhrm; an example of a luminescent material includes liuninol; and examples of biohmrurescent materials include Inelfemse, luetferm, and aequorin. ffl329J In another aspect, the raveutio» features coropositioas, e.g., pharmaceutically acceptable compositions, which include an anti~G€€ antibody molecule or irmsunoconjugate thereof as described herein, formulated together with a pfeanaaceutieally acceptable earner, In embodiments, the aati-GCC antibody molecule is Gas with emnptery features smumarixed in Tables 1 and 2. p330f As used herein, ‘‘pharmaceutically acceptable carrier'5 iadtafes any mi all solvents, dispersion media, isotonic and absorption delaying agents, and tbs like that am physiologically eon^atihle. The earner can be.statable for intmveocaas, intramuscular, sabcotaieoas, parenteral, rectal, spinal or epidermal administration (e,g,, by injection or infusion), The pharmaoentical composition can include one or more additional excipients, e,g„ salts, buffers, tonicity moditiers, lyoprotectants, nemoalc detergeuls, surfactants, md preservatives, f 0331 | The compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g,, injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Some typical compositions are m the fern of injectable or infusible solution, intended for parenteral administration (e,g., htttavenous, subcutaneous, intraperitoneal, intmmuscukr). In some embodiments, the antibody is administered by intravenons infusion or injection. In other embodiments, the antibody is administered by intramuscular or subcutaneous injection. (0332} The phrases 5<patcnteml administration” md “administered parentemlly” as used herein means modes of administration other than enteral and topical administration, usually fey injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, mtraoapsnlar, iuhraorfeital, mfracardiae, mtedsrmai, intmperhoueal, transtracheal, subcutaneous, subcuticular, intraartiatiar, subcapsular, subarachnoid, mfraspinat, epidural and intrasternal injection and infusion. 103331 1» some embodiments, foe pharmaceutical composition is sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, ntierocmulsion, dispersion, liposome, micro^shere, or other ordered structure suitable to high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (Is,, antibody or antibody portion) in tire required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, Mowed by sterilisation, e.g., by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic the ease of sterile powders for the preparation of sterile injectable solutions, the provided methods of preparsrioa a*» vacuum dbytsag and feene-drymg that yields a powder of the active ingredient plus say additional desired ingredient from a previously stedle-fQtesed solution thereof. The proper fluidity of a solution can be maintained, for example* by the use of a coating such as lecithin, by the maiatenunce of the required particle ms in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by inchidimg in the GomptBlhon an agent that delays absorption, for example, monosieatate salts and gelatin. 113341 The antibodies and antigen binding fragments of the present invention can be adnnmstesed by a variety of methods known in the art, although for many therapeutic applications, the ronte/mode of administration is intravenous injection or Infusion. As will . be appreciated by the skilled, artisan* the mute and/or mode of admmistadon will vary depending upon the desired results. In certain embodiments, the active compoaad may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, iransdermai patches* and such as ethylene vinyl acetate, polyanhydrides, polyglycolic add, collagen, poIyorthoesterSj and polylactic acid Many methods for the preparation o f such formations are patented or generally know» to those skilled in the art. See* e.g.%

Sustained and CcmPvUed Release Dmg Deiivety $p·.stmis, J.R, Robinson, e&amp;, Marcd Dekker, Inc., New York, 1978. |Θ335| la certain asbetohneats* an antM3CC antibody molecule or immrmoeoiyugate described herein may be orally administered, &amp;r example, with an mert diluent or an assimilable edible carrier, The compound (and other ingredients if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, buccal tablets, troches, capsules, eiixim. suspensions, syrups, wafers, md the like. To administer an antibody or an antibody fragment of tlie invention by other than parenteral administration, it may be accessary to coat tee «anpeusd with, or co-admmister the componnd wife, a material, to prevent its inactivation. (¢336] Therapeutic compositions can be administered with medical devices known.

In the art. For example, pharmaceutical preparations can be disposed witMn a. device, e.g.s an air- or liquid-tight cxmfemer, which, contains one or more dodges. Examples of delivery devices include, without limitation, vials, cannulas, needles, drip hags, and lines. The invention also provides methods of placing an antibody molecule or imimmoconjngafc described herein into such a device. |©33T| In some embodiments, fee invention provides an aaii-GCC antibody molecule or mununoeonjiigaie described herein, which is formulated tn a liposome composition, hi some emhodimmts, the liposome is coated with antibody molecule. la some such embodiments, die liposome is filled with a therapeutic agent, Lipoaomie delivery can allow for the delivery of an agent, e.g.s a therapeutic agent, that is not linked to the antibody. This approach can housed to deliver an agent, e.g., a therapeutic agent, that is not amenable to cross-iinMng to the antibody molecule or an agent, e,g„ a fherapentie agent, which is to be sequestered, or which contact with non-target cells should be minimised. In particular embodiments, the liposome is filled with a cytostatic or cytotoxic agent In certain particular emhodimeuts, fee feempentic agent is selected horn the group consisting of maytaasmoida, an auristatms, dolasiafiM, duocamiycius, cryptophyems, taxanes, DMA alkylating agents calicheamieins, and derivatives of the foregoing. In other embodiments, the liposome is filled with nucleic acid sequence comprising RNA interference molecules, e,g.? antisense molecules, slRNA, hsRNA or miRNA molecules, which are capable of diminishing G€C expression or the expression of another gene, e.g., an oncogene, in cells expressing GCC In some other embodiments, the liposome is coated or filled with an iornmnoconjugaie comprising an. and-GCC antibody Tnolecuie and a therapeutic agent or label. |®338J Dosage regimens are adjusted to px-ovide the optimum desired response (eg., a therapeidic response). For example, a single bolus may be administered, several divided doses may he administered over time or the dose may be pmportionaily reduced or increased as indicated by fee exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral expositions in dosage unit form for ease of admimstration md tmifermliy of dosage. 'The term “dosage unit form,” as used herein, refers to physically discrete units suited as unitary dosages for fee subjects to he treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect In association wife fee required pharmaceutical carrier. The specification for the dosage unit tens of fee invention axe dictated by and dimctly dependent on (a.) the unique characteristics of fee active compound and fee particular therapeutic effect to be achieved, an&amp;(b) the IfouMfons inherent m the art of impounding such aft active compound for the treatment of sensifivity to toditoduals.

[0339] An exemplary, aoa-limitmg range for a therapeutically or pmphyi&amp;otie&amp;lly effective amount of an antibody or an antigen binding fragment of the invention is 0.1-20 rrsg/kff . or 1-10 0¾¾¾. It to to be acted that dosage values may wsy with, the type and severity of the conditio» to be alleviated. It to to be forte tmtetood that for any particular subject, specific dosage regimens should he adjusted over time according to the individual need and the professional judgment of foe person administering or supervising foe administration of the compositions, and that dosage ranges 8¾ forth ham are exemplary only and are oof intended to limit the scope or practice of the claimed composition, [03#j The pharmaoendeal compositions of foe invention may include a 'therapeutically effective" amount of an antibody or an antigen binding fragment of foe foventtoa A foberapenticaliy effective” amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, A therapeutic-ally effective amount of foe modified antibody or antibody fragment may vary according to factors such as foe disease state, age, sex, and weight of the individual, and foe ability of foe anfibo^ or antibody portion to dSidi a desired response in the individual. A foerapeutically dTective amount is also one to which any toxic or detrimental effects of foe modified antibody or antiboefy ffagmeni is outwtoghed by foe foempeuttoally beneficial effects, A "therapeutically effective dosage” preferably foMhits a measurable parameter (e.g„ tumor growth rate) to. treated snbjects by at least about 20%, at least about 40%, at least about 60%, and in some embodiments at least about 80%, relative to untreated subjects. Hie ability of a compound to inhibit a measurable parameter, e.g., cancer, can be evaluated m m animal model system predictive of efficacy to. human turnons.

Alternatively, this property of a composition can be evaluated by examining foe ability foe compound to inhibit, such inhibition in vitm by assays known to the skilled practitioner,

[03411 Also within foe scope of the invention are Mis comprising an anii~G€C antibody molecule or tmmunocosjugaf e as described herein. Further included ate kite errmprising liposome compositions comprising an aati-GCC antibody molecule or immanoeonjugaie, The kit can include one or more other elements including; fostroefions far use; other magenta, e.g,, a label, δ therapmttie agent, or m agent useful lor chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective eemposifros; devices or other materials forptspring the aabhcdy ior adj-nisidrabotu gteinpprScaBg acceptable earners; and devices or other materials for adtnm^^^ subject instTUCttoda for use can rndude mstmedous for diagnostic applications of the &amp;φύ££ Mtibodg ffibieoile or immtmoconiupie to delect OCOj in vitro ^ e.g., in a sample, e.g., a biopsy or celkfront a padent h^&amp;ga eracer, or Mvim. The instewcdoas can Include guidance let tberapende ^plicadon i»cla#»g sugge^ed dosages and/or modes of administration, e,g« in a paddat wds a e®ep (&amp;j|., a cancer of gastroi»tesdnai origin, such as, fix enampln, colon. cancer). Other tnsteuetioas can include instacrioos o»'C®^i^#ise;.a^|©dy to a chelator, a label or a therapeutic agent, or for purification o£a eim|ttg8ted ani%dys c,g., fern, wareected cerpgation components. As discussed above, the kit can include a label, e.g., any of the lab# described herein., As discussed, above, the kit can include a theapeutic agent, e.g,, a tkempeatfc agent described herein, In some appUcaftoasihe antibody will be reacted with other components, .9-4., a chelator or a label or therapeutic agent, mg., a radioisotope, e,g., yttrium: or iuiefium:. in such eases the kit can include one or more of a reaction vessel to carry but tbs reaction or a separation device, e,g„ a chromatographic column, for use in separating the finished product from starting maim# or reaction, intermediates. f$3d2] Hie kit can further costal» at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a di agnostie orthp^eude agod as described herein, and/or one or more additional anti AjCC antibody molecules or iiumeaoconjogates, foimuiated as appropriate, in one or mom separate phamt&amp;ceu&amp;al pmparaiions. |§3431 The lit can further contain a radioproteetant The radiolyiic nature of isotopes, e.g., Yttrium (^Y) is known, In order to overcome thm radiolysis, radiopmiectants may be Included, e.&amp;, m die rsacte bnSer, as long as such radioprotecfaiifs are benign, meaning that they do not inhibit or otherwise adversely affect the labeling reaction, e.g„ of an isotope, such as of to the antibody. The formulation %ffer of te pmscot taveinion may inch# such as human serum albumin (HSA) or ascorbate, which miaimiae radiolysis due to yttrium or other strong mikmschdes, Ottier radioproiectani® are krsown in the art and can also be used in the iimmlstion buffer of die present invention, he,, iree mdical scavemgets Cphenol, sulfites, glutathione, cysteine, gentisle acid, nicotinic add, asoorbyl palmiiate, HOP{:D)HsI glycerol, sodium formaldehyde sdfoxylate, Ntfe&amp;O., NaaSzQs» end 80¾ etc.), P344f A provided Mt is one useful for rsdiokbeling a chelator* eopjugated protein or peptide with a therapeutic radioisotope for adntimslmtion to a patient. The kit includes (i) a vial containing cbelatorHmjfugated antibody, (») a vM containing formulation buffer for stabilizing and admimstensg the radiolabeled antibody to a patient» and (iii) instructions for performing the radfoiabeifog procedure. The Mt provides for eatpastog a chektor-eonjugateti antibody to the radioisotope or a. salt thereof tor a suMdeat amount of time under amiable coalitions, e,g,, as rcctanmcnded to the instructions. A radiolabeled antibody having sufficient purity, specific activity and binding specificity is produced.

The radiolabeled antibody may be diluted to an appropriate concentration, e.g., to formulation buffer, and adntotistered directly to the patient wife or without further purification, lire chelator- conjugated antibody may be supplied to iyopMlteed torn.

Uses |0345| The anti-GCC antibody molecules described ham have in vitro and in vivo diagnostic, prognostic, imaging, therapeutic and prophylactic utilities. For example, these antibody molecules can be administered to ceils la culture, e.g. in vitro ot&amp;t vim, or administered la a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders. {03451 The antibody molecules, knmimoeoniugaten, and fusion proteins described herein can be used can modulate au activity or function of a OCC protein, such as ligand binding (e.g,5 binding of ST or guanylin), GCC-medkfcd signal iransduefion, maintenance offotestfesl fluid electrolyte homeostasis, intracellular calclnm release (calcium flux), cell differentiation, cell proliferation, or cell acti vation. 183471 In oae aspect, Ac invention features a method of JdSItog, inhibiting car modulating the growth of* or interfering with the metabolism of, a CXX-expressing cell.

In one embodiment, toe invention provides a method of «debiting GCC-medtafed eel signaling or a method of.killing a cell. The method may be used with any cell or tissue which expresses OCC, such as a cancerous cell (e.g,, a cel! from a cancer of toe gastrointestinal system, such as, for example, a cancer of the colon, stomach, or esophagus, or a pmrereatic cell), or a metastatic lesion. Nodimittog examples of GCC-expmsstog cells include T84 kwma colonic adetecarcinoma cells, fresh or ffezea colonic tenor cells, and cells comprising a recombinant nucleic acid encoding GCC or a portion thereof. 10348] Methods of the invention Include the steps of contacting fee cell with as anti-GCC antibody molecule or inumnoeonjugafe thereof, as described herein, in an effective amount, ie,, amount sntEcient to inhibit GCC-mediated ceil signaling or m amount snfBdent to ME fee cell The mefeod can fee used on cells in culture, e.g. in vim), in vim, ex v/vo, or .in situ. For example, cells feat express GCC (c,g., cells collected by biop%’ of a tenor or metastatic legion; cells from an established cancer ceil line; or recombinant ceils), can he cultured in vitro in culture medium and fee contacting step can he effected fey adding the anti-GCC antibody molecule or immtmoconjugate to the culture medium, la methods of kfllaig a cell, fee mefeod comprises using a naked anii~G€C antibody molecule, or art immtmoeonjugate comprising an anti-GCC antibody molecule and a cytotoxic agent. The method will result in Mllrng of cells expressing GCC, including in particular tumor cells expressing GCC (e.g., colonic tumor cells). |034$1 Reference to Table 7 can serve as a guide to select an aniibody(tes) to use tor various methods. For example. Table 7 indicates which antibodies were confirmed to feternafee after binding GCC. Such antibodies would fee useful when baked to a cytotoxic moiety or a moiety for cell imaging. Antibodies which do uot internalize can be used for diagnostic purposes or therapeutic methods using naked antibody designed to elicit an antibody-dependant cell-mediated cytotoxic response, or perhaps for liposome delivery methods. mm Anti-GCC antibody molecules of fee present invention bind to extracellular domains of GCC or portions thereof in cells expressing fee antigen. As a result, when prafeicing fee methods of fee present invention to kill, suppress, or detect cancerous cells, fee antibodies or antigen binding fragments, bind to all such cells, not only to cells which are feed or ceils whose intracellular antigenic domains am otherwise exposed to fee extracellular emdmntneni Consequently, binding of fee antibodies or antigen binding fragments, is concentrated in areas where there are cells expressing GCC, «respective of whether these cells are feed or unfixed, viable or necrotic. Additionally or alternatively, the anti-GCC antibody molecules, bind to and are internalised with GCC upon bin ding cells expressing fee antigen. pISIf Tiie method also can be performed on cells present in a object, as pari of an in vim protocol. In one embmliment, the subject is a human subject Alternatively, the subject can be a mammal expressing a GCC antigen with which an antl-OCC antibody molecule disclosed herein cross-reacis. An and-OCC antibody moiecnle or immunotxmjugste thereof can be administered to a human subject lor therapeutic purposes. An anii~G€C antibody molecule or immunoconjugate also cm be administered to a non-human mammal expressing the GCOlike antigen with which the antibody cross-reacts (e.g>„ a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).

For in vivo embodiments, the contacting step is effected, in a subject and includes administering an anti«GCC antibody molecule or immunoconjugate thereof to the subject under conditions effective to permit both balding of tire antibody molecule to the extracellular domain of GOC expressed on the cell, and the treating of the cell 11152] In one embodiment, the invention provides a method of treating cancer by administering an aaii~GCC antibody molecule or an immunoconjugate comprising an anti-GCC antibody molecule and a cytotoxic agent to a patient in need of such treatment The method can he used for the treatment of any cancerous disorder which includes at least some cells that express the GCC antigen* As used herein, the term “cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tisanes, or organs. Irrespective of histopathologic type or stage of mvasiveness. The terms “cancer” and ‘humor” may be used interchangeably (e,g.:> when used in the context of treatment methods, ‘ireataient of a cancer” and “tmatment of a tumor* have the same meaning).

[0353] In embodiments, the treatment is sufScient to reduce or inhibit the growth of the subject's tumor, induce the number or erne of metastatic lesions, reduce tumor load, reduce primary tumor load, reduce Invasiveuess, prolong survival time, or maintain or improve the qualify of life. $354] Examples of cancerous disorders include, hut are not limited to, solid tumors, soil tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g,, sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting colon and pancreas, Adenocamnomas include malignancies such m son-smalt ceil carcinoma of the hmg. Metastatic lesions of the aforeaieationed cancers can also he treat®! or prevented using the methods and compositions of the invmtioft. Itt some embodiments, the cancer to be treated is a cancer of the gastrointestinal system (e.g,5 colorectal cancer, esophageal cancer, or stomach cancer). In some embodiments, tbe pancreatic cancer. $355] In one embodiment, die cancer is a colorectal case®:, e.g., colorectal adenocarcinoma, colorectal leiomyosarcoma, colorectal lymphoma, colorectal melanoma, or a colorectal neumesdoerise tumor, fit a partiealar embodiment, the cancer is metastatic colon cancer. In another embodiment, the cancer is a stomach cancer (e.g„ gastric adenocarcinoma, lymphoma, or sarcoma), or metastasis thereof. 1« another emhodiateni, the cancer is an esophageal cancer (¢.8.. a squamous cell carcinoma or adenocarcinoma of the esophagus), mm The method can be useful in heating a relevant disorder at any stage or subclasstfieatiou. For example, method can be used to treat early or late stage colon cancer, or colon cancer of any of stages 0,!, HA, HE, MA, BIB, IIIC, and IV. $357] In some embodiments, the method for treating cancer (e.g., colorectal, esophageal, or stomach cancer) comprises administering to a patient in need ofsnch treatment a naked anli-GCC antibody molecule described herein, in other embodiments, the method, comprises administering an immanoconjugate comprising an aati-GCC antibody molecule described herein and a cytotoxic agent In some such embodiments, the immunoconjugste is characterized by formula (I), as described herein. In certain embodiments, theimmunoeonjugate is characterised by formnia (Ι-I), (J~3), (/-5). (1-4% (/-5). (J~5), or (1-7) as described herein. In parrieulsrembodiments, the iismuaoeorpgaie is characterized by formula (/), (M% (/-3), ¢/-5), (//), (/-5), (/-5), or (/-7), wherein the variable Ab is an antibody molecule with features summarized In Tables 1 to 6, In cert ain embodiments, toe variable Ab is a 5F9 antibody molecule or an Abx-229 antibody molecule. In certain particular embodiments, the immunoconjugate is characterized by formula (/-5) or (1-7), wherein the variable Ab is a 5F9 antibody molecule. $358] Methods of administering antibody molecules and irnmnnoconjugates are described above. Suitable dosages of toe molecules used will depend on fee age and weight of the sufejiect and the particular compound used. {I35§| In some entfsodMaeats, the aaii»GCC antibody molecule or immmrnetmjugaie is administered m fteafraeaf cycles. A “treatment cycle” consists of a treatment period, dmtog which toe aati-GCC antibody molecule or immimoeonfugate is administered as described above, followed by a rest period, daring which no anti-CCC antibody molecule or immimocoujupte is administered The treatment cycle can be repeated as necessary to achieve the desired effect £63601 The aafi-GCC antibodies described hereto (e.g., naked anti-GCC antibody molecules or immxmoconjugaies ormprising m anii~G€C antibody molecule and a therapeutic agent) may be used in combination with other therapies. For example, toe combination therapy can include a composition of the present invention co-formulated with, and/or co-admtoistered with, one or more additional therapeutic agents, e.g., one or more anti-cancer agents, e.g,, cytotoxic or cytostatic agents, hormone treatment, vaccines, and/or other inttnunotherapies. &amp; other embodiments, the anti-GCC antibodies are administered in combination with other therapeutic treatment modalities, inclndmg surgery, radiation, cryosurgery, and/or thermotherapy. Such combination therapies may advantageously utilise lower dosages of toe administered therapeutic agents, tons avoiding possible toxicides or complications associated with the various monotoetapies, f§3«l Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g,, the- two or more ireatoents are delivered alter the subject has been diagnosed with the disorder and before toe disorder has been cured or eliminated, In. some embodiments, the delivery of one treatment is still occurring when toe delivery of the second begins, so that there is overlap. This is sometimes referred to herein as “simultaneous” or “coocmrraai deliver.” to other embodiments, toe delivery of one treatment ends before the delivery of the other treatment begins, to some embodiments of either case, the treatment is more effective because of combined administration. For example, toe second treatment is more effective, e.g„ an equivalent effect is seen with less of toe second treatment, or the second treatment reduces symptoms to a greater extent, than would he «sen if the second treatment were administered in toe absence of toe first treatment, or the analogous situation is seen with toe first treatment, to some embodiments, delivery is such that the reduction In a symptom, or other parameter related to toe disorder is greater than what would he observed with one treatment delivered in the absence of the other. Hie effect of toe two treatments can bo partially additive, wholly additive» or greater than additive. The delivery cm be such that m defect of die first treatment delivered is still detectable when die second is delivered, [93621 lit smite embodiments, the aati-OCC antibody molecule or immimoeoaj'tigafe thereof is used m combination wi.ih a chemotherapeutic agent Non-limiting examples ofDNA damaging chemotherapeutic agents include topdsomerase I inhibitors {&amp;&amp;» irmotecan, topoteeaa, ma&amp;tofa&amp;m and analogs or metabolites thereof» and doxorubicin); topoisomerase II inhibitors (e,g., etoposide, itmipisside, and daunorubiem); alkylating agents (e,g., mdphalan, chlorambucil, busul&amp;n, thiotepSj ifosfamide, earmustine, iomustiae, senmstine, streptossoek, decarbame» methotrexate, mitomycin C, and cyclophasphamide); DMA iuferc&amp;iators (e.g,* eisplatin, oxatiplatia, and earboplatin); DNA interealatars and free radical generators such as bleomycin; md nucleoside mimetics (e.g,s S-fhioromadi, capccitibine» gemcitahme, ilodarabine, eytarshme, mercaptopnrin e, Ihtogu&amp;ome, pentosiatin, and hydroxyurea), |1M3| Chemotherapeutic agents that disrupt ceil replication include: paditaxel, docetaxel, and related analogs; vmci&amp;tiine, vinblastin, and related analogs; fhatidooiMe, lenalidomide, md related analogs (e.g,, CC-5013 and CC-4G47); protein tyrosine kinase inhibitors ie.g,, imatfeib mesylate and gefitinib); proteasoine inhibitors (s.g., bortezomih); MF-kB inhibitors, including inhibitors of IkB kinase; antibodies which Mad to proteins overexpressed in cancers and thereby downregulsto cell replication (e.g., irantuzumab, ntoimab, cetuximab, and bevadxumab); and other inhibitors of proteins or enzymes known to be upregalated, over-expressed or activated in cancers, the inhibition of which dowmregulates cell replication.

[13641 The selection of therapeutic ages t(s) or treatment modality to be comb ined with an. anti-GOC antibody molecule or annumoconjugate of the invention will depend on. the disorder to be treked The additional s.gent{s) or treatment modality may include, for example, standard approved therapies for the indication being treated For example, when the anti-GCC antibody molecule or immmtoconfugate thereof is used to treat cote cancer, it may be used in combination with, e.g., surgery; radiation therapy; 5~fluorouticil (5-FU), capoeitibin% leucovorm, hinoteeaa, oxaiiplatin, bevactetmab, cetetimab, paaitiimtim, or otiubinations thereof (e.g,, oxaHpiatim'eapedtibhie (XELOX), S-fluoronficil/ienoworim'-oxalipiatm (FOIFOX), S41norouoeil/Iencovoriu/innoiecan (FGLFIRI), FQLFOX plus bevaeixumab, or POLPOG pins bevacizumab).

[03SS] in another aspect, the invention features the nse of as anti-GCX antibody molecule or inmurooconjugats as described herein ία fee manufacture of a mediesmeat la m embodiment, fee medicament»' for treating cancer, e.g., a g&amp;tirointestinai cancer, la some enfeodrmenls, the medicament comprises a« anti~GCC antibody molecule having features suaanarfeed in Tables 1«6. la some embodiments, fee medicament comprises a 5F9 antibody molecule or Abx-229 antibody molecule. flBtitiJ Anti-G€€ antibodies aad immuaoconjugafe described herein cars, be used ίο detect feepreseoee of GCC, e.g., to detect fee presence of GOD in a biological sample, or to delect fee presence or diaftibutioa of GCC in a subject. The term “detectm^* as used herein encompasses quantitative or qualitative detection. Detecting GCC or GCC protein, as used herein, means detecting intact GCC protein or detecting a portion of fee GCC protein feat comprises fee epitope to which the aaM-OCC antibody molecule binds.

[§3$7f Accordingly, in another aspect, the invearion features, a method of detecting GCC protein, e.g., detecting a GCC expressing cell or tissue, e.g., a tumor cell, or a tumor having cells, that express GCC. The method comprises: contacting a material, e.g., a cel! or tissue, e.g., a sample of a tumor which expresses GCC, wife an anti-GCC antibody molecule, e.g., an anfi-GCC antibody molecule described herein, under conditions which allow formation of a complex between the anfeGCC antibody molecule and GCC protein; aad detecting formation of a complex between antibody molecule and GCC protein, to thereby detect fee presence of GCC proteia, e,g,, to detect a GCC expressing ceil or tenor.

[0338} In m embodiment fee anti-GCC antibody molecule is an immiuMtconjugafe comprising a detectable labei.

[03#! fit certain embodiments, fee tissues include normal aad/or caacetous tissues feat express OCX’ at higher levels relative to other tissues, for example other tissue such as B cells and/or B ceil associated tissues, [03 71} Methods of detection described herein, whether in vitro or in vivo, can be used to evaluate a subject. In an embodiment fee method &amp; performed in vivo, aad can be used, e.g., for imaging, staging, evaluation or diagnosis of a patient. In eertein embodfmoate, fee disorder is a cell proliferative disorder, such as a cancer or a tumor, e.g., colon cancer. |8371] 1¼¾ in aaother aspect, the invention provides, a method tor defecting the passaace of GCC pratem in vitro {e.g,,io a bfologjbaf sample» such as &amp; tissue biopsy, eg.* front a turner tissue, torn a subject) orM vivo (e.g., by in vivo imaging in a subject), The method comprises; (i) contacting a sample with m antf-GCC an tibody molecule or inmumoeonjugate thereof, or adamtisteting to a Mibjects an anti~GOC antibody molecule or immunoconjEgate thereof; and (ii) detecting formation of a complex between the aoti~G€€ antibody molecule and GCC protein. Complex fermation is indicative of the presence or level of GCC, ($312} In embodiments the level of complex detected in the sample or subject is compared wife a tefereoce value, e.g,s a value for complex formation or level of GCC, In m embodiment a level of GCC which exceeds a mferen.ee value is indicative of a GCC-mediated disorder, 183731 In an embodiment tire method comprises contacting a reference sample, e,g.s a control nipple (e.g., a control biological s&amp;tnple, such as plasma, tissue, biopsy) or a control subject) with an aaii-OCC antibody molecule or immmioeonjugate thereof and comparing the level of complex detected therein with the level detected in the sample or subject, [8374] In certain embodiments, a test cell or tissue is obtained from an indi vidual suspected of hasting a disorder associated with increased expression of GCC, [8375] hi an. embodiment the level of GCC, in a sample from the subject, or in the subject, is compared with a reference level, e,g., the level of OCX) in a control material, e.g,, a normal cell of tire same tissue origin as the subject’s cell or a cel! having OCC at levels comparable to such a normal ceil. The method can comprise, e.g„ responsive to the detected level of GCC, providing a diagnosis, &amp; prognosis, a» evahmtion of the efficacy of treatment, or the staging of a disorder, A higher level of GCC in. the sample or sublet, «««pared to the control material, Indicates ike presence of a disorder associated with increased expression of GCC, A higher level of GCC in the sample or subject, as compared to the control material, can also Indicate, tire relative lack of efficacy of a treatment, a relatively poorer prognosis, or a later stage of disease. The level of GCC Cim also be used to evaluate or select future treatment, e.g, the need tor mom or leas aggressive treatment, or the need to switch, from one treatment regimen to another. |i3Wj The level ©f GCC can also fee used to select or evaluate patients. E,g,, at embodiments patients whose tumor cells express high amounts of GCC on their surfaces would fee considered good candidates for treatment with toxin-conjugated and-GCC antibody molecules. Da embodiments patients whose tumor cells express low amounts of GCC on their surfaces would not fee as good candidates for this or might be candidates for combating the aad-GCC antibody molecule with m additional treatment method, or he candidates tor naked antibody therapy, in another example, the dose of the toxin-conjugated aoti-GCC antibody molecule could be adjusted to reflect the number of GCC molecules expressed on the surfaces of tumor cells. Patients with high numbers of GCC molecules on fetor tumor cell surfaces might be treated with lower doses than patients with, low' numbers of GCC molecules. Detecting the presence of GCC-expressing tumor cells in vivo can allow identfficatfon of tissues into the primary G€C>expressmg tenor has metastasized. Knowledge of which tissues fee metasiases can lead to targeted application of tumor therapy, [03??| As discussed above, the antibody moleenl&amp;s described herein permi t assessment of the presence of a GCC protein in normal versus neoplastic tissues, through which fee presence or severity of disease, disease progress and/or the efficacy of thmspy can be assessed. For example, therapy can. fee monitored and efficacy assessed. M one example, a GCC protein, cm be detected and/or measured is a first sample obtained Sum a subject having m inflammatory disease and therapy can be initiated. Later, a second sample can fee obtained from tbe subject and GCC pmteto m the sample can fee detected and/or measured, A decrease to the quantity of GCC protein detected or measured In the second sample can fee indicative of therapeutic efficacy.

[®3781 Exemplary cell prolifcrati ve disorders that may fee evaluated, e.g,, diagnosed, using an antibody disclosed herein include a proliferative disorder including, but not limited to, colon cancer, stomach cancer, esophageal cancer, |I3?9J In cerium embodiments, a method, such as those described above, comprises detecting binding of an anh-GCC antibody to GCC expressed on fee surface of a cell or in a membrane preparation obtained from a cell expressing GCC on its surface, in certain, embodiments, the method comprises contacting a cell with an anti-OCC aatfeody under couditioss permissi ve for binding of the asti-GCC antibody to GCC, and detecting whether a complex is fermed between fee aod-GOC antibody and GCC on the cell surface.

An exemplary assay for detecting blading of an anri-GCC antibody to GCC expressed on foe surface of a cell, is a “EA€S?> assay. 103801 Exemplasy1' samples for met bods described herein comprise tissue or body fluid, such as an inflammatory exudate, blood, serum, bowel fluid, stool sample, or biopsy. In one example, a sample (e.g,, tissue anri-br body fluid) can be obtained from an individual and a suitable immunological method eats be used to delect and/or measure GCC protein expression. Satiable immunological methods for detecting or measuring GCC protein expression include etsyme-imkedl «mnaaosorbent assays (EUSA), radioimmnnoassay, immunoMstology, flow cytometry, and the like. 10381) Anfi-GCC antibody moleouies used in methods described herein, e.g., is. the in vivo and in vitro detection, e.g., diagnostic, staging, or imaging methods, can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound binding agent. Suitable detectable substances include various biologically active enzymes, ligands, pmstheric groups, Suoreseent materials, hmunescent materials, diemilummesoeni materials, biolumineseest materials, cbromopboric materials, electron dense materials, paramagnetic (e.g., nuclear magnetic resonance active) materials, and radioactive materials, 1« some embodiments, the anti-GCC antibody molecule is coupled to a radioactive ion, e.g., fedium (mIa)s iodise (htl or I2'1), yltriunt (3¾ iutetium (u Xn), actinium {^5Ac), bismuth (il2Bi or nsBi), sulfitr (%), eathon (i4C), tritium (3H), rhodium (188Rh), technetium ("mTc), praseodymium, or phosphorous (32P); or a poritron-emitring radionuclide, e.g., o&amp;rixm-ll (ilCJ), potassium»# niirogen-D (¾¾ oxygen-15 (i50), fluorine-18 (?SF), and iodine-121 (mI), 10382} Exemplary' labels include fluorophores such as rare earth chelates or fluoteseda and its derivatives, rhodamuie and Its derivatives, daasyi, mnhellifemne, luceriferases, e.g., Smfly Inciferase and bacterial Inciferase (U.S. Pat. Mo. 4,737,456), iudferia, and 2,3~diliydrophthalaanedioaes Other exemplary labels Include horseradish peroxidase (HRP), alkaline phosphatase, galactosidase, gtwcoamykse, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose 6~phospkafo dehydrogenase, hrieroeycllc oxidases such as uncase and xanthine oxidase, coupled with an eazyme that employs hydrogen peroxide to oxidize a dye precursor such as HEP, lactopetoxidasc, or microperoxidase, biotin/avidia, spin labels, bacteriophage labels, stable free radi cals, and the like.

[03831 Fluorophore and chrompphore labeled antibody molecules cm be prepared .fem standard moieties kaow» m die art, Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to bane substantial. absorption at wavelengths drove 310 nm and preferably above 400 am. A variety of suitable fluorescent atmpounds and chronrophores are described fey Stryer Segment 162:526 (1968) and Brand, L. et at, Annual Review tfJMechmtstiy, 4.1:843-868 (1972). The antibodies can be labeled with fluorescent chromophore groups by eonvenrienal pocedures such as those disclosed in US. Patent K;os. 3,940,475,4,289,747, and 4,376,110, [0384.1 One gtoup of fluorescers having a number of the desi rable properties described above is the xanthene dyes, which include the fluoresceins derived from 3,6-<hhydroxy-9"henylxanthhydrol and resarmnes and rhodamines derived from S^dimino-^-ph^tylxanthy&amp;oI and iissanime rtiodamme B. The rhsdamine and fluorescein derivatives of 9-o-carboxyphenykmfebydrel have a 9-o-carboxyphenyl group. Fluorescein compounds having neacrive coupling groups such as amino and isothiocyanate groups such as fluorescein isothiocyanate and fluorescamineare readily available.

Another group of fluorescent compounds are the naphthylamines, having an atmno group in the a or β position, 10385] Labeled antifeo% molecules can be used, for example, diagnostically and/or experimentally in a number of contexts, Including (1) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or hnmunoprcciptation; (fi) to detect a predetermined antigen {e.g,s in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein; (in) to monitor protein levels in tissue as part of a clinical testing procedure, e.g,, to determine the efficacy of a given treatment regimen

Certain other methods can be used to detect binding of anti-GCC antibodies to CICC, Such methods include, but are mi limited to, antlgm-feinding assays that are known in the art, such as western blots, radiommmoassays, BLISA (enzyme linked immunosorbent assay), “sandwich" Immunoassays, immimopreeipitation assays, fluorescent hmnunoassays, protein A immunoassays, and immuaohisfochemistry (IHC).

[0387J Complex formation between the aati-G€€ antibody molecule and GCC can be detected by measuring or visualizing either the antibody (or antibody fragment) bound to the GCC antigen or unbound antibody molecule. Conventional detection assays can be used, e.g„, western blots, mdtoantmnmassays, ELISA (enzyme Snied'imnmoosorbast assay), Sandwich” imnnmoassays, immtmopmdpitatian assays, fluorescent immtraasssays., protein A immunoassays, aadS immmmbktocbetmsby (S3C) or radfoimmunoassay (RTA). §8388! Alternative to labeling the anti-GCC antibody molecule, the presence of GCC can be assayed in a sample by a compctitioa immunoassay utilising standards labeled with a. detectable substance and an aalabeled anti-GCC antibody molecule, In this assay, foe biological sample, foe labeled standards and the GCC binding agent are combined, and fee amount of labeled standard bound to the unlabeled antibody is determined. The amount of GCC in foe sample is inversely proportional to fee amount of labeled standard : bound to foe GCC binding agent, (0389| It is also possible to directly detect GCC to anti-GCC antibody molecule complex formation without further manipulation or labeling of either component (GCC or antibody molecule), for example by utilizing fee teetaique of fluorescence energy transfer (FET, see, for example, Lakowiczefot, U.S, Patent No. 5*631,169; Stavrianopoufos, et aL, CIS. Patent No. 4,868,103), A flnomphore label on the Erst, ‘donor’ molecule is selected such feat, upon excitation wife incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in tom is able to fluoresce due to fee absorbed energy. Alternately, the ‘donor* protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen feat emit different wavelengths of tight, such feat foe ‘acceptor’ molecule label may be differentiated from feat of fee ‘donor5. Since the effleteney of energy transfer between fee labels is related to the distance separating fee molecules, spatial lelatiouships between foe molecules can be assessed In a situation in which binding occurs between the molecules, foe fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e,g,, using a fluorimeter). f$399] la another example, deteratinatioa of fee ability of am antibody 'molecule to recognize GCC can be accomplished without labeling either assay component (<3CC or aatibody molecule) by utilizing a technology such as real-time Btomoleoukr Istaractioa Analysis (BIA) (see, e.&amp;, Sjolaadbr, S. aMUrbamczky, C., 1991, Ami Ckem. 63:2338-2345 and Szabo &amp;tal, 3995, Cum Opin. Struct* Biol 5:699-705), As used herein, “BIA” or “surface plasmon resonance” is a technology for sCmlying biospecifk· interactions in. real time, without labeling any of the inferaofanfs ([&amp;g.t BMCORE™}. Changes in fee mass et the binding surface (indicative of a binding event) result in alterations of the rofractive index of light near- the surface (fee optical phenomenon of surface plasmon resonance (SPR)), resul ting in a detectable signal which can he used as as indication of teal-time reactions between biological molecules. |939lf In still another embodiment, the Invention provides a method .tor detecting the presence of GCC-expressing tumor tissues in vivo, The method includes 0) administering to a subject (e,g;s a patient having a cancer) an aoii-G€€ antibody or antigen binding fragment thereof, preferably a antibody or antigen binding fragment thereof conjugated to a detectable label or marker, (ii) exposing the subject to a means for detecting said detectable label or marker to the GCC-exfirossing tissues or cells.

[9392-] Examples of labels useful for diagnostic imaging in aecordaace with the present invention are radiolabels such as 1λ1Ι, lu in, ^Ga, I1SI, ss*"Tc, i2SI, 3H, I4C, and t88Rh, fluorescent labels such as fluorescein and rhodamme, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a single photon emission computed tomography (“SPECT”) detector or position emission tomography (“PET*) scanner, chemsluminescets such as hutiferm, and enzymatic markers such m peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes, such as a trsnsrectsi probe, can also be employed. The antibody can be labeled with such reagents using tecimiques knowu in fee art For example, see Weasel ami Mean» (1983) Rsdioimmummaging mtdRadhimmum&amp;terapp, Elsevier, New York, for teclmiques relating to the radfolabelmg of antibodies. See also, D. Coleher et al Mesh. EnzymoL 121:802-816 (198b.

[$393] in the case of a radiolabeled antibody, the antibody Is adntinisterod to the pattest, is localized to fee tumor bearing fee antigen wife which the antibody reacts, and is detected or ^imaged" in viw using known techniques such as radionudear scanning using e,g,, agemtma camera or emission tomogtophy m competed tomogmphy. See e.g., Ajt Bradwed ei at, “Development in Antibody imaging”, Afmtotiomii Aniibo$eiJhr Crncer DerimUon muiTkerapjK JLW, Baldwin ei ah, (eds,), pp 65-85 (Academic Press 1985). ό transaxM tomography scanner, sods as designated Pet ¥! located at Btxudthaven Wallonat Laboratory, eaa be used where the mdioiabe! emits gositroBS (e.g., nC> l% i5Os and nH), |I394] In. other embodiments, the invention provides methods for determining the dose, e,g. , radiation dose, that ditibreai tissues are exposed to who» a subject, e.g.»a himtan subject, is 8d»ii»i^ered m imthGCC antibody molecule that is cotgttjpfed to a mdioaetive isoa^&amp; ^he method tseliides: (i) admmisterihg aaanti<K3C antibody molecule as described herein, e.g., a a»ii-GCC antibody molecule, that is labeled with a radioactive isotope to a subject; (ii) measuring the amount of radioactive isotope located in different tissues, 4g., tomor, orblood, ai various time points until some or alt of the x»d»i«<^ve:^ic^.hi«:ibe»''i^^^i!d.fiKtoi the body of the subject; and (hi) calculating the total dose of ratMarios received by each, tissue analysed. The measurements can be taken at scheduled time points, 0¾ day 1, 2,3,5, ?, aud 12, tolio wing srindmstmtk)» (st !0!-labeled anti-GCC antibody mpiecale to the subject. %e eoncentimtton ofmdioisoiope present hi a given tissue, integrated over time, a»d multiplied by the speeiito activity of tie radioisotof e can be used to calculate the dose that a give» tissue receives. Phammcologicai ffiformatioa generated using anti-GCC antibody moleoilesIgbeled with one radioactive isotope, e.g., a gamma-emitter, e.g., luIn, can be used to caliculate the expected dose that the same tissue would receive from a different radioactive iscfcpe which cannot be easily measured, e.g., a beta-emitter, e.g., *Ύ.

MiiCCMtee^s f 0395J Asti-GCC antibodies were gen erated, by Severn], methods, as is discussed. ip mom detail hi the Examples, Briefiy, mouse monoclonal antibodies 3G1 8 FI and 10B8 were generated by traditional itorntmteatio» technology I» coavMtiottai mice. Bums» monoclonal antibodies 1D2,5F9,5H3:i 6H8, |C2S and 10C10 were generated, using timsgpmc micethat generate fully human IgG2 antibodies, utilising Ahgeuix ΧΕΜΟΜΟϋΕΕ asasgeme technology, and Isolated using hybridoma tecfetofogy- Hinaaa mAh Abx-012, mAb Abx-020, mAh Ahx-106, mAh Ahx~198, raAb Abx-22I?mAb Abx-229, mAh Abx~338 mi mAb Abx-393 were generated toing ttansgeaic mice that generate folly -hamaa Ig<32 afoibodies. Slagle Mfibodies were isolated usrag Abge&amp;ix SLAM teetaology, These were used to make Miy human IgO! aufohefoes, Spec-Mc&amp;y of the-aotifeodies agaiastQCC was tested by ELISA mi flow ^ytometiy (PCM). A subset of foe gsasrated aabbedks was selected fbr ferfoer efowaoterisa&amp;w. |#39ij Table 1 below sommames for semi aofoGCC atfobodies foe Mtlbody desigMtfos, foe immuaogett used to geaetate foe autibady, foe amma! used, foe so«rees the species aad the isotype isolates.

Table 1

1¾¾ sequences of the light and heavy chain variable regions were determined. Table 2 below is a summary of the SEQ ID NOs for the variable regions of several antibodies. The amino add and nucleic add sequences for the variable regions of each of the heavy trad light drains iht* murine sad human aati-GCC antibodies are shown in Tables 3 and 4, respecti vely. 19398J The amino acid aud nucleic acid sequences tor each of the CDRs of the heavy and light chains for sati-GCC antibodies are shown in Tables 5 and 6, respectively. P399] For example. Table 3, row 9 is an il lustration of the encoded amino acid sequence o f the mature heavy chain variable region of mAh 5F9 (SBQ ID NO; 18); and the nucleic acid sequence encoding the mature heavy chain variable region of mAh 5F9 (SBQ ID NO: 17) is depicted in Table 4, row 9. The encoded amino acid sequences of mAh 5F9 heavy clmiu CDE. (CDE.) I (SEQ ID NO:lil6)s CDE2 (SEQ ID NQT0&amp;) and CDRS (SEQ ID NOT 10) are depicted ία Table 5, tows 25-27» respectively; and the nucleic acid sequences of mAh 5F9 heavy chain CDRI (SEQ ID NO :105)» CDE2 (SBQ ID NOT07) and CDRS (SEQ ID NO: 109) are shown m Table 6S mws 25-27, respectively. (94001 Table 3, row 10 is an illustration of the encoded amino acid sequence of the mature light chain variable region ofmAb 5F9 (SEQ ID NO:20); and the nucleic acid sequence encoding the mature kappa light chain variable region ofmAb SF9 (SEQ ID NO; 19) is depicted in Table 4, row 10. lire encoded amino acid sequences of mAh 5F9 light cham CBR (CDE) I (SEQ ID NOT 12), CDR2 (SEQ ID NOT 14)and CDRS (SEQ ID NOT 16) are depicted in Table 5* rows 28-30, respectively; and the nucleic acid sequences of mAh 5F9 light chain CDRI (SEQ ID NOT 11), CD&amp;2 (SEQ ID NO: 113) and CDR3 (SBQ ID NOT 15) are shown k Table 6, rows 28-30, respectively. (0#I| Sequencing of the CDRs allowed detmnmation of tire abundance of residues that might serve as toxin conjugation sites. An unpaired free cysteine in the antigen binding region could be a site for auristaiin conjugation and a lysine could be a site for maytanrine conjugation, Toxin conjugation to an amino acid of fixe COR would raise fie concern of altering the binding affinity of fie antibody to G€€, Thus, in embedments the CDRs lack m mam aeM winch tan be coiyogated to a therapeotie agent

|044I2] Expression vectors were created as described above which contain coding secpeoce for both the heavy aad light chela of each of mAb 5P9 and Abx-229. |®403) Tbs iaveatieH. is Illustrated by the following examples, which should not he construed as further limiting.

EXAMPLES EXAMPLE D Geaerailoiiofauti-GCCaatlbotifesasitl eharaetnrfeMfon [0404] The generation of GCC protein tor immunization and screening was performed as foSows. GCC antigen was prepared by subclonkg a portion of the GCC geae encoding a sequence comprising the following GCC sequence (signal sequence and extracellular domain) into m. expression vector,

MKTllLmALWSLLFQPGmSFSSGVSQNCHNGSYm:SVLhMiM$,4.FAB-PLEHLEDAVNEaLEIVR

GM^NACtiWTENAmfySDGLtHNSCTCRSST<^GLDI,LSK?SMAqRMtjC\TIGPSCIYS’IPGMY

LD'XEiSY'PMISACtSFCOCDYKBTLI^LMSEASKLMTFLYNFWKTNDLFFKIYSWSTSYWjKNG’i'E

TEDCF%ATNALRASVShTSIffiLGFKWLRQi«EFQDh.hroiMgKSmimiCXlGPEFLYKLKCiDAAV

Amwuvvιmnΐκ^¥SΏm^A^ΌYMκmΊmm,spσm^msΐsmιs?τκm>fALAnMύlLL·Έ

GHMLKMTNGENITTPKFiYHAFENLTFEGYTX>PVTLDDWerDVDSThn.'TLYTSVBIKKYrK\XLW imAmiYPTOMSPTFTVVKNSKL (SBQ ID NO:229) f©4H5J The expression vector {pLKTOK107) provided a CTemuaal IgGIFc region to fuse with, the GCC sequence. This vector comprised m exon with the IgGI hinge, CH2 and CH3 domains, mutated to eliminate an unpaired cysteine from the CHI fragment i« the exon. Tins IgGIFc region was further mutated at lysine 235 and glycine 237 to alanines. The construct was expressed recombiaantty in human embryonic kidney (BEK) 293 cells transfected with the gene for SV40 T-ashgea as secreted GCC sequence (amine add residues 24 to 430 of SEQ ID N0228) fused to a Cderrainal human IgGI Fc. The protein, named TOK1074% (all name h0€C4ECD/MgG.i Fc, SEQ ID NO;317), was purified by protein A ehmmatography and size exclusion chromatography. 10406) GCC antigen was also prepared by snbclouing the above fusion protein into an expression vector such as pLKTOKl .1.1, which allows for fusion of the marine lgG2a transmembtaae region onto the C-terminus. When ibis construct is expressed moombmantly in CMD cells, the GCC extracellular domain is detected on the cell surface. High cell surface expression of the GCC4g fusion protein (SEQ ID NO :318) is achieved when the pLKTOK.111 vector is ecMransfected with gLK.TQKl.23, which, comprises murine CD79&amp; (MB«1) wad €07% (B29). Gene #27 tea this frausfectkn (CHO-GOC#2?) w used as immunogen, HT»29^GO€#2 ceils also were used as hmmmogen.

[Θ407| For sramtkg of hybridoma supernatants sad purified mAbs by ELISA* the nucleic add encoding a GCC fusion construct was cloned Into a pCMVI expression vector (Sigma). Perificafioa tags; FLAG-teg (k the N-tenmnus) and HiliMag (kthe CAermkus) were closed kto the construct as well. The fusion protein construct was traasfected into 293 cels* expressed* and recombinant protein was purified over and Aa&amp;FtAG® M2-Agarose Affinity column (Sigma), (0408f Hoageots and Cel lines, HEK293 cells* CBO, and T84 human colon cancer cells and were obtained from ATCC and maintained according to ATCC protocols. (04091 .Mice; Female C57BL/6 mice, 4-6 weeks old, were purchased from Tasonie Farms, Inc. (Germantown, NY) for the generation of the murine hykideom Xeommee, bred in-house until 4-6 weeks old, pmdudng human Ig02 antibodies were obtained from Ahgenix, Inc. (Fremont, CA) for the generation of the human hybridomas. All animals were acquired and maintained according to the guidelines of the Institutional Animal Care and Use Committee of Millennium Pharmaceuticals, Inc, (04101 Cell lines: The cell lines used for functional assays were ceil pairs of GCC transfected ceils and vector control HEK293 or HT29 cells. HT29 cells were transfected with the fell length GCC under control of the EF~1 a promoter or empty vector (pIKTOKA) and selected m G418. The GCC in these ceils was confirmed to have a cGMF response when contacted with the ST peptide (1-1S or 5-18). HEK293 cells were transfected, full length GCC under control of the CMY promoter or empty vector (pN8iwycSV40) end selected in hiastieidin, The GCC k these cells has a raye tag. The clones selected for highest GCC expression were 293-GCC42, HT29“G€€#2 and f!T29~ GCC#5. The 1ST29“G€C#2 also were used as immmogem for generating anti-GCC antibody molecules. Additional GCC-expresskg cells are CT26 cells. To develop the GCC~®pres&amp;mg CT26 cell line, pTOKSSD vector was used. Full length GCC was cloned into the site normally used for heavy chain cloning and iuciferase was cloned into tire site normally used for light chain eloukg, Alter tramfection into CT26 ceils, independent expression of both GCC and foeifemse was confirmed. Surface expression rtf' GCC was confirmed by fiow cytometry' using the 5F9 antibody. Clone #32 was selected for further studies.

[®4111 %s TS4 colon ernes* cell line· endogenously exposes GCC, Taqmast analysis of GCC in a toad cell Line paid revealed that T84 was the only cell line that express isENA fbr GCC, Staining for GCC with a GCC selective mAh on cell pdlds of T84 cells showed sigmfieant GCC protein expression.

[#4121 Quantitation of GCC receptor levels with, radiolabeled Hgaad (ST-toxfe) suggested that the 293-GCC#2 cell expressed more GCC than T84 cells -while the ΕΪ29* GCC#2 or #5 expressed the fewest GCC molecules per cell,

[¢413] Generation of marine mAJhs by protein Immunlxation: Human GCC extracellular domain/htanan Ig felon protein (TOK107~Mg, 58 ug) was suspended in Dalbecco’s phosphate buffered saline (PBS; GIBCG» Grand Island., NY) and emidsiSed with an equal volume of complete Freunds adjuvant (Sigma Chemical Co., St. Loots, MO)< €5 781/¾ mice were Immtozed by injection of the emulsion at three subcutaneous sites and one intr^eritoneal (ip.) site, Two weeks after the imbai immunization, the mice were given a booster mtnuunxabo» ip. with 25 g.g TOKIOT-Mg in incomplete Preundss adjuvant, One week later, a small amount of blood was collected from to tail vein and the scrum binding activity against TOK107»Ig was titered by ELISA. Mice were selected for fusion when their titer exceeded 1:24,300 by ELISA or 1:500 by FACS'. The selected mice wore boosted by injection of 25 pg TOK107~hIg in FES, Four days later, one moose was cudianized and a spleen cell suspension was prepared and washed with PBS for felon with P3 cells. One «tooth later, spleen cells from another mouse were pupated for felon with K cells. The toed cells were tested for production of antibodies which specifically hound to GCC by ELISA for binding T0ICI07-Mg compared to a nooGCC antigen or to to Pc region of IgG aod by FACS for binding ίο T84 cells, or Caco-2 cells or HT-29 clone M cells compared to vector control and compared to noo-GCOexpressing MCF-7 cells. Isotype was determined using ISOSTRIP® mouse monoclonal antibody isoiypiag kit (Roche Diagnostics Mannheim Germany). These inimuslstion schemes andhybridmna fusions produced the 1D3, 8E12,3GI sad 10.88 mnine auti-CCC antibody molecules, {#4141 Gm&amp;stim of httmtm mAM, XENGMQUSB genetically engineered mice (Abgeniz, Fremont, CA) (8 to 10 weeks old) ware immmdzed for production of hnman monoclonal antibodies. See, Mendez et &amp;L Nature Genetics 15:!dA4Sh (1997), Green sad Jaliobowts /. £xp, Med. 188:483-495 (1998). Several immunization schemes were employed* in one scheme, one hundred anotograms of human. CfC-C extracellular domaixi/tautonlg fusion protein (TOK!07-hIg) w ere suspended ia Dulbecco’s phosphate buffered saline (PBS; GIBCG, Grand Island, NY) and emulsified with m eqttai volume of complete Freund's adjuvant (Sigma Chemical Co., St, Lotus, MO). XMOMQUSEm were immunized by Injection of the emulsion at three subcutaneous sites, base of tail and one mtapsrheiaeai (ip.) site. Fourteen days after the initial immuakatioa, the mtee were given a booster immunization, with 50 pg TGK10?-Mg in breomplete Freund's adjuvant Sera testing indicated insufficient liter so after a few weeks rest, &amp; second booster of 50 pg human TOK.107»Mg was given. Two weeks later» a small amount of blood was collected horn the tail vela and the serum activity against TOK107-Ig was titered by ELISA and against ffI29-<3€C#2 cells by FACS, Mice were selected fer fusion whea their liter exceeded 1:24,300 by ELISA or 1:500 by FACS. Neatly three months after that boost, mice wore boosted with 107 HT-29 #2 cells and the next day boosted with 50 pg Τ0ΚΪ 07-big, both in incomplete Freund's adjuvant A mouse immunized with this scheme produced the 3F9 and the 1D2 human aati-GCC antibody molecules. A mouse immunised with this scheme produced the SF9 and the 1D2 human anti-GCC antibody molecules.

Four days later, the mice were euthanized and spleen cell suspensions were prepared and washed with PBS for fee fusion. The fused cells were tested for production of antibodies which specifically bound to GCC by ELISA for binding TOK107~Mg comparedto a nonGCC antigen or to the Fc region of IgG and by FACS for binding to T84 cells or HT-29 clone #2 cells compared to vector control and compared to non-GCC-expressing MCF-7 cells. Isolype was determined using ELISA or by FACS using IgG or IgM specific secondaty antibodies. A mouse immunized with this .scheme ptodneed the 5F9 and the 1D2 human anti-GCC antibody molecules. IMIS) la another scheme, €HO-GCC#27 cells (5 x 1#) ecanpristog the pkTO$Q 11. vector aad expressing the GCC exftacelMar domain on their surfaces were used as die immunogen two times with two weeks between B JP (base of toil -+ htoaperitoaesi) immunisations. After sampling blood to identify a&amp;ti-GCC reactivity by ELISA against TDK ΙΘ7-!% the mice were boosted with HT-29 GCC #2 cells (either three weeks or more tfaaa two months since the previous boost). Four days after the last boost, the respective spleen was harvested tor cell fusion. The feed cells woe tested for production of antibodies wMch specifically bound to GCC by ELBA fer binding TOKlG7“Mg compared to a noaGCC antigen or to the Fc region of IgG and by FACS tor binding to HT-29 clone #2 cells compared to vector control, 184 cells or non-GCC-expressing MCF“7 cells. Isotype was determined usmgBLISA. These inmtmnmtion schemes «id hyhridoma fusions produced the 5H3,6Η8»8C2,10C1G, 10D3 and I €9 human anti-OC€ antibody molecules.

[9416] Mybri dooms that produce human mAh: Spleen cells were counted and mixed with SP 2/0 myeloma cells (A.TCC No. CBL8-006, Rockville, MX>) that are incapable of secreting either heavy or light chain immunoglobulin chains at a spteen:myeloma ratio of 2:1. Cells were feed with polyethytoe glycol 1450 (ATCC) in 12 96-well tissue culture plates in HAT selection medium, according to standard procedures. Between 10 and 21 days after felon, hyhridoma colonies became visible and culture supernatants were harvested then screened by ELISA and FACS.

[941,71 SLAM isehnotogy-tosed aatlhody genemfi©»: Monocioaal antibodies were also isolated by Abgeaix’s SLAM tecfeology (Babcook etal PNAS 93:7843-7848 (1996))- The initial step in this method was to tmmunme XEN0MGO5E mice «?«» immunised with GCC antigen by a scheme among those described below. Then the SLAM (Selected Lymphocyte Antibody Method) step involves Erst identifying within a large population of lymphoid cells a single lymphocyte that is producing an antibody with a desired specificity or function, and then rescuing ftom that lymphocyte the genetic information that encodes the specificity of the antibody. The variable region of such a lymphocyte (initially producing an IgG2 or 1^34 antibody) is amplified and transferred to a vector bearing the IgG I isotype.

[Ml§J hnmrmkkion seiieines fe the SLAM wtfba#k&amp; (e.g. s Α1χό229, *01 :¾ -221, -(^-338,--106,-108 or -393} included using TOK-hlg or TOK~Mg conjugated to keyhole limpet hemoeyani». Immunizations were either through the foot jades'a axnhmatkn of (lie base of the tail and iiiWpedteeal. 'Hie initial minimisation of 10 gg immunogen inciuded either TITBMLAX® gold adjuvant or complete FreundTadjuvmf Sk to eight boosts with 5 gg of immunogenwere performed* iisotg eMrer Ate, T|PfiSpA3® gold a^uwit m it«$^ete IfTFIEEMAX gold waa the iaitiai adjuvaat, shun was the boost scgavptf «id boosts were peribrmed at 3 to 4 day itefemis. If the initial hoommaatioa aud boosts aosployed eoaiFfetethm meompiete keund’s adjuf smts, mtervals between boosts wet® performed at shoot two week interv als. Sera tests offers at the fourth to sixth boost sometimes were .followed by additional boosts. The final boost prior to harvest fmr days later employed the iminmegen in BBS.

[04 I#j Analysis of mAh by ELISA, Bigh-proteia biadmg 96-well El A plates (CoMgjASuumg, i»e - C&amp;ttmig, NT) were coated with 50 pl/weli of a 2 μ$Μ soluhon 0) J gg/weli) of 1X5KI§?~Mg sad kcobgted overnight #.4¾. 1As excess solution .was aspirated and the plates wore washed, with FB£/0<05% Tween~20 (three times), then bteoked w#r 1% bovine serum alhtimis (BSA, fiaction ¥* Sigma Chemical Co., MO) tbr 1 hr at roemtemperato'e p.T) to kMhh soo-i|iocific binding. The BSA solution was removed and 50 each ksion plate well wem added-

The plates were thmiaeukhed f©r.:4J min. at 32 *fc and washed. three times wdlh FBS/6,0351 Tween-tO, Ifctseradish peroxidase 0RP>ooKjiogaied gwt Mti-mouseo'r a#tb tehattlg© B(afe)2(13^L) paoks^feaearctii Laboratories, Inc·., West Grove PA) dinted 1:4900 ia 1% BSATBS was added to each well mi ken the plates were incubated for 45 min. at %TC,. Aider washing, 50 μΙ/welf of ABTS sointion jTlpsed, South lAaoeiseo,

CA) was added. The Intensity of the green odor of positive w ells at 405 mn was assessed op a Vxsax nucroiitre plate reader (Molecular Devices Chip., Sunnyvale, €A), All hybddmm wdls that gave a posi tiv e tesponse w®e then expanded to 24-well cultures, sabi^.edhy lhmt^ The three heA producing subetones were expanded further. 104201 Analysts of mAh By flow eyPnmetry, plow eytomelry (FACS) setting was done on all the fusion plate sap ernatanis in pandlel to the 1L1SA seswening. BT-29 clone #2 or tinPnnsfeeted MT--29 cells ww grown in T22S Basks (Costar/Coming, tux*

Coining, NY) in DMBM (CSBCO) supplemented 10% fetal bovine aecum (OIBCO). Cells were detached ism the flask surface using Versene (G1BC0), collected and washed twice wMt DMBM, then wee with 1% BSA/PBS solution. The cells wexere-stupended in 1% BSA/PBS and 2x10s cells were added to each well of Y~boitomed 96-well plates (Costar) md centrifuged for 5 min, at 2500 EPM (wash). The wash solution was discarded and 50 μΙ/weil of supern atant from each felon plate well wash added A plate sealer (Lmbm/MF Biomedicals, LL€S Solos, OH) was applied and the plates were then gently vortexed to resuspend and mix the cells with the supernatants and meubated at 4*0 (on Ice) for 30 mim The plates were then washed with cold 1% BSA/PBS (three times) and 50 μί/well FTTC-eeojugafed donkey anti-mouse IgG F(Ah)2 (H&amp;L) or FITC-conjugated goal anti-human igG F(Ab)2 (H&amp;L) (Jackson) diluted .1 ;50 was added to each well for 30 ma at 4°C (on ice in dark). The plates worn again washed three times in cold 1 % BSA/PBS and fixed In cold 1% paraformaldehyde (Signm)/PBS, The cells were transferred to cluster tubes (Cosxar) md analyzed mi a FACSatiibur flow cytontteer (Bectoa Dickenson, San Jose, CA), Any hyhridoma wells that showed a. positive shift were then expanded to 24-well cultures, subcloned by limiting dilution.

[04211 foternalfeatlon assay, Memalizatkm of anii-GCC antibody molecules was tested in both GCC»expresskg cells and vector control cells, using immtMofluorescence microscopy. Cells were grow» on coverslips and placed on lee for 10 minutes prior to incubation with 10 ggritl antibody in cold culture medium for 20 minutes on ice. For internalization, antibody-containing medium was replaced with fresh culture medium and the cells were shifted to 37°€ for 2-3 hours or maintained m ice.

After rinsing m PBS md a brief fixation in 4% paraformaldehyde at room temperature, cells were permeabifteed for 15 min in 0/5% TRITON X-100. The localization of the test antibody was determined using a ftoeseently labeled miti-IgG antibody by laser scanning coafoed microscopy. Antibody molecules localized to the cell surface of GCC-cxpsssing cells when on ice. Upon incubation at 37*0,5F9 showed punctuate staining within the cell membrane, indicative of iitiemdizatfon. No intekteation was detected with, vector cells.

[0422} Summary of Properties of Antl-GCC Antibody Meleewfes. Most of the antibodies generated herein were tested in a number of the assays described above.

Table ? summarizes the in vitm properties for each. (T84«humaa colon tumor cells, MCFT^hamaai bamst tumor cells, WB^westem btot, nHhmBaopsre^itstkjn, iat®aslfeatIosiised T84 ©sils compared to MCF~? ceils)

||423I AikKtioBslly, some tested for foe&amp;dMhty fo isMbitiie ST pepildd^dcced calefom ion flux in <KXi*^«p«ssssg.:c#Si - The cCIMP assay was performed hi HT20~GC€# 1S cells in the presence of 50 nM ST in the presence or absence ofaafo-GCC antibody motedes, There Ion

Bm by 5F9, Other antibodfos, SH3 and Abx-338 also hddbitedfos caksum Ion flax aKfocedbySi; pM$4 j Estimate of relative afinlty of astiGCC antibody «lateenles. The relative afSeldds fEC-50; antibody eoiicentrarkm for half maxima? binding) of some anti" OCC aailbody nxdeenles Were esdmaied fism 1LISA mcesuremeids against TOK107-Mg and by FACS jne^sat^ments with OC&amp;es|?r8Ssiag c&amp; The following table displays some reSelts» T»ble|, ECSD of aatl-GCC antibody meleettte

jl425| Measurement of aiSidty of antl-GCC antibody malectoes* A BIACORE™ '1100 system (GE Healthcare, Piseataway, NJ) was used to measure pie affinity of ssdi~G€C 5Iffi antibody at 22¾.

[IWI Step !; MAb 5P9(Prep A) was diluted to 20 pg/mL ie lO mMspdiimi acetate, pH 4,0 andltefkenee SF9 MAb ψΐβρ 3) was dilated to IP |ig0sf to 30 mM sodiuni acetate., pH 4M Each mAh was coamtetely ionsbh&amp;ed to several €M4 BIACORE chips using standard amine coupling. For each CM4 chip prepared, Imp A

two-flaw c^:if«totto4:?54(K) RtJ while Prep B 5E9 was iMnohilfeed to cne flow cell at arotmd 7$~80 EfJ, The m«iammg fourth Sow ceil of each €M4 chip was used as the reference SowHcelL

[0427} Step2: The stock runcmtralfoaofGCCffiClT.Fe (FOK.Hl?-hIg)was deteimmed using the methods derailedfey FaeeteM. in Protein Science, 4:2411 0995), and Face a«d Citimsiey rn Gurrmt Pmmmis in Mgtein Science 3. U -3.1 <9 (2003).

[0421] Step 3: For each prepared CM4 chip described la Step I, GCC-ECD-Fc was irpeett^ fer 2 minutes at a concentration range of 202 nM ~ 1,6 nM (2x serial dilution) followed by a 7 minute dissoctetion, Samples were randomly injected in triplicate with sevjsml baiter tojcgffttf&amp;sgmed $>f ;d0«ble tetot^teg, To otoamomtehigmSctoit ai&amp;tm decay data, three additional 101 nM OCC- ECD-Fc mf^doas andSiree additipna] buffer flections were performed with a 2 minnte mjectfoa and a 4 bour diteoekioa fhne, A Sow mte trf 100 pL/mte was used for ad experiments and all arrfaces were regeoemfed with a 20 mvnd pulse of lOmM Diyeine-HCi (pH 2,0), All samples wereptepred in the bmniag buffer· which was Hs|»^uf&amp;toi^'.is^iae* 0,005% polysorfeate 20, pH 7,4 (I3BS-I) with iOOp^/mL ofB$A added. (9429} Step 4: Alisps^^^C#!^ wem pmeesaadvteto Swabber 2 j software (BioLogfe Software, C2amphel| Attetimlia) and globally htte a I; f iteemcion: model inchidtog a term tor the mass tratispft constant 1¾¾ using CIAMFm software {Mysdea andMortoa Trends Bioehmn. $eit 23 ft 49» 150 (1998)). 11430} Ihe 1; 1 model pamded a very good, fit to the date as long as the mAh jumohilkation labels were kept low' enough so that tec 1W «waiting from the sltibal saalysls of the senscsgrata data was at least below 12 RO for each surface. la most eases» one of the two Prep A §F9 surfaces had an too low (below 2 RX3) for reliable kmefie measurements,. Data from two Sow cells of G€€-E€D~Pe binding to Peep A 5F9 item the same CM4 chip were simultaneously St whenever possible, however. When mAb sscfeces were prepared resalting m a higher R®ax{> 12 RU), sensorgmms steady showed complex kisedes sad thus a 1:1 model fit file date poorly. This MfT sarprisiag doe to tie fact that GCC«R€D*Fc Is a bivalent coastmei aad a. higher surface density of immobilised raAb most likely increases the probability that the GOO~ECB-Fc binds avidly to fire stirfiee. Replicates reported for this study iociy.de only those .data that it wall to the 1; 1 interaction model. The resulting Ku’s and rate constants of nil replicates for Prep A 5F9 sad the Prep B reference .mAh are listed inTable 9 aad Table 10» respectively.

j®43.1| Maytansines, Mouse aoix~h«msa(MAH)“!gG“DMl, and anii-GCC-DMl were generated according to a one-step process for the production of cytotoxic conjugates of maytansmoids as described in XJ.S. Patent No, 6.441163. |0432f Briefly, maytarinoids were conjugated to antibodies using published piocedureSv DM1 was· conjugated to antibodies using SMCC heterobifim&amp;ionai crosslinker (sncdoiOTit}d-4-(N-mald»idoTnetby!)cyclobexane~lHiarboxylMe; Chari et al Cmeer Research 52:127-13.1 (1992). DM4 was ««^ugstedto eaisbodiesnssig SP.DB heterobifuaetioaai crossliuker (Wtddison ct al. J. Med Ckem, 49:4392-4408 (2006)).

Conjugated antibody is separated from unreacted reaction by-products by gel filtration chromatography «sing a SBPHADEX™ 0-25 column. |0433| Anristetios, Conjugation by auristadns can be performed using published preeedures (e.g., Domnina ei ei„ Nature Biotech., 21: 778-784 (2003)). In general, anristatins are linked to cysteines of antibody chains. Linkage to cysteines is ’ accomplished first by reduction of disulfide bonds in the antibody mo iecufc. Control of the redaction process seeks to limit the reduction to some, bat not necessarily all* interchain disulfide bonds. Consequently, auristadns are able to bind, at the free cysteines.

Quenching of the conjugation reaction is followed by removal of reaction byproducts and buffer exchange to the desired formulation, |iN34j la kief, an aafi-GCC antibody molecule at 7,6 mg/mL is pre-equilibrated at 37*0, and then a 15% volume of500 mM sodium horato, pH S J is added to .raise the pH to 7,5-8.0. The solution also amtains 1 ®M DTPA, Ihe antibody h partially reduced by adding 2,6 equivalents of .tm(2--cari>axyetliyl^bo^hine (TCBP) per mole of anri-OCC antibody molecule and stirring at 37 °€, After 2S mtnutes, the solution of reduced anii~ GCC antibody molecule is placed on ice, then treated immediacy with 4.8-4.9 molar equivalents (relative to anri-GCC antibody molecule) of drug linker (e.g.s mc-vo-MMAF ormc-vc-MMAE or mc-MMAF) as a 20.5 .mM sohdton in DMSO. Additional DMSG is iutmdnced to bring the nikture to 10% DMSO by volume, The reaction mixture is sited e® ice for -90 minutes before hvahncnrwiih a 5~fe!d molar excess olN-naVyl cysteine (relative to mc-ve~MMAF). lie conjugate is isolatedby tangential Sow filtration, first being couetpitstM to ~!0 tiegfixL* teodiafil toted w8h -40 dfeslumes o£FB&amp; Hfc resulting atebodSy drug conjugates bad an average

'uni^::^-t^e#*-'Fcr.«mvem«as^.iiii:ll^ Mlowmg Ixamf lesmd attached Figures, antistate iimnmoconjugates are refers to in the fhliewasg tibrevsatM format,, mespeottve of drag iotkhhg: teb-wcfMMAF” refers to M atte<3CXi antibody teteeule amjugaied with mo-vc-hfeteFr^h-vc^MMAE” wfem to m anti-GCC antibody molecule cosjugated with mcwc~MMAE; and telvmc~MMAFs’ refers to an ate-GCC tetbody molecule <»t§ogated with mc-MMAP Jmni.tmoco^iigate® emnpristng specific anti-GClC antibody molecules are refeed to &amp; the same ibrmal, .e.g, 5F9-VC-MMAF, 5F9-vc-ΜΜΑΒ» aad5F9mto- MMAE |#435| To prepare asibody drug conjugates with an average drug loading of about two dmg-tinte units pc? afeody, the pmtoso! (abovej is tsodiged by reducing the fefet ofTCEF by 50%, The amount of drug Imker is also reduced by 50%, The corr «spending aahbody drag om^ugsife ig'ablifmtfe^/ltstAl^MMAi^).

ifewmfei &amp;f$0 vcMMME |943d| Using a method similar to the general method set forth above, the 5F9 mAh was conjugated to an. aimsiatin derivative designated MMAB {Pemada (Mil)} using a vc f^aMStj hufe described herein to create die imsnuiocenjugate designated SF9 voMMAE. The conjugation ofthe vc linker to MMAB (Seattle Genetics, Inc.» BiAheil* WA) was complied as previously d^crlibed' ·· [M37j Briefly a 17,8 mg/mL solution of the SF9 mAh in 100 mM acetate at pH 5,8 was adjimted fe pH I with 0.3 M sodium phosphate dibasic» yielding a final mAh concentration of 11,3 ®g/ml Then, DTPA was acted for a I niM feal concentration irr the reactfemifem. The mAb was then partially reduced by adding 2.28 molar epk]^hs'0fir{3?; (relative to moles ofm Afe), and the® ..steed at 39¾ far 1.5 hours.

The partially mducad niAb solution was then coded to 4¾ wrd 4.4 molar equivalents of voMMAB (mlative to moles of antibody) were added as a 303 rnM solution in DM$0.

The mixfe was stirred for 30 minutes &amp;%%% the® Tor 15 additional minutes iMJowmg the addhionof 5 feiar equivalents of N^aeetyleystesm (relative to moles of vcMMAB),

Excess qnemised tcMMAE and other reaction osmposeaismrs {m^vedlsy Mfti^fesib^diafitodonofilis homn&amp;ocoejngaie with 10 diavokmesofPSS,fsB 7,4. "Hie i^plf&amp;g immunoeoojegate was designated $F9 veMMAE and has the following Ibrpmia:

where Ab Is the 509 mAh, and m is fims I to 8. The ayersge ^rag lcs>admg (k) was ahoat 3,6. f§43Sj Cytotoxic! iy assays* To measure mk aB^body^· ability1' to bind, iwtemi&amp;s m4MI target expmsskg cells, cytotoxicity assays were performed. In this assay, cells were incubated with, various confutations of the imconpgatsd primary aniKKX antibody and a fixed non-toxic co^cestratkm Eo secondary aatl tody{icifeect cytotoxicity) or with various ecmcesteatioas of fexia ecmjugaied ash-OCC mAh (direct cytofokeity). CetlViahiBiy was measured by WST sssay after 4 days iscabatioo, The mlaiive ixitericy of harm» hoiMiCC aaftbodies on 293"C|CC#2 cells is shown in Table $ mifWM defenbBed teng a DM! conjugated mouse tei-lmum Ig<3 mAh (MAtMgft was ptefied horn clone HP607(CRL 1753, ATC€). 509 and 229 arc the with LP59f s of 26 sod 71 f M, Although, cot shown tee, ihs error is gcmmfty within 29% of these averages, m tas^ur^:%iMge»^iss|#ceisa,«r standard: dteation of > 2 replicates.

P439J Cell sarlac® binding* Binding of uaconj ugaied 5F9 of SF9 conjugated to anBsteks, was evaluated by in indirect ijiaBmioSuOTescence assay using flow cytofeeity> 3 X 19,f eetei well were plated teaVdteiotn ψ$ well plate and teeubated on lee far 1 hoar wife serial anybody dilutions of l~0Ml ggktl Cels w«'e washed iwiee wife 3% ESS1«. fee cold PBS and incubated wife 1;200' inouse kfedsutmn 1¾ lgG{So«ihem Biotech 2®43-§9) for 1 hoar on ice. Cells were washed again andanalyzed by flow cytenetry on a 80 FACS Canto 11 Bow cymometer. Data was analysed «sing FACS Canto ΙΪ system soiware andmean inoresceaee i»teasMes%¥«redetemifeed. $1440] Eplfefo mapping. Multiple sfeafegfes were initiated to i&amp;nii$ epitopes.

Fepddeairaw ovq^ware generated, tpptiing fee: esiraceifelar domain (ECO) into fee iraossiienfeTOe domainof GCC (aa 1440) BepMes were synfeeslzed and provided as «rays,, inimofeilfeed on glass slid·®, &amp;m$&amp; were. hybrfdbMwife eaebpf fee aafeClCCbmfebGdiea to determine if linear peptides were sufficient for fending. Ate-198 bound peptides 55 and 56» while 3GI} 8F1 and 10B8 antibodies board peptides 55,56 and 5?. The sequence, ILVDISMH^ (SEQ 1DN0;225) to spanned by fee

Aregionofoverlapamong the peptideskllSWlHIY {SEPTOHO:3I4|. Abx-012, AfexG38 and Abx-106 bound peptides 71 and 72. lie sequence, FAHAFRmTOeTDGFVTLDDWQDVYSEDIB HQ;22% and the sequeaoe PM.TFEO¥DGF¥TLD (SEQ ID M5:315) euedaps the twn j^ddes.

[S44i| Cel snrfk* Mnding to GCC temmtjm· mntsnito Truncation mutants of tie GCC BCD wets generated (FL taatore peptide sad 8imie#ona (Al-32, MA% Αϊ-94 A1-I2S, Δ1-177, Δ1-224 Δ1-279, A1-229 ami A1-379), as FLAG tagged constructs tp>FLAG-CMV“3)iiepteseniag apptoximately 50 as deletion iaoiemeots. Constructs were expressed in 293 cells, followed by issaaaoprectpladloa by dte aaSGCC satiisody molecule and Western blotting for the BLAG epitope in lysates of 291 eeiisirausfevtsd with lie GCC BOD mutants. Aadbo# 5F9 binds eetts wx&amp; &amp;e Al^-BiPtahioit» ladpcd cells with the Δ.1-49 motatloa The balding of 50 to GCC was lost when the protein is truncated between j* 33-50 snggeking that this region is involved k the tecogwtioa of 5F9 to its bkdlag epitope on GCC. However, since the rat and moose GCC sequences are ideatidal to human GCC la this region, aad $F§ does not hind mouse or tafc GCC, 5F9 antibody likely Mi# a eoofbdnattoM! -tfee of amino acids 33 to 30 of Imam GCC. EX4MJPBE 2, Toith-ilh her selection / ADC characterisation [0442J I» so mdbody thug eosifugate (ADf^krafegy, &amp; coaingkionof highly potent toxins to anisbodi es, the cytotoxicity of the toxin can be directed to tenors k a targeBspeaic manner, delivering the toxin to ^Ed%i^:a^a^ngti^&amp;r pells without aiuotkg antigen negadrto oeilxin normal tissues fe reducing syMemto toxicity.

Auristsfe (analog of dolastatk !0) and nu^tesfee class toxins were evaluated as-ADCs with aatl-GCC mAh. tlsse toxins am all yWktok'of'mimshtlndppoljanerkatto^ acting as ankiniidties. Tests in which ceils were rxs&amp;tacted with freetoxka indicated fhai the cytotoxicity of free toxins with GCC expression and. control cells without GCC, These fee toxins were potent against the 293-vector, 293-GCG#2 cells Jo Hf 29-vecier vs, BT29-GCm cells as stewakMIs 9.

10443} Th^ ^ am €^ty ia^ateai So aatifeodi©» differs sadaf&amp;&amp;lixfer sra^lip .Linker stability affects fee therapeutic window by iaftacimg drag teiessesn the fcnipr tissues m dr^^ ideal AiJG lir^er has Mgh stability while in fee hbod> but efficient release upon target mediated eel! entry-Aunstaims fW44} wet^er-ev^iiat^U. To'iM ev&amp;loaie these conjugates in vim and then determine which toxiudinkcr for large scale in hvo studies, 5F9 was a>nj «gated to vcMMAB, vcMMAF and. mcMMAF £2§mgs pet con|«gste). 11445} Atmhaffnd are synthetic toxins related to tie natural product dolastatin 10. MMAE and MMAF dil^sr subtly, wife fee MMAF: ffen baying a carboxylic sold group in fee R2 position* reducing eeE penneabiity and potency m free toxin. MMAE is a Pgp drug pursp substrate, while MMAF is not immi Auriha&amp;s are eonjugated to fcfessd^ hpiocess of partial antibody i-eduetlon, reaction wife a nmleiniMo drug dedwdiyej quenching with excess cysteine, coueeutration end bnffer exchange into PBS, The aoristatfes caa be ao&amp;ifeed wt# aeafeepsfeB smsMw dipepiide Suker, which is cellular uptake* m wife anoncfceysble linker, P44?j la the vc^aoMetMAltffststfe linker, ayaMue ditmllitie dipejfede Irnkage &amp; attached to the iteg dimr^h a p~araa» ben^^ aud io fee ao.ti.bedy through a caproyl conjugation group. Upon ktemlizatiou, the dipeptide ifefeer is deaved by the lysosomal protease cabiepsln B, the PAB group self destraeis, and free toxin is released. ThisitBibsr stpiiKfy while maxirammg intracellular drag release by cafhepslu B. I044i| Aumtatias can also be linked to antibodies through noncleavabfe linkers

such as MMAF directly attached to the amleimido ronmgation group, with no peptidase sensitiveIrofeer, MC eonjttgated ADC's are also efehive at target mediated ceil MIL 11440! The raedrapisro of drug release for noncteavable auristatln conjugates is ileughi to be through general antibody degradation la 'fysommm. Ίψ$νφϊ$€/Μ$ Msdies it has be® reported that AboncMMAF copjgg^es release toxin in the form of a single c^teine-addact,

Mkg.og|id^^ |044®!' All of the 5F9 antibody drug «ipingates lioirod to 293-GCC#2 cells equally well. Table 10 shows the mean tluoresceace tetensity of 5F9 eonittgates at increasing concentrations 293 -GC€M cells. Ote studies detemimed that ti^.|prSPDB-Difsl4' coipgate bound 293~G€C#2 cells te a cooceateadorodspc®dmt 'Mamet* whereas the 209-SFDB-.DM4 antibody did not bind. 1¾Me 13. Chart ofMFls front binding assay of SF9 and 5F9 conjugates I» 293 6CC #2 cells;

IMP! 1¼. direet $ytefo*»s|!| imp of a Tsrk^f ofodis which been transfected with GCC nucleic acid arid selected for a^passioa of GCC. A survey of foe level of sfofoce expression of 0(¾ found foal 293 G€C#2 cellsexpress htgb asaodntsof GCC; EDD 29 #2 ,a»dCT MM3 cells e*press GOD at tomtelB low levd^.:'CT 26 #32 cells express GCC at high levels; andBT 29 GGC #5 mi HI 29 GOG #18 express low amounts of GCC. Table II shows a cornpiUthn of oiidfode sfodiei? yielding cytotoxicity data for foe foree «foafo&amp; eofo^ates Its. cells eapressfog target, or m wild-type cells or vector control cells- Tanget enhancedfoiling is observed m all cases of on 293 GCC #2 cells, vdfo a gmslly increased window when using MMAf vs. MM All. Both foe cleavable and noncteavabis forms of MMAF were similarly potent As a negative control antibody drug conjugates were also made with sc2<39 antibody, which is a tanaa I|G! foooodoaa! mibodyralsed against an «arefated target, wife no reacdvity to GCC, Direct cytofoxicify assays with 209 ADCs vs. 5F9 vcMMAF ADC showed forget enhanced cell foil m foe 293 cell model and the HT29 sell model, A comparisoa of SF9fo0tt|agaied toxin aobviiy amat^ foe ceil lines indicates AM the level Of cyloioximty has some con'd anon with foe amount of GCC pressed by the cells* These data sag^stfost at least some cell linos sxpmssfog foe noost GCC were mote ^scqfoblo to the cytotoxic aebvity of foe conjugate tban were celfe e^fosstt^fowexiusisunfo of GCC. See Example 1 forreladve GGC me&amp;m per celL Aaofoer factor in fotYermeesof cytotoxicity levels may be ditTemoces in inla-naifoation or ii^eup^ltiler which isay y^:-am(a%:mM'f^pe' a® linen.

Tabfold. Cyfoforfoity of aaiTGCC afodsfotte ADCs.

{0452| If&amp;ese peim0&amp; femskie m vim, me and 'V'cMMAF, the Mgh^pr^iGte#;MTD for moMMAF would suggest a te^er kerapeatic window for this conjugate, M&amp;ymminm i®453{ 2%>5F9-ma>4imiae ADCs showed potek tkfk eali«sioed Idllkg itt pe. 293»GC^i^ ^{sfFabie 12). fetreestisgly, the 3F9-®a^ssme conjugates both showed iarge&amp;^fc»^ MII^ffiifee293t«oisi9 Jd’Iti&amp;s ·ν*^·©^«©^ί?«^' ftt toe FFF29 teodoi» raising concern alsoutihe o^ky of tom inodd tor k vivo evkoatom of if® coigygaies:. Again, the reasons fef this toffatenee are not readily explicable* kd re«y fee khibuied to difierent recep tor densities and/or dipsMces: itt the iotemaikadott or preceding of the eosgagates within ceils. As s negative cokrol, antibody drug conjugates were also Matte with sc209 antibody,· which is a· human Ig<H monoclonal antibody to m unrelated target, withnO'macriyiiyto G€€. B&amp;eet cj'tototeeity assays with 2# ABCs m 5F9 ABCa show target enhanced cell MU hi the 293 ceil model with all 5F9 conjugates, la the HT29 cell humM, 5F9-DMx emsjugates and 209-DMx coafagaies Mi! eqaally well. Indicating a non-target specific niedtanlsm of kilikg In those cells.

EXAMPLE 3? In vim evaMMldn |Θ4$4| Is vivo eytofciteeily of SF9 ABCs were evaluated ia mouse xenograft models. The initial in vim work was done with the IiT29AKXl#5 and #18 cell tines. The 293-0(1012 cell line also was lasted for in vivo growth and was developed m a serially transplantable trocar model |M§§1 To address the question ofwhether the G€€ expression; level in the xenograftmodel was relevant to the level of GCC expression &amp; patients with metastatic csoloacssieer» GCC exjaessten levels were eftmp&amp;t'ed by IHC analyses of xenograft tissue, human jsimary colon tutuo» and metasiases, A panel of fresh. Ilmen ceil lines sad tissues were utilised for GGC quantitation by iIHC with.a mouse mAh to GOC 3G1, For .IHC qaaatltatlon, scoring was done -using a senu-qnnntlmhve 0-3 score system. If tamer model GOC levels < dmieal GCC levels, modeling wifi likely he accurate or overestimate the exposure needed clinically, If tumor model GCC levels > drum! GCC levels, modeling may naderestimate the exposure needed clinically, fMSSjj "While· there was mm variability m the expression of GCC hi metastatic samples»·expression hy the HT29-G€C#5 and #18 ceils was in the range of many of the metastatic samples. By IHC, 'the staining of GCC on the HT20~GCG#5 and #18 cells was equivalent or lower than on the melastade cells. This data, suggests that ear tumor models express GCC at levels comparable to the le vels found In clinical sampler of metCilC, [9457) Table 1 § represents the soiatilktlon counts for various tissues harvested at the 192 hour timepoint with averages for three aniomls represented, SF9 preferentially aeemmdatsd miiT29~G€C#5 tumors vs, Hl'29-vectorinmom, while 209 did not show much diffe-ential accumulation. This result provided support that a SF9 antibody drug conjugates could be expected to -accumulate is GCC expressing tumors. fe all other tissues evaluated» there was little difference in the levels of 5F9 vs, mAh 209 antibody accumulation.

In vivo distribution ofmdiokhekd SF9 in HT29~@€€U5 if HT29~mmr mm&amp;r-hearmg mice f§45§f A mdiotmagisg shidy in tumor bearing mice was performed to evaluate tumor targeting and m vivo hiodistrihution of the anti-GCC antibody 5F9 and a negative control antibody se-209 {human IgGI ntonocional antibody tergetmg an unrelated cell Surface target). The antibodies were radiolabeled with J35In using DTPA m a hifunetlonal chelator. The In vim behavior, including tumor iargeting and biodisirlhotiou is normal tissues overtime, was investigated with a murine dual-tumor model with both GC€{») usd GCG^temote mtohfef vms tw$p αφρΙοα^&amp;φ^ msolteon. P4S9| Saboteneoas tenors were grotto m'&amp;ude mice* wife 1029wector tomom oa the right md OT29~GCC#5 tern® on the M. Aat&amp;ottes .w®re.-it«s^'ei'43.iHa «15 ggjte gahmi. 11«i»teereaa^ wemhanrestodat 1¾. 2¾ 4th, 72h:s iaO&amp; ami 15>2h. fteh®] A survey of tsssuss from animals- in the !92fe group indicated that both 5F9 gad 209 seannulaied to a similar degree in most normal tissues (e.g.> blood, heart, stomach, small miestkec large intestine, muscle ate skk) and lit ΗΓ29·· vector control txmm,: Afe 209goosmula^dle sillily feiglier l«TCls tfeaa 5F9 in liver md 5F9 itetmteatod to slightly higher levels than 209 in hmgs, spleen sad kidneys, la the BT29-8€C#5 tetets, 50 p^eteally acestedatei at level more tern twofold higfeerfh® tie 209 levels. Tins mtehpbviied support that. s 5F9 arstibody dmg coajagatos coaid fee expected to aeeemtede m GOG exp^ipg htetek f$te i 1 To' yaderstmd tie kmeties temhltody teeumuktios is tenors, tenor data for each antibody was obtained for all lime points throughout the study. The only hsste tfest stewed acctomiistiort is tfee 30 antibody in tise GCC mpmssiag teste. The mditeciMty level mall ether tissues remained rdahvejy fte, with little difference between the 50 levels md 20 levels. 50 pteetoteaOy ecateti&amp;ted m ΗΤ2ΜΪΟΟ#5 tumors, syhile 209 did not stew soy accumulation, This result provided support that a 5F9 »^bo%:-#Ea®::Oi»gttgtes:tewW be expected to aoeuptetes in OGC tursoa

Table 11 Aeatmutotta of mfe~M}gted €€€ specific isAb but mt central mAh % t^om^prsssital <3CC1

|§442| Aecmuukisoa of radiolabeled 5F9 in QCC expressing tumors over '7 days supported a once weekly dosing schedule.

Pitot effime?studies m :ΕΏ9~ΰ€€#5 trnncm |$443| Safe were performed to deimame efficacious conjugates <md dosage repmetss mmioefefeto£ili29A3€^^ single or multiple #ses. fesestofeferfeedfetlKireTO^^ levels of toym feugate (e.g., ISO ug/kg 3P9ycMMAF oa a q3d x 5 schedule}, Another study dstOTi&amp;ed fe ql4d s 5 schedule was too mfeuent in this model to allow some to&amp;ia coafugates io show sigoificaat efficacy vs, emtools., A#ib«mfe a PD study with mayimmitmid-aaubfe cotyugfe m this model demcmstmted doserfepemlmt fesphoMitoie accmaulafe with the DM# fofefeiheBMl toxin. Another study ia ttos model staved some tomor growth febMoa by tbemm-'Specilic 209 ~4oxia conjugate. These results suggested that other la vivo models as## to be evaluated. PM/PD ntu^wUk SF9 ADCs in mice carrying Μ3-ΰ€€$2 tumors. 14444) Aa alternative tumor model as# 293-Q€€#2 ceils. A PD study for 5P9 ABCs in 293-430(¾ tumor beafthgimee was perttouoi MiM wsue dosed with slagld doses of SJfeMMAF at 75ug/kg or ISOug/kg a# mttmms takes at tlfeomto ftom. 1 Hr through 4 lays ihr PD aaslysls of yltosplioMstone F13 to test aaatmtetie effects of the toxia ou tumor cells, Phospbo-Msioae M3 was detected % antibody (Upstate

Bioiedffiofogy, sow Mi!lipaisf Billerica, MA) staining of paraffin embedded sections of tumors. Hie dais in Table 17 shows that each of the ADCs caused a significant increase m the pH3 positive cell population m the teas indicating that each of them at both 75 and 150 pg/kg toxin dose equivalents was able to teach the tamos» and have the desired anti-mitotic effect on the tumor cells.

Table 17» The PD response as assessed by arrest of c«Bs 1« mitosis (% j»H3 positive tumor eels) fellowlng a single lv dose of 5FS ABCs,

(04S5) Similar studies measured the phosphoMskme levels iti 29343€€#2 tumor-bearing mm tested with 5F9v»MMAFf SFMFDMM4 and 5F9«SMCC4>M1. Mice were dosed with single doses at. ISOug&amp;g and serum was taken at tlmepoists fiom lhr through 21 days for P&amp;,analysis of feoife fetal antibody and ioxm-ccmjugsted antibody. The percentage of phnspbdmtoae B3 positive cells in 293~GCC#2 susots· inarmed in, refuse to ail three ADCS: 5F9vcMMAF, 5F9~SMC€-BM1 and 5F9-SFDB~DM4, Maximal phospbohlsione H3 levels -were 3- to 5-Md increased over· baseline, with peaks at 24 hours post~mjectiua £βααψ$ίΜφ with- SF9wMMAFmd 5F9-1ML·in 293~GCCm sx. nmors

IM&amp;$1 5F9~SFDB»DM4, 5F9-=SMCC-DM1 and 5F9vcMMAF were tested for efficacy m the 293-GCC#2 tumor model at two doses (75 ,»g/kg and 150 pg /kg toxin), on a q!4d % 5 schedule, Specifically, this study included veMcteteated control, Sc209~DMl (150 pg/kgDMl eq}? SC209-DM4 (150 pg%DM4 eq)5 So209~vcMMAF(iS0 jtgfcg-MMAF eq), SF9-DM1 (150 DM! eq)> 5F9“BMi (75 gg/kg DM1 eq)t 5F9-DM4 (ISO gg/kg DM4 eg), 5F9-DM4 (75 pg/kg DM4 eg), 5F9~vcMMAF (ISO pg/kg MMAF eg), asd 5F9-vcMMAF (75 pg/kg MMAP eq>. Taeomefemates mice heating 293»OC€#2 cells (10 mice per group) were used fMS?| Figure I deplete tamer grawtifc in 293»GHX#2 bearing SCI0 m&amp;e treated with 5F9vc*MMAFs *DMl, sad, «DM4 on a ql4d schedule. Dose-dependent efficacy was observed with 5F9-SPDB-DM4 k the 293~Q€€m model, while the 209-SPDB43M4 control had -as ©fleet SF9~SM€€~DM1 was also eileacioas, however less so than 5F9-SFDB~DM4 at ISCteg/kg, 5F9veMMAP (?5pg$g and !5tog%g) was the most effieackiia, however 209vcMMAF also had some activity, Themfure at these doses md schedule, SF9~SPDB-DM4 had the greatest efficacious differential from its control conjugate. .Efficacy study with 5F9vcMMAF md SF9~DMx in 293~GC€%2 s.c. timmt [i4dSI 5MFDB-DM4 and 5F9-SMCC-DMI were tested for efficacy k the 293-8C€#2 tumor model at two doses (75a$% aad 156ug/kg toxin) oa &amp; q7d x 5 schedule. Specifically» this study included veMde-trealed control, 5F9 alone (15 rag/kgk DM! (366 gg/kg>, DM4 ¢380 j*g%), Sc2fi9-DM1 (156 gg%DMi eq), Sc209-BM4 (150 ng/kg DM4 eq), Sc269~wMMAF (150 gg'kgMMAF eq}, SF9-DM1 (150 p.g/fcgBMl eg), 5F9~ DM1 (75 gg/kgDM1 eq), 5F9-BM4 (ISO gg/kg DM4eq), sad5F9-DM4 (75 pgfkg DM4 sq), Tannic females mice hearing 293~GC€#2 ceils (10 mice per group) were ased.

Efficacy study with atmstadn conjugates in 293 (1C€M tenors |04i9J 293 6CC42 mmor-bearing SOD mice was treated with SF9 conjugates with vc MMAE, vcMMAF or mcMMAF at three doses k comparison with 209 conjugates of these toxins or with fee® toxins or vehicle control. Doses were administered iv ο» a q7d x 4 schedule, Tumors wet© harvested at days 3, % 10,13 and 17, The tumors in mice tested with control reagents demonstrated a continual increase m volume, Inmorn tested with. 3F9 atrrktalin conjugates showed dose- md time-dependent Inhibition of tills tamo* growth. Table IB .provides a stktaiaty of the mstilis CTGFkimnr growth iuhiliMon, T/C^«ateeai/<mtei, TGDto;ra.«f growth delay, CR/PR=complete mspoase/partial response; ;p vnlae^neasuie to j udge statistical significance, NS-not sigmfieantj.

Tafefe 18. Analysis »f Amhi^tin ABCs m 293 QCCM mmQr-b&amp;rmg sake,

|Wtf All three of toe ADCs were effitoms m the 293 G€€#2 mode! m&amp;qU schedule. 5F9-eeMi\14F md 5F9-eicMMAF ere store potest to 5F9weMMAE<, wMch eorreteies with m viw PD (pHM13> asd in vitro· eyioiokdty data, Εβοαψ study with SFVvcMMAf and SI^-lWx in T84 s<e, tumors- [0471] 5F9-SFDB*DM4 and 5F9~SMCC~DM 1 were tested for efficacy k the T84 tumor model si two doses (7$ug/kg aad ISOeg/kg toxin) oa a g'7d x 5 schedule,

SpedHealty, this study included vehide-tre&amp;tcd control,, 5P9 alone (15 :mg%g)s DM1 (300 gg/kg}9 DM4 (300 jigftg), Sc20M>MI (150 ggdcgDMl eq), Sc20M>M4 (150 gg% mm Sm&amp;mMMM {.150 5F9-DM1(150 pg/kgBMi «&amp; 5F9- DM1 (75 jig&amp;gDMl eq), 5EADM40S0 gg%DM4 eq), aad 5F9-BM4 (75 ggrirg 0M4 eq). Taeomc females mice bearing T84 cells (10 mice per grwp) wemiised

ImmumhMmkemistry |i4?2| Bkecrioa and measwsment : of relative amounts o f O CC in tissues- qacfe as bicgses andxenogrA east be periamedby immimoWstocOeaiisfey, Flosses tissue sections am Saed in a 1:1 solution of acetone imdmeihanol for 20mik> atroom tmiperatere. Slides am -washed in 1 x PBS for 5 minutes. Slides ate processed with m automatic staining de?ieeypsb as Ventans KseoveryTCI1 mtonated stainer (Ventea Medieal Systems, Toes*®, A2f using mannfteumk suggested, reaction buffet. AntK3€€ antibodies are diluted tod pg/mi to. 5?Agoat serusi* &amp;g«, 5F9, delecting secondary antibody is a 1:500 sohfeon of goat anti-human biotinylated antibody in Date Bluteia Ido# (D|ko, Carpinteria, CA), After autestsiner process the antibody inaction* the slides ate removed from the aiUostemer, rinsed in reaction buffer, dehydrated through standard series untiD3ty!en»^4j^vm..c©t^csitl|iS- w&amp;fr mqundng media,

Efflmoy study in upmmry tumor model {04731 Models of primary colorectal oareinoma and gastric cancer are developed as subcutaneous toots in mice* 5FSM?cMMAE and Sl^m#iMAF ADCls are tested in FHTX-l le primary tumor-bearing mice. Doses are administered, iv on a qld x 4 schedule. Conjugates of toxins with 209 am administered to control mice. dimmer Activity of 'MDCs in, a pmmy tumor model [0474J Two aimiiar studies were conducted to deiennine thein vivo sutitmnor activity of 5F0~vcMMA'E and to compare the anritrunot acSvfty of 5F0~veMMAE to ri ce toxlriMMAB and to uon-mnm.me veMMAE antibody toxin conjugate (209-veMMAE) in colon tumor xenograft mice at kirioi^ldlc^^-doskg·· schedules and to dkermtoe dm re»growth .kinetics following treatment. Female €B-17 SOD mice (eight weeks oldl were moenteadsuteu^^ the Sank with

Ml9C“0e tumor fragments (2 mm x 2 mm), Tumor growth was monitored twee per week using vernier caliphs and the· mean tmnor volume was calculated using the formula (0.5 x [length κ width2]). Whsa the mean tumor volume reached appmxlmately 150 he3 (Study A) or 160 nms (Study B), animals were randomised Mo treatment groups (a - 10/groap for Study A and a ~ 9/group for Study B).

[04?$] Mice were treated (Study A) on a ease weekly (QW) dosing schedule (3 doses) with 0,938,1.875,3.75» or 75 mg/kg 5F 9~νοΜΜ&amp;Ε intravenously (IV) for 20 days orcoum&amp;te, whteh included vehicle (0,9% saline), 0,075 or 0.15· ntg/kg MMAEIV m a QW schedule, or 1.875 or 3,75 mg/kg 209-vcMMAE IVon QW dosing schedules for .20 Days, In ths second study (Study B)> mice were'treated with 0,938,1,875,3.75,7.5, or 10.9 mg/kg 5F9-VCMMABIV on a QW schedule (3 doses) or 3.75 mg/kg IV on a twice weekly (BIW) .schedule (6 doses), or controls induding vehicle, 7.5 or 19 mg/kg 209» veMMAB,.or 0,135 or 9,18 mg/kg MMAE administered IV on a QW schedule for 20 days. Doses were adauaistered os Days 1, 8, and 15 for the QW schedule and Days 1,4,8» II, 15, and 18 for the BIW schedule. The dose of free MMAE ms e&amp;teulaied to match the amount of MMAE ia the immnnoeonjapfo doses by the .Mowing rationale: Hie sqiuvaleaf dose of MMAE is 1.8% of the MLN9264 dose. The equivalent dose of linker + MMAE is 4% of die 5F9*voMMAE dose. These calculations are based on a mean 3.9 MMAE molecules per antibody and a free antibody mokcalar weight of 150 kD,

Actual antibody molecular weight will vary slightly due to degree ofglycosyiatfen, [9476] Tumor volume and body weight were measured twice weekly aad were continued ’beyond the treatment period to measure regfwoth kinetics, as evidenced by tumor growth delay (TGD), Taper volume measuremeais were continued until tumor volume reached 10% of the body weight fo a skglc tnouse within ^treatment group» at which lime the group was tormluatcd, The percentage of tumor growth inMbiiion (TGl) ([mean tumor -volume of the control group mesa tumor volume of a heated group] / meau tumor volume of the control group; a-T/C ratio) was Mmasm&amp; m Day 20. The T.C ratios across a treatment group were compared to the T/C ratios of the control group «sag a turn-tailed Welch* » t-tesi Because the cadre group was tmaiaated if one tumor reached the size limit (approx. 1000 mm3), TGD could not be calculated for groups where the average regmwfh was slow,

{§4771 The differences in the tumor growth trends over time between pairs of treatment groups were assessed using iiaear mixed elects regression models. These models account for the fact that each animal was measured at multiple time points, A separate model was fk for each comparison, sad the areas under the curve (AUC) for each treatosttf group ware calculated using the predicted'values from the model. The percent decrease m ADC (dAOC) relative to the reference gawp was the» calculated. A statistically sipdfreant P value (< 0.05) suggests that the treads over time for die two treatment gamps were different Results are summarized la Tables 19 arid 28, below, [t47S| Astitamsr activity vvm observed ia all Sf 9-vcMMAE~irealed groups in bed» studies md die effect was shown, to he dose^ependeni The results of the two sfedies were comparable, In mice treated with SI^-vcMMAE at 0.93S mg/kg. l¥ on a QW schedule, TGI 20,,7-21,4%, p value was <0.05 eorapareit with vehicle group. Is the 1,875 mg/kg treated grasp administered IV, on a QW schedule TGI was 413-447%, p value ms <0,001, In 3. % rng/fcg treated group administered TV, m a QW schedule TGI ms 653-65,7% (p <0,001) compared with vehicle group, 5F0-voMMAE adatmistercd at 7,5 mg/kg IV QW yielded a TGI of 843-84,3% #<0,003) md 10 mg/kg W QW (Study B only) yielded a TGI of 91,2% #¢0.001). When 3.75 mg/kg wm administered IV on a BIW schedule (S), significant kMMfioa was observed with a TGI of 84.9% #«0.001).

[0479| Moderate antitumor activity was observed at the higher doses of 209-veMMAE of 7,5 -ml 10.0 mg/kg wife TGI of 35.7 and 45.4%, respectively #«0.00.1) but low doses of 209-vcMMAE (1,875 and 3.75 mg/kg) exhibited so inhibition #>0.05). the antitumor activity observed by the high 209-vcMMAE is likely due m nonspecific acdvfty by the MMAE portion of the inammoeonjugate. 114»! Adnihusiradon of tree toxiuMMAE yielded mixed results: 0,075,0,135, md 0,15 mg/kg administered l¥ Q W yielded so tumor growth inhibition #>0.05) bat administration of 0.18 mg/kg IV QW resulted in TGI of 50,4% #«0,001), [04813 Tbs greatest maximum body weight loss observed during treatment period was 2.3% m day 7 in the free toxin 0.18 mg/kg MMAE group of Study B and fee 0.938.mg/kg 5F9-vcMMAE group of the mm study. This indicates the drag was well- 104823 Tamor volume measurem.ents were continued beyond fe e treatment period until tumor volume reached 10% of the body weight in a single moose within a treatment group arid thm the treatment group wm terminated. In these studies, tumor re-growth appeared to he dose-dependent

Table 19. Stody A results of treatmeM of primary human cote, tew Xenograft In SCH) mice.

Table 20, Study B results of treatment of primary taaaa cote trnmr Xenograft in SCID mice.

Μ»/ φοβ®. m4y with m&amp;ed $F9.mtl&amp;0€C\mtiimdy inCMbhOCCMm §32 dmeminaM mode! {haib/c mm } PM831 ms model tests the ability of naked awtibofe to Mndio GCC-expressiftg femur «setts in fee and prevent Bem#e Falh/e xmm wereinoculated %tv. with CT26 hOCC/Iue #32 cells at \xlQs/mmm md Sxi&amp;'kmm,· ¥sfcicfe 0,9% Na€l: radam^speslfitt 209) were »ίΐ^4ί88·:?^.'6ϊ^αβ(ί3^. (in feepLKTOK58 vector) in have fee IgOi isoiyps, so their Fc regiom could elicit an auiikfey-d«pen.dem esiknediafed cytotoxic response after binding to ceil stffface antigens (Ee,, OCC tor 5F9 and an imrelaied target for 209),

Busing was started one day be&amp;sre iBOcaiafem by iv, wife a dosing schedule of oiteeAyeek hr x4 (£}7d x 4)v Ifemur growth was monitored % Nenogen imaging system twice a week. Body weight and ^survival were monitored twice a week as well Long wei ght and images including MR! images wr«re taken at the end of tins study, |0#4| As slatwo in Flph*· -¾ both 5F9 gmnps (4(teg/kg and Itagfegf {^ιφί^.ύί^ύί} is 0.04 to 0,05), T/C for fee 5f 9 group is 0.1$ to 1,14 on day 34p4 cmnpamd to fee 209 poup, T/C for the 209 :.40tn^kg' group is 0 J4 compared to 0,99¾ N&amp;Q (Nonml l Saline) group. No benefit was seen wife 5F9 la fet id5 groups. p4S5j Lung weight of each poop at the end of tins study was shown in Figure 3,

TfesbvehMe vs, 209 46mg% P=4},4; vehiclem 5F9 :40mg/fcg P<0,05; vehicle vst 5F9 20mgfeg P <0 Ji. Visual inspectibo of lungs confirmed fever tumor nodules in fee SF9- tested group than is the vehicle or 209~treated groups. In vivo MRl el the mice shewed massive teg turner aid heart chspiaeem^t k vehicle- treated mire. la a 5F9 40 mg&amp;gdreated mouse, normal hmg presentatioa was seen without evidence of tumor. |04§§} A surtdvsl ctm'e fe shows m Figure 4. SigaMtaat message is survive! with 5F9 trmtsdgffi^s{lxl0^ wss observed and there was tm dtFfermee hotweea SF9 10 sad #0%¾ groups. EX&amp;MFLl 4: deaerating m antibody grodaegon evil Hue |04h?| To generate a stable OHO cell ike clone expressing 5F9 with a jsodnetety of >600utg/Lj exgmssdon vectors for 3.F9 were generated by snbeloamg light chain variable region (SEQ ID NO: 19) md heavy chsia variable region (SEQ El NO: 17} into the pLICFOKSS egression vector, eoMakingTW human TgGi Pc sad the neomycin resktsace gene. Expression, of the 5Έ9 variable icgion-lgOl fusion prodnet is under control of the IF- la promoter.

Ckning mdseqmmsing qftkn unirGCC kmtmjmnochna!antiiwfy 5F9 mri&amp;Me regions |848$1 Total RNA was isdiated (Qisgsp % ENeasy Ml) tea htkm hyhriddsta 46.5F9 snbelone 8.2, This liyh«ddma carries the ^slandard” puhltsheil Kappa eonstpl region of the tight ehaia (GeaEahJi accession # AW38362S Ψ. EM9 J £519) arid the “sksdsrd” published Ig<$E constant region of the heavy ebak penBaak accession # BX640623, or AJ294731). 5f race-ready, poiy-θ tailed eDNA was sp&amp;esked by tradhiaaai. methods (Nature Methods 2,629-630 {200$», The light chain variable region was FOR amplified item eDMA by 55 race using apoly-C anchor oligo in comMuatiim with &amp; reverse primer specific for the Kappa constant regioa. The heavy chain variable r^<».wa8::8Wfljiled.'wi&amp; a levetsepimerispeeiiic for theigGI constant regioa in multiple combinations with forward primers specific to the known heavy chain leader sequeaees, F€R pudonts were T0FQ® cloned (IuviirogenTW, Life Technologies, Inc.) and sequenced with MOP and:M131 primers.

Construction qfmammalian expression vectors carrying unihGCC immn monoclonal antibody 5F9 |04g9| Mammalian oppression vectors carrying the 5F9 light and heavy variable regions were constructed to generate production CEO cell lines. For the native construct, the variable regions of the SF9 light and heavy chains were sub-cloned info pLKTOKSSD (US Patent Application # 20(140033561). This vector carries two mammalian selection markets: neomycin resistance and .DEFR/methotvexafe (ibr arapkiication). Tkcvector allows co-expression of both light and heavy chains font tandem EFI alpha promoters, each located upstream of the vectors leader-Kappa constant and leader-IgGl (wild type Fc) constant regions. For sub-cloning, the variable regions of the light and heavy chains were PCR amplified from sequence-confirmed TOPO clones with gene-specific primers containing unique restriction sites for directional cloning into the junctions of the respective kader-Kappa and leader-IgGl regions of the vector. The sequences of the primers are as follows (5F9 variable region-specific sequences ia bold font):

Native SF9 light chain leader-variable primers: forward Not! ymrnm®m£mKm:M£c^merATGGAmTmmxMTceTem:imGTAseAACA ^SId£MQTQT€X:ACmCX2AAATAGJGATGACG€AGTCI€:€iAG(X!Am-:T€i~V (SEQ ID NO: 234) reverse BsiWl 5*- ^S^ffilfiOTTGAITTCCMIGITGGTCCCWGOCCGAACGTC-S^ (SEQ ID NO:235 ) primers

forward EcaRI 5(e^QA4XIiCCIQMlCAT£iOOATOOAGCTOTATCATCCTC1TCITGGTAGCAACAGCT 4GMii33^.C£A£TCCCAGGTGC AG€TACAG€AGTGGGG€G€AGGA€-3 ’ (SEQ ID NO:236) reverse Blpl NO: 237} jiftlffff Closes were confirmed by doable stranded DNA sequencing of both the light and heavy chains,

[0# t| Two transfection methods were used to introduce the constructs into CHO cdis: the tefifiotaal ΜΡΪ process and the Crucell process, CHO cell transfections were initiated with the native 5F9 construct using the traditional MPI process, linearised and nonlkearized DMAs were used, with either electroporation or Lipopfeetamine 2000 CD transfection. Approximately 30 stable pools were generated tlnongh selection in 0418, non nucleoside medium and 5nM methotrexate. Based on FMAT analysis of antibody induction levels, three stable pools were chosen fi>r cloning, The pool with the highest pRxiiictiori secreted antibody at I2,2ug/tul, These three pods have been frozen down.

[04921 Cruceli STAR dements can he evaluated to make 5F9 expression vectors containing a STAR element, SFWMgGl heavy chain nucleotide sequence is: <5AATTCCTCAC^ATCKjGATCKiAiiCTOTATCATCCTCn,C'n,G(H'ACiCAACA.(K?rACAG<TPGTiX;

ACTC€€AtXTrG€AGCTA€AO€AGlGG<XT€GCAC>GA€TGlTGAAGC€TT€G€tAGA€C€TGTC€€T

CACCT<KX5CTGTCTlTOGTGGGTCTTTCAGTGGTTACTACTtK3At3Cr<K*ATCXX3iXiAGCCCCCAO

GGAACRjCKsCTtSCfAGTCiCfAITGGtjCiAAA'KhVAIttATCGTCjGAAACACXiAACXsACAACXXjGTCCXI

TCAAChUyrCGAGTCACXt,ATATCAGTAGACACOTC5CAAGAACCAGTTCGCCCTi3,4AGCTCiAGtTC

TGTGAGO3CGG0uGACACGGCT(irrTATmCTGliKC?AGAGAACGTCRlAiACACCIAr€A}TAAC

TTTGA<XACTGG<KXXlAGGGAACX]CTGGTCACCGICAGCTCA(KXrrOCA<XAAGGi3CCCATCGG

TCtTCXXICCmiCACCC'RXTCCAAGAGCACCrGTl'GGGGCtCACAOCCACCCl'GGG'CrGCCTCRST

CAAGGACTAClTCXiCCGAACCGCTrGACGGTGTCXsTGCfAACTCAtXi'CGCiXTrGACCACiCGGCGT

G€ACACCXltXXGGCTG1X:CTACACGCX7m4G(l:ACrCTAGTaXTCAGCAGCGIGGTCMCCGTG

CCCTCXiAi3CAGCTTGGGCACtC.!CAGACCTACAl'CrGGXACGTGAATCACA\GCCXlAGCAACACC

AAiXiKKjACAAGAAAGTTGAGCGCAAATCTTGTGACAAAACrCACACATGCXXACOGTCCCCA CCACCTOAACTCCKXXXXlGACCGTCAGIXimiCTCl’TCX'CCCCiAAAACCCAAGG'ACACa.ITCA .

'TGATCTC0CGGACCCCXGAGGTCAClATGCG7XX)TGGIGGACGTGAGCCAC<iAA.GACCCTGAGG 7tIAAGlTCAA€TGGTACXnt3GA€GCXiGTGC?AGGTGCATAATGCSCAAGACAAAGCCGCXXKiA<K3

AGCAGTACAACAGCACXjTACCG’FGTCRtTCAGCGTCCTCACCGT'CCTGCACCAGGACTGiX'TGAA

TGGCAACKfAGTACAAOTGCAACiCiTCTCCAACAAAGCCCTCOCACiiXXlCCATCGAGAAAACXtAT

CTCCAAAGOCAAACKXiCAGCXDCCGAGAACCACAGGTGTACACCCTGCCCCCATCXXGGGATGA

GCTGACCAAGAACCAG<m:AGCCTGACCTCKXJiGGTCAAAGGCTTaA:rCCCAeC<MCAIGGCC

QT(KMGT8CKjAGAC^])AAf(K^GA<KXXKM.GAACAA{TACAA<M<XA<3GiXrrOCOCnX3CI1GGfAC TCCGAC<i<KTCCTTClTCX^TCTACACK^AA,CjC’IX'ACCGTGGACAAGA<jCAGGTGGCACICAGGGCi

AACGTCTIC'K^TGCIO^T(3ATGCAI^AGX3CTCTGCA.CAACCACTACACCiCAGAAGA£jCCTCT CXCTGTCTCCGGGTAAATAATAGGGATAACAGGCfTAATACTAGAO· (SEQ IDKO:230)

MGWSCflLFLVATATGVHSQVQIQQWOAGIIiC^ETLSLTCAVFCKJSFSGYyWSWRQFPGKGIjE

WIGBINHRCiNTMi^SyCSRVnSVIW^KNQFALKLSSVTAADTAVTyCARERGYTVGNFDHWGQ

{xFLVTV'SSASTKGPSyFPLAPSSKSTSGGTAALGCiLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSWWVPSSSLGTQTYICMVNHKPSKTK.V'DICiCVEF.KSCBICIHTCFi’CPAPELICICiPSVFLFP FKPSDlXMSRTFEVTCW¥D¥SHEDPE\«Mima>c3WWMAKTKP5lEEQl:'KS’nil.m^\iLTVL· '

HQDWiJS’GiCBYKCKVSNKALI’APrEKTiSKAKGQHiBPQVYTIJiPSRDBLTKN'QVBLTCLVKGFYFS

OMyBWESNGQFEKK^lim^LDSDGSFFLYSKLWDKjSlWQQaWFSCSYMHEALHNHWQICS

LSLSKjK (SEQ if) M():23 S) 5F9i%Kappa light chain nucleotide sequence is:

6CG6lXiXCrCACC^TCiG<MTGCrACK:hSTATCATCX2TCTTCITG<ITAGCAACAGCrACAGG-TGT

CXiAC?IO^AAAXAGTOAlGACXK^GTCTOCA(KX]A<XX]T<HCr<3TCTCICCAG(KKSAAAGAGOC

ACXX^TCTCCTCTCACj€^JCCAGIC^GA(JTt}TTA<»CAGAAAC2rTAGCCTG0TATCACK;ACtAAAC€2rG

CiCCAiKsCTCCCAijGCTCCTCATCTATtKjTGCATCCACCACjGGCCACTCKJAAICCCACCCAGCnT

CAGT<KK^AfiTKXJGTClGiXiACAiM.OTTCACTCICACCATOG€iCAjGCCTGCAGTCTGAAOATi’TT a^GTiTATTACn'GTCAGCAGTATAAAACCTCKilXTCiK?ACXrTTCGGC€AACtGGi\CCAACXrfGCl

AAATCAAACGTACGGTGGCrFGCACCAl€TCjlUrTCATCTTCCXGOCATCTGATG'AGCAOTKiAA

AlCTGGAACIXKXiTCrGTTGTGTGCCrGC'rGAATAACTrCTAlXXXAGACiAGGCCAAAGTACAG

IGGAAGOTGGATAMXJCCXilCOAlXXKXyrAACSDOGAiKrAGAG'RjFCACAiGAGCAGGACAGC aa.ggacagcacctacagcctcagcagcacxjctgaccctgagcaaagcaoactacgagaaacac

AAAGICTAiXNXIGOGAAGTCACiXATCAGGCiCCTGAGCTOGCCCXiXCACAAAGAGCTTCAAC

ACCXiGAGAGTGTTAC/rCTAGA (SEQ 10190:232} 5F9&amp;Kappa light chain protein sequence is: MGW^IMLVATAltJVHSEIVMK^PAlLSV'SPG^RA‘IlSCSASQSVSHMAW1f,Q<^BGQAPSXl«i

TCASm4I«iFAlFSGSGSGTBFTLtIGSLC^FI>FA\^CQQYKTWPRm3QGTHVBii:miAAi,SVF ilYPSDEQLICSGTASVVCLIHNFYKiEAKA?QWlCVI>NALQSGNSQBSYiliQXMO>8TySLSSXLTLS.&amp;

ADWJCHKVTACEVTHQGLSSF'VTiCSFKRGEC CSBQIDM>;233) f##3| The hesvy and light chain nucleic acid sequences for 5F9 listed below were inserted into pTOK.58B vector: oiccagg gga^s-gagecaccclctcctgcagggccagtcagagtgttagcagaaadtagcetggsaicagca^aacctggccagg· ctcccaggctceicatctatggigcaiccaccagggccactggaaicccagccaggltcaglggeagtgggtagggacs gsgttcaetcicsecateggcagctttgtif^togsagsititgoagttMiaeigtcagcagistaaasectggoctcg pcgtteggtx;aaggg8cc^cgtggaaatcaaacgtacggl.ggctgcs.ecaicigicttea.tottccogceatetgatg ageagllgaaasciggaaetgcctetgttglgtgectgctgaataaciteiaioceagagaggccaa^lacaglggaag gtggat^cgncciccaaicggglsacisncaggagaglgtcaeagagcaggacagcaaggacagcacciacagcctcag egecegieacaaagagcttoaacaggggagaglgtt&amp;g (SEQ U> N():308) acaggtgtccaclcceaggtgcagctacagci^tggggcg€aggactgttgasgecitegga.gaccctgicc0teaectg cgctgtihtiggtgggtotttcagtggttaciactggsgetggatecgoeagccaxaggpagg^ctggagtggattg gggana&amp;taaicaicgtgpaacaecaaegaeaaocegtctxt^gs^cgagteaccidaicagtagaeacgtceaag aaccagttcgecoi^aget^gttctglgaccgccgcggacac^ct^ttMiact^gcgaga^ae^^atacac ctatggtwWgaccaetggggwagggMcctdg^iaccgtcagcfcagcetccaceMgggeceatcggtetfcc axtggc3e€ctectccsagsg«acrfclgggggcacagcggcccl:gggctgcctggtca-aggactacitccccgfxaccg gigacggigtcgtgpacicaggcgccctgaccagcggcgigsaeaccitijccggclgteciacagicx'fca^aotcia etccetcagcagcgtggigaecgigcociocageapag^cacccagacctacatx-iigcaacgtgaatcaeaagccca gcaacaccaaggtggacaagaaa-gttgage;ccaaaU;ti:gtgacaaaactcacacaigcecacxigtgvxca.geacctg-ia cfcct^ggggacc^c^tettcetettccccceaa^cccaaggacaccctcatgatctcccggaccccfgag^cae at§^WBEiMs^gagccacgaagaccctgaggicaagiecaaciggtacg^acggcgtgga^%cataaig ocaagacaaagccgegg^gpgcagiaaaacagcac^accgtgtggicag^icscicacogjcctgcaccaggac^g c^aiggoaagg^tacaagtgcaaggtatecaacaaagccctcoca^eccoMcgagaaaaocaietccaaageaaa a^gcagcoccppaccacaggigtacscectgcceccaicecgggalgagetgaccaapaixaggca^ctgaajt gcctggicaaaggcttiitateceagcgacatcgecgtggagtgggaga^aatgggcagccggagaacaactacaa^cc acgcctccog^ctggactccgac^ctcoitcttcetciacagcaagctcaccgt^acaagagca^tggcagcaggg gaacgtcttcicatgcfccgtgaigca%aggctctgcacaaceaciaeaegcagaagagecfctccct^ctccgggia aafaa (SEQ ID NO:309) f W94] The sequences encoding the Afex-229 heavy and light chains sequence below were inserted into pTOKSSB vector: atggagttt^gctgggetggeiixttettgggdatt ta.aaaggtgiccagtgtpggtgcagc%ttggagtctgggggaggcttggtecagcciggggggtecetgagactete ctgtgcagcctetggattcacctttagccgctatgceat^aa.gggtecgecagg<hce8gggaaggggelggagtg^: teteaggiattagtgggagtggtggtaggacatactacgcagactccgtgaagggccggtteaccatetccagagacaat ieeaagaaeaeactaiatclgcaaatgascagceigagagecgaggacacggcegtataiiaetgtgcgaaagaiegcga tttftgga^ggl:ccatttg8.ctaeiggggeeagggaaccctggteaccgtcagcteagcctocaceaagggcccaic® tcttecceetggcaceeiveteeaagageacotetgggggeaeapggeeeigggctgcctggtcaaggaciacttcccc gaaccggtg^?ggtgtegt^acte^c^ceotga.cc.ageggjgigeacacdiceeggetgtcetaeagicctea® actetectacctcagcagcgtggtgacegtgccetocagcagetlg^eacccagacctacatctgcaaogtgaateaca ageecagcaaoaccaa^tggacaagaaagiip.geceaaateitgtpeaaaacleaeaeaigcecaeegtgcccagca cxii@.actefgg§^gaccgicagtc^ccicttcceccc88^sccca«^acaceetcatgiitcteccgg8occciga ataatgccaagacasagecgcgggsggagcsgiacascagcacgtsecg^t^osgcgtccteaccgicfilgesficag agscaaagggcagccecgagaaccacaggigtaeaccctgceeccaicccgggaigagctgacfiaagaaccaggteagcc igaccigcct^icasiaggctictafwcagcg^catcgccg^^gigggagagcaaig^cagCQggapseaacrac aagaccacgccteccgigeiggaQtccg80^0tcttiett'fici«iaGageaagateaciogigga«aagagCiiggtggca ic^§igaa^c^^^%ctccgig8tgcatga^cici:gcaesmce8ci8esicgcagaagagcetei€:cctgict€ egggtaaataa (SBQIDNO:310) ai^ggeicccigcteagcliBie ttecicetgctacici^«icceagataccactggagMatagtgatgacgecgtcttcagccae{JcigtBtgigictcc aggctcccaggctcctcaietalggfgcatccacca^gecaci^lateccagccaggticaglggaagi^gfatggg

aeapaitcactc^cacoMaagcagcC'tgsagtcigaagattttgcagtttatfaetgteaccagtatagtaactggM gigca^ttiggccagggga«caagctggagat®aaacgtac^t^c%caccalctgtcttcatcitcccgceatcig aaggtggateacgccctccaaicgggeaacieccaggagagfgtacagagcaiiacagcaaggacagcaccfec&amp;gciJi cagcagoacccigacccigagcaaageagac-fecgapaacaeaaagtciaegeetgcpagteacccatcagggceiga gctegaic^tcacaaagagciicaaeaggggagagigttag fSBQ ID NO:3 Π) ftM^S] WMie this ffivesfio» has been showa md described with refemnoes to giwided embodiments thereof, it will be woderstood by those skilled is ike art that various ctmges* m form and details may be made tela without departing frost tfee scope of th e isveodoa eacompassed by the appended claims.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises” or "comprising", will be understood to Imply the inclusion of a stated element or integer or method step or group of dements or integers or method steps but not die exclusion of any element or integer or method step or group of elements or integers or method steps.

Reference to any prior art in this specification Is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms pari of the common general knowledge in any country''-

Claims (51)

1. CLAIMS:

   1. An isolated anti-GCC antibody molecule that binds to the same epitope on GCC as, or competes for binding to GCC with, an antibody molecule comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), wherein the light chain variable region comprises: an LCDR1 comprising the amino acid sequence of SEQ ID NO:112, an LCDR2 comprising the amino acid sequence of SEQ ID NO:114 and an LCDR3 comprising the amino acid sequence of SEQ ID NO:l 16; and wherein the heavy chain variable region comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 106, an HCDR2 comprising the amino acid sequence of SEQ ID NO:108 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 110.
2. 2. The antibody molecule of Claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:20, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18.
3. 3. The antibody molecule of Claim 1 or Claim 2 that binds to, or competes for binding to, an epitope on GCC, wherein the epitope resides within, or comprises: (a) amino acid residues 1 to 50 of SEQ ID NO:228; (b) amino acid residues 25 to 50 of SEQ ID NO:228; (c) amino acid residues 33 to 50 of SEQ ID NO:228; (d) amino acid residues 24 to 420 of SEQ ID NO:228; and/or (e) amino acid residues 24 to 75 of SEQ ID NO:228.
4. 4. The antibody molecule of Claim 3, wherein the epitope comprises one or more amino acid residues from amino acid residues 50 to 1073 of SEQ ID NO:228.
5. 5. The antibody molecule of Claim 3 or Claim 4, wherein the epitope is a conformational epitope.
6. 6. The antibody molecule of Claim 1 or Claim 2, wherein the antibody molecule binds a fragment of GCC comprising: (a) amino acid residues 1 to 50 of SEQ ID NO:228; (b) amino acid residues 25 to 50 of SEQ ID NO:228; (c) amino acid residues 33 to 50 of SEQ ID NO:228; (d) amino acid residues 24 to 420 of SEQ ID NO:228; and/or (e) amino acid residues 24 to 75 of SEQ ID NO:228.
7. 7. An isolated anti-GCC antibody molecule comprising a light chain variable region and a heavy chain variable region and an Fc region, wherein the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRl, HCDR2, and HCDR3), wherein the light chain variable region comprises: an LCDR1 comprising the amino acid sequence of SEQ ID NO:112, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 114 and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 116; wherein the heavy chain variable region comprises: an HCDRl comprising the amino acid sequence of SEQ ID NO: 106, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 108 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 110; and wherein the Fc region is mutated to modify effector function.
8. 8. The antibody molecule of Claim 7, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:20, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:18.
9. 9. A bispecific antibody molecule having binding specificity to GCC and a second antigen, wherein the bispecific antibody molecule comprises a light chain variable region and a heavy chain variable region forming a binding site for GCC, wherein the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDRl, HCDR2, and HCDR3), wherein the light chain variable region comprises; an LCDR1 comprising the amino acid sequence of SEQ ID NO:112, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 114 and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 116; and wherein the heavy chain variable region comprises: an HCDRl comprising the amino acid sequence of SEQ ID NO: 106, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 108 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 110.
10. 10. The bispecific antibody molecule of Claim 9, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:2Q, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18.
11. 11. The bispecific antibody molecule of Claim 9 or Claim 10, comprising: (a) two antibody molecules, one with binding specificity to GCC and another with binding specificity to a second antigen; (b) a single antibody molecule that comprises one chain with binding specificity to GCC and a second chain with binding specificity to a second antigen; or (c) a single chain antibody molecule that has binding specificity to GCC and a second antigen.
12. 12. An anti-GCC antibody molecule conjugated to a radiolabel, wherein the antibody molecule comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), wherein the light chain variable region comprises: an LCDR1 comprising the amino acid sequence of SEQ ID NO: 112, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 114 and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 116; and wherein the heavy chain variable region comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 106, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 108 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 110.
13. 13. The antibody molecule of Claim 12, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:20, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18.
14. 14. The antibody molecule of Claim 12 or Claim 13, wherein the radiolabel comprises an α-emitter, a β-emitter, or a positron-emitting radionuclide.
15. 15. The antibody molecule of Claim 14, wherein the α-emitter is 21jBi or 225Ac 90 177 and the β-emitter is Y or Lu.
16. 16. An immunoconjugate of formula (I):

    (/) or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody molecule of any one of Claims 1 to 15; X is a linker moiety which connects Ab and Z; Z is a therapeutic agent or a label; and m is the number of-X-Z moieties per antibody molecule in the immunoconjugate of formula (I), and m is an integer from about 1 to about 15.
17. 17. The immunoconjugate of Claim 16, wherein the linker -X- has the formula -Aa-Ww-Yy-, and the immunoconjugate is characterized by formula (II):

    (//) or a pharmaceutically acceptable salt thereof, wherein: -A- is a Stretcher unit; a is 0 or 1; each -W- independently is an Amino Acid unit; w is an integer ranging from 0 to 12; -Y- is a self-immolative Spacer unit; y is 0, 1, or 2; Z is a therapeutic agent or label; and m is an integer from about 1 to about 15.
18. 18. The immunoconjugate of Claim 16 or Claim 17, wherein Z is a detectable label or a radiolabel.
19. 19. The immunoconjugate of Claim 18, wherein the radiolabel comprises an a-emitter, a β-emitter, or a positron-emitting radionuclide. 075
20. 20. The immunoconjugate of Claim 19, wherein the α-emitter is " Bi or “ Ac and the β-emitter is 90Y or 177Lu.
21. 21. The immunoconjugate of Claim 16 or Claim 17, wherein Z is a cytostatic or cytotoxic agent.
22. 22. The immunoconjugate of Claim 21, wherein the cytostatic or cytotoxic agent is selected from an antimetabolite; an alkylating agent; an anthracycline; an antibiotic; an antimitotic agent; a topoisomerase inhibitor; and a proteasome inhibitor.
23. 23. The immunoconjugate of Claim 22, wherein the antimetabolite is selected from azathioprine, 6-mercaptopurine, 6-thioguanine, fludarabine, pentostatin, cladribine, 5-fluorouracil (5-FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), methotrexate, trimethoprim, pyrimethamine, and pemetrexed; the alkylating agent is selected from cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, thiotepa/chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, dibromomannitol, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, dacarbazine, mitozolomide, and temozolomide; the anthracycline is selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin; the antibiotic is selected from dactinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, mitomycins, duocarmycins, and calicheamicins; the antimitotic agent is selected from maytansinoids, auristatins, dolastatins, cryptophycins, vinca alkaloids, taxanes, and colchicines; the topoisomerase inhibitor is selected from irinotecan, topotecan, amsacrine, etoposide, teniposide, and mitoxantrone; and the proteasome inhibitor is a peptidyl boronic acid.
24. 24. The immunoconjugate of Claim 23, wherein the mitomycin is mitomycin C; the duocarmycin is CC-1065; the vinca alkaloid is selected from vincristine, vinblastine, vindesine, and vinorelbine; and the taxane is paclitaxel or docetaxel.
25. 25. The immunoconjugate of Claim 16 or Claim 17, wherein Z is a maytansinoid or maytansine, a dolastatin, or an auristatin.
26. 26. The immunoconjugate of Claim 25, wherein the maytansinoid or 2' 2' 21 maytansine is N -deacetyl-N -(4-mercapto-l-oxopentyl)maytansine (DM3), N -deacetyl- Nz -(3-mercapto-1 -oxopropylj-maytansine (DM1), or N" -deacetyl-N -(4-mercapto-4- methyl-1 -oxopentyljmaytansine (DM4); and the auristatin is AEB, AEVB, AFP, MMAE, or MMAF.
27. 27. The immunoconjugate of any one of Claims 16, 25, or 26, wherein (a) the immunoconjugate has formula (1-1):

    {1-1) or a pharmaceutically acceptable salt thereof, wherein m is an integer from about 1 to about 15; or (b) the immunoconj ugate has formula (1-2):

    (1-2) or a pharmaceutically acceptable salt thereof, wherein m is an integer from about 1 to about 15; or (c) the immunoconj ugate has formula (/-3):

    (/-3) or a pharmaceutically acceptable salt thereof, wherein m is an integer from about 1 to about 15.
28. 28. The immunoconjugate of any one of Claims 16 to 27, wherein m is an integer from about 1 to about 10, about 3 to about 7, or about 3 to about 5.
29. 29. The immunoconjugate of any one of Claims 16 to 28, wherein m is about 4.
30. 30. An antibody molecule of any one of Claims 1 to 15, or an immunoconjugate of any one of Claims 16 to 29, for use in a method of treating a cancer in a subject.
31. 31. Use of the antibody molecule of any one of Claims 1 to 15, or an immunoconjugate of any one of Claims 16 to 29, in the manufacture of a medicament for treating a cancer in a subject.
32. 32. The antibody molecule, immunoconjugate, or use of Claim 30 or Claim 31, wherein the cancer is a cancer of the gastrointestinal system or a metastatic lesion thereof, a primary or metastatic colorectal cancer, a gastric cancer, an esophageal cancer, a pancreatic cancer, or a lung cancer.
33. 33. The antibody molecule, immunoconjugate, or use of any one of Claims 30 to 32, wherein the antibody or immunoconjugate is used in combination with one or more additional therapeutic agents.
34. 34. The antibody molecule, immunoconjugate, or use of Claim 33, wherein the one or more additional therapeutic agents comprise 5-fluorouracil (5-FU), capecitibine, leucovorin, irinotecan, oxaliplatin, bevacizumab cetuximab, panitumum, or any combination thereof.
35. 35. The antibody molecule, immunoconjugate, or use of Claim 34, wherein the combination is selected from oxaliplatin/capecitibine (XELOX), 5-FU/leucovorin/oxaliplatin (FOLFOX), 5-FU/leucovorin/irinotecan (FOLFIRI), FOLFOX plus bevacizumab, and FOLOFIRI plus bevacizumab.
36. 36. The antibody molecule, immunoconjugate, or use of Claim 33, wherein the one or more additional therapeutic agents comprise a chemotherapeutic agent.
37. 37. The antibody molecule, immunoconjugate, or use of Claim 36, wherein the chemotherapeutic agent is a DNA damaging agent.
38. 38. The antibody molecule, immunoconjugate, or use of Claim 37, wherein the DNA damaging agent is selected from a topoisomerase I inhibitor; a topoisomerase II inhibitor; an alkylating agent; a DNA intercalator; a free radical generator; a nucleoside mimetic; and an agent that disrupts cell replication.
39. 39. The antibody molecule, immunoconjugate, or use of Claim 38, wherein the topoisomerase 1 inhibitor is selected from irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin; the topoisomerase II inhibitor is selected from etoposide, teniposide, and daunorubicin; the alkylating agent is selected from melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide; the DNA intercalator is selected from cisplatin, oxaliplatin, and carboplatin; the free radical generator is bleomycin; the nucleoside mimetic is selected from 5-fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea; and the agent that disrupts cell replication is selected from paclitaxel, docetaxel, related analogs of paclitaxel or docetaxel, vincristine, vinblastine, related analogs of vincristine or vinblastine, thalidomide, lenalidomide, related analogs of thalidomide or lenalidomide, protein tyrosine kinase inhibitors, proteasome inhibitors, NF-kB and IkB kinase inhibitors, antibodies which bind to proteins overexpressed in cancers and thereby downregulate cell replication, and other inhibitors of proteins or enzymes known to be upregulated, over-expressed, or activated in cancers, the inhibition of which downregulates cell replication.
40. 40. The antibody molecule, immunoconjugate, or use of Claim 39, wherein the related analog of thalidomide or lenalidomide is CC-5013 or CC-4047; the protein tyrosine kinase inhibitor is imatinib mesylate, or gefitinib; the proteasome inhibitor is bortezomib; and the antibody which binds to proteins overexpressed in cancers and thereby downregulates cell replication is selected from trastuzumab, rituximab, cetuximab, and bevacizumab.
41. 41. The antibody molecule, immunoconjugate, or use of any one of Claims 30 to 40, wherein the cancer has been determined to express a GCC molecule.
42. 42. A method of treating a subject for a GCC-expressing cancer, comprising administering to said subject a therapeutically effective amount of the antibody molecule of any one of Claims 1 to 15, or the immunoconj ugate of any one of Claims 16 to 29.
43. 43. The method of Claim 42, wherein the cancer is a cancer of the gastrointestinal system or a metastatic lesion thereof, a primary or metastatic colorectal cancer, a gastric cancer, an esophageal cancer, a pancreatic cancer, or a lung cancer.
44. 44. A pharmaceutical composition comprising the antibody molecule of any one of Claims 1 to 15, or the immunoconj ugate of any one of Claims 16 to 29, and a pharmaceutically acceptable carrier.
45. 45. A container comprising the pharmaceutical composition of Claim 44.
46. 46. A kit comprising the antibody molecule of any one of Claims 1 to 15, or the immunoconj ugate of any one of Claims 16 to 29.
47. 47. A method of producing an immunoconjugate, comprising providing the antibody molecule of any one of Claims 1 to 11; and contacting the antibody molecule with a non-antibody moiety comprising a therapeutic agent or label under conditions that allow the production of an immunoconjugate, thereby producing the immunoconjugate.
48. 48. An expression vector comprising isolated nucleic acid sequences encoding the antibody molecule of any one of Claims 1 to 11.
49. 49. A cell comprising the expression vector of Claim 48.
50. 50. A method of producing an antibody molecule of any one of Claims 1 to 11, comprising culturing the cell of Claim 49 under conditions that allow the production of an antibody molecule, thereby producing the antibody molecule of any one of Claims 1 to 11.
51. 51. A method of detecting a GCC molecule comprising contacting the GCC molecule with an antibody molecule of any one of Claims 1 to 15 and determining if the antibody molecule binds to the GCC molecule.