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119) United States112) Patent Application Publication 110) Pub. No.: US 2009/0311257 A1

Masat et al. (43) Pub. Date: Dec. 17, 2009

154) Related U.S. Application DataHUMAN ANTIBODIES SPECIFIC FORGASTRIN MATERIALS AND METHODS

160) Provisional application No. 60/784,501, filed on Mar.20, 2006.Linda Masat, Walnut Creek, CA

1US); Marina Roell, Concord, CA1US)

175) Inventors:

Publication Classification

Assignee: XOMA Technology Ltd., Hamilton1BM)

173)152) U.S. Cl.....

Appl. No.:121)157) ABSTRACT

PCT Filed:122)The present invention relates to materials and methods forhuman antibodies specific for the peptide hormone gastrinand uses of these antibodies in the treatment of subjectshaving cancer and other conditions or disorders related togastrin expression.

186) PCT No.:

) 371 1c)11),12), 14) Date: Jun. 3, 2009

Correspondence Address:MARSHALL, GERSTEIN 4 BORUN LLP233 SOUTH WACKER DRIVE, 6300 SEARSTOWERCHICAGO, IL 60606-6357 (US)

12/293,890

Dec. 13, 2006

PC T/US2006/047S40

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US 2009/0311257 A1 Dec. 17, 2009

HUMAN ANTIBODIES SPECIFIC FORGASTRIN MATERIALS AND METHODS

[0001] This application claims the priority benefit of U.S.Provisional Patent Application No. 60/784,501, filed Mar. 20,2006, hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to materials and methods forhuman antibodies specific for the peptide hormone gastrinand uses of these antibodies in the treatment of subjectshaving cancer and other conditions or disorders related togastrin expression.

BACKGROUND OF THE INVENTION

[0003] Gastrin is a peptide hormone that signals throughthe G-protein coupled receptor (GPCR)CCK2R, and has avariety of effects including stimulation of gastric epithelialcell proliferation and acid secretion by parietal cells (Yassin RR, Peptides 20:885-98, 1999). It has also been characterizedas a factor in the progression of gastric cancers and presents apotential target for therapies that neutralize its function(Smith et al. Gut 47:820-24, 2000).[0004] Gastrin is a hormone produced in the digestive tractof many species, including humans. Normal adults producegastrin in only one cell type the G cells which line thegastric mucosa in the antral portion of the stomach (Ganong,Review of medical physiology. Norwalk, Conn., Appleton &Lange, 1995). Food intake stimulates the G cells to producegastrin. Specifically, distension of the lumen of the stomachor the presence of peptides and amino acids in the stomachstimulate gastrin secretion. There is also a neural pathway forgastrin release as the sight or smell of food may stimulate(through the Vagus nerve) release of gastrin.[0005] Once secreted, gastrin has a range of activities onthe digestive tract. The primary roles of gastrin in a normaladult are to stimulate acid production by the parietal cells ofthe stomach and to act as a trophic factor for cells lining thegastrointestinal tract. Gastrin also serves other secondaryroles in the digestive tract such as stimulating pepsin andpancreatic enzyme release, and gall bladder contraction andsmall intestine motility. Gastrin is produced in a precursorform of 101 amino acids called pre-pro-gastrin. This proteingoes through a series of cleavage steps to generate severaldifferent proteins of varying length (Mulholland et al., Sur-gery 103:135-47, 1988). Additional post-translational stepsinclude glycine addition and amidation. Gastrin may beexpressed as pre-pro-gastrin, pregastrin, gastrin 34 (G34,having 34 amino acids), gastrin 17 (G17, having 18 aminoacids) and gastrin 14 (G14, having 14 amino acids). Gastrin(G34) stimulates stomach acid secretion and has a trophiceffect on gastrointestinal tract (G.I) mucosa. Glycine-Ex-tended Gastrin (Gly-G17) and amidated Gastrin (G17-NH2)also stimulate stomach acid secretion and exhibit a trophiceffect on G.I. mucosa.[0006] Gastrin functions in healthy adults are limited topreparing the gastrointestinal tract for the process of digest-ing ingested food. However, much recent research has impli-cated gastrin as a growth factor for some types of cancer(Baldwin et al., Gut 42:581-4, 1998; Smith et al., AlimentPharmacal Ther 14:1231-47, 2000) including pancreatic,gastric, and colorectal carcinoma. Expression of gastrin and

gastrin receptors has been demonstrated in primary tumorstaken from cancer patients. Since some tumor types appear toproduce and secrete their own gastrin, gastrin can act tostimulate tumor growth via autocrine and paracrine pathwaysas well as via an endocrine pathway. Several studies pub-lished in the literature have demonstrated that tumors takenfrom cancer patients both produce gastrin and express highlevels of gastrin receptors (Schmitz et al., Eur J Clin Invest31:812-20. 2001; Finley et al., Cancer Res 53:2919-26. 1993;Weinberg et al., J Clin Invest 100:597-603, 1997; Caplin etal., Br J Surg 87:1035-40, 2000).[0007] The therapeutic approach of disrupting the gastrin-mediated mitogenesis of cancer cells has been tried in theclinic using small molecule antagonists to the gastrin recep-tor. Several small molecule antagonists for gastrin receptorshave been tested in clinical trials for oncology indications.[0008] Antibodies represent a powerful approach to neu-tralize therapeutic targets due to their high degree of speci-ficity and affinity. Monoclonal antibodies specific for murinegastrin peptides have been disclosed in U.S. Pat. Nos. 6,861,510 and 5,688,506. However, these antibodies do not possessthe desired specificity for human gastrin as needed for clinicaltherapy.[0009] Thus there remains a need in the art to developspecific antibodies against human gastrin to use in the treat-ment of cancers and other conditions or disorders associatedwith gastrin expression.

SUMMARY OF THE INVENTION

[0010] The materials and methods of the present inventionfulfill the aforementioned and other related needs in the art.[0011] In one embodiment the invention provides antigen-binding compounds, including functional fragments, havingthe amino acid sequences set forth in SEQ ID NOs: 1-12 and23-33. In a related embodiment, an aforementioned antigenbinding compound is selected from the group consisting of afully assembled tetrameric antibody, a polyclonal antibody, amonoclonal antibody including a HUMAN ENGI-NEEREDâ„¢ antibody; a humanized antibody; a human anti-body; a chimeric antibody; a multispecific antibody, an anti-body fragment, Fab, F(ab')~; Fv; scFv or single-chainantibody fragment; a diabody; triabody, tetrabody, minibody,linear antibody; chelating recombinant antibody, a tribody orbibody, an intrabody, a nanobody, a small modular immunop-harmaceutical (SMIP), a binding-domain immunoglobulinfusion protein, a camelized antibody, a V~~ containing anti-body, or a variant or derivative of any one of these antibodies,that comprise one or more CDR sequences of the inventionand exhibit the desired biological activity. The antigen bind-ing compounds of the invention preferably retain bindingaffinity of at least 10, 10, 10 M or higher as measured bysurface plasmon resonance.[0012] In one aspect, the antibodies of the invention com-prise the antibodies. XPA061, XPA063, XPA065, XPA067and XPA081 set out in amino acid sequences SEQ ID NO:1-10. It is further contemplated that the antibodies may com-prise all or part of the antibodies set out in the above aminoacid sequences. In one embodiment, the antibodies compriseat least one of CDRI, CDR2, or CDR3 of the heavy chain ofSEQ ID NOs: I, 3, 5, 7, 9 and 11, or at least one of CDRI,CDR2 or CDR3 of the light chain of SEQ ID NOs: 2, 4, 6, 8,10 and 12.[0013] In another embodiment of the invention, variants ofthe aforementioned antibody are provided, comprising a vari-

US 2009/0311257 A1 Dec. 17, 2009

able heavy chain amino acid sequence which is at least 60, 65,70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%homologous to the amino acid sequence set forth in SEQ IDNOs: I, 3, 5, 7, 9 and 11. In a related embodiment, theantibody comprises a variable light chain amino acidsequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% homologous to the amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10 and 12.[0014] One aspect of the invention provides an antibodythat binds target antigen comprising a heavy chain that com-prises any one, two, and/or three of the heavy chain CDRsequences of the invention described below.[0015] Preferably the heavy chain comprises an amino acidsequence identified as a heavy chain CDR3 sequence. Such a"heavy chain CDR3 sequence" includes an amino acidsequence identified as a heavy chain CDR3 sequence set outin FIG. 1 or FIG. 3 and SEQ ID NOs: I, 3, 5, 7, 9, 11 and23-33. Alternatively, the heavy chain CDR3 sequence com-prises an amino acid sequence that contains one or moreamino acid changes compared to any heavy chain CDR3amino acid sequence identified in FIG. 1 or FIG. 3, i.e. asubstitution, insertion or deletion. Preferable substitutionsinclude a substitution to an amino acid at the correspondingposition within another heavy chain CDR3 of FIG. 1 or FIG.3. Alternatively, the heavy chain CDR3 sequence may com-prise a consensus amino acid sequence of heavy chain CDR3shown in FIG. 1 or FIG. 3.[0016] The heavy chain comprising a heavy chain CDR3sequence of the invention described above may further com-prise a "heavy chain CDRI sequence" of the invention, whichincludes any of the amino acid sequences identified as a heavychain CDRI in FIG. 1 or FIG. 3, amino acid sequences thatcontain one or more amino acid changes compared to anyheavy chain CDRI identified in FIG. 1 or FIG. 3, preferablya substitution to an amino acid at the corresponding positionwithin another heavy chain CDRI of FIG. 1, or a consensussequence of heavy chain CDRI shown in FIG. 1 or FIG. 3.[0017] Alternatively, the heavy chain comprising a heavychain CDR3 sequence of the invention described above mayfurther comprise a "heavy chain CDR2 sequence" of theinvention, which includes any of the amino acid sequencesidentified as a heavy chain CDR2 in FIG. 1 or FIG. 3, aminoacid sequences that contain one or more amino acid changescompared to any heavy chain CDR2 identified in FIG. 1 orFIG. 3, preferably a substitution to an amino acid at thecorresponding position within another heavy chain CDR2 ofFIG. 1 or FIG. 3, or a consensus sequence of heavy chainCDR2 shown in FIG. 1 or FIG. 3.[0018] The heavy chain comprising a heavy chain CDR3sequence of the invention described above may also compriseboth (a) a heavy chain CDRI sequence of the inventiondescribed above and (b) a heavy chain CDR2 sequence of theinvention described above.[0019] Any of the heavy chain CDR sequences describedabove may also include amino acids added to either end of theCDRs. Preparation of variants and derivatives of antibodiesand antigen-binding compounds of the invention, includingaffinity maturation or preparation of variants or derivativescontaining amino acid analogs, is described in further detailbelow. Exemplary variants include those containing a conser-vative or non-conservative substitution of a correspondingamino acid within the amino acid sequence, or a replacementof an amino acid with a corresponding amino acid of a humanantibody sequence.

[0020] Antibodies comprising any one of the heavy chainsdescribed above may further comprise a light chain, prefer-ably a light chain that binds to target antigen, and most pref-erably a light chain comprising light chain CDR sequences ofthe invention described below.[0021] Another aspect of the invention provides an anti-body that binds target antigen comprising a light chain thatcomprises any one, two, and/or three of the light chain CDRsequences of the invention described below.[0022] Preferably the light chain comprises an amino acidsequence identified as a light chain CDR3 sequence. Such a"light chain CDR3 sequence" includes an amino acidsequence identified as a light chain CDR3 sequence in FIG. 2and within SEQ ID NOs: 2, 4, 6, 8 10 and 12. Alternatively,the light chain CDR3 sequence comprises an amino acidsequence that contains one or more amino acid changes com-pared to any light chain CDR3 amino acid sequence identifiedin FIG. 2, i.e. a substitution, insertion or deletion. Preferablesubstitutions include a substitution to an amino acid at thecorresponding position within another light chain CDR3 ofFIG. 2. Alternatively, the light chain CDR3 sequence maycomprise a consensus amino acid sequence of light chainCDR3 shown in FIG. 2.[0023] The light chain comprising a light chain CDR3sequence of the invention described above may further com-prise a "light chain CDRI sequence" of the invention, whichincludes any of the amino acid sequences identified as a lightchain CDRI in FIG. 2, amino acid sequences that contain oneor more amino acid changes compared to any light chainCDRI identified in FIG. 2, preferably a substitution to anamino acid at the corresponding position within another lightchain CDRI of FIG. 2, or a consensus sequence of light chainCDRI shown in FIG. 2.[0024] Alternatively, the light chain comprising a lightchain CDR3 sequence of the invention described above mayfurther comprise a "light chain CDR2 sequence" of the inven-tion, which includes any of the amino acid sequences identi-fied as a light chain CDR2 in FIG. 2, amino acid sequencesthat contain one or more amino acid changes compared to anylight chain CDR2 identified in FIG. 2, preferably a substitu-tion to an amino acid at the corresponding position withinanother light chain CDR2 of FIG. 2, or a consensus sequenceof light chain CDR2 shown in FIG. 2.[0025] The light chain comprising a light chain CDR3sequence of the invention described above may also compriseboth (a) a light chain CDRI sequence of the inventiondescribed above and (b) a light chain CDR2 sequence of theinvention described above.[0026] Antibodies comprising any one of the light chainsdescribed above may further comprise a heavy chain, prefer-ably a heavy chain that binds to target antigen, and mostpreferably a heavy chain comprising heavy chain CDRsequences of the invention described above.[0027] In yet another embodiment, the antibody or antigen-binding compound comprises a constant region and one ormore heavy and light chain variable framework regions of ahuman antibody sequence. In a related embodiment, the anti-body comprises a modified or unmodified constant region ofa human IgGI, IgG2, IgG3 or IgG4. In an exemplary embodi-ment, the constant region is optionally modified to enhance ordecrease certain properties. For example, modifications to theconstant region, particularly the hinge or CH2 region, mayincrease or decrease effector function, including ADCC and/or CDC activity. In other embodiments, an IgG2 constant

US 2009/0311257 A1 Dec. 17, 2009

region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constantregion, particularly the hinge region, may reduce the forma-tion of half-antibodies.

[0028] In exemplary embodiments, the antibody of theinvention is derived from, based on, or contains part of thehuman antibody consensus sequence, human germlinesequence, human consensus germline sequence, or any one ofthe human antibody sequences in Kabat, NCBI Ig Blast(http: //www.ncbi.nlm.nih.gov/igblast/showGermline.cgi),which enables searching all Ig sequences in the database,including germline sequences (maintained by the NationalCenter for Biotechnology Information); Kabat Databasehttp: //www.bioinf.org.uk/abs/sexiest.html, Martin, A.C.R."Accessing the Kabat Antibody Sequence Database by Com-puter" Proteins: Structure, Function and Genetics, 25 (1996),130-133;

[0029] ImMunoGeneTics database (Montpellier France)(http: //imgt.cines.fr/), Lefranc, M.-P. et al., Nucleic AcidsResearch, 27, 209-212, 1999;[0030] V-Base, Tomlinson, I. M., Williams, S. C., Ignatov-ich, O., Corbett, S. J. & Winter, G. (1996) VBASE SequenceDirectory (Medical Research Council Centre for ProteinEngineering, Cambridge, UK);[0031] Zurich University (http: //www.biochem.unizh.ch/antibody[0032] /Sequences/index. html), Burmester, et al., Selec-tion, characterization and X-ray structure of anti-ampicillinsingle chain Fv fragments from phage-displayed murine anti-body libraries. J. Mol. Biol., 309 (2001) 671-685;

[0033] The Therapeutic Antibody Human HomologyProject (TAHHP) (http: //www.path.cam.ac.uk/-mrc7/hu-manisation/TAHHP.html), "Reshaping antibodies fortherapy" Edward G. Routledge, Scott D. Gorman and MikeClark, in Protein Engineering of Antibody Molecules forProphylactic and Therapeutic Applications in Man pp. 13-44(1993), Academic Titles, Nottingham, England;[0034] Humanization by design (http: //people.cryst.bbk.ac.uk/-ubcg07s/), Bendig, M. M., Kettleborough, C. A.,Jones, S. T., Maeda, H. and Saldanha, J. (1993), "The humani-sation of mouse monoclonal antibodies by CDR-grafting:Examples with anti-viral and anti-tumour cell antibodies", inMonoclonal Antibodies 2: Applications in Clinical Oncologyed. A. A. Epenetos, pp 119-140, Chapman & Hall MedicalPublishers; Leger, O. J. P. and Saldanha, J. W. (2000), "Prepa-ration of recombinant antibodies from immune rodentspleens and the design of their humanization by CDR graft-ing", in Monoclonal Antibodies: A Practical Approach eds. P.Shepherd and C. Dean, pp 25-69, Oxford University Press;[0035] Antibody Resources (http: //www.antibodyre-source.corn/educational. html), Antibody Engineering (byTT Wu), Humana Press.

[0036] In yet another embodiment of the invention, theaforementioned antibody has an affinity Kd of at least 10[ ]M. In a related embodiment, the antibody has an affinity Kd ofat least 10[ ]M.

[0037] In one aspect, the invention specifically contem-plates sterile compositions of isolated monoclonal antibodythat binds to gastrin with an affinity Kd ranging from about10 M to 10 M, or about 10 M to 10 M, or 10 M to10 " M; in a related aspect, the invention contemplates theuse of such compositions to treat disorders associated withgastrin expression.

[0038] Yet another aspect of the invention provides non-immunoglobulin-like recombinant polypeptides or othercompounds that comprise any of the heavy chain or lightchain CDR sequences of the invention described above, orany combinations of these CDR sequences. For example,such compounds may comprise a CDR sequence of the inven-tion as a single copy or in multiple copies in, for example, atandemly repeated or multivalent configuration. Such com-pounds may further comprise other CDR sequences in singleor multiple copies. Such compounds may also include non-peptidyl linkages.[0039] In still another embodiment of the invention, anisolated nucleic acid is provided comprising a nucleic acidsequence encoding the aforementioned antibody. In a relatedembodiment, the isolated nucleic acid comprises a heavychain nucleic acid sequence which is at least 60, 65, 70, 75,80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical tothe heavy chain nucleotide sequence set forth in SEQ ID NO:13, 15, 17, 19 and 21. In yet another related embodiment, theisolated nucleic acid comprises a light chain nucleic acidsequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% identical to the light chain nucle-otide sequence set forth in SEQ ID NOs: 14, 16, 18, 20 and 22.[0040] In another embodiment, a vector comprising theaforementioned isolated nucleic acid is provided. In a relatedembodiment, the aforementioned vector is provided whereinthe isolated nucleic acid is operably linked to a regulatorycontrol sequence. In still another embodiment, a host cell isprovided comprising the aforementioned vector.[0041] Numerous methods are contemplated in the presentinvention. For example, a method of producing an aforemen-tioned antibody is provided comprising culturing the afore-mentioned host cell such that the isolated nucleic acid isexpressed to produce the antibody. In a related embodiment,the method further comprises the step of recovering the anti-body from the host cell culture. In a related embodiment, anisolated antibody produced by the aforementioned method isprovided.[0042] A further aspect of the invention addresses the por-tions of the compounds of the invention that do not bind thetarget antigen but instead are responsible for other functions,such as circulating half-life, direct cytotoxic effect, detectablelabeling, or activation of the recipient's endogenous comple-ment cascade or endogenous cellular cytotoxicity. Antibodiesof the invention may comprise all or a portion of the constantregion and may be of any isotype, including IgA (e.g., IgAI orIgA2), IgD, IgE, IgG (e.g. IgGI, IgG2, IgG3 or IgG4), orIgM. In addition to, or instead of, comprising a constantregion, antigen-binding compounds of the invention mayinclude an epitope tag, a salvage receptor epitope, a labelmoiety for diagnostic or purification purposes, or a cytotoxicmoiety such as a radionuclide or toxin.[0043] In another embodiment of the invention, a pharma-ceutical composition is provided comprising any one of theaforementioned antibodies and a pharmaceutically suitablecarrier, excipient or diluent. Preferably the antibodies andcompounds of the invention are administered in a therapeu-tically effective amount, i.e., an amount sufficient to amelio-rate a clinical sign or symptom of a condition or disorderassociated with the target protein expression, to a subject inneed of such treatment. In a related embodiment, the phar-maceutical composition further comprises a second therapeu-tic agent. In yet another related embodiment, the pharmaceu-tical composition is provided wherein the second therapeutic

US 2009/0311257 A1 Dec. 17, 2009

agent is a growth factor, a cytokine, a chemotherapeuticagent, or a radiotherapeutic agent. In another embodiment thesecond therapeutic agent is another antibody.[0044] In another embodiment of the invention, the afore-mentioned methods are provided wherein the subject is amammal. In a related embodiment, the mammal is human.[0045] In another embodiment, the aforementioned meth-ods are provided wherein the antibody inhibits the interactionbetween the target and a binding partner. In yet anotherembodiment, the aforementioned methods are providedwherein the antibody is administered at a dose between about2 pg/kg to 50 mg/kg, 0.1 mg/kg to 30 mg/kg, or 0.1 mg/kg to10 mg/kg.[0046] In another embodiment of the invention, the use ofan antibody of the invention is contemplated in the manufac-ture of a medicament for preventing or reducing a conditionor disorder associated with target protein expression, asdefined herein.

[0047] In any of the aforementioned uses, the medicamentis coordinated with treatment using a second therapeuticagent.[0048] In another embodiment of the invention, the use of asynergistic combination of an antibody of the invention forpreparation of a medicament for treating a patient exhibitingsymptoms of a condition or disorder disclosed herein whereinthe medicament is coordinated with treatment using a secondtherapeutic agent is contemplated. In a related embodiment,the second therapeutic agent is a chemokine, a cytokine, agrowth factor, a chemotherapeutic agent, a radiotherapeuticagent, or radiation therapy.[0049] Embodiments of any of the aforementioneduses arecontemplated wherein the amount of antibody in the medica-ment is at a dose effective to reduce the dosage of secondtherapeutic agent required to achieve a therapeutic effect.[0050] The amount of antibody in any of the aforemen-tioned medicaments may be at a dose between about 2 pg/kgto 50 mg/kg body weight. In a related embodiment, theamount of antibody in the medicament is at a dose betweenabout 0.1 mg/kg to 30 mg/kg body weight. In still anotherembodiment, the amount of antibody in the medicament is ata dose between about 0.1 mg/kg to 10 mg/kg body weight.[0051] Kits are also contemplated by the present invention.In one embodiment, a kit comprises a therapeutically effec-tive amount of a composition of the invention, packaged in acontainer, such as a vial or bottle, and further comprising alabel attached to or packaged with the container, the labeldescribing the contents of the container and providing indi-cations and/or instructions regarding use of the contents of thecontainer to prevent or reduce a condition or disorder associ-ated with target protein expression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows the heavy chain amino acid sequencesof anti-gastrin antibodies XPA061, XPA063, XPA065,XPA067, XPA081 and a consensus sequence. CDRs areunderlined and Chothia numbering for all sequences isincluded beneath the consensus sequence.

[0053] FIG. 2 shows the light chain amino acid sequencesof anti-gastrin antibodies XPA061, XPA063, XPA065,XPA067, XPA081 and a consensus sequence. CDRs areunderlined and Chothia numbering for all sequences isincluded beneath the consensus sequence.

[0054] FIG. 3 shows the heavy chain amino acid sequencesof the XPA.067 affinity matured antibodies (SEQ ID NOs:23-33). CDRs are underlined.[0055] FIG. 4 is a comparison of the heavy chain CDRregions of the originating XPA067 antibody and the affinitymatured antibodies.[0056] FIG. 5 illustrates the improved gastrin neutraliza-tion capacity of reformatted, affinity matured antibodiesXPA067.06 and XPA067.18 compared to parent antibodyXPA067.[0057] FIG. 6 shows the neutralization of anti-gastrin anti-bodies in a gastric pH famotidine mouse model. F: Fainoti-dine, G: h-G17 (human gastrln), XPA067: parental a-gastrinmAb, XPA067.06: affinity mature u-gastrin mAb.[0058] FIG. 7 shows the neutralization of anti-gastrin anti-bodies in a gastric pH telenzepine mouse model. T: Telen-zepine, G: h-G17 (human gastrln), XPA067: parental u-gas-trin mAb, XPA067.06: affinity mature u-gastrln mAb.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention addresses a need in the art todevelop therapeutics to treat conditions or disorders associ-ated with target antigen expression. The present inventionprovides molecules or agents that interact with the target toeliminate signaling through binding partners of the target.[0060] In order that the invention may be more completelyunderstood, several definitions are set forth.[0061] As used herein, "target" or "target antigen" refers tothe gastrin peptide hormone. Gastrin may be the 34 aminoacid gastrin peptide or may be a shorter version of the peptide,such as the 17 amino acid or 14 amino acid variant of gastrin.[0062] In a preferred embodiment the gastrin is humangastrin.[0063] As used herein, the "desired biological activity" ofan anti-target antibody is the ability to bind to gastrin andinhibit its functional effects.[0064] As used herein, a "condition" or "disorder associ-ated with target expression" is a condition or disorder inwhich target activity is detrimental and includes diseases andother disorders in which high levels o f target have been shownto be or are suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes toa worsening of the disorder, as well as diseases and otherdisorders in which high levels of target expression are asso-ciated with undesirable clinical signs or symptoms. Suchdisorders may be evidenced, for example, by an increase inthe levels of target secreted and/or on the cell surface orincreased signalling inthe affected cells ortissues ofa subjectsuffering from the disorder. The increase in target levels maybe detected, for example, using an target specific antibody asdescribed above.[0065] Exemplary conditions or disorders associated withtarget expression include cancers, such as pancreatic cancer,esophageal cancer, gastric cancer, colorectal cancer, andsmall lung cell carcinoma, as well as gastric ulcer, duodenalulcer, other ulcers or conditions associated with H. Pylori,gastroesophageal reflux disease, autoimmune gastritis, atro-phic body gastritis, Zollinger-Ellison syndrome associatedwith tumor of the pancreas (gastrinoma), and inflammatorybowel disease.[0066] An "immunoglobulin" or "native antibody" is a tet-rameric glycoprotein. In a naturally-occurring immunoglo-bulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one "light" (about 25

US 2009/0311257 A1 Dec. 17, 2009

kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsiblefor antigen recognition. The carboxy-terminal portion of eachchain defines a constant region primarily responsible foreffector function. Human light chains are classified as kappa(K) and lambda P.) light chains. Heavy chains are classified asmu (p), delta (A), gamma (7), alpha (u), and epsilon (e), anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable andconstant regions are j oined by a "I" region of about 12 or moreamino acids, with the heavy chain also including a "D" regionof about 10 more amino acids. See generally, FundamentalImmunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all pur-poses). The variable regions of each light/heavy chain pairform the antibody binding site such that an intact immuno-globulin has two binding sites.[0067] Each heavy chain has at one end a variable domain(V~) followed by a number of constant domains. Each lightchain has a variable domain at one end (Vz) and a constantdomain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain,and the light chain variable domain is aligned with the vari-able domain of the heavy chain. Particular amino acid resi-dues are believed to form an interface between the light andheavy chain variable domains (Chothia et al., J. Mol. Biol.196: 901-917, 1987).[0068] Immunoglobulin variable domains exhibit the samegeneral structure of relatively conserved framework regions(FR) joined by three hypervariable regions or CDRs. From¹erminus to C-terminus, both light and heavy chains com-prise the domains FRI, CDRI, FR2, CDR2, FR3, CDR3 andFR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Pro-teins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk,(J. Mol. Biol. 196:901-917, 1987); Chothia et al., (Nature342: 878-883, 1989).[0069] The hypervariable region of an antibody refers to theCDR amino acid residues of an antibody which are respon-sible for antigen-binding. The hypervariable region com-prises amino acid residues from a CDR [i.e., residues 24-34(Ll), 50-56 (L2) and 89-97 (L3) in the light chain variabledomain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in theheavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5' Ed.Public Health Service, National Institutes of Health,Bethesda, Md. (1991)] and/or those residues from a hyper-variable loop (i.e., residues 26-32 (Ll), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (HI), 53-55(H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917(1987)].[0070] Framework or FR residues are those variabledomain residues other than the hypervariable region residues.[0071] "Heavy chain variable region" as used herein refersto the region of the antibody molecule comprising at least onecomplementarity determining region (CDR) of said antibodyheavy chain variable domain. The heavy chain variable regionmay contain one, two, or three CDRs of said antibody heavychain.[0072] "Light chain variable region" as used herein refersto the region of an antibody molecule, comprising at least one

complementarity determining region (CDR) of said antibodylight chain variable domain. The light chain variable regionmay contain one, two, or three CDRs of said antibody lightchain, which may be either a kappa or lambda light chaindepending on the antibody.[0073] The term "antibody" is used in the broadest senseand includes fully assembled antibodies, monoclonal anti-bodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), antibody fragments that can bind anti-gen (e.g., Fab', F'(ab)~, Fv, single chain antibodies, diabod-ies), and recombinant peptides comprising the forgoing aslong as they exhibit the desired biological activity. Antigen-binding portions may be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intactantibodies. Antibody fragments or antigen-binding portionsinclude, inter alia, Fab, Fab', F(ab')~, Fv, domain antibody(dAb), complementarity determining region (CDR) frag-ments, single-chain antibodies (scFv), single chain antibodyfragments, chimeric antibodies, diabodies, triabodies, tetra-bodies, minibody, linear antibody; chelating recombinantantibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a antigen-binding-domain immunoglobulin fusion protein, a camelizedantibody, a V~~ containing antibody, or a variant or a deriva-tive thereof, and polypeptides that contain at least a portion ofan immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide, such as a CDR sequence, as longas the antibody retains the desired biological activity.[0074] "Monoclonal antibody" refers to an antibodyobtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occur-ring mutations that may be present in minor amounts.[0075] "Antibody variant" as used herein refers to an anti-body polypeptide sequence that contains at least one aminoacid substitution, deletion, or insertion in the variable regionof the natural antibody variable region domains. Variants maybe substantially homologous or substantially identical to theunmodified antibody.[0076] A "chimeric antibody," as used herein, refers to anantibody containing sequence derived from two differentantibodies (see, e.g., U.S. Pat. No. 4,816,567) which typicallyoriginate from different species. Most typically, chimericantibodies comprise human and rodent antibody fragments,generally human constant and mouse variable regions.[0077] A "neutralizing antibody" is an antibody moleculewhich is able to eliminate or significantly reduce an effectorfunction of a target antigen to which it binds. Accordingly, a"neutralizing" anti-target antibody is capable of eliminatingor significantly reducing an effector function, such as enzymeactivity, ligand binding, or intracellular signaling.[007S] An "isolated" antibody is one that has been identi-fied and separated and recovered from a component of itsnatural environment. Contaminant components of its naturalenvironment are materials that would interfere with diagnos-tic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-pro-teinaceous solutes. In preferred embodiments, the antibodywill be purified (I) to greater than 95% by weight of antibodyas determined by the Lowry method, and most preferablymore than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of ¹erminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homo-geneity by SDS-PAGE under reducing or nonreducing con-

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ditions using Coomassie blue or, preferably, silver stain. Iso-lated antibody includes the antibody in situ withinrecombinant cells since at least one component of the anti-body's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least onepurification step.

[0079] As used herein, an antibody that "specifically binds"is "target specific", is "specific for" target or is "immunore-active" with the target antigen refers to an antibody or anti-body substance of the invention that binds the target antigenwith greater affinity than with similar antigens. In one aspect,the target-binding polypeptides of the invention, or frag-ments, variants, or derivatives thereof, will bind with a greateraffinity to human target as compared to its binding affinity totarget of other, i.e., non-human, species, but binding polypep-tides that recognize and bind orthologs of the target are withinthe scope of the invention.

[00SO] For example, a polypeptide that is an antibody orfragment thereof "specific for" its cognate antigen indicatesthat the variable regions of the antibodies recognize and bindthe polypeptide of interest with a detectable preference (i.e.,able to distinguish the polypeptide of interest from otherknown polypeptides of the same family, by virtue of measur-able differences in binding affinity, despite the possible exist-ence of localized sequence identity, homology, or similaritybetween family members). It will be understood that specificantibodies may also interact with other proteins (for example,S. aureus protein A or other antibodies in ELISA techniques)through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constantregion of the molecule. Screening assays to determine bind-ing specificity of an antibody for use in the methods of theinvention are well known and routinely practiced in the art.For a comprehensive discussion of such assays, see Harlow etal. (Eds), Antibodies A I aborato~ Manual; Cold Spring Har-bor Laboratory, Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies for use in the invention can be produced using anymethod known in the art.

[00S1] The term "epitope" refers to that portion of anymolecule capable of being recognized by and bound by aselective binding agent at one or more of the antigen bindingregions. Epitopes usually consist of chemically active surfacegroupings of molecules, such as, amino acids or carbohydrateside chains, and have specific three-dimensional structuralcharacteristics as well as specific charge characteristics.Epitopes as used herein may be contiguous or non-contigu-ous. Moreover, epitopes may be mimetic (mimotopes) in thatthey comprise a three dimensional structure that is identical tothe epitope used to generate the peptibody, yet comprise noneor only some of the amino acid residues found in the targetthat were used to stimulate the peptibody immune response.As used herein, a mimotope is not considered a differentantigen from the epitope bound by the selective bindingagent; the selective binding agent recognizes the same three-dimensional structure of the epitope and mimotope.

[00S2] The term "derivative" when used in connection withantibody substances and polypeptides of the invention refersto polypeptides chemically modified by such techniques asubiquitination, conjugation to therapeutic or diagnosticagents, labeling (e.g., with radionuclides or variousenzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion orsubstitution by chemical synthesis of amino acids such as

ornithine, which do not normally occur in human proteins.Derivatives retain the binding properties of underivatizedmolecules of the invention.[00S3] "Detectable moiety" or a "label" refers to a compo-sition detectable by spectroscopic, photochemical, biochemi-cal, immunochemical, or chemical means. For example, use-ful labels include P, S, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins forwhich antisera or monoclonal antibodies are available, ornucleic acid molecules with a sequence complementary to atarget. The detectable moiety often generates a measurablesignal, such as a radioactive, chromogenic, or fluorescentsignal, that can be used to quantitate the amount of bounddetectable moiety in a sample.[00S4] The term "therapeutically effective amount" is usedherein to indicate the amount of target-specific compositionof the invention that is effective to ameliorate or lessen symp-toms or signs of disease associated with target protein expres-sion.

[00S5] The present invention provides a target-specificantibody, which may comprise those exemplary sequencesset out in FIGS. 1 and 2, fragments, variants and derivativesthereof, pharmaceutical formulations including a target-spe-cific antibody recited above, methods of preparing the phar-maceutical formulations, and methods of treating patientswith the pharmaceutical formulations and compounds.

[00S6] Depending on the amino acid sequence of the con-stant domain of their heavy chains, immunoglobulins can beassigned to different classes, IgA, IgD, IgE, IgG and IgM,which may be further divided into subclasses or isotypes, e.g.IgGI, IgG2, IgG3, IgG4, IgAI and IgA2. The subunit struc-tures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Different iso-types have different effector functions; for example, IgGI andIgG3 isotypes have ADCC activity. An antibody of the inven-tion, if it comprises a constant domain, may be of any of thesesubclasses or isotypes.

[00S7] The antibodies of the present invention may exhibitbinding affinity to antigen of a Ka of greater than or equal toabout 10 M ', greater than or equal to about 10 M ', orgreater than or equal to about 10 M ', or greater than or equalto about 10 M ', or greater than or equal to about 10 M ',10' M ',10"M 'or10' M '.Suchaffinitiesmaybereadilydetermined using conventional techniques, such as by equi-librium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radio-immunoassay using ' I labeled target antigen; or by anothermethod known to the skilled artisan. The affinity data may beanalyzed, for example, by the method of Scatchard et al., (AnnN.Y. Acad. Sci., 51:660, 1949).

Antibody Polypeptides of the Invention

[00SS] The present invention encompasses amino acidmolecules encoding target specific antibodies. In exemplaryembodiments, a target specific antibody of the invention cancomprise a human kappa (K) or a human lambda P.) lightchain or an amino acid sequence derived therefrom, or ahuman heavy chain or a sequence derived therefrom, or bothheavy and light chains together in a single chain, dimeric,tetrameric or other form. In some embodiments, a heavychain and a light chain of a target specific immunoglobulinare different amino acid molecules. In other embodiments,

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the same amino acid molecule contains a heavy chain variableregion and a light chain variable region of a target specificantibody.

[0089] In some embodiments, the amino acid sequence ofthe human anti-target antibody comprises one or more CDRsof the amino acid sequence of the light chain variable region(V~) of antibodies XPA061, XPA063, XPA065, XPA067, andXPA081 set out in FIG. 2 or variants thereof. In some embodi-ments, the V~ comprises the amino acid sequence from thebeginning of the CDRI to the end of the CDR3 of the lightchain of any one of the foregoing antibodies.

[0090] In one embodiment, the target specific antibodycomprises a light chain CDRI, CDR2 or CDR3, each ofwhich are independently selected from the CDRI, CDR2 andCDR3 regions of an antibody having a light chain variableregion comprising the amino acid sequence of the V~ regionset out in SEQ ID NOs: 2, 4, 6, 8, 10 and 12, or encoded by anucleic acid molecule encoding the V~ region set out in SEQID NOs: 14, 16, 18, 20 and 22. In one aspect the light chainCDRI is from approximately residues 24-34, CDR2 is fromapproximately residues 50-56 and CDR3 extends fromapproximately residues 89-97, according to Chothia number-ing. A polypeptide of the target specific antibody may com-prise the CDRI, CDR2 and CDR3 regions of an antibodycomprising the amino acid sequence of the V~ region selectedfrom the group consisting of XPA.061XPA063, XPA065,XPA067, and XPA081.

[0091] In some embodiments, the human target specificantibody comprises one or more CDRs of the amino acidsequence of the heavy chain variable region (V~) of antibodyXPA.061, XPA063, XPA065, XPA067, and XPA081 set outin FIG. 1 or FIG. 3 or variants thereof. In some embodiments,the V~ comprises the amino acid sequence from the begin-ning of the CDRI to the end of the CDR3 of any one of theheavy chain of the foregoing antibodies.

[0092] In one embodiment, the target specific antibodycomprises a heavy chain CDRI, CDR2 or CDR3, each ofwhich are independently selected from the CDRI, CDR2 andCDR3 regions of an antibody having a heavy chain variableregion comprising the amino acid sequence of the V~ regionset out in SEQ ID NOs: I, 3, 5, 7, 9 and 11, or encoded by anucleic acid molecule encoding the V~ region set out in SEQID NO: 13, 15, 17, 19 and 21. It is further contemplated thata target specific antibody comprises a heavy chain CDRI,CDR2 or CDR3, each of which are independently selectedfrom the CDRI, CDR2 and CDR3 regions of an antibodyhaving a heavy chain variable region comprising the aminoacid sequence of the V~ region set out in SEQ ID NOs: 23-33.In one aspect the heavy chain CDRs are located according toChlothia numbering set out in FIG. 1: CDRI is from approxi-mately residues 31-35, CDR2 is from approximately residues50-65 and CDR3 extends from approximately residues95-102. A polypeptide of the target specific antibody maycomprise the CDRI, CDR2 and CDR3 regions of an antibodycomprising the amino acid sequence o f the V~ region selectedfrom the group consisting of XPA061, XPA063, XPA065,XPA067, and XPA081.

[0093] In another embodiment, the antibody comprises alight chain as disclosed above and a heavy chain as disclosedabove.

[0094] It is contemplated that a variant of the antibodysequence refers to amino acid sequences, comprising a vari-able heavy chain or a variable light chain amino acid sequence

which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99% homologous to any of the amino acidsequences set forth herein.[0095] It is further contemplated that the CDR of the anti-body heavy and light chains comprise variant amino acidsequences which may improve antibody binding affinity andare derived through, for example, affinity maturation. In oneaspect it is contemplated that an antibody of the inventioncomprises a heavy chain CDR2 sequence having about 35%identity to a CDR2 of a parent antibody sequence set out inSEQ ID NOs: I, 3, 5, 7, 9, 11 or 23-33. In a related aspect itis contemplated that an antibody of the invention comprises aheavy chain CDR3 sequence having about 50% identity to aCDR3 of a parent antibody sequence set out in SEQ ID NOs:I, 3, 5, 7, 9, 11 or 23-33.

Antibody Nucleic Acids of the Invention

[0096] The present invention also encompasses nucleicacid molecules encoding target specific antibodies. In someembodiments, different nucleic acid molecules encode aheavy chain variable region and a light chain variable regionof a target specific antibody. In other embodiments, the samenucleic acid molecule encodes a heavy chain and a light chainvariable regions of a target specific antibody.[0097] In one embodiment, the nucleic acid encodes a tar-get specific antibody of the invention.[0098] In one aspect, a nucleic acid molecule of the inven-tion comprises a nucleotide sequence that encodes the V~amino acid sequence of antibodies XPA061, XPA063,XPA065, XPA067, and XPA081 set out in SEQ ID NOs: 2, 4,6, 8 and 10 or a portion thereof. In a related aspect, the V~amino acid sequence is a consensus sequence set out in SEQID NO: 12. In some embodiments, the nucleic acid encodesthe amino acid sequence of the light chain CDRs of saidantibody. In some embodiments, said portion is a contiguousportion comprising CDRI-CDR3. In one embodiment, saidportion comprises at least one, two or three of a light chainCDRI, CDR2, or CDR3 region.[0099] In a related aspect, the nucleic acid molecule com-prises a nucleotide sequence that encodes the light chainamino acid sequence of one of SEQ ID NOs: 2, 4, 6, 8, 10 and12 or a portion thereof. In one embodiment, the nucleic acidmolecule comprises the light chain nucleotide sequence ofany one of SEQ ID NOs: 14, 16, 18, and 22 or a portionthereof.[0100] In some embodiments, the nucleic acid moleculeencodes a V~ amino acid sequence that is at least 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96 97, 98 or 99% identicalto a V~ amino acid sequence set out in SEQ ID NOs: 2, 4, 6,8, 10 and 12. Nucleic acid molecules of the invention includenucleic acids that hybridize under highly stringent condi-tions, such as those described herein, to a nucleic acidsequence encoding the light chain variable region amino acidsequence of SEQ IDNOs: 2,4, 6, 8, 10 and 12, orthathas thelight chain variable region nucleic acid sequence of SEQ IDNOs: 14, 16, 18, 20 and 22.[0101] It is further contemplated that a nucleic acid mol-ecule of the invention comprises a nucleotide sequence thatencodes the V~ amino acid sequence of any one of antibodiesXPA061, XPA063, XPA065, XPA067, and XPA081, or aportion thereof. In some embodiments, the nucleic acidencodes the amino acid sequence of the heavy chain CDRs ofsaid antibody. In some embodiments, said portion is a con-tiguous portion comprising heavy chain CDRI-CDR3. In one

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embodiment, said portion comprises at least one, two or threeof a heavy chain CDRI, CDR2, or CDR3 region.[0102] In a related aspect, the nucleic acid molecule com-prises a nucleotide sequence that encodes the heavy chainamino acid sequence of one of heavy chain of SEQ ID NOs:I, 3, 5, 7, 9 and 11 or a portion thereof. In one embodiment,the nucleic acid molecule comprises the heavy chain nucle-otide sequence of SEQ ID NO: 13, 15, 17, 19 and 21 a portionthereof.[0103] In some embodiments, the nucleic acid moleculeencodes a V~ amino acid sequence that is at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to aV~ amino acid sequence XPA061, XPA063, XPA065,XPA067, and XPA081 set out in SEQ ID NOs: I, 3, 5, 7 and9. In a related aspect, the V~ amino acid sequence is a con-sensus sequence set out in SEQ ID NO: 11. Nucleic acidmolecules of the invention include nucleic acids that hybrid-ize under highly stringent conditions, such as those describedabove, to a nucleic acid sequence encoding the heavy chainvariable region amino acid sequence of SEQ ID NOs: I, 3, 5,7, 9 and 11, or that has the heavy chain variable region nucleicacid sequence of any one of SEQ ID NO: 13, 15, 17, 19 and21.[0104] It is further contemplated that the nucleic acids ofthe invention encode a full-length light chain or heavy chainof an antibody selected from XPA061, XPA063, XPA065,XPA067, and XPA081 wherein a full-length light chain orfull-length heavy chain comprises a light chain constantregion or a heavy chain constant region, respectively.[0105] In one aspect, the full length light chain antibodycomprises the sequences set out in SEQ ID NOs: 2, 4, 6, 8, 10and 12. It is further contemplated that the nucleotide encodingthe full-length light chain encodes the sequences SEQ IDNOs: 2, 4, 6, 8, 10 and 12, and comprises the nucleotidessequence set forth in SEQ ID NOs: 14, 16, 18, 20 and 22.[0106] In one aspect, the full length heavy chain antibodycomprises the sequences in any one of SEQ ID NOs: I, 3, 5,7, 9 and 11. It is further contemplated that the nucleotideencoding the full-length heavy chain encodes the sequencesheavy chain of SEQ ID NOs: I, 3, 5, 7, 9 and 11 and comprisesthe nucleotides sequence set forth in any one of SEQ ID NO:13, 15, 17, 19 and 21.

Polyclonal Antibodies

[0107] Polyclonal antibodies are preferably raised in ani-mals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the relevant antigen and an adjuvant. Animproved antibody response may be obtained by conjugatingthe relevant antigen to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin,serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, forexample, maleimidobenzoyl sulfosuccinimide ester (conju-gation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydrideor other agents known in the art.[0108] Animals are immunized against the antigen, immu-nogenic conjugates, or derivatives by combining, e.g., 100 pgor 5 pg of the protein or conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's: complete adjuvantand injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with 'ts to (fraction('bio) ) the original amount of peptide or conjugate in Freund'scomplete adjuvant by subcutaneous injection at multiple

sites. At 7-14 days post-booster injection, the animals are bledand the serum is assayed for antibody titer. Animals areboosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conju-gated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinantcell culture as protein fusions. Also, aggregating agents suchas alum are suitably used to enhance the immune response.

Monoclonal Antibodies

[0109] Monoclonal antibody refers to an antibody obtainedfrom a population of substantially homogeneous antibodies.Monoclonal antibodies are generally highly specific, and maybe directed against a single antigenic site, in contrast to con-ventional (polyclonal) antibody preparations that typicallyinclude different antibodies directed against different deter-minants (epitopes). In addition to their specificity, the mono-clonal antibodies are advantageous in that they are synthe-sized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteris-tics.

[0110] Monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridomamethod first described by Kohler et al., (Nature, 256:495-7,1975), or may be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiesmay also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., (Nature352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581-597, 1991).

[0111] In the hybridoma method, a mouse or other appro-priate host animal, such as a hamster or macaque monkey, isimmunized as herein described to elicit lymphocytes thatproduce or are capable of producing antibodies that will spe-cifically bind to the protein used for immunization. Alterna-tively, lymphocytes may be immunized in vitro. Lympho-cytes then are fused with myeloma cells using a suitablefusing agent, such as polyethylene glycol, to form a hybri-doma cell (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)).

[0112] The hybridoma cells thus prepared are seeded andgrown in a suitable culture medium that preferably containsone or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine gua-nine phosphoribosyl transferase (HGPRT or HPRT), the cul-ture medium for the hybridomas typically will include hypox-anthine, aminopterin, and thymidine (HAT medium), whichsubstances prevent the growth of HGPRT-deficient cells.

[0113] Preferred myeloma cells are those that fuse efft-ciently, support stable high-level production of antibody bythe selected antibody-producing cells, and are sensitive to amedium. Human myeloma and mouse-human heteromy-cloma cell lines also have been described for the productionof human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Produc-tion Techniques and Applications, pp. 51-63 (Marcel Dekker,Inc., New York, 1987)). Exemplary murine myeloma linesinclude those derived from MOP-21 and M.C.-II mousetumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653cells available from the American Type Culture Collection,Rockville, Md. USA.

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[0114] Culture medium in which hybridoma cells are grow-ing is assayed for production of monoclonal antibodiesdirected against the antigen. Preferably, the binding specific-ity of monoclonal antibodies produced by hybridoma cells isdetermined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of themonoclonal antibody can, for example, be determined byScatchard analysis (Munson et al., Anal. Biochem., 107:220(1980)).[0115] After hybridoma cells are identified that produceantibodies of the desired specificity, affinity, and/or activity,the clones may be subcloned by limiting dilution proceduresand grown by standard methods (Goding, Monoclonal Anti-bodies: Principles and Practice, pp. 59-103 (Academic Press,1986)). Suitable culture media for this purpose include, forexample, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in ananimal. The monoclonal antibodies secreted by the subclonesare suitably separated from the culture medium, ascites fluid,or serum by conventional immunoglobulin purification pro-cedures such as, for example, protein A-Sepharose, hydroxy-lapatite chromatography, gel electrophoresis, dialysis; oraffinity chromatography.[0116] It is further contemplated that antibodies of theinvention may be used as smaller antigen binding fragmentsof the antibody well-known in the art and described herein.

Antibody Fragments

[0117] Antibody fragments comprise a portion of an intactfull length antibody, preferably an antigen binding or variableregion of the intact antibody. Examples of antibody fragmentsinclude Fab, Fab', F(ab')~, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules (e.g., scFv);multispecific antibody fragments such as bispecfic, trispe-cific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies);minibody; chelating recombinant antibody; tribodies orbibodies; intrabodies; nanobodies; small modular immunop-harmaceuticals (SMIP), binding-domain immunoglobulinfusion proteins; camelized antibodies; Vuu containing anti-bodies; and other polypeptides formed from antibody frag-ments.[0118] Papain digestion of antibodies produces two identi-cal antigen-binding fragments, called "Fab" fragments,monovalent fragments consisting of the Vz, Vu, Cz and Cudomains each with a single antigen-binding site, and aresidual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab')~ fragment,a bivalent fragment comprising two Fab fragments linked bya disulfide bridge at the hinge region, that has two "Single-chain Fv" or "scFv" antibody fragments comprise the Vu andVz domains of antibody, wherein these domains are present ina single polypeptide chain. Preferably, the Fv polypeptidefurther comprises a polypeptide linker between the Vu and Vzdomains that enables the Fv to form the desired structure forantigen binding, resulting in a single-chain antibody (scFv),in which a Vz and Vu region are paired to form a monovalentmolecule via a synthetic linker that enables them to be madeas a single protein chain (Bird et al., Science 242:423-426,1988, and Huston et al., Proc. Natl. Acad. Sci. USA. 85:5879-5883, 1988). For a review of sFv see Pluckthun, in The Phar-macology of Monoclonal Antibodies, vol. 113, Rosenburgand Moore eds., Springer-Verlag, New York, pp. 269-315(1994). An Fd fragment consists of the Vu and Cul domains.

[0119] Additional antibody fragment include a domainantibody (dAb) fragment (Ward et al., Nature 341:544-546,1989) which consists of a Vu domain. Diabodies are bivalentantibodies in which Vu and Vz domains are expressed on asingle polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen bindingsites (see e.g., EP 404,097; WO 93/11161; Holliger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak etal., Structure 2:1121-1123, 1994). Diabodies can be bispe-cific or monospecific.[0120] Functional heavy-chain antibodies devoid of lightchains are naturally occurring in nurse sharks (Greenberg etal., Nature 374:168-73, 1995), wobbegong sharks Outtall etal., MolImmunol. 38:313-26, 2001) and Camelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen et al., J.Mol. Biol. 275: 413, 1998), such as camels, dromedaries,alpacas and llamas. The antigen-binding site is reduced to asingle domain, the Vuu domain, in these animals. These anti-bodies form antigen-binding regions using only heavy chainvariable region, i.e., these functional antibodies arehomodimers of heavy chains only having the structure H~L~(referred to as "heavy-chain antibodies" or "HCAbs"). Cam-elidVuureportedly recombines with IgG2 and IgG3 constantregions that contain hinge, CH2, and CH3 domains and lacka CHI domain (Hamers-Casterman et al., supra). Forexample, llama IgGI is a conventional (H~L~) antibody iso-type in which Vu recombines with a constant region thatcontains hinge, CHI, CH2 and CH3 domains, whereas thellama IgG2 and IgG3 are heavy chain-only isotypes that lackCH I domains and that contain no light chains. Camelid Vuudomains have been found to bind to antigen with high affinity(Desmyter et al., J. Biol. Chem. 276:26285-90, 2001) andpossess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Classical sonly fragments are difficultto produce in soluble form, but improvements in solubilityand specific binding can be obtained when framework resi-dues are altered to be more VH~like. (See, e.g., Reichman, etal., I Immunol Methods 1999, 231:25-38.) Methods for gen-erating antibodies having camelid heavy chains are describedin, for example, in U.S. Patent Publication Nos. 20050136049and 20050037421.

[0121] Because the variable domain of the heavy-chain-antibodies is the smallest fully functional antigen-bindingfragment with a molecular mass of only 15 kDa, this entity isreferred to as a nanobody (Cortez-Retamozo et al., CancerResearch 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described inConrath et al., (Antimicrob Agents Chemother 45: 2807-12,2001) or using recombinant methods as described in[0122] Production of bispecific Fab-scFv ("bibody") andtrispecific Fab-(scFv)(2) ("trlbody") are described inSchoonjans et al. (J Immunol. 165:7050-57, 2000) andWillems et al. (J Chromatogr B Analyt Technol Biomed LifeSci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH, (Fd) chains, e.g., to produce a tribody two scFvs arefused to C-term of Fab while in a bibody one scFv is fused toC-term of Fab.

[0123] A "minibody" consisting of scFv fused to CH3 via apeptide linker (hingeless) or via an IgG hinge has beendescribed in Olafsen, et al., Protein Eng Des Sel. 2004 April;17(4):315-23.

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[0124] Intrabodies are single chain antibodies which dem-onstrate intracellular expression and can manipulate intrac-ellular protein function (Biocca, et al., EMBO J. 9:101-108,1990; Colby et al., Proc Natl Acad Sci U SA. 101:17616-21,2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody contruct in intracellular regions,may be produced as described in Mhashilkar et al (EMBO J14:1542-51, 1995) and Wheeler et al. (FASEB J. 17:1733-5.2003). Transbodies are cell-permeable antibodies in which aprotein transduction domains (PTD) is fused with singlechain variable fragment (scFv) antibodies Heng et al., (Med.Hypotheses. 64:1105-8, 2005).

[0125] Further contemplated are antibodies that are SMIPsor binding domain immunoglobulin fusion proteins specificfor target protein. These constructs are single-chain polypep-tides comprising antigen binding domains fused to immuno-globulin domains necessary to carry out antibody effectorfunctions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

[0126] One or more CDRs may be incorporated into a mol-ecule either covalently or noncovalently to make it an immu-noadhesin. An immunoadhesin may incorporate the CDR(s)as part of a larger polypeptide chain, may covalently link theCDR(s) to another polypeptide chain, or may incorporate theCDR(s) noncovalently. The CDRs permit the immunoad-hesin to specifically bind to a particular antigen of interest.

[0127] Thus, a variety of compositions comprising one,two, and/or three CDRs of a heavy chain variable region or alight chain variable region of an antibody may be generatedby techniques known in the art.

Multispecific Antibodies

[0128] In some embodiments, it may be desirable to gen-erate multispecific (e.g. bispecific) anti-target antibody of theinvention having binding specificities for at least two differ-ent epitopes of the same or different molecules. Exemplarybi specific antibodies may bind to two different epitopes o f thetarget molecule. Alternatively, a target-specific antibody armmay be combined with an arm which binds to a cell surfacemolecule, such as a T-cell receptor molecule (e.g., CD2 orCD3), or Fc receptors for igG (FcyR), such as FcyRI (CD64),FcyRII (CD32) and FcyRIII (CD16) so as to focus cellulardefense mechanisms to the target. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells whichexpress or take up the target. These antibodies possess atarget-binding arm and an arm which binds the cytotoxicagent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricinAchain, methotrexate or radioactive isotope hapten). Bispecificantibodies can be prepared as full length antibodies or anti-body fragments (e.g., F(ab')2 bispecific antibodies).

[0129] According to another approach for making bispe-cific antibodies, the interface between a pair of antibodymolecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell cul-ture. The preferred interface comprises at least a part of theCu3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger sidechains (e.g., tyrosine or tryptophan). Compensatory "cavi-ties" of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g.,alanine or threonine). This provides a mechanism for increas-

ing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO96/27011 publishedSep. 6, 1996.

[0130] Bispecific antibodies include cross-linked or "het-eroconjugate" antibodies. For example, one of the antibodiesin the heteroconjugate can be coupled to avidin, the other tobiotin. Heteroconjugate antibodies may be made using anyconvenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat.No. 4,676,980, along with a number of cross-linking tech-niques.

[0131] Techniques for generating bispecific antibodiesfrom antibody fragments have also been described in theliterature. For example, bispecific antibodies can be preparedusing chemical linkage. Brennan et al., (Science 229:81-83,1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab')~ fragments. Thesefragments are reduced in the presence of the dithiol complex-ing agent sodium arsenite to stabilize vicinal dithiols andprevent intermolecular disulfide formation. The Fab' frag-ments generated are then converted to thionitrobenzoate(TNB) derivatives. One of the Fab'-TNB derivatives is thenreconverted to the Fab'-thiol by reduction with mercaptoet-hylamine and is mixed with an equimolar amount of the otherFab'-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for theselective immobilization of enzymes. In yet a further embodi-ment, Fab'-SH fragments directly recovered from E. cali canbe chemically coupled in vitro to form bispecific antibodies.(Shalaby et al., J. Exp. Med. 175:217-225 (1992))

[0132] Shalaby et al., J. Exp. Med. 175:217-225 (1992)describe the production of a fully humanized bispecific anti-body F(ab')~ molecule. Each Fab' fragment was separatelysecreted from E. cali and subjected to directed chemicalcoupling in vitro to form the bispeefic antibody. The bispe-cific antibody thus formed was able to bind to cells overex-pressing the HER2 receptor and normal human T cells, aswell as trigger the lytic activity of human cytotoxic lympho-cytes against human breast tumor targets.

[0133] Various techniques for making and isolating bispe-cific antibody fragments directly from recombinant cell cul-ture have also been described. For example, bispecific anti-bodies have been produced using leucine zippers. (Kostelnyet al., J. Immunol. 148:1547-1553, 1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab'portions of two different antibodies by gene fusion. The anti-body homodimers were reduced at the hinge region to formmonomers and then re-oxidized to form the antibody het-erodimers. This method can also be utilized for the produc-tion of antibody homodimers. The "diabody" technologydescribed by Hollinger et al. (Proc. Natl. Acad. Sci. USA90: 6444-48, 1993) has provided an alternative mechanism formaking bispecific antibody fragments.

[0134] The fragments comprise a heavy chain variableregion (Vu) connected to a light-chain variable region (Vz) bya linker which is too short to allow pairing between the twodomains on the same chain. Accordingly, the Vu and Vzdomains of one fragment are forced to pair with the comple-mentary Vz and Vu domains of another fragment, therebyforming two antigen-binding sites. Another strategy for mak-ing bispecific antibody fragments by the use of single-chainFv (sFv) dimers has also been reported. See Gruber et al., J.Immunol. 152: 5368 (1994).

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[0135] Alternatively, the bispecific antibody may be a "lin-ear antibody" produced as described in Zapata et al. ProteinEng. 8:1057-62 (1995). Linear antibodies comprise a pair oftandem F d segments (Vu-Cul -Vu-Cu1) which form a pair o fantigen binding regions. Linear antibodies can be bispecificor monospecific.[0136] In a further embodiment, the bispecific antibodymay be a chelating recombinant antibody (CRAb). A chelat-ing recombinant antibody recognizes adjacent and non-over-lapping epitopes of the target antigen, and is flexible enoughto bind to both epitopes simultaneously (Neri et al., J Mol.Biol. 246:367-73, 1995).[0137] Antibodies with more than two valencies are alsocontemplated. For example, trispecific antibodies can be pre-pared. (Tutt et al., J. Immunol. 147:60, 1991).

Chimeric and Humanized Antibodies

[0138] Because chimeric or humanized antibodies are lessimmunogenic in humans than the parental mouse monoclonalantibodies, they can be used for the treatment of humans withfar less risk of anaphylaxis. Thus, these antibodies may bepreferred in therapeutic applications that involve in vivoadministration to a human.[0139] In particular, a rodent antibody on repeated in vivoadministration in man, either alone or as a conjugate, willbring about an immune response in the recipient against therodent antibody; the so-called HAMA response (Human AntiMouse Antibody). The HAMA response may limit the effec-tiveness of the pharmaceutical if repeated dosing is required.The immunogenicity of the antibody may be reduced bychemical modification of the antibody with a hydrophilicpolymer such as polyethylene glycol or by using the methodsof genetic engineering to make the antibody binding structuremore human like.[0140] Chimeric monoclonal antibodies, in which the vari-able Ig domains of a mouse monoclonal antibody are fused tohuman constant Ig domains, can be generated using standardprocedures known in the art (See Morrison et al., Proc. Natl.Head. Sci. USA 81, 6841-6855 (1984); and, Boulianne et al,Nature 312, 643-646, (1984)). Although some chimericmonoclonal antibodies have proved less immunogenic inhumans, the mouse variable Ig domains can still lead to asignificant human anti-mouse response.[0141] Humanized antibodies may be achieved by a varietyof methods including, for example: (I) grafting the non-human complementarity determining regions (CDRs) onto ahuman framework and constant region (a process referred toin the art as humanizing through "CDR grafting"), or, alter-natively, (2) transplanting the entire non-human variabledomains, but "cloaking" them with a human-like surface byreplacement of surface residues (a process referred to in theart as "veneering"). In the present invention, humanized anti-bodies will include both "humanized" and "veneered" anti-bodies. These methods are disclosed in, e.g., Jones et al.,Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Head.Sci., US.A., 81:6851-6855 (1984); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immunol. 31:169-217 (1994); andKettleborough et al., Protein Eng. 4:773-83 (1991) each ofwhich is incorporated herein by reference.[0142] CDR grafting involves introducing one or more ofthe six CDRs from the mouse heavy and light chain variableIg domains into the appropriate four framework regions of

human variable Ig domains. This technique (Riechmann, etal., Nature 332:323-27 (1988)), utilizes the conserved frame-work regions (FRI-FR4) as a scaffold to support the CDRloops which are the primary contacts with antigen. A disad-vantage of CDR grafting, however, is that it can result in ahumanized antibody that has a substantially lower bindingaffinity than the original mouse antibody, because aminoacids of the framework regions can contribute to antigenbinding, and because amino acids of the CDR loops caninfluence the association of the two variable Ig domains. Tomaintain the affinity of the humanized monoclonal antibody,the CDR grafting technique can be improved by choosinghuman framework regions that most closely resemble theframework regions of the original mouse antibody, and bysite-directed mutagenesis of single amino acids within theframework or CDRs aided by computer modeling of the anti-gen binding site (e.g., Co et al., J. Immunol. 152, 2968-2976(1994)).[0143] One method of humanizing antibodies comprisesaligning the non-human heavy and light chain sequences tohuman heavy and light chain sequences, selecting and replac-ing the non-human framework with a human frameworkbased on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing tothe conformation of the parent antibody. This process is fol-lowed by repeated back mutation of residues in the CDRregion which disturb the structure of the CDRs until thepredicted conformation of the humanized sequence modelclosely approximates the conformation of the non-humanCDRs of the parent non-human antibody.

Human Engineeringâ„¢

[0144] HUMAN ENGINEERINGâ„¢ of antibody variabledomains has been described by Studnicka [See, e.g., Stud-nicka et al. U.S. Pat. No. 5,766,886; Studnicka et al., (ProteinEngineering 7: 805-814, 1994)] as a method for reducingimmunogenicity while maintaining binding activity of anti-body molecules. According to the method, each variableregion amino acid has been assigned a risk of substitution.Amino acid substitutions are distinguished by one of threerisk categories: (I) low risk changes are those that have thegreatest potential for reducing immunogenicity with the leastchance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity,but have a greater chance of affecting antigen binding orprotein folding; (3) high risk residues are those that are impor-tant for binding or for maintaining antibody structure andcarry the highest risk that antigen binding or protein foldingwill be affected. Due to the three-dimensional structural roleof prolines, modifications at prolines are generally consid-ered to be at least moderate risk changes, even if the positionis typically a low risk position.[0145] Variable regions of the light and heavy chains of arodent antibody are HUMAN ENGINEEREDâ„¢ as follows tosubstitute human amino acids at positions determined to beunlikely to adversely effect either antigen binding or proteinfolding, but likely to reduce immunogenicity in a humanenvironment. Amino acid residues that are at "low risk" posi-tions and that are candidates for modification according to themethod are identified by aligning the amino acid sequences ofthe rodent variable regions with a human variable regionsequence. Any human variable region can be used, includingan individual Vu or Vz sequence or a human consensus Vu orVz sequence or an individual or consensus human germline

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sequence. The amino acid residues at any number of the lowrisk positions, or at all of the low risk positions, can bechanged. For example, at each low risk position where thealigned murine and human amino acid residues differ, anamino acid modification is introduced that replaces the rodentresidue with the human residue. Alternatively, the amino acidresidues at all of the low risk positions and at any number ofthe moderate risk positions can be changed. Ideally, toachieve the least immunogenicity all of the low and moderaterisk positions are changed from rodent to human sequence.

[0146] Synthetic genes containing modified heavy and/orlight chain variable regions are constructed and linked tohuman 7 heavy chain and/or K light chain constant regions.Any human heavy chain and light chain constant regions maybe used in combination with the HUMAN ENGINEEREDâ„¢antibody variable regions, including IgA (of any subclass,such as IgAI or IgA2), IgD, IgE, IgG (of any subclass, suchas IgGI, IgG2, IgG3, or IgG4), or IgM. The human heavy andlight chain genes are introduced into host cells, such as mam-malian cells, and the resultant recombinant immunoglobulinproducts are obtained and characterized.Human Antibodies from Transgenic Animals

[0147] Human antibodies to target protein can also be pro-duced using transgenic animals that have no endogenousimmunoglobulin production and are engineered to containhuman immunoglobulin loci. For example, WO 98/24893discloses transgenic animals having a human Ig locuswherein the animals do not produce functional endogenousimmunoglobulins due to the inactivation of endogenousheavy and light chain loci. WO 91/00906 also discloses trans-genic non-primate mammalian hosts capable of mounting animmune response to an immunogen, wherein the antibodieshave primate constant and/or variable regions, and whereinthe endogenous immunoglobulin encoding loci are substi-tuted or inactivated. WO 96/30498 and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system to modify theimmunoglobulin locus in a mammal, such as to replace all ora portion of the constant or variable region to form a modifiedantibody molecule. WO 94/02602 discloses non-humanmammalian hosts having inactivated endogenous Ig loci andfunctional human Ig loci. U.S. Pat. No. 5,939,598 disclosesmethods of making transgenic mice in which the mice lackendogenous heavy chains, and express an exogenous immu-noglobulin locus comprising one or more xenogeneic con-stant regions. See also, U.S. Pat. Nos. 6,114,598 6,657,103and 6,833,268.[0148] Using a transgenic animal described above, animmune response can be produced to a selected antigenicmolecule, and antibody producing cells can be removed fromthe animal and used to produce hybridomas that secretehuman monoclonal antibodies. Immunization protocols,adjuvants, and the like are known in the art, and are used inimmunization of, for example, a transgenic mouse asdescribed in WO 96/33735. This publication discloses mono-clonal antibodies against a variety of antigenic moleculesincluding IL-6, IL-8, TNFa, human CD4, L selectin, gp39,and tetanus toxin. The monoclonal antibodies can be testedfor the ability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO96/33735 discloses that monoclonal antibodies against IL-8,derived from immune cells of transgenic mice immunizedwith IL-8, blocked IL-8 induced functions of neutrophils.Human monoclonal antibodies with specificity for the anti-gen used to immunize transgenic animals are also disclosed in

WO 96/34096 and U.S. patent application no. 20030194404;and U.S. patent application no. 20030031667.[0149] Additional transgenic animals useful to makemonoclonal antibodies include the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429 and Fish-wild, et al. (Nat. Biotechnol. 14:845-851 (1996)), which con-tains gene sequences from unrearranged human antibodygenes that code for the heavy and light chains of humanantibodies. Immunization of a HuMAb-MOUSE® enablesthe production of fully human monoclonal antibodies to thetarget protein.[0150] Also, Ishida et al. (Cloning Stem Cells. 4:91-102(2002)) describes the TransChromo Mouse (TCMOUSEâ„¢)which comprises megabase-sized segments of human DNAand which incorporates the entire human immunoglobulin(hig) loci. The TCMOUSEâ„¢ has a fully diverse repertoire ofhIgs, including all the subclasses of IgGs (IgGI-G4). Immu-nization of the TCMOUSEâ„¢ with various human antigensproduces antibody responses comprising human antibodies.[0151] See also Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggennann et al., Year in Immunol., 7:33 (1993);and U.S. Pat. No. 5,911,699, U.S. Pat. No. 5,899,699, U.S.Pat. No. 5,44,,077; and U.S. Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125describes methods for biasing the immune response of ananimal to the desired epitope. Human antibodies may also begenerated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275).Human Antibodies from Display Technology[0152] The development of technologies for making reper-toires of recombinant human antibody genes, and the displayof the encoded antibody fragments on the surface of filamen-tous bacteriophage, has provided a means for making humanantibodies directly. The antibodies produced by phage tech-nology are produced as antigen binding fragments-usually Fvor Fab fragments in bacteria and thus lack effector functions.Effector functions can be introduced by one of two strategies:The fragments can be engineered either into complete anti-bodies for expression in mammalian cells, or into bispecificantibody fragments with a second binding site capable oftriggering an effector function.[0153] The invention contemplates a method for producingtarget-specific antibody or antigen-binding portion thereofcomprising the steps of synthesizing a library of human anti-bodies on phage, screening the library with target protein or aportion thereof, isolating phage that bind target, and obtain-ing the antibody from the phage. By way of example, onemethod for preparing the library of antibodies for use in phagedisplay techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci withtarget antigen or an antigenic portion thereof to create animmune response, extracting antibody producing cells fromthe immunized animal; isolating RNA from the extractedcells, reverse transcribing the RNA to produce cDNA, ampli-fying the cDNA using a primer, and inserting the cDNA intoa phage display vector such that antibodies are expressed onthe phage. Recombinant target-specific antibodies of theinvention may be obtained in this way.[0154] Phage-display processes mimic immune selectionthrough the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One suchtechnique is described in WO 99/10494, which describes the

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isolation of high affinity and functional agonistic antibodiesfor MPL and msk receptors using such an approach. Antibod-ies of the invention can be isolated by screening of a recom-binant combinatorial antibody library, preferably a scFvphage display library, prepared using human Vz and VucDNAs prepared from mRNA derived from human lympho-cytes. Methodologies for preparing and screening such librar-ies are known in the art. See e.g., U.S. Pat. No. 5,969,108.There are commercially available kits for generating phagedisplay libraries (e.g., the Pharmacia Recombinant PhageAntibody System, catalog no. 27-9400-01; and the StratageneSurfZAP.â„¢ phage display kit, catalog no. 240612). There arealso other methods and reagents that can be used in generatingand screening antibody display libraries (see, e.g., Ladner etal. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al PCT Publication No. WO 92/20791;Markland et al. PCT Publication No. WO 92/15679; Breitlinget al. PCT Publication No. WO 93/01288; McCafferty et al.PCT Publication No. WO 92/01047; Garrard et al. PCT Pub-lication No. WO 92/09690; Fuchs et al. (1991) Bio JTechnol-ogy 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybrido-mas 3:81-85; Huse et al. (1989) Science 246:1275-1281;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/'Technology 9:1373-1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) Proc. Natl. Acad. Sci. USA 89:7978-7982.

[0155] In one embodiment, to isolate human antibodiesspecific for the target antigen with the desired characteristics,a human Vu and Vz library are screened to select for antibodyfragments having the desired specificity. The antibody librar-ies used in this method are preferably scFv libraries preparedand screened as described herein and in the art (McCafferty etal., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J.12:725-734 (1993)). The scFv antibody libraries preferablyare screened using target protein as the antigen.

[0156] Alternatively, the Fd fragment (V~Cul) and lightchain (Vz-Cz) of antibodies are separately cloned by PCR andrecombined randomly in combinatorial phage display librar-ies, which can then be selected for binding to a particularantigen. The Fab fragments are expressed on the phage sur-face, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for theFab encoding sequences, which can be amplified subse-quently. Through several rounds of antigen binding and re-amplification, a procedure termed panning, Fab specific forthe antigen are enriched and finally isolated.

[0157] In 1994, an approach for the humanization of anti-bodies, called "guided selection", was described. Guidedselection utilizes the power of the phage display technique forthe humanization of mouse monoclonal antibody (See Jes-pers, L. S., et al., BioJTechnology 12, 899-903 (1994)). Forthis, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library,and the resulting hybrid Fab library may then be selected withantigen. The mouse Fd fragment thereby provides a templateto guide the selection. Subsequently, the selected human lightchains are combined with a human Fd fragment library.Selection of the resulting library yields entirely human Fab.

[0158] A variety of procedures have been described forderiving human antibodies from phage-display libraries (See,for example, Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol, 222:581-597 (1991); U.S.Pat. Nos. 5,565,332 and 5,573,905; Clackson, T., and Wells,J. A., TIBTECH 12, 173-184 (1994)). In particular, in vitroselection and evolution of antibodies derived from phagedisplay libraries has become a powerful tool (See Burton, D.R., and Barbas III, C. F., Adv. Immunol. 57, 191-280 (1994);Winter, G., et al., Annu. Rev. Immunol. 12, 433-455 (1994);U.S. patent publication no. 20020004215 and WO 92/01047;U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.[0159] Watkins, "Screening of Phage-Expressed AntibodyLibraries by Capture Lift," Methods in Molecular Biology,Antibody Phage Display: Methods and Protocols 178:187-193 (2002), and U.S. patent publication no. 20030044772,published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding mol-ecules by capture lift, a method involving immobilization ofthe candidate binding molecules on a solid support.[0160] Fv fragments are displayed on the surface of phage,by the association of one chain expressed as a phage proteinfusion (e.g., with M13 gene III) with the complementarychain expressed as a soluble fragment. It is contemplated thatthe phage may be a filamentous phage such as one of the classI phages: fd, M13, fl, lfl, Ike, ZJ/Z, Ff and one of the class IIphages Xf, Pfl and Pf3. The phage may be M13; or fd or aderivative thereo f.[0161] Once initial human Vz and Vu segments areselected, "mix and match" experiments, in which differentpairs of the initially selected Vz and Vu segments are screenedfor target binding, are performed to select preferred Vz/Vupair combinations. Additionally, to further improve the qual-ity of the antibody, the Vz and Vu segments of the preferredVz/Vu pair(s) can be randomly mutated, preferably withinthe any o f the CDR I, CDR2 or CDR3 region o f Vu and/or Vz,in a process analogous to the in vivo somatic mutation processresponsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturationcan be accomplished by amplifying Vz and Vu regions usingPCR primers complimentary to the Vu CDRI, CDR2, andCDR3, or Vz CDRI, CDR2, and CDR3, respectively, whichprimers have been "spiked" with a random mixture o f the fournucleotide bases at certain positions such that the resultantPCR products encode Vz and Vu segments into which randommutations have been introduced into the Vu and/or Vz CDR3regions. These randomly mutated Vz and Vu segments can berescreened for binding to target antigen.[0162] Following screening and isolation of an target spe-cific antibody from a recombinant immunoglobulin displaylibrary, nucleic acid encoding the selected antibody can berecovered from the display package (e.g., from the phagegenome) and subcloned into other expression vectors by stan-dard recombinant DNA techniques. If desired, the nucleicacid can be further manipulated to create other antibodyforms of the invention, as described below. To express arecombinant human antibody isolated by screening of a com-binatorial library, the DNA encoding the antibody is clonedinto a recombinant expression vector and introduced into amammalian host cell, as described herein.

[0163] It is contemplated that the phage display methodmay be carried out in a mutator strain of bactaria or host cell.A mutator strain is a host cell which has a genetic defect

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which causes DNA replicated within it to be mutated withrespect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut Tl.[0164] It is also contemplated that the phage displaymethod may be carried out using a helper phage. This is aphage which is used to infect cells containing a defectivephage genome and which functions to complement the defect.The defective phage genome can be a phagemid or a phagewith some function encoding gene sequences removed.Examples of helper phages are M13K07, M13K07 gene IIIno. 3; and phage displaying or encoding a binding moleculefused to a capsid protein.[0165] Antibodies are also generated via phage displayscreening methods using the hierarchical dual combinatorialapproach as disclosed in WO 92/01047 in which an individualcolony containing either an H or L chain clone is used to infecta complete library of clones encoding the other chain (L or H)and the resulting two-chain specific binding member isselected in accordance with phage display techniques such asthose described therein. This technique is also disclosed inMarks et al, (Bio/'Technology, 10:779-783 (1992)).[0166] Methods for display of peptides on the surface ofyeast and microbial cells have also been used to identifyantigen specific antibodies. See, for example, U.S. Pat. No.6,699,658. Antibody libraries may be attached to yeast pro-teins, such as agglutinin, effectively mimicking the cell sur-face display of antibodies by B cells in the immune system.[0167] In addition to phage display methods, antibodiesmay be isolated using ribosome mRNA display methods andmicrobial cell display methods. Selection of polypeptideusing ribosome display is described in Hanes et al., (Proc.Natl. Acad Sci USA, 94:4937-4942 (1997)) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome dis-play is also useful for rapid large scale mutational analysis ofantibodies. The selective mutagenesis approach also providesa method of producing antibodies with improved activitiesthat can be selected using ribosomal display techniques.

Amino Acid Sequence Variants

[0168] It is contemplated that modified polypeptide com-positions comprising one, two, three, four, five, and/or sixCDRs of an antibody are generated, wherein a CDR is alteredto provide increased specificity or affinity to the target mol-ecule. Sites within antibody CDRs are typically modified inseries, e.g., by substituting first with conservative choices(e.g., hydrophobic amino acid substituted for a non-identicalhydrophobic amino acid) and then with more dissimilarchoices (e.g., hydrophobic amino acid substituted for acharged amino acid), and then deletions or insertions may bemade at the target site. For example, using the conservedframework sequences surrounding the CDRs, PCR primerscomplementary to these consensus sequences are generatedto amplify the antigen-specific CDR sequence locatedbetween the primer regions. Techniques for cloning andexpressing nucleotide and polypeptide sequences are well-established in the art [see e.g. Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor, N.Y. (1989)]. The amplified CDR sequences areligated into an appropriate plasmid. The plasmid comprisingone, two, three, four, five and/or six cloned CDRs optionallycontains additional polypeptide encoding regions linked tothe CDR.[0169] Antibody substances comprising the modifiedCDRs are screened for binding affinity for the original anti-

gen. Additionally, the antibody or polypeptide is furthertested for its ability to neutralize the activity of the targetantigens. For example, antibodies of the invention may beanalyzed as set out in the Examples to determine their abilityto interfere with the biological activity of target antigen.

[0170] Modifications may be made by conservative or non-conservative amino acid substitutions described in greaterdetail below. "Insertions" or "deletions" are preferably in therange of about I to 20 amino acids, more preferably I to 10amino acids. The variation may be introduced by systemati-cally making substitutions of amino acids in an antibodypolypeptide molecule using recombinant DNA techniquesand assaying the resulting recombinant variants for activity.Nucleic acid alterations can be made at sites that differ in thenucleic acids from different species (variable positions) or inhighly conserved regions (constant regions). Methods foraltering antibody sequences and expressing antibodypolypeptide compositions useful in the invention aredescribed in greater detail below.

[0171] Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from oneresidue to polypeptides containing a hundred or more resi-dues, as well as intra-sequence insertions of single or multipleamino acid residues. Examples of terminal insertions includean antibody with an ¹erminal methionyl residue or theantibody (including antibody fragment) fused to an epitopetag or a salvage receptor epitope. Other insertional variants ofthe antibody molecule include the fusion to a polypeptidewhich increases the serum half-life of the antibody, e.g. at the¹erminus or C-terminus.

[0172] The term "epitope tagged" refers to the antibodyfused to an epitope tag. The epitope tag polypeptide hasenough residues to provide an epitope against which an anti-body there against can be made, yet is short enough such thatit does not interfere with activity of the antibody. The epitopetag preferably is sufficiently unique so that the antibody thereagainst does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues(preferably between about 9-30 residues). Examples includethe flu hemagglutinin (HA) tag polypeptide and its antibody12CA5 (Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988));the c-myc tag and the 8F9, 3C7, 6EIO, G4, B7 and 9EIOantibodies thereto (Evan et al., Mol. Cell. Biol. 5:3610-16(1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering3:547-53 (1990)). Other exemplary tags are a poly-histidinesequence, generally around six histidine residues, that per-mits isolation of a compound so labeled using nickel chela-tion. Other labels and tags, such as the FLAG® tag (EastmanKodak, Rochester, N.Y.), well known and routinely used inthe art, are embraced by the invention.

[0173] As used herein, the term "salvage receptor bindingepitope" refers to an epitope of the Fc region of an IgGmolecule (e.g., IgG„ IgG~, IgG3, or IgG4) that is responsiblefor increasing the in vivo serum hal f-life o f the IgG molecule.

[0174] Another type of variant is an amino acid substitutionvariant. These variants have at least one amino acid residue inthe antibody molecule removed and a different residueinserted in its place. Substitutional mutagenesis within any ofthe hypervariable or CDR regions or framework regions iscontemplated. Conservative substitutions involve replacingan amino acid with another member of its class. Non-conser-

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vative substitutions involve replacing a member of one ofthese classes with a member of another class.[0175] Conservative amino acid substitutions are made onthe basis of similarity in polarity, charge, solubility, hydro-phobicity, hydrophilicity, and/or the amphipathic nature ofthe residues involved. For example, nonpolar (hydrophobic)amino acids include alanine (Ala, A), leucine (Leu, L), iso-leucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine(Phe, F), tryptophan (Trp, W), and methionine (Met, M);polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr,Y), asparagine (Asn, N), and glutamine (Gln, Q); positivelycharged (basic) amino acids include arginine (Arg, R), lysine(Lys, K), and histidine (His, H); and negatively charged(acidic) amino acids include aspartic acid (Asp, D) andglutamic acid (Glu, E).[0176] Any cysteine residue not involved in maintainingthe proper conformation of the antibody also may be substi-tuted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant crosslinking. Con-versely, cysteine bond(s) may be added to the antibody toimprove its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).[0177] Affinity Maturation[0178] Affinity maturation generally involves preparingand screening antibody variants that have substitutions withinthe CDRs of a parent antibody and selecting variants that haveimproved biological properties such as binding affinity rela-tive to the parent antibody. A convenient way for generatingsuch substitutional variants is affinity maturation using phagedisplay. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutionsat each site. The antibody variants thus generated are dis-played in a monovalent fashion from filamentous phage par-ticles as fusions to the gene III product of M13 packagedwithin each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity).See e.g., WO 92/01047, WO 93/112366, WO 95/15388 andWO 93/19172.[0179] Current antibody affinity maturation methodsbelong to two mutagenesis categories: stochastic and nonsto-chastic. Error prone PCR, mutator bacterial strains (Low etal., J. Mol. Biol. 260, 359-68 (1996)), and saturationmutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20(2000); Chowdhury, P. S. Methods Mol. Biol. 178, 269-85(2002)) are typical examples o f stochastic mutagenesi s meth-ods (Rajpal et al., Proc Natl Acad Sci USA. 102:8466-71(2005)). Nonstochastic techniques often use alanine-scan-ning or site-directed mutagenesis to generate limited collec-tions of specific variants. Some methods are described infurther detail below.[0180] Affinity maturation via panning methods Affinitymaturation of recombinant antibodies is commonly per-formed through several rounds of panning of candidate anti-bodies in the presence of decreasing amounts of antigen.Decreasing the amount of antigen per round selects the anti-bodies with the highest affinity to the antigen thereby yieldingantibodies of high affinity from a large pool of starting mate-rial. Affinity maturation via panning is well known in the artand is described, for example, in Huis et al. (Ca neer ImmunolImmunothen 50:163-71 (2001)). Methods of affinity matura-tion using phage display technologies are described else-where herein and known in the art (see e.g., Daugherty et al.,Proc Natl Acad Sci USA. 97:2029-34 (2000)).

[0181] Look-through mutagenesis Look-throughmutagenesis (LTM) (Rajpal et al., Proc Natl Acad Sci USA.102:8466-71 (2005)) provides a method for rapidly mappingthe antibody-binding site. For LTM, nine amino acids, repre-sentative of the major side-chain chemistries provided by the20 natural amino acids, are selected to dissect the functionalside-chain contributions to binding at every position in all sixCDRs of an antibody. LTM generates a positional series ofsingle mutations within a CDR where each "wild type" resi-due is systematically substituted by one of nine selectedamino acids. Mutated CDRs are combined to generate com-binatorial single-chain variable fragment (scFv) libraries ofincreasing complexity and size without becoming prohibitiveto the quantitative display of all variants. After positive selec-tion, clones with improved binding are sequenced, and ben-eficial mutations are mapped.[0182] Error-prone PCR Error-prone PCR involves therandomization of nucleic acids between different selectionrounds. The randomization occurs at a low rate by the intrin-sic error rate of the polymerase used but can be enhanced byerror-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783(1999)) using a polymerase having a high intrinsic error rateduring transcription (Hawkins et al., JMol Biol. 226:889-96(1992)). After the mutation cycles, clones with improvedaffinity for the antigen are selected using routine mehods inthe art.[0183] DNA Shuffling Nucleic acid shuffling is a methodfor in vitro or in vivo homologous recombination of pools ofshorter or smaller polynucleotides to produce variant poly-nucleotides. DNA shuffling has been described in U.S. Pat.No. 6,605,449, U.S. Pat. No. 6,489,145, WO 02/092780 andStemmer, Proc. Natl. Acad. Sci. USA, 91:10747-51 (1994).Generally, DNA shuffling is comprised of 3 steps: fragmen-tation of the genes to be shuffled with DNase I, randomhybridization of fragments and reassembly or filling in of thefragmented gene by PCR in the presence of DNA polymerase(sexual PCR), and amplification of reassembled product byconventional PCR.[0184] DNA shuffling differs from error-prone PCR in thatit is an inverse chain reaction. In error-prone PCR, the numberof polymerase start sites and the number of molecules growsexponentially. In contrast, in nucleic acid reassembly or shuf-fling of random polynucleotides the number of start sites andthe number (but not size) of the random polynucleotidesdecreases over time.[0185] Inthe case of an antibody, DNA shuffling allows thefree combinatorial association of all of the CDRI s with all ofthe CDR2s with all of the CDR3s, for example. It is contem-plated that multiple families of sequences can be shuffled inthe same reaction. Further, shuffling generally conserves therelative order, such that, for example, CDRI will not be foundin the position of CDR2. Rare shufflants will contain a largenumber of the best (e.g. highest affinity) CDRs and these rareshufflants may be selected based on their superior affinity.[0186] The template polynucleotide which may be used inDNA shuffling may be DNA or RNA. It may be of variouslengths depending on the size of the gene or shorter or smallerpolynucleotide to be recombined or reassembled. Preferably,the template polynucleotide is from 50 bp to 50 kb. Thetemplate polynucleotide often should be double-stranded.[0187] It is contemplated that single-stranded or double-stranded nucleic acid polynucleotides having regions of iden-tity to the template polynucleotide and regions of heterologyto the template polynucleotide may be added to the template

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polynucleotide, during the initial step of gene selection. It isalso contemplated that two different but related polynucle-otide templates can be mixed during the initial step.[01SS] Alanine scanning Alanine scanning mutagenesiscan be performed to identify hypervariable region residuesthat contribute significantly to antigen binding. Cunninghamand Wells, (Science 244:1081-1085 (1989)). A residue orgroup of target residues are identified (e.g., charged residuessuch as arg, asp, his, lys, and glu) and replaced by a neutral ornegatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids withantigen. Those amino acid locations demonstrating func-tional sensitivity to the substitutions then are refined by intro-ducing further or other variants at, or for, the sites of substi-tution.[01S9] Computer-aided design Alternatively, or in addi-tion, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points betweenthe antibody and antigen, or to use computer software tomodel such contact points. Such contact residues and neigh-boring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are gen-erated, the panel of variants is subjected to screening asdescribed herein and antibodies with superior properties inone or more relevant as says may be selected for further devel-opment.[0190] Altered Glycosylation[0191] Antibody variants can also be produced that have amodified glycosylation pattern relative to the parent antibody,for example, deleting one or more carbohydrate moietiesfound in the antibody, and/or adding one or more glycosyla-tion sites that are not present in the antibody.[0192] Glycosylation of antibodies is typically eitherN-linked or 0-linked. N-linked refers to the attachment o f thecarbohydrate moiety to the side chain of an asparagine resi-due. The tripeptide sequences asparagine-X-serine and aspar-agine-X-threonine, where X is any amino acid except proline,are the recognition sequences for enzymatic attachment of thecarbohydrate moiety to the asparagine side chain. The pres-ence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glyco-sylation sites may be added to an antibody by altering theamino acid sequence such that it contains one or more of thesetripeptide sequences. 0-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galac-tose, or xylose to a hydroxyamino acid, most commonlyserine or threonine, although 5-hydroxyproline or 5-hydroxy-lysine may also be used. 0-linked glycosylation sites may beadded to an antibody by inserting or substituting one or moresenne or threonine residues to the sequence of the orginalantibody.[0193] Also contemplated are antibody molecules withabsent or reduced fucosylation that exhibit improved ADCCactivity. A variety of ways are known in the art to accomplishthis. For example, ADCC effector activity is mediated bybinding of the antibody molecule to the Fc7RIII receptor,which has been shown to be dependent on the carbohydratestructure of the N-linked glycosylation at the Asn-297 of theCH2 domain. Non-fucosylated antibodies bind this receptorwith increased affinity and trigger Fc7RIII-mediated effectorfunctions more efficiently than native, fucosylated antibod-ies. For example, recombinant production of non-fucosylatedantibody in CHO cells in which the alpha-1,6-fucosyl trans-ferase enzyme has been knocked out results in antibody with

100-fold increased ADCC activity (Yamane-Ohnuki et al.,Biotechnol Bioeng. 87:614-22 (2004)). Similar effects can beaccomplished through decreasing the activity of this or otherenzymes in the fucosylation pathway, e.g., through siRNA orantisense RNA treatment, engineering cell lines to knockoutthe enzyme(s), or culturing with selective glycosylationinhibitors (Rothman et al., Mol Immunol. 26:1113-23(1989)). Some host cell strains, e.g. Lec13 or rat hybridomaYB2/0 cell line naturally produce antibodies with lower fuco-sylation levels. (Shields et al., J Biol. Chem. 277:26733-40(2002); Shinkawa et al., J Biol Chem. 278:3466-73 (2003)).An increase in the level ofbisected carbohydrate, e g. throughrecombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCCactivity (Umana et al., Nat. Biotechnol. 17:176-80 (1999)). Ithas been predicted that the absence of only one of the twofulcose residues may be sufficient to increase ADCC activity(Ferrara et al., Biotechnol Bioeng. 93:851-61 (2006)).[0194] Variants with Altered Effector Function[0195] Other modifications of the antibody are contem-plated. In one aspect, it may be desirable to modify the anti-body of the invention with respect to effector function, forexample, to enhance the effectiveness of the antibody in treat-ing cancer. One method for modifying effector functionteaches that cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formationin this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., (J. Exp Med.176: 1191-1195 (1992)) and Shopes, B. (J. Immunol. 148:2918-2922 (1992)). Homodimeric antibodies with enhancedanti-tumor activity may also be prepared using heterobifunc-tional cross-linkers as described in Wolff et al., (CancerResearch 53: 2560-2565 (1993)). Alternatively, an antibodycan be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. SeeStevenson et al., (Anti-Cancer Drug Design 3: 219-230(1989)). In addition, it has been shown that sequences withinthe CDR can cause an antibody to bind to MHC Class II andtrigger an unwanted helper T-cell response. A conservativesubstitution can allow the antibody to retain binding activityyet lose its ability to trigger an unwanted T-cell response. Alsosee Steplewski et al., (Proc Natl Acad Sci USA. 85:4852-56(1998)), which described chimeric antibodies wherein amurine variable region was joined with human gamma I,gamma 2, gamma 3, and gamma 4 constant regions.[0196] In certain embodiments of the invention, it may bedesirable to use an antibody fragment, rather than an intactantibody, to increase tumor penetration, for example. In thiscase, it may be desirable to modify the antibody fragment inorder to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers,including polysaccharide polymers, to antibody fragments toincrease the half-life. This may also be achieved, for example,by incorporation of a salvage receptor binding epitope intothe antibody fragment (e.g., by mutation of the appropriateregion in the antibody fragment or by incorporating theepitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, e.g., WO96/32478).[0197] The salvage receptor binding epitope preferablyconstitutes a region wherein any one or more amino acidresidues from one or two loops of a Fc domain are transferred

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to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops ofthe Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g.,of an IgG) and transferred to the CHI, CH3, or VH region, ormore than one such region, of the antibody. Alternatively, theepitope is taken from the CH2 domain of the Fc region andtransferred to the C~ region or V~ region, or both, of theantibody fragment. See also International applications WO97/34631 and WO 96/32478 which describe Fc variants andtheir interaction with the salvage receptor.

[0198] Thus, antibodies of the invention may comprise ahuman Fc portion, a human consensus Fc portion, or a variantthereof that retains the ability to interact with the Fc salvagereceptor, including variants in which cysteines involved indisulfide bonding are modified or removed, and/or in whichthe a met is added at the ¹erminus and/or one or more of the¹erminal 20 amino acids are removed, and/or regions thatinteract with complement, such as the Clq binding site, areremoved, and/or the ADCC site is removed [see, e.g., Sam-may et al., Mo/ec. Immunol 29: 633-9 (1992)].

[0199] Previous studies mapped the binding site on humanand murine IgG for FcR primarily to the lower hinge regioncomposed of IgG residues 233-239. Other studies proposedadditional broad segments, e.g. Gly316-Lys338 for human Fcreceptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lowerhinge, e.g., Asn297 and Glu318 for murine IgG2b interactingwith murine Fc receptor II. The report of the 3.2-A. crystalstructure of the human IgGI Fc fragment with human Fcreceptor IIIA delineated IgGI residues Leu234-Ser239,Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor IIIA. It has been suggestedbased on crystal structure that in addition to the lower hinge(Leu234-Gly237), residues in IgG CH2 domain loops FG(residues 326-330) and BC (residues 265-271) might play arole in binding to Fc receptor IIA. See Shields et al., (J. Biol.Chem., 276:6591-604 (2001)), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptorbinding sites can result in altered effector function, such asaltered ADCC or CDC activity, or altered half-life. Asdescribed above, potential mutations include insertion, dele-tion or substitution of one or more residues, including substi-tution with alanine; a conservative substitution, a non-con-servative substitution, or replacement with a correspondingamino acid residue at the same position from a different IgGsubclass (e.g. replacing an IgGI residue with a correspondingIgG2 residue at that position).

[0200] Shields et al. reported that IgGI residues involved inbinding to all human Fc receptors are located in the CH2domain proximal to the hinge and fall into two categories asfollows: I) positions that may interact directly with all FcRinclude Leu234-Pro238, Ala327, and Pro329 (and possiblyAsp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgGIresidues that affected binding to Fc receptor II are as follows:(largest effect) Arg255, Thr256, Glu258, Ser267, Asp270,Glu272, Asp280, Arg292, Ser298, and (less effect) His268,Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307,Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A andD270A reduced binding. In addition to the residues identifiedabove for all FcR, additional IgGI residues that reducedbinding to Fc receptor IIIA by 40'ro or more are as follows:

Ser239, Ser267 (Gly only), His268, Glu293, Gln295, Tyr296,Arg301, Va1303, Lys338, and Asp376. Variants that improvedbinding to FecRIIIA include T256A, K290A, S298A,E333A, K334A, and A339T. Lys414 showed a 40'ro reductionin binding for FcRIIA and FcRIIB, Arg416 a 30'ro reductionfor FcRIIA and FcRIIIA, Gln419 a 30'ro reduction to FeRIIAand a 40'ro reduction to FcRIIB, and Lys360 a 23'ro improve-ment to FcRIIIA. See also Presta et al., (Biochem. Soc. Trans.30:487-490, 2001), incorporated herein by reference in itsentirety, which described several positions in the Fc region ofIgGI were found which improved binding only to specific Fcgamma receptors (R) or simultaneously improved binding toone type of Fc gamma R and reduced binding to another type.Selected IgGI variants with improved binding to Fc gammaRIIIa were then tested in an in vitro antibody-dependentcellular cytotoxicity (ADCC) assay and showed an enhance-ment in ADCC when either peripheral blood mononuclearcells or natural killer cells were used.

[0201] For example, U.S. Pat. No. 6,194,551, incorporatedherein by reference in its entirety, describes variants withaltered effector function containing mutations in the humanIgG Fc region, at amino acid position 329, 331 or 322 (usingKabat numbering), some of which display reduced C1q bind-ing or CDC activity. As another example, U.S. Pat. No. 6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fe-gamma-receptor bindingcontaining mutations in the human IgG Fc region, at aminoacid position 238, 239, 248, 249, 252, 254, 255, 256, 258,265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,414, 416, 419, 430, 434, 435, 437, 438 or 439 (using Kabatnumbering), some of which display receptor binding profilesassociated with reduced ADCC or CDC activity. Of these, amutation at amino acid position 238, 265, 269, 270, 327 or329 are stated to reduce binding to FcRI, a mutation at aminoacid position 238, 265, 269, 270, 292, 294, 295, 298, 303,324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435,438 or 439 are stated to reduce binding to FcRII, and amutation at amino acid position 238, 239, 248, 249, 252, 254,265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301,303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416,434, 435 or 437 is stated to reduce binding to FcRIII.[0202] U.S. Pat. No. 5,624,821, incorporated by referenceherein in its entirety, reports that Clq binding activity of anmurine antibody can be altered by mutating amino acid resi-due 318, 320 or 322 of the heavy chain and that replacingresidue 297 (Asn) results in removal of lytic activity.[0203] U.S. Patent Publication No. 20040132101, incorpo-rated by reference herein in its entirety, describes variantswith mutations at amino acid positions 240, 244, 245, 247,262, 263, 266, 299, 313, 325, 328, or 332 (using Kabat num-bering) or positions 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299,313, 325, 327, 328, 329, 330, or 332 (using Kabat number-ing), of which mutations at positions 234, 235, 239, 240, 241,243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296,297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 mayreduce ADCC activity or reduce binding to an Fc gammareceptor.[0204] Chappel et al. (Proc Natl Acad Sci USA. 88:9036-40(1991)), incorporated herein by reference in its entirety,report that cytophilic activity of IgGI is an intrinsic property

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of its heavy chain CH2 domain. Single point mutations at anyof amino acid residues 234-237 of IgGI significantly loweredor abolished its activity. Substitution of all of IgGI residues234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entireELLGGP sequence (residues 233-238) was observed to bemore active than wild-type IgGI.[0205] Isaacs et al. (JImmunol. 161:3862-9 (1998)), incor-porated herein by reference in its entirety, report that muta-tions within a motif critical for Fc gammaR binding(glutamate 233 to proline, leucine/phenylalanine 234 tovaline, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to ala-nine eliminated effector function of mouse IgG2b and alsoreduced the potency of human IgG4.[0206] Armour et al. (Mol Immunol. 40:585-93 (2003)),incorporated by reference herein in its entirety, identifiedIgGI variants which react with the activating receptor,FcgammaRIIa, at least 10-fold less efficiently than wildtypeIgGI but whose binding to the inhibitory receptor, Fcgam-maRIIb, is only four-fold reduced. Mutations were made inthe region of amino acids 233-236 and/or at amino acid posi-tions 327, 330 and 331. See also WO 99/58572, incorporatedby reference herein in its entirety.

[0207] Xu et al. (JBio/ Chem. 269:3469-74 (1994)), incor-porated by reference herein in its entirety, report that mutatingIgGI Pro331 to Ser markedly decreased Clq binding andvirtually eliminated lytic activity. In contrast, the substitutionof Pro for Ser331 in IgG4 bestowed partial lytic activity(40'zo) to the IgG4 Pro331 variant.

[0208] Schuurman et al. (Mol Immunol. 38:1-8 (2001)),incorporated by reference herein in its entirety, report thatmutating one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, to serine resulted in amore stable inter-heavy chain linkage. Mutating the IgG4hinge sequence Cys-Pro-Ser-Cys to the IgGI hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent inter-action between the heavy chains.

[0209] Angal et al. (Mo/Immunol. 30:105-8 (1993)), incor-porated by reference herein in its entirety, report that mutatingthe serine at amino acid position 241 in IgG4 to proline (foundat that position in IgGI and IgG2) led to the production of ahomogeneous antibody, as well as extending serum half-lifeand improving tissue distribution compared to the originalchimeric IgG4.

Covalent Modifications

[0210] Covalent modifications of the antibody are alsoincluded within the scope of this invention. They may bemade by chemical synthesis or by enzymatic or chemicalcleavage of the antibody, if applicable. Other types of cova-lent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of theantibody with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminalresidues.

[0211] Cysteinyl residues most commonly are reacted withu-haloacetates (and corresponding amines), such as chloro-acetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also arederivatized by reaction with bromotrlfluoroacetone, .alpha.-bromo-D-(5-imidozoyl)propionic acid, chloroacetyl phos-phate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,

methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chlo-romercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.[0212] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is rela-tively specific for the histidyl side chain. Para-bromophena-cyl bromide also is useful; the reaction is preferably per-formed in 0.1 M sodium cacodylate at pH 6.0.[0213] Lysinyl and amino-terminal residues are reactedwith succinic or other carboxylic acid anhydrides. Derivati-zation with these agents has the effect of reversing the chargeof the lysinyl residues. Other suitable reagents for derivatiz-ing .alpha.-amino-containing residues include imidoesterssuch as methyl picolinimidate, pyridoxal phosphate, pyri-doxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-cata-lyzed reaction with glyoxylate.[0214] Arginyl residues are modified by reaction with oneor several conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.Derivatization of arginine residues requires that the reactionbe performed in alkaline conditions because of the high pKof the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as thearginine epsilon-amino group.[0215] The specific modification of tyrosyl residues may bemade, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly,N-acetylimidizole and tetranitromethane are used to form0-acetyl tyrosyl species and 3-nitro derivatives, respectively.Tyrosyl residues are iodinated using ' I or ' 'I, to preparelabeled proteins for use in radioimmunoassay.[0216] Carboxyl side groups (aspartyl or glutamyl) areselectively modified by reaction with carbodiimides (R N.dbd.C.dbd.N R'), where R and R' are different alkyl groups,such as I-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiim-ide or I -ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiim-ide. Furthermore, aspartyl and glutamyl residues are con-verted to asparaginyl and glutaminyl residues by reactionwith ammonium ions.[0217] Glutaminyl and asparaginyl residues are frequentlydeamidated to the corresponding glutamyl and aspartyl resi-dues, respectively. These residues are deamidated under neu-tral or basic conditions. The deamidated form of these resi-dues falls within the scope of this invention.[0218] Other modifications include hydroxylation of pro-line and lysine, phosphorylation of hydroxyl groups of serylor threonyl residues, methylation of the u-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton,Proteins: Structure and Molecular Properties, W.H. Freeman& Co., San Francisco, pp. 79-86 (1983)), acetylation of the¹erminal amine, and amidation of any C-terminal carboxylgroup.[0219] Another type of covalent modification involveschemically or enzymatically coupling glycosides to the anti-body. These procedures are advantageous in that they do notrequire production of the antibody in a host cell that hasglycosylation capabilities for N- or 0-linked glycosylation.Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulfhydryl groups such as those of cysteine,(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phe-

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nylalanine, tyrosine, or tryptophan, or (fJ the amide group ofglutamine. These methods are described in WO87/05330 andinAplin and Wriston, (CRC Crit. Rev. Biochem., pp. 259-306(1981)).[0220] Removal of any carbohydrate moieties present onthe antibody may be accomplished chemically or enzymati-cally. Chemical deglycosylation requires exposure of theantibody to the compound trifluoromethanesulfonic acid, oran equivalent compound. This treatment results in the cleav-age of most or all sugars except the linking sugar (N-acetyl-glucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described byHakimuddin, et al., (Arch. Biochem. Biophys. 259: 52 (1987))and by Edge et al., (Anal. Biochem. 118: 131 (1981)). Enzy-matic cleavage of carbohydrate moieties on antibodies can beachieved by the use of a variety of endo- and exo-glycosidasesas described by Thotakura et al., (Meth. Enzymol. 138: 350(1987)).[0221] Another type of covalent modification of the anti-body comprises linking the antibody to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, polyoxyethylated polyols, polyoxy-ethylated sorbitol, polyoxyethylated glucose, polyoxyethy-lated glycerol, polyoxyalkylenes, or polysaccharide poly-mers such as dextran. Such methods are known in the art, see,e.g. U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106, 4,179,337, 4,495,285,4,609,546 or EP 315 456.

Derivatives

[0222] As stated above, derivative refers to polypeptideschemically modified by such techniques as ubiquitination,labeling (e.g., with radionuclides or various enzymes), cova-lent polymer attachment such as pegylation (derivatizationwith polyethylene glycol) and insertion or substitution bychemical synthesis of amino acids such as ornithine. Deriva-tives of the antibody substance of the invention, such as abispecific antibody, are also useful as therapeutic agents andmay be produced by the method of the invention

[0223] The conjugated moiety can be incorporated in orattached to an antibody substance either covalently, orthrough ionic, van der Waals or hydrogen bonds, e.g., incor-poration of radioactive nucleotides, or biotinylated nucle-otides that are recognized by streptavadin.

[0224] Polyethylene glycol (PEG) may be attached to theantibody substances to provide a longer half-life in vivo. ThePEG group may be of any convenient molecular weight andmay be linear or branched. The average molecular weight ofthe PEG will preferably range from about 2 kiloDalton("kD") to about 100 kDa, more preferably from about 5 kDato about 50 kDa, most preferably from about 5 kDa to about10 kDa. The PEG groups will generally be attached to theantibody substances of the invention via acylation or reduc-tive alkylation through a natural or engineered reactive groupon the PEG moiety (e.g., an aldehyde, amino, thiol, or estergroup) to a reactive group on the antibody substance (e.g., analdehyde, amino, or ester group). Addition of PEG moieties toantibody substances can be carried out using techniques well-known in the art. See, e.g., International Publication No. WO96/11953 and U.S. Pat. No. 4,179,337.

[0225] Ligation of the antibody substance with PEG usu-ally takes place in aqueous phase and can be easily monitoredby reverse phase analytical HPLC. The PEGylated sub-

stances are purified by preparative HPLC and characterizedby analytical HPLC, amino acid analysis and laser desorptionmass spectrometry.[0226] Antibody Conjugates[0227] An antibody may be administered in its "naked" orunconjugated form, or may be conjugated directly to othertherapeutic or diagnostic agents, or may be conjugated indi-rectly to carrier polymers comprising such other therapeuticor diagnostic agents.[0228] Antibodies can be detectably labeled through theuse of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alka-line phosphatase, etc.) fluorescent or luminescent or biolumi-nescent labels (such as FITC or rhodamine, etc.), paramag-netic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Stern-berger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970);Bayer, E.A. et al., Meth. Enzym. 62:308 (1979); Engval, E. etal., Immunol. 109:129 (1972); Goding, J. W. J. Immunol.Meth. 13:215 (1976)).[0229] Conjugation of antibody moieties is described inU.S. Pat. No. 6,306,393. General techniques are alsodescribed in Shih et al., Int. J. Cancer 41:832-839 (1988);Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih etal., U.S. Pat. No. 5,057,313. This general method involvesreacting an antibody component having an oxidized carbo-hydrate portion with a carrier polymer that has at least onefree amine function and that is loaded with a plurality of drug,toxin, chelator, boron addends, or other therapeutic agent.This reaction results in an initial Schiff base (imine) linkage,which can be stabilized by reduction to a secondary amine toform the final conjugate.[0230] The carrier polymer may be, for example, an ami-nodextran or polypeptide of at least 50 amino acid residues.Various techniques for conjugating a drug or other agent tothe carrier polymer are known in the art. A polypeptide carriercan be used instead of aminodextran, but the polypeptidecarrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At leastsome of the amino acids should be lysine residues orglutamate or aspartate residues. The pendant amines of lysineresidues and pendant carboxyl ates of glutamine and aspartateare convenient for attaching a drug, toxin, immunomodulator,chelator, boron addend or other therapeutic agent. Examplesof suitable polypeptide carriers include polylysine, poly-glutamic acid, polyaspartic acid, co-polymers thereof, andmixed polymers of these amino acids and others, e.g., serines,to confer desirable solubility properties on the resultantloaded carrier and conjugate.[0231] Alternatively, conjugated antibodies can be pre-pared by directly conjugating an antibody component with atherapeutic agent. The general procedure is analogous to theindirect method of conjugation except that a therapeutic agentis directly attached to an oxidized antibody component. Forexample, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.[0232] Alternatively, a therapeutic agent can be attached atthe hinge region of a reduced antibody component via disul-fide bond formation, or using a heterobifunctional cross-linker, such as ¹uccinyl 3-(2-pyridyldithio)proprionate(SPDP). Yu et al., Int. J. Cancer 56:244 (1994). Generaltechniques for such conjugation are well-known in the art.See, for example, Wong, Chemistry Of Protein Conjugationand Cross-Linking (CRC Press 1991); Upeslacis et al.,

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"Modification of Antibodies by Chemical Methods," inMonoclonal Antibodies: Principles and Applications, Birchet al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price,"Production and Characterization of Synthetic Peptide-De-rived Antibodies," in Monoclonal Antibodies: Production,Enineering and Clinical Application, Ritter et al. (eds.), pages60-84 (Cambridge University Press 1995). A variety ofbifunctional protein coupling agents are known in the art,such as ¹uccinimidyl-3-(2-pyrldyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imi-doesters (such as dimethyl adipimidate HCL), active esters(such as disuccinimidyl suberate), aldehydes (such as glu-tareldehyde), bis-azido compounds (such as bis (p-azidoben-zoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates(such as tolyene 2,6-diisocyanate), and bis-active fluorinecompounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0233] Antibody Fusion Proteins[0234] Methods of making antibody fusion proteins arewell known in the art. See, e.g., U.S. Pat. No. 6,306,393.Antibody fusion proteins comprising an interleukin-2 moietyare described by Boleti et al., Ann. Oncol. 6:945 (1995),Nicolet et al., Cancer Gene Ther. 2: 161 (1995), Becker et al.,Proc. Nat'I Acad. Sci. USA 93:7826 (1996), Hank et al., Clin.Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res.56:4998 (1996). In addition, Yang et al., (Hum. AntibodiesHybridomas 6:129 (1995)), describe a fusion protein thatincludes an F(ab')~ fragment and a tumor necrosis factor alphamoiety.[0235] Methods of making antibody-toxin fusion proteinsin which a recombinant molecule comprises one or moreantibody components and a toxin or chemotherapeutic agentalso are known to those of skill in the art. For example,antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brirlk-mann et al., Proc. Nat'I Acad. Sci. USA 88:8616 (1991), Batraet al., Proc. Nat'I Acad. Sci. USA 89:5867 (1992), Friedmanet al., J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can.60:137 (1995), Fominaya et al., J. Biol. Chem. 271:10560(1996), Kuan et al., Biochemistry 35:2872 (1996), andSchmidt et al., It. J. Can. 65:538 (1996). Antibody-toxinfusion proteins containing a diphtheria toxin moiety havebeen described by Kreitman et al., Leukemia 7:553 (1993),Nicholls et al., J. Biol. Chem. 268:5302 (1993), Thompson etal., J. Biol. Chem. 270:28037 (1995), and Vallera et al., Blood88:2342 (1996). Deonarain et al., Tumor Targeting I:177(1995), have described an antibody-toxin fusion protein hav-ing an RNase moiety, while Linardou et al., Cell Biophys.24-25:243 (1994), produced an antibody-toxin fusion proteincomprising a DNase I component. Gelonin was used as thetoxin moiety in the antibody-toxin fusion protein of Wang etal., Abstracts of the 209th ACS National Meeting, Anaheim,Calif., Apr. 2-6, 1995, Part I, BIOTOOS. As a furtherexample, Dohlsten et al., Proc. Nat'I Acad. Sci. USA 91:8945(1994), reported an antibody-toxin fusion protein comprisingStaphylococcal enterotoxin-A.

[0236] Illustrative of toxins which are suitably employed inthe preparation of such conjugates are ricin, abrin, ribonu-clease, DNase I, Stapbylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif. ACancer Journal for Clinicians 44:43 (1994). Other suitabletoxins are known to those of skill in the art.

[0237] Antibodies of the present invention may also beused in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidylchemotherapeutic agent, See WO81/01145) to an active anti-cancer drug. See, for example, WO88/07378 and U.S. Pat.No. 4,975,278.

[0238] The enzyme component of the immunoconjugateuseful for ADEPT includes any enzyme capable of acting ona prodrug in such a way so as to covert it into its more active,cytotoxic form.

[0239] Enzymes that are useful in the this inventioninclude, but are not limited to, alkaline pho sphatase useful forconverting phosphate-containing prodrugs into free drugs;arylsulfatase useful for converting sulfate-containing pro-drugs into free drugs; cytosine deaminase useful for convert-ing non-toxic 5-fluorocytosine into the anti-cancer drug,5-fluorouracil; proteases, such as serratia protease, thermol-ysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypepti-dases, useful for converting prodrugs that contain D-aminoacid substituents; carbohydrate-cleaving enzymes such as[3-galactosidase and neuraminidase useful for converting gly-cosylated prodrugs into free drugs; [3-Iactamase useful forconverting drugs derivatized with [3-Iactams into free drugs;and penicillin amidases, such as penicillin V amidase or peni-cillin G amidase, useful for converting drugs derivatized attheir amine nitrogens with phenoxyacetyl or phenylacetylgroups, respectively, into free drugs. Alternatively, antibodieswith enzymatic activity, also known in the art as abzymes, canbe used to convert the prodrugs of the invention into freeactive drugs (See, e.g., Massey, Nature 328: 457-458 (1987).Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

[0240] The enzymes above can be covalently bound to theantibodies by techniques well known in the art such as the useof the heterobifunctional crosslinking reagents discussedabove. Alternatively, fusion proteins comprising at least theantigen binding region of an antibody of the invention linkedto at least a functionally active portion of an enzyme of theinvention can be constructed using recombinant DNA tech-niques well known in the art (See, e.g., Neuberger et al.,Nature 312: 604-608 (1984))

Recombinant Production of Antibodies

[0241] DNA encoding a monoclonal antibody of the inven-tion may be isolated and sequenced from the hybridoma cellsecreting the antibody using conventional procedures (e.g.,by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains ofthe monoclonal antibodies). Sequence determination willgenerally require isolation of at least a portion of the gene orcDNA of interest. Usually this requires cloning the DNA or,preferably, mRNA (Le., cDNA) encoding the monoclonalantibodies. Cloning is carried out using standard techniques(see, e.g., Sambrook et al. (1989) Molecular Cloning: ALaboratory Guide, Vols 1-3, Cold Spring Harbor Press, whichis incorporated herein by reference). For example, a cDNAlibrary may be constructed by reverse transcription of polyA+mNA, preferably membrane-associated mRNA, and thelibrary screened using probes specific for human immunoglo-bulin polypeptide gene sequences. Nucleotide probe reac-tions and other nucleotide hybridization reactions are carried

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out at conditions enabling the identification of polynucle-otides which hybridize to each other under specified condi-tions.

[0242] One exemplary set of conditions is as follows: strin-gent hybridization at 42' C. in 50'zo formamide, SxSSC, 20mM Na.PO4, pH 6.8; and washing in IxSSC at 55' C. for 30minutes. Formula for calculating equivalent hybridizationconditions and/or selecting other conditions to achieve adesired level of stringency are well known. It is understood inthe art that conditions of equivalent stringency can beachieved through variation of temperature and buffer, or saltconcentration as described Ausubel, et al. (Eds.), Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to6.4.10. Modifications in hybridization conditions can beempirically determined or precisely calculated based on thelength and the percentage of guanosine/cytosine (GC) basepairing of the probe. The hybridization conditions can becalculated as described in Sambrook, et al., (Eds.), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labora-tory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51

[0243] In a preferred embodiment, however, the poly-merase chain reaction (PCR) is used to amplify cDNAs (orportions of full-length cDNAs) encoding an immunoglobulingene segment of interest (e.g., a light chain variable segment).The amplified sequences can be readily cloned into any suit-able vector, e.g., expression vectors, minigene vectors, orphage display vectors. It will be appreciated that the particu-lar method of cloning used is not critical, so long as it ispossible to determine the sequence of some portion of theimmunoglobulin polypeptide of interest. As used herein, an"isolated" nucleic acid molecule or "isolated" nucleic acidsequence is a nucleic acid molecule that is either (I) identifiedand separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the naturalsource of the nucleic acid or (2) cloned, amplified, tagged, orotherwise distinguished from background nucleic acids suchthat the sequence of the nucleic acid of interest can be deter-mined, is considered isolated. An isolated nucleic acid mol-ecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are dis-tinguished from the nucleic acid molecule as it exists innatural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordi-narily express the antibody where, for example, the nucleicacid molecule is in a chromosomal location different fromthat of natural cells.

[0244] One source for RNA used for cloning and sequenc-ing is a hybridoma produced by obtaining a B cell from thetransgenic mouse and fusing the B cell to an immortal cell. Anadvantage of using hybridomas is that they can be easilyscreened, and a hybridoma that produces a human mono-clonal antibody of interest selected. Alternatively, RNA canbe isolated from B cells (or whole spleen) of the immunizedanimal. When sources other than hybridomas are used, it maybe desirable to screen for sequences encoding immunoglobu-lins or immunoglobulin polypeptides with specific bindingcharacteristics. One method for such screening is the use ofphage display technology. Phage display is described furtherherein and is also well-known in the art. See e.g., Dower et al.,WO 91/17271, McCafferty et al., WO 92/01047, and Catonand Koprowski, (Proc. Natl. Acad. Sci. USA, 87:6450-54(1990)), each of which is incorporated herein by reference. Inone embodiment using phage display technology, cDNAfrom an immunized transgenic mouse (e.g., total spleen

cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immu-noglobulin polypeptide, e.g., CDR regions, and the amplifiedsequences are inserted into a phage vector. cDNAs encodingpeptides of interest, e.g., variable region peptides with desiredbinding characteristics, are identified by standard techniquessuch as panning.[0245] The sequence of the amplified or cloned nucleic acidis then determined. Typically the sequence encoding an entirevariable region of the immunoglobulin polypeptide is deter-mined, however, it will sometimes by adequate to sequenceonly a portion of a variable region, for example, the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at leastabout one-third or at least about one-half of the length of thevariable region will be sequenced.[0246] Sequencing can be carried out on clones isolatedfrom a cDNA library, or, when PCR is used, after subcloningthe amplified sequence or by direct PCR sequencing of theamplified segment. Sequencing is carried out using standardtechniques (see, e.g., Sambrook et al. (1989) Molecular Clon-ing: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press,and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). Bycomparing the sequence of the cloned nucleic acid with pub-lished sequences of human immunoglobulin genes andcDNAs, one of skill will readily be able to determine, depend-ing on the region sequenced, (I) the germline segment usageof the hybridoma immunoglobulin polypeptide (including theisotype of the heavy chain) and (ii) the sequence of the heavyand light chain variable regions, including sequences result-ing from ¹egion addition and the process of somatic muta-tion. One source of immunoglobulin gene sequence informa-tion is the National Center for Biotechnology Information,National Library of Medicine, National Institutes of Health,Bethesda, Md.[0247] Once isolated, the DNA may be placed into expres-sion vectors, which are then trans fected into host cells such asE. co/i cells, simian COS cells, human embryonic kidney 293cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglo-bulin protein, to obtain the synthesis of monoclonal antibod-ies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

[0248] Expression control sequences refers to DNAsequences necessary for the expression of an operably linkedcoding sequence in a particular host organism. The controlsequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilizepromoters, polyadenylation signals, and enhancers.[0249] Nucleic acid is operably linked when it is placed intoa functional relationship with another nucleic acid sequence.For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed asa preprotein that participates in the secretion of the polypep-tide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or aribosome binding site is operably linked to a coding sequenceif it is positioned so as to facilitate translation. Generally,operably linked means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contigu-ous and in reading phase. However, enhancers do not have tobe contiguous. Linking is accomplished by ligation at conve-

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nient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordancewith conventional practice.[0250] Cell, cell line, and cell culture are often used inter-changeably and all such designations herein include progeny.Transformants and transformed cells include the primarysubject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progenymay not be precisely identical in DNA content, due to delib-erate or inadvertent mutations. Mutant progeny that have thesame function or biological activity as screened for in theoriginally transformed cell are included. Where distinct des-ignations are intended, it will be clear from the context.[0251] In an alternative embodiment, the amino acidsequence of an immunoglobulin of interest may be deter-mined by direct protein sequencing. Suitable encoding nucle-otide sequences can be designed according to a universalcodon table.[0252] Amino acid sequence variants of the desired anti-body may be prepared by introducing appropriate nucleotidechanges into the encoding DNA, or by peptide synthesis.Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within theamino acid sequences of the antibodies. Any combination ofdeletion, insertion, and substitution is made to arrive at thefinal construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also mayalter post-translational processes of the antibody, such aschanging the number or position of glycosylation sites.[0253] Nucleic acid molecules encoding amino acidsequence variants of the antibody are prepared by a variety ofmethods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case ofnaturally occurring amino acid sequence variants) or prepa-ration by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of theantibody.[0254] The invention also provides isolated nucleic acidencoding antibodies of the invention, optionally operablylinked to control sequences recognized by a host cell, vectorsand host cells comprising the nucleic acids, and recombinanttechniques for the production of the antibodies, which maycomprise culturing the host cell so that the nucleic acid isexpressed and, optionally, recovering the antibody from thehost cell culture or culture medium.[0255] For recombinant production of the antibody, thenucleic acid encoding it is isolated and inserted into a repli-cable vector for further cloning (amplification of the DNA) orfor expression. DNA encoding the monoclonal antibody isreadily isolated and sequenced using conventional proce-dures (e.g., by using oligonucleotide probes that are capableof binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. Thevector components generally include, but are not limited to,one or more of the following: a signal sequence, an origin ofreplication, one or more selective marker genes, an enhancerelement, a promoter, and a transcription terminationsequence.[0256] (I) Signal Sequence Component[0257] The antibody of this invention may be producedrecombinantly not only directly, but also as a fusion polypep-tide with a heterologous polypeptide, which is preferably asignal sequence or other polypeptide having a specific cleav-

age site at the ¹erminus of the mature protein or polypep-tide. The signal sequence selected preferably is one that isrecognized and processed (ke., cleaved by a signal peptidase)by the host cell. If prokaryotic host cells do not recognize andprocess the native antibody signal sequence, the signalsequence may be substituted by a signal sequence selected,for example, from the group of the pectate lyase (e.g., peIB)alkaline phosphatase, penicillinase, Ipp, or heat-stableenterotoxin II leaders. For yeast secretion the native signalsequence may be substituted by, e.g., the yeast invertaseleader, a factor leader (including Saccharomyces andEluyveromyces o.-factor leaders), or acid phosphatase leader,the C. albicans glucoamylase leader, or the signal describedin WO90/13646. In mammalian cell expression, manmaliansignal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available.[025S] The DNA for such precursor region is ligated inreading frame to DNA encoding the antibody.[0259] (2) Origin of Replication Component[0260] Both expression and cloning vectors contain anucleic acid sequence that enables the vector to replicate inone or more selected host cells. Generally, in cloning vectorsthis sequence is one that enables the vector to replicate inde-pendently of the host chromosomal DNA, and includes ori-gins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmidpBR322 is suitable for most Gram-negative bacteria, the 2pplasmid origin is suitable for yeast, and various viral originsare useful for cloning vectors in mammalian cells. Generally,the origin of replication component is not needed for mam-malian expression vectors (the SV40 origin may typically beused only because it contains the early promoter).[0261] (3) Selective Marker Component[0262] Expression and cloning vectors may contain a selec-tive gene, also termed a selectable marker. Typical selectiongenes encode proteins that (a) confer resistance to antibioticsor other toxins, e.g., ampicillin, neomycin, methotrexate, tet-racycline, G418, geneticin, histidinol, or mycophenolic acid(b) complement auxotrophic deficiencies, or (c) supply criti-cal nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.[0263] One example of a selection scheme utilizes a drug toarrest growth of a host cell. Those cells that are successfullytrans formed with a heterologous gene produce a protein con-ferring drug resistance and thus survive the selection regimen.Examples of such dominant selection use the drugs methotr-exate, neomycin, histidinol, puromycin, mycophenolic acidand hygromycin.[0264] Another example of suitable selectable markers formammalian cells are those that enable the identification ofcells competent to take up the antibody-encoding nucleicacid, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosinedeaminase, ornithine decarboxylase, etc.[0265] For example, cells transformed with the DHFRselection gene are first identified by culturing all of the trans-formants in a culture medium that contains methotrexate(Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.[0266] Alternatively, host cells (particularly wild-typehosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibody of the

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invention, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3'-phosphotransferase (APH)can be selected by cell growth in medium containing a selec-tion agent for the selectable marker such as an aminoglyco-sidic antibiotic, e.g., kanamycin, neomycin, or G418. SeeU.S. Pat. No. 4,965,199.[0267] A suitable selection gene for use in yeast is the trp Igene present in the yeast plasmid YRp7 (Stinchcomb et al.,Nature, 282: 39 (1979)). The trp1 gene provides a selectionmarker for a mutant strain of yeast lacking the ability to growin tryptophan, for example, ATCC No. 44076 or PEP4-1.Jones, (Genetics 85:12 (1977)). The presence of the trpllesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in theabsence of tryptophan. Similarly, Leu2-deficient yeast strains(ATCC 20,622 or 38,626) are complemented by known plas-mids bearing the Leu2 gene. Ura3-deficient yeast strains arecomplemented by plasmids bearing the ura3 gene.[0268] In addition, vectors derived from the 1.6 pm circularplasmid pKDI can be used for transformation of Eluyvero-myces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chyrnosin was reportedfor E. lactis Van den Berg, (Biol'Technology, 8:135 (1990)).Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains ofEluyveromyces have also been disclosed (Fleer et al, Biol'Technology, 9:968-975 (1991)).[0269] (4) Promoter Component[0270] Expression and cloning vectors usually contain apromoter that is recognized by the host organism and is oper-ably linked to the antibody-encoding nucleic acid. Promoterssuitable for use with prokaryotic hosts include the arabinose(e.g., arab) promoter phoA promoter, [I-Iactamase and lactosepromoter systems, alkaline phosphatase, a tryptophan (trp)promoter system, and hybrid promoters such as the tac pro-moter. However, other known bacterial promoters are suit-able. Promoters for use in bacterial systems also will containa Shine-Dalgamo (S.D.) sequence operably linked to theDNA encoding the antibody of the invention.[0271] Promoter sequences are known for eukaryotes. Vir-tually all eukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genesis a CNCAAT region where N may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence thatmay be the signal for addition of the poly A tail to the 3' endof the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.[0272] Examples of suitable promoting sequences for usewith yeast hosts include the promoters for 3-phosphoglycer-ate kinase or other glycolytic enzymes, such as enolase, glyc-eraldehyde-3-phosphate dehydrogenase, hexokinase, pyru-vate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucoseisomerase, and glucokinase.[0273] Other yeast promoters, which are inducible promot-ers having the additional advantage of transcription con-trolled by growth conditions, are the promoter regions foralcohol dehydrogenase 2, isocytochrome C, acid phos-phatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and

galactose utilization. Suitable vectors and promoters for usein yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promot-ers.[0274] Antibody transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtainedfrom the genornes of viruses such as Abelson leukemia virus,polyoma virus, fowlpox virus, adenovirus (such as Adenovi-rus 2), bovine papilloma virus, avian sarcoma virus, mostpreferably cytomegalovirus, a retrovirus, hepatitis-D virus,Simian Virus 40 (SV40), from heterologous mammalian pro-moters, e.g., the actin promoter or an immunoglobulin pro-moter, from heat-shock promoters, provided such promotersare compatible with the host cell systems.[0275] The early and late promoters of the SV40 virus areconveniently obtained as an SV40 restriction fragment thatalso contains the SV40 viral origin of replication. The imme-diate early promoter of the human cytomegalovirus is conve-niently obtained as a HindIII E restriction fragment. A systemfor expressing DNA in mammalian hosts using the bovinepapilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S.Pat. No. 4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) on expression of human [3-interferon cDNA inmouse cells under the control o f a thymidine kinase promoterfrom herpes simplex virus. Alternatively, the rous sarcomavirus long terminal repeat can be used as the promoter.[0276] (5) Enhancer Element Component[0277] Transcription of a DNA encoding the antibody ofthis invention by higher eukaryotes is often increased byinserting an enhancer sequence into the vector. Manyenhancer sequences are known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukary-otic cell virus. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100-270), the cytome-galovirus early promoter enhancer, the polyoma enhancer onthe late side of the replication origin, and adenovirus enhanc-ers. See also Yaniv, Nature 297:17-18 (1982) on enhancingelements for activation o f eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' tothe antibody-encoding sequence, but is preferably located ata site 5' from the promoter.[027S] (6) Transcription Termination Component[0279] Expression vectors used in eukaryotic host cells(yeast, fungi, insect, plant, animal, human, or nucleated cellsfrom other multicellular organisms) will also containsequences necessary for the termination of transcription andfor stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3', untranslatedregions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encodingantibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. SeeWO94/11026 and the expression vector disclosed therein.Another is the mouse immunoglobulin light chain transcrip-tion terminator.[02SO] (7) Selection and Transformation of Host Cells[02S1] Suitable host cells for cloning or expressing theDNA in the vectors herein are the prokaryote, yeast, or highereukaryote cells described above. Suitable prokaryotes for thispurpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as

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Escherichia, e.g., E. coli, Enterobacter, Erwinia, Elebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia,e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis41 P disclosed in DD 266,710 published Apr. 12, 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446),although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.[0282] In addition to prokaryotes, eukaryotic microbessuch as filamentous fungi or yeast are suitable cloning orexpression hosts for antibody-encoding vectors. Saccharo-myces cerevisi ae, or common baker's yeast, is the most com-monly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains arecommonly available and useful herein, such as Schizosaccha-romyces pombe; Eluyveromyces hosts such as, e.g., E. lactis,E. fragilis (ATCC 12,424), E. bulgaricus (ATCC 16,045), E.wiclreramii (ATCC 24,178), E. waltii (ATCC 56,500), E.drosophilarum (ATCC 36,906), E. thermotolerans, and E.marx/anus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neuro-spora crassa; Schwanniomyces such as Schwanniomycesoccidentalis; and filamentous fingi such as, e.g., Neurospora,Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.[0283] Suitable host cells for the expression of glycosy-lated antibody are derived from multicellular organisms.Examples of invertebrate cells include plant and insect cells.Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly),and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-Ivariant of Autographa californica NPV and the Bm-5 strainof Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularlyfor trans fection of Spodoptera frugiperda cells.[0284] Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, tobacco, lemna, and other plant cells can alsobe utilized as hosts.

[0285] Examples of useful mammalian host cell lines areChinese hamster ovary cells, including CHOKI cells (ATCCCCL61), DXB-I I, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CVI line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture,(Graham et al., J. Gen Virol. 36: 59, 1977); baby hamsterkidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkeykidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A,ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TRI cells (Mather etal., Annals N. I'. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).[0286] Host cells are transformed or transfected with theabove-described expression or cloning vectors for antibody

production and cultured in conventional nutrient media modi-fied as appropriate for inducing promoters, selecting trans-formants, or amplifying the genes encoding the desiredsequences. In addition, novel vectors and transfected celllines with multiple copies of transcription units separated bya selective marker are particularly useful and preferred for theexpression of antibodies that bind target.[0287] (8) Culturing the Host Cells[02SS] The host cells used to produce the antibody of thisinvention may be cultured in a variety of media. Commer-cially available media such as Ham's FIO (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma),and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al., (Meth. Enz. 58: 44,1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. Re. No.30,985 may be used as culture media for the host cells. Any o fthese media may be supplemented as necessary with hor-mones and/or other growth factors (such as insulin, transfer-rin, or epidermal growth factor), salts (such as sodium chlo-ride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN drug), trace elements(defined as inorganic compounds usually present at final con-centrations in the micromolar range), and glucose or anequivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture con-ditions, such as temperature, pH, and the like, are those pre-viously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.[0289] (9) Purification of Antibody[0290] When using recombinant techniques, the antibodycan be produced intracellularly, in the periplasmic space, ordirectly secreted into the medium, including from microbialcultures. If the antibody is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed frag-ments, is removed, for example, by centrifugation or ultrafil-tration. Better et al. (Science 240:1041-43, 1988; ICSU ShortReports 10:105 (1990); and Proc. Natl. Acad. Sci. USA90:457-461 (1993) describe a procedure for isolating anti-bodies which are secreted to the periplasmic space of E. coli.[See also, (Carter et al., Bio/'Technology 10:163-167 (1992)].[0291] The antibody composition prepared from microbialor mammalian cells can be purified using, for example,hydroxylapatite chromatography cation or avian exchangechromatography, and affinity chromatography, with affinitychromatography being the preferred purification technique.The suitability of protein A as an affinity ligand depends onthe species and isotype of any immunoglobulin Fc domainthat is present in the antibody. Protein A can be used to purifyantibodies that are based on human 71, 72, or 74 heavy chains(Lindmark et al., J. Immunol. Meth. 62: 1-13, 1983). ProteinG is recommended for all mouse isotypes and for human 73(Guss et al., EMBO J. 5:15671575 (1986)). The matrix towhich the affinity ligand is attached is most often agarose, butother matrices are available. Mechanically stable matricessuch as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times thancan be achieved with agarose. Where the antibody comprisesa CH 3 domain, the Bakerbond ABXâ„¢ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques

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for protein purification such as fractionation on an ion-ex-change column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparinSEPHAROSEâ„¢ chromatography on an anion or cationexchange resin (such as a polyaspartic acid column), chro-mato focusing, SDS-PAGE, and ammonium sulfate precipita-tion are also available depending on the antibody to be recov-ered.

Screening Methods

[0292] Effective therapeutics depend on identifying effica-cious agents devoid of significant toxicity. Antibodies may bescreened for binding affinity by methods known in the art. Forexample, gel-shift assays, Western blots, radiolabeled com-petition assay, co-fractionation by chromatography, co-pre-cipitation, cross linking, ELISA, and the like may be used,which are described in, for example, Current Protocols inMolecular Biology (1999) John Wiley & Sons, NY, which isincorporated herein by reference in its entirety.[0293] In one embodiment of the invention, methods ofscreening for antibodies which modulate the activity of atarget antigen comprise contacting test antibodies with a tar-get polypeptide and assaying for the presence of a complexbetween the antibody and the target ligand. In such assays, theligand is typically labeled. After suitable incubation, freeligand is separated from that present in bound form, and theamount of free or uncomplexed label is a measure of theability of the particular antibody to bind to the target ligand.[0294] In another embodiment of the invention, highthroughput screening for antibody fragments or CDRs havingsuitable binding affinity to a target polypeptide is employed.Briefly, large numbers of different small peptide test com-pounds are synthesized on a solid substrate. The peptide testantibodies are contacted with a target polypeptide andwashed. Bound polypeptides are then detected by methodswell known in the art. Purified antibodies of the invention canalso be coated directly onto plates for use in the aforemen-tioned drug screening techniques. In addition, non-neutraliz-ing antibodies can be used to capture the target and immobi-lize it on the solid support.

Combination Therapy

[0295] If more than one antibody effective at binding totarget antigen is identified, it is contemplated that two or moreantibodies to different epitopes of the target antigen may bemixed such that the combination of antibodies together toprovide still improved efficacy against a condition or disorderto be treated associated with the target polypeptide. Compo-sitions comprising one or more antibody of the invention maybe administered to persons or mammals suffering from, orpredisposed to suffer from, a condition or disorder to betreated associated with the target polypeptide.[0296] Concurrent administration of two therapeuticagents does not require that the agents be administered at thesame time or by the same route, as long as there is an overlapin the time period during which the agents are exerting theirtherapeutic effect. Simultaneous or sequential administrationis contemplated, as is administration on different days orweeks.[0297] A second agent may be other therapeutic agents,such as cytokines, growth factors, other anti-inflammatoryagents, anti-coagulant agents, agents that will lower or reduceblood pressure, agents that will reduce cholesterol, triglycer-

ides, LDL, VLDL, or lipoprotein(a) or increase HDL, agentsthat will increase or decrease levels of cholesterol-regulatingproteins, anti-neoplastic drugs or molecules. For patientswith a hyperproliferative disorder, such as cancer or a tumor,combination with second therapeutic modalities such asradiotherapy, chemotherapy, photodynamic therapy, or sur-gery is also contemplated.

[0298] It is contemplated the antibody of the invention andthe second agent may be given simultaneously, in the sameformulation. It is further contemplated that the agents areadministered in a separate formulation and administered con-currently, with concurrently referring to agents given within30 minutes of each other.

[0299] In another aspect, the second agent is administeredprior to administration of the antibody composition. Prioradministration refers to administration of the second agentwithin the range of one week prior to treatment with theantibody, up to 30 minutes before administration of the anti-body. It is further contemplated that the second agent isadministered subsequent to administration of the antibodycomposition. Subsequent administration is meant to describeadministration from 30 minutes after antibody treatment up toone week after antibody administration.

[0300] It is further contemplated that when the antibody isadministered in combination with a second agent, wherein thesecond agent is a cytokine or growth factor, or a chemothera-peutic agent, the administration also includes use of a radio-therapeutic agent or radiation therapy. The radiation therapyadministered in combination with an antibody composition isadministered as determined by the treating physician, and atdoses typically given to patients being treated for cancer.

[0301] A cytotoxic agent refers to a substance that inhibitsor prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes(e.g., I' ', I', Y and Re' ), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fun-gal, plant or animal origin or synthetic toxins, or fragmentsthereof. A non-cytotoxic agent refers to a substance that doesnot inhibit or prevent the function of cells and/or does notcause destruction of cells. A non-cytotoxic agent may includean agent that can be activated to be cytotoxic. A non-cytotoxicagent may include a bead, liposome, matrix or particle (see,e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Suchagents may be conjugated, coupled, linked or associated withan antibody according to the invention.

[0302] Chemotherapeutic agents contemplated for use withthe antibodies of the invention include, but are not limited tothose listed in Table I:

TABLE I

Alkylating agentsNiuogen mustards

mechlore&aminecyclophosphamideifosfamidemelphalanchlorambucilNiu osoureas

carmustine (BCNUilomustine (CCNUisemustine (methyl-CCNUi

26

Antiandrogens

flutamideNatural productsAntimitotic drugsTaxanes

busulfanTriazines

etoposideteniposideAntibiotics

L-asparaginaseL-arginaseRadiosensitizers

cisplatinCarboplatinoxaliplatinAnthracenedionemitoxantroneSubstituted urea

tamoxifenAndrogens

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TABLE I-continued

Ethylenimine/Methyl-melamine

flmefltyienemeiamine (TEMitriethylene thiophosphoramide(thiotepa)hexamethylmelamine(HMM, altretamine)Alkyl sulfonates

dacarbazine (DTICiAntimetabolitesFolic Acid analogs

methouexateTrimetrexatePemetrexed(Multi-targeted antifoiateiPyrimidine analogs

5-fluorouracifluorodeoxyuridinegemcitabinecytosine arabinoside(AraC, cytarabinei5-azacytidine2,2udifluorodeoxy-cytidinePurine analogs

6-mercaptopurine6-fluoguanineazathioprine2udeoxycoformycin(pentostatinierythrohydroxynonyl-adenine (EHNAifludarabine phosphate2-chlorodeoxyadenosine(ciadribine, 2-CdAiType I Topoisomerase Inhibitors

camptothecintopotecanirinotecanBiological response modifiers

G-CSFGM-CSFDifferentiation Agents

retinoic acid derivativesHormones and antagonistsAdrenocorticosteroids/antagonists

prednisone and equivalentsdexamethasoneainoglutethimideProgestins

hydroxyprogesterone caproatemedroxyprogesterone acetatemegestrol acetateEstrogens

diethylstilbestrolethynyl estradiol/equivalentsAntiestrogen

testosterone propionatefluoxymesterone/equivalents

TABLE I-continued

flutamidegonadotropin-releasinghormone analogsleuprolideNonsteroidal antiandrogens

paclitaxelVinca alkaloidsvinblastine (VLBivincristinevinorelbineTaxotere ® (docetaxeiiestramustineestramustine phosphateEpipodophylotoxins

actimomycin Ddaunomycin (rubido-mycinidoxorubicin (adria-mycinimitoxantroneidarubicinbleomycinsplicamycin (mithramycin)mitomycinCdactinomycinaphidicolinEnzymes

metronidazolemisonidazoledesmethylmisotridazolepimonidazoleetanidazolenimorazoleRSU II369E09RB 6145SR4233nicotinamide5-bromodeozyuridine5-iododeoxyuridinebromodeoxycytidineMiscellaneous agentsPlatinium coordination complexes

hydroxyureaMethylhydrazine derivatives

N-methylhydrazine (MIHiprocarbazineAdrenocortical suppressant

mitotane (o, pu DDDiainoglutethimide

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TABLE I-continued

Cytokines

interferon (ct, P, yiinterleukin-2Photosensitizers

hematoporphyrin derivativesPhoto frin ®benzoporphyrin derivativesNpe6tin etioporphyrin (SnET21pheoboride-abacteriochlorophyll-anaphthalocyaninesphthalocyanineszinc phthalocyaninesRadiation

X-rayultraviolet lightgamma radiationvisible lightinfrared radiationmicrowave radiation

Treatment of Disorders Using the Methods and Compositionsof the Invention

[0303] In another embodiment, the invention provides amethod for inhibiting target activity by administering a tar-get-specific antibody to a patient in need thereof. Any of thetypes of antibodies described herein may be used therapeuti-cally. In exemplary embodiments, the target specific antibodyis a human, chimeric or humanized antibody. In anotherexemplary embodiment, the target is human and the patient isa human patient. Alternatively, the patient may be a mammalthat expresses a target protein that the target specific antibodycross-reacts with. The antibody may be administered to anon-human mammal expressing a target protein with whichthe antibody cross-reacts (ke. a primate) for veterinary pur-poses or as an animal model of human disease. Such animalmodels may be useful for evaluating the therapeutic efficacyof target specific antibodies of the invention.

[0304] Exemplary conditions or disorders associated withtarget expression that can be treated with an antibody sub-stance according to the present invention include cancers,such as pancreatic cancer, esophageal cancer, gastric cancer,colorectal cancer, and small lung cell carcinoma. Other con-ditions or disorders that may be treated using antibodies of theinvention include, gastric ulcer, duodenal ulcer, other ulcersor conditions associated with H. Pylori, gastroesophagealreflux disease, autoimmune gastritis, atrophic body gastritis,Zollinger-Ellison syndrome associated with tumor of the pan-creas (gastrlnoma), and inflammatory bowel disease.[0305] Gastrin expression and cell activation has beenfound in several models of pancreatic cancer (Smith et al., AmJPhysiol 268(1 Pt 2):R135-41 (1995)), gastric cancer KWat-son et al., Br J Surg 75:342-5 (1988)) (Watson, et al., Br JCancer 59:554-8 (1989)) (Piontek and Hengels, AnticancerRes 13:715-20 (1993)) (Szabo et al., JPhysiol Paris. 94:71-4(2000)), and colorectal cancer (Ahmed et al., FEBS Lett 556:199-203 (2004)) (Stepan et al., Mol Med 5:147-59 (1999))(Smith et al., Am J Physiol 271(3 Pt 2):R797-805 (1996)).

[0306] Pancreatic cancer Pancreatic cancer is the fifthleading cause of cancer death in the United States. Surgical

resection remains the primary treatment since, on occasion,resection can lead to long-term survival and provides effec-tive palliation. For decades, 5-fluorouracil (5-FU) was themost widely used chemotherapeutic agent in metastatic pan-creatic cancer. Today, gemcitabine is the current standard ofcare for patients with locally advanced and metastatic pan-creatic cancer (Tempero et al., J Clin Oncol. 21:3402-8(2003)) (Berlin et al., Clin Oncol 20:3270-5 (2002)). Theunique mechanism of action and favorable toxicity profile ofgemcitabine have allowed exploration of many novel gemcit-abine-based combination regimens as treatment for pancre-atic cancer (see e.g., Rocha Lima et al., JClin Oncol 20:1182-91 (2002)).[0307] Gastric Cancer Stomach cancer is the secondleading cancer. Cure for gastric cancer is available, only forthose patients in whom a complete surgical resection can beperformed; this is possible for only 30'zo to 35'zo of allpatients, and even in these patients, relapse is common. Thelevel of lymph-node dissection is an important prognosticfactor: when the en bloc lymph node dissection is performed,5-year survival is nearly 40'zo, which is much better than thatwithout lymph node dissection. The estimated 5-year relativesurvival rates by Stage are as follows: 65'zo for Stage I, 20'zofor Stage II, 10'zo for Stage III and <I'zo for Stage IV.Advanced disease is incurable and patients are treated bychemotherapy. Current thereapies for gastric cancer includeFAM, a combination of 5-FU, mitomycin C, and low doses ofdoxorubicin (see e.g., Rougier et al., 1994), FAMTX (FAMwith mitomycin replaced by a high dose of methotrexate),etoposide plus LV and 5-PU (ke., ELF), and ECF (epirubicinplus 5-FU as a protracted infusion and cisplatin).[0308] Colorectal cancer (commonly referred to as "colon"cancer) develops in the lower part of the digestive system,also referred to as the gastrointestinal, or GI, system. Thiscancer usually develops from precancerous changes orgrowths in the lining of the colon and rectum. Colon canceraccounts for about 10 percent of cancer deaths this year in theUnited States. Surgery is the most common form of treatmentfor colon cancer. For cancers that have not spread, it fre-quently controls the disease.[0309] Chemotherapy or chemotherapy with radiationtreatment is given before or after surgery to most patientswhose cancer has spread into the bowel wall or to the lymphnodes. Treatments include, but are not limited to the follow-ing agents, alone or in combination with each other: 5Fluo-rouracil, docetaxel, Campto sar/Irinotecan, capecitabine,oxaliplatin, and anti-Vascular Endothelial Growth Factorantibody (AVASTIN®).[0310] Small lung cell carcinoma Small lung cell carci-noma (SLCC), an aggressive (fast-growing) cancer that usu-ally forms in tissues of the lung and spreads to other parts ofthe body, accounts for approximately 20'zo of all lung cancers.It is characterized by its origin in large central airways andhistological composition of sheets of small cells with scantycytoplasm. Small cell carcinoma is a tumor of neuroendo-crine origin which metastasizes often. The cancer cells looksmall and oval-shaped when looked at under a microscope.Common treatments include surgery to remove the affectedtissue, as well as radiation thereapy and chemotherapy. Che-motherapeutic agents used to treat SLCC include, but are notlimited to, etoposide, cisplatin, and vincristine.[0311] Gastroesophageal reflux disease Acid reflux irri-tates the walls of the esophagus, inducing a secondary peri-staltic contraction of the smooth muscle, and may produce the

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discomfort or pain known as heartburn. Most episodes of acidreflux are asymptomatic. Secondary peristalsis returnsapproximately 90'zo of the acid and food to the stomach.Digested food in the stomach chemically stimulates therelease of gastrin from G cells located in the antrum of thestomach. Distention of the stomach causes release of acetyl-choline from the vagus nerve, and this further stimulates theG cells to produce gastrin. Gastrin travels through the blood-stream and binds to the gastrin receptor on the parietal cells,located in the gastric body and fundus. When gastrin binds toits receptor, the parietal cell's permeability to calcium ions(Ca++) is altered so that the ions move into the cell. Theintracellular increase in Ca++activates the intracellular pro-tein phosphokinases. The increase in protein phosphokinasesresults in the translocation of H+-K+-ATPase to the secretorycanaliculus where the extracellular aspect of the pump isexposed to potassium ions (K+).[0312] Some common treatments of GERD include antac-ids; foaming agents which coat thestomach; H~ blockers,such as cimetidine, famotidine, nizatidine, and ranitidine,impede acid production; proton pump inhibitors such as ome-prazole, lansoprazole, pantoprazole, rabeprazole, and esome-prazole; prokinetics, which help strengthen the esophagealsphincter and makes the stomach empty faster, such asbethanechol and metoclopramide;[0313] Gastric and Duodenal Ulcer A gastric ulcer is abreak in the normal tissue lining the stomach. A duodenalulcer is a break in the normal tissue lining the duodenum (thefirst part of the small bowel). Benign gastric ulcers are causedby an imbalance between the secretion of acid and an enzymecalled pepsin and the defenses of the stomach's mucosallining. For people with Helieobaeter pylori infection, themain goal is eradication of the organism that causes the prob-lem. Multiple regimens are effective and usually includeeither an H2 receptor antagonist such as famotidine or niza-tidine, or a proton pump inhibitor such as omeprazole oresomeprazole to suppress acid, combined with antibiotics.For people without H. pylori infection, ulcer-healing medi-cations such as antacids, H2 receptor antagonists, or protonpump inhibitors are usually effective. A vagotomy (cuttingthe vagus nerve, which controls the stomach's production ofgastric acid) or a partial gastrectomy (removal of part of thestomach) may be necessary.[0314] Autoimmune gastritis Autoimmune gastritis isassociated with serum antiparietal and anti-intrinsic factor(IF) antibodies. The gastric corpus undergoes progressiveatrophy, IF deficiency occurs, and patients may develop per-nicious anemia. Two types of IF antibodies are detected, ie,types I and II. Type I IF antibodies block the IF-cobalaminbinding site, thus preventing the uptake of vitamin B-12.Cell-mediated immunity also contributes to the disease.T-cell lymphocytes infiltrate the gastric mucosa and contrib-ute to epithelial cell destruction and resulting gastric atrophy.

[0315] Atrophic body gastritis Atrophic body gastritis(ABG) is characterized by atrophy of the gastric bodymucosa, hypergastrinemia, and hypo/achlorhydrla. Likeautoimmune gastritis, it is association with pernicious ane-mia. Body gastritis is also associated with H. pylori infectionand hypergastrinemia, or increased gastrin levels (Delle Faveet al., Dig Liver Dis. 34:270-8 (2002)).[0316] Zollinger-Ellison syndrome (ZES) is a rare disorderthat causes tumors in the pancreas and duodenum and ulcersin the stomach and duodenum. The tumors secrete gastrinwhich causes the stomach to produce too much acid causing

stomach and duodenal ulcers (Gibrll et al., Curr Gastroen-terol Rep. 7:114-21 (2005)). The ulcers caused by ZES areless responsive to treatment than ordinary peptic ulcers. Theprimary treatment for ZES is medication to reduce the pro-duction of stomach acid, including proton pump inhibitorsand H-2 blockers.

[0317] Inflammatory bowel disease-Inflammatory boweldisease includes two conditions known as ulcerative colitis(UC) and Chron's disease (CD). Gastrin receptor expressionis decreased in inflamed and non-inflamed colon of CD, butnot in UC (ter Beck et al., J Cli n Pathol. 57:1047-51 (2004)).However, other studies have shown that the levels of serumgastrin are elevated in patients with Crohn's disease whilepatients with ulcerative colitis exhibited no significant differ-ences compared to normal controls (Triantafillidis et al.,Hepatogastroenterology. 50 Suppl 2:cccxv-cccxvii, (2003)).[0318] In one embodiment, treatment of these disorders orconditions in an animal in need of said treatment, comprisesadministering to the animal an effective amount of a compo-sition comprising an antibody substance of the invention.[0319] The conditions treatable by methods of the presentinvention preferably occur in mammals. Mammals include,for example, humans and other primates, as well as pet orcompanion animals such as dogs and cats, laboratory animalssuch as rats, mice and rabbits, and farm animals such ashorses, pigs, sheep, and cattle.

Non-therapeutic Uses

[0320] The antibodies of the invention may be used asaffinity purification agents for target or in diagnostic assaysfor target protein, e.g., detecting its expression in specificcells, tissues, or serum. The antibodies may also be used for invivo diagnostic assays. Generally, for these purposes the anti-body is labeled with a radionuclide (such as ' "In, Tc, ' C,

Iy Iy Hy P or S) so that the antibody can be localizedusing immunoscintiography.

[0321] The antibodies of the present invention may beemployed in any known assay method, such as competitivebinding assays, direct and indirect sandwich assays, such asELISAs, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRCPress, Inc. 1987). The antibodies may also be used for immu-nohistochemistry, to label tissue or cell samples using meth-ods known in the art.

[0322] The target specific antibodies can be used in a con-ventional immunoassay, including, without limitation, anELISA, an RIA, FACS, tissue immunohistochemistry, West-ern blot or immunoprecipitation, which are all techniqueswell-known in the art. The antibodies of the invention can beused to detect target in humans and other mammals. Theinvention provides a method for detecting target in a biologi-cal sample comprising contacting a biological sample with atarget specific antibody of the invention and detecting thebound antibody. In one embodiment, the target specific anti-body is directly labeled with a detectable label. In anotherembodiment, the target specific antibody (the first antibody)is unlabeled and a second antibody or other molecule that canbind the target specific antibody is labeled. As is well knownto one of skill in the art, a second antibody is chosen that isable to specifically bind the particular species and class of thefirst antibody. For example, if the target specific antibody is ahuman IgG, then the secondary antibody could be an anti-human-IgG. Other molecules that can bind to antibodies

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include, without limitation, Protein A and Protein G, both ofwhich are available commercially, e.g., from Pierce ChemicalCo.

[0323] It is contemplated that the immunoassays disclosedabove are used for a number of purposes. For example, thetarget specific antibodies can be used to detect target in cellsor on the surface of cells in cell culture, or secreted into thetissue culture medium. The target specific antibodies can beused to determine the amount of target on the surface of cellsor secreted into the tissue culture medium that have beentreated with various compounds. This method can be used toidentify compounds that are useful to inhibit or activate targetexpression or secretion. According to this method, onesample of cells is treated with a test compound for a period oftime while another sample is left untreated. I f the total level o ftarget is to be measured, the cells are lysed and the total targetlevel is measured using one of the immunoassays describedabove. The total level of target in the treated versus theuntreated cells is compared to determine the effect of the testcompound.

Labels

[0324] In some embodiments, the antibody substance islabeled to facilitate its detection. A "label" or a "detectablemoiety" is a composition detectable by spectroscopic, pho-tochemical, biochemical, immunochemical, chemical, orother physical means. For example, labels suitable for use inthe present invention include, radioactive labels (e.g., P),fluorophores (e.g., fluorescein), electron-dense reagents,enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens as well as proteins which can be madedetectable, e.g., by incorporating a radiolabel into the haptenor peptide, or used to detect antibodies specifically reactivewith the hapten or peptide.

[0325] Examples of labels suitable for use in the presentinvention include, but are not limited to, fluorescent dyes(e.g., fluorescein isothiocyanate, Texas red, rhodamine, andthe like), radiolabels (e.g., H, ' I, S, ' C, or P), enzymes(e.g., horse radish peroxidase, alkaline phosphatase and oth-ers commonly used in an ELISA), and calorimetric labelssuch as colloidal gold, colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.).

[0326] The label may be coupled directly or indirectly tothe desired component of the assay according to methods wellknown in the art. Preferably, the label in one embodiment iscovalently bound to the biopolymer using an isocyanatereagent for conjugation of an active agent according to theinvention. In one aspect of the invention, the bifunctionalisocyanate reagents of the invention can be used to conjugatea label to a biopolymer to form a label biopolymer conjugatewithout an active agent attached thereto. The label biopoly-mer conjugate may be used as an intermediate for the synthe-sis of a labeled conjugate according to the invention or may beused to detect the biopolymer conjugate. As indicated above,a wide variety of labels can be used, with the choice of labeldepending on sensitivity required, ease of conjugation withthe desired component of the assay, stability requirements,available instrumentation, and disposal provisions. Non-ra-dioactive labels are often attached by indirect means. Gener-ally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable

or covalently bound to a signal system, such as a detectableenzyme, a fluorescent compound, or a chemiluminescentcompound.[0327] The compounds of the invention can also be conju-gated directly to signal-generating compounds, e.g., by con-jugation with an enzyme or fluorophore. Enzymes suitable foruse as labels include, but are not limited to, hydrolases, par-ticularly phosphatases, esterases and glycosidases, or oxidot-ases, particularly peroxidases. Fluorescent compounds, i.e.,fluorophores, suitable for use as labels include, but are notlimited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Further examples ofsuitable fluorophores include, but are not limited to, eosin,TRITC-amine, quinine, fluorescein W, acridine yellow, lissa-mine rhodamine, B sulfonyl chloride erythroscein, ruthenium(tris, bipyrldinium), Texas Red, nicotinamide adeninedinucleotide, flavin adenine dinucleotide, etc. Chemilumi-nescent compounds suitable for use as labels include, but arenot limited to, luciferin and 2,3-dihydrophthalazinediones,e.g., luminol. For a review of various labeling or signal pro-ducing systems that can be used in the methods of the presentinvention, see U.S. Pat. No. 4,391,904.[032S] Means for detecting labels are well known to thoseof skill in the art. Thus, for example, where the label isradioactive, means for detection include a scintillationcounter or photographic film, as in autoradiography. Wherethe label is a fluorescent label, it may be detected by excitingthe fluorochrome with the appropriate wavelength of lightand detecting the resulting fluorescence. The fluorescencemay be detected visually, by the use of electronic detectorssuch as charge coupled devices (CCDs) or photomultipliersand the like. Similarly, enzymatic labels may be detected byproviding the appropriate substrates for the enzyme anddetecting the resulting reaction product. Colorimetric orchemiluminescent labels may be detected simply by observ-ing the color associated with the label. Other labeling anddetection systems suitable for use in the methods of thepresent invention will be readily apparent to those of skill inthe art. Such labeled modulators and ligands can be used inthe diagnosis of a disease or health condition.

Formulation of Pharmaceutical Compositions

[0329] To administer antibody substances of the inventionto human or test animals, it is preferable to formulate theantibody substances in a composition comprising one or morepharmaceutically acceptable carriers. The phrase "pharma-ceutically or pharmacologically acceptable" refer to molecu-lar entities and compositions that do not produce allergic, orother adverse reactions when administered using routes well-known in the art, as described below. "Pharmaceuticallyacceptable carriers" include any and all clinically useful sol-vents, dispersion media, coatings, antibacterial and antifun-gal agents, isotonic and absorption delaying agents and thelike.[0330] In addition, compounds may form solvates withwater or common organic solvents. Such solvates are contem-plated as well.[0331] The antibody is administeredby any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapul-monary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintravenous, intraarterial, intraperitoneal, intramuscularintradermal or subcutaneous administration. In addition, theantibody is suitably administered by pulse infusion, particu-

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larly with declining doses of the antibody. Preferably thedosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether theadministration is brief or chronic. Other administration meth-ods are contemplated, including topical, particularly trans-dermal, transmucosal, rectal, oral or local administration e.g.through a catheter placed close to the desired site. Injection,especially intravenous, is preferred.[0332] Pharmaceutical compositions of the present inven-tion containing an antibody substance of the invention as anactive ingredient may contain pharmaceutically acceptablecarriers or additives depending on the route of administration.Examples of such carriers or additives include water, a phar-maceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, car-boxymethylcellulose sodium, polyacrylic sodium, sodiumalginate, water-soluble dextran, carboxym ethyl starchsodium, pectin, methyl cellulose, ethyl cellulose, xanthangum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin,propylene glycol, polyethylene glycol, Vaseline, paraffin,stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, a pharmaceutically acceptablesurfactant and the like. Additives used are chosen from, butnot limited to, the above or combinations thereof, as appro-priate, depending on the dosage form of the present invention.[0333] Formulation of the pharmaceutical compositionwill vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition com-prising the antibody to be administered can be prepared in aphysiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles caninclude sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride, lactated Ringer's or fixed oils. Intrave-nous vehicles can include various additives, preservatives, orfluid, nutrient or electrolyte replenishers.

[0334] A variety of aqueous carriers, e.g., sterile phosphatebuffered saline solutions, bacteriostatic water, water, bufferedwater, 0.4'zo saline, 03'zo glycine, and the like, and mayinclude other proteins for enhanced stability, such as albumin,lipoprotein, globulin, etc., subjected to mild chemical modi-fications or the like.

[0335] Therapeutic formulations of the antibody are pre-pared for storage by mixing the antibody having the desireddegree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remingon's Pharmaceuti-cal Sciences 16th edition, Osol, A. Ed. (1980)), in the form oflyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients atthe dosages and concentrations employed, and include buff-ers such as phosphate, citrate, and other organic acids; anti-oxidants including ascorbic acid and methionine; preserva-tives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzetho-nium chloride; phenol, butyl or benzyl alcohol; alkyl para-bens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydro-philic polymers such as polyvinylpyrrolidone; amino acidssuch as glycine, glutamine, asparagine, histidine, arginine, orlysine; monosaccharides, disaccharides, and other carbohy-drates including glucose, mannose, or dextrins; chelating

agents such as EDTA; sugars such as sucrose, mannitol, tre-halose or sorbitol; salt-forming counter-ions such as sodium;metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICSâ„¢ orpolyethylene glycol (PEG).[0336] The formulation herein may also contain more thanone active compound as necessary for the particular indica-tion being treated, preferably those with complementaryactivities that do not adversely affect each other. Such mol-ecules are suitably present in combination in amounts that areeffective for the purpose intended.[0337] The active ingredients may also be entrapped inmicrocapsule prepared, for example, by coacervation tech-niques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidaldrug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocap-sules) or in macro emulsions. Such techniques are disclosed inRemingon's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).[0338] The formulations to be used for in vivo administra-tion must be sterile. This is readily accomplished by filtrationthrough sterile filtration membranes.[0339] Aqueous suspensions may contain the active com-pound in admixture with excipients suitable for the manufac-ture of aqueous suspensions. Such excipients are suspendingagents, for example sodium carboxymethylcellulose, meth-ylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dis-persing or wetting agents may be a naturally-occurring phos-phatide, for example lecithin, or condensation products of analkylene oxide with fatty acids, for example polyoxyethylenestearate, or condensation products of ethylene oxide withlong chain aliphatic alcohols, for example heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol suchas polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides, for example polyethylenesorbitan monooleate. The aqueous suspensions may also con-tain one or more preservatives, for example ethyl, or n-propyl,p-hydroxybenzoate.[0340] The antibodies of the invention can be lyophilizedfor storage and reconstituted in a suitable carrier prior to use.This technique has been shown to be effective with conven-tional immunoglobulins. Any suitable lyophilization andreconstitution techniques can be employed. It will be appre-ciated by those skilled in the art that lyophilization and recon-stitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.[0341] Dispersible powders and granules suitable forpreparation of an aqueous suspension by the addition of waterprovide the active compound in admixture with a dispersingor wetting agent, suspending agent and one or more preser-vatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above.[0342] The concentration of antibody in these formulationscan vary widely, for example from less than about 0.5'zo,usually at or at least about I'zo to as much as 15 or 20'zo byweight and will be selected primarily based on fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. Thus, a typical pharmaceutical com-position for parenteral injection could be made up to contain

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I ml sterile buffered water, and 50 mg of antibody. A typicalcomposition for intravenous infusion could be made up tocontain 250 ml of sterile Ringer's solution, and 150 mg ofantibody. Actual methods for preparing parenterally admin-istrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed.,Mack Publishing Company, Easton, Pa. (1980). An effectivedosage of antibody is within the range of 0 01 mg to 1000 mgper kg of body weight per administration.

[0343] The pharmaceutical compositions may be in theform of a sterile injectable aqueous, oleaginous suspension,dispersions or sterile powders for the extemporaneous prepa-ration of sterile injectable solutions or dispersions. The sus-pension may be formulated according to the known aft usingthose suitable dispersing or wetting agents and suspendingagents which have been mentioned above. The sterile inject-able preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Thecarrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, pro-pylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, vegetable oils, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or sus-pending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addi-tion, fatty acids such as oleic acid find use in the preparationof injectables.

[0344] In all cases the form must be sterile and must be fluidto the extent that easy-syringability exists. The proper fluiditycan be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. It must bestable under the conditions of manufacture and storage andmust be preserved against the contaminating action of micro-organisms, such as bacteria and fungi. The prevention of theaction of microorganisms can be brought about by variousantibacterial an antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be desirable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorp-tion of the injectable compositions can be brought about bythe use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0345] Compositions useful for administration may be for-mulated with uptake or absorption enhancers to increase theireflicacy. Such enhancer include for example, salicylate, gly-cocholate/Iinoleate, glycholate, aprotinin, bacitracin, SDS,caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

[0346] Antibody compositions contemplated for use toinhibit target activity, including binding of the target to itscognate receptor or 1igand, target-mediated signaling, and thelike. In particular, the compositions exhibit inhibitory prop-erties at concentrations that are substantially free of sideeffects, and are therefore useful for extended treatment pro-tocols. For example, co-administration of an antibody com-position with another, more toxic, cytotoxic agent can achievebeneficial inhibition of a condition or disorder being treated,while effectively reducing the toxic side effects in the patient.

[0347] In addition, the properties of hydrophilicity andhydrophobicity of the compositions contemplated for use inthe invention are well balanced, thereby enhancing their util-ity for both in vitro and especially in vivo uses, while othercompositions lacking such balance are of substantially lessutility. Specifically, compositions contemplated for use in theinvention have an appropriate degree of solubility in aqueousmedia which permits absorption and bioavailability in thebody, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membrane to aputative site of action. Thus, antibody compositions contem-plated are maximally effective when they can be delivered tothe site of target antigen activity.

Administration and Dosing

[0348] In one aspect, methods of the invention include astep of administration of a pharmaceutical composition.

[0349] Methods of the invention are performed using anymedically-accepted means for introducing a therapeuticdirectly or indirectly into a mammalian subject, including butnot limited to injections, oral ingestion, intranasal, topical,transdermal, parenteral, inhalation spray, vaginal, or rectaladministration. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, and intracisternalinjections, as well as catheter or infusion techniques. Admin-istration by, intradermal, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or sur-gical implantation at a particular site is contemplated as well.

[0350] In one embodiment, administration is performed atthe site of a cancer or affected tissue needing treatment bydirect injection into the site or via a sustained delivery orsustained release mechanism, which can deliver the formula-tion internally. For example, biodegradable microspheres orcapsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included inthe formulations of the invention implanted near the cancer.

[0351] Therapeutic compositions may also be delivered tothe patient at multiple sites. The multiple administrations maybe rendered simultaneously or may be administered over aperiod of time. In certain cases it is beneficial to provide acontinuous flow of the therapeutic composition. Additionaltherapy may be administered on a period basis, for example,hourly, daily, weekly or monthly.

[0352] Also contemplated in the present invention is theadministration of multiple agents, such as an antibody com-position in conjunction with a second agent as describedherein.

[0353] The amounts of antibody composition in a givendosage will vary according to the size of the individual towhom the therapy is being administered as well as the char-acteristics of the disorder being treated. In exemplary treat-ments, it may be necessary to administer about I mg/day, 5mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day, 100mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or1000 mg/day. These concentrations may be administered as asingle dosage form or as multiple doses. Standard dose-re-sponse studies, first in animal models and then in clinicaltesting, reveal optimal dosages for particular disease statesand patient populations.

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[0354] It will also be apparent that dosing may be modifiedif traditional therapeutics are administered in combinationwith therapeutics of the invention.

Gene Therapy

[0355] The nucleic acid molecules of the present inventioncan be administered to a patient in need thereof via genetherapy. The therapy may be either in vivo or ex vivo. In oneembodiment, nucleic acid molecules encoding both a heavychain and a light chain are administered to a patient. Inanother embodiment, the nucleic acid molecules are admin-istered such that they are stably integrated into chromosomesof B cells because these cells are specialized for producingantibodies. In a related embodiment, precursor B cells aretransfected or infected ex vivo and re-transplanted into apatient in need thereof. In a further embodiment, precursor Bcells or other cells are infected in vivo using a virus known toinfect the cell type of interest.[0356] Delivery of a functional gene encoding a polypep-tide of the invention to appropriate cells is effected ex vivo, insitu, or in vivo by use of vectors, including viral vectors (e.g.,adenovirus, adeno-associated virus, or a retrovirus), or exvivo by use of physical DNA transfer methods (e.g., lipo-somes or chemical treatments). See, for example, Anderson,Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998).For additional reviews of gene therapy technology see Fried-mann, (Science, 244: 1275-1281 (1989)); Verma, (ScientiJcAmerican: 263:68-72, 81-84 (1990)); and Miller, (Nature,357: 455-460 (1992)). Introduction of any one of the nucle-otides of the present invention or a gene encoding a polypep-tide of the invention can also be accomplished with extrach-romosomal substrates (transient expression) or artificialchromosomes (stable expression). Cells may also be culturedex vivo in the presence of proteins of the present invention inorder to proliferate or to produce a desired effect on, oractivity in, such cells. In another embodiment, cells compris-ing vectors expressing the polynucleotides or polypeptides ofthe invention may be cultured ex vivo and administered to anindividual in need of treatment for overexpression of targetprotein.[0357] In one aspect, the gene therapy method comprisesthe steps of administering an isolated nucleic acid moleculeencoding the heavy chain or an antigen-binding portionthereof of a target specific antibody and expressing thenucleic acid molecule. In one embodiment, the gene therapymethod comprises the steps of administering an isolatednucleic acid molecule encoding the light chain or an antigen-binding portion thereof of a target specific antibody andexpressing the nucleic acid molecule. In another embodi-ment, the gene therapy method comprises the steps of admin-istering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof and an isolatednucleic acid molecule encoding the light chain or the antigen-binding portion thereof of a target specific antibody of theinvention and expressing the nucleic acid molecules. Thegene therapy method may also comprise the step of adminis-tering another second agent to the patient receiving genetherapy.[0358] After infection either in vivo or ex vivo, levels ofantibody expression can be monitored by taking a samplefrom the treated patient and using any immunoas say known inthe art or discussed herein.

Kits

[0359] As an additional aspect, the invention includes kitswhich comprise one or more compounds or compositions

packaged in a manner which facilitates their use to practicemethods of the invention. In one embodiment, such a kitincludes a compound or composition described herein (e.g., acomposition comprising a target-specific antibody alone or incombination with a second agent), packaged in a containersuch as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of thecompound or composition in practicing the method. Prefer-ably, the compound or composition is packaged in a unitdosage form. The kit may further include a device suitable foradministering the composition according to a specific route o fadministration or for practicing a screening assay. Preferably,the kit contains a label that describes use of the antibodycomposition.[0360] Additional aspects and details of the invention willbe apparent from the following examples, which are intendedto be illustrative rather than limiting.

EXAMPLES

Example I

Methods for Isolating Target-specific Antibodies

[0361] To isolate a panel of antibodies able to neutralize theactivity of the human growth factor gastrin, three humanantibody phage display libraries, expressing etiher scFv orFab fragments, were investigated in parallel. The target usedfor the library panning was a short 10 amino acid peptide fromthe N terminal portion of Gastrin 17. The peptide was bioti-nylated at its C-terminus and used in a soluble panningapproach.[0362] Selection of target specific antibody from phagedisplay was carried out according to methods described byMarks et al. (Methods Mol. Biol. 248: 161-76 (2004)). Briefly,the phage display library was incubated with 100 pmo1 s o f thebiotinylated peptide at room temperature for I hr and thecomplex formed was then captured using 100 pl of Strepta-vidin beads suspension (DYNABEADS® M-280 Streptavi-din, Invitrogen). Non specific phages were removed by wash-ing the beads with wash buffer (PBS+5'zo Milk). Boundphages were eluted with 0.5 ml of 100 nM Triethyleamine(TEA) and immediately neutralized by addition of an equalvolume of IM TRIS-CI pH 7.4. Eluted phage pool was usedto infect TGI E cali cells growing in logarithmic phase, andphagemid was rescued as described (Marks et al., MethodsMol. Biol. 248:161-76 (2004)). Selection was repeated for atotal of three rounds. Single colonies obtained from TGI cellsinfected with eluted phage from the third round of panningwere screened for binding activity in an ELISA assay. Briefly,single colonies obtained from the TGI cell infected witheluted phage were used to inoculate media in 96-well plates.Microcultures were grown to an OD«o=0.6 at which pointexpression of soluble antibody fragment was induced byaddition of I mM IPTG following overnight culture in ashaker incubator at 30' C. Bacteria were spun down andperiplasmic extract was prepared and used to detect antibodybinding activity to biotinylated gastrin immobilized onStreptavidin microplates (REACT-BINDâ„¢ StreptavidinHBC, Pierce) following standard ELISA protocol providedby the microplate manufacturer.

Example 2

Methods for Off-rate Ranking of Antibody Frag-ments

[0363] Thousands of binders were identified from thephage libraries, requiring an improved method of reducing

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the number of binders through a screening funnel. Thescreening funnel employed a high-throughput off-rate rank-ing method to allow rapid prioritization of peptide binders.[0364] Clones were analyzed for their relative off-rate andrelative mass bound to peptides using BIACORE®. Biotiny-lated peptides were captured on a Streptavidin sensor chip(BIACORE®). Peripreps containing the antibody fragmentswere injected over the chip, resulting in the binding of up to1500 Response Units (RU). Raw data were transferred intoScrubber software, which was used to calculate dissociationrates of individual samples. The amount of sample bound toeach test surface (specific, nonspecific, or no peptide) wascalculated using BiaEval software. Samples which bound tocontrol surfaces (streptavidin alone, or control peptide) at aratio of approximately 13 that of specific peptides were elimi-nated from consideration, as were samples that resulted in<20 RU of binding to specific peptides. The remainingsamples were ranked according to dissociation rates.[0365] By focusing on clones with the slowest offrates, thenumber of binders was narrowed to about 65 for purificationand single-point cell-based functional assays. Cell basedfunctional assays include measuring inhibition of gastrin-induced increases in intracellular calcium flux and detectingthe levels of phosphorylation and activation of ERKI/2(pERKI/2) in CCK2R expressing cells.

Example 3

Flow Cytometric Measurement of Intracellular Cal-cium Flux to Select for Neutralizing Antibodies to

GPCRs

[0366] Binding of gastrin to CCK2R results in phospholi-pase C (PLC)-mediated intracellular calcium flux. The pri-mary functional assay for screening and characterization oftarget neutralizing antibodies employed flow cytometry andthe fluorescent calcium indicator dyes Fluo-3 and Fura Red tomeasure target stimulated intracellular calcium flux in aCCK2R overexpressing cell line (AGS- TR). Because of theiropposite responses to calcium binding, Fluo-3 increases influorescence while Fura Red decreases in fluorescence, andtheir different emission spectra, use of these dyes in combi-nation results in generation of a pseudo-ratiometric signalthat can reduce the influence of variables such as differencesin cell size and intracellular dye concentration on the overallcalcium flux response (Novak et al., Cytometry 17:135-141(1994)). Real-time injection of stimulus during kinetic analy-sis was performed using the Cytek Time Zero Calcium FluxMeasurement System.[0367] Cell loading of AGS-TR cells with Fluo-3 AM andFura Red AM was optimized for concentration, time, andtemperature to ensure the highest possible signal in responseto increases in intracellular calcium concentrations. Dyeloaded cells were kept on ice until needed. Prior to analysis,cells were pre-incubated to 37' C. before addition of gastrinstimulant. Cells were acquired using the Cytek Time Zerocalcium flux measurement system. Approximately 10 sec-onds of events were acquired prior to stimulus injection toestablish a baseline. Upon stimulus injection, approximately40 seconds of events were acquired to monitor the changes inintracellular calcium concentrations by detecting the changesin fluorescence of Fluo-3 and Fura Red.[0368] Lead antibody selection to identify neutralizingantibodies follows the following screening cascade. Antibod-ies were initially screened for neutralization activity at a 100x

molar excess over gastrin which is typically used at the EC50concentration (I nM) then further characterized at a lowerconcentration.[0369] Mouse hybridoma-derived anti-gastrin antibodies(Aphton Corporation, Philadelphia, Pa.) and purifie ELISA-positive phage display-derived anti-gastrin scFv and F(ab)fragments were screened for gastrin neutralizing activity atroughly 100 fold molar excess over gastrin (I nM). Antibod-ies XPA061, XPA081, XPA065, XPA067 all demonstratedapproximately 80'zo Ca flux neutralization in cells stimulatedwith gastrin. Antibodies XPA071, XPA063, XPA064,XPA088 and XPA0116 all showed greater than 70'zo neutral-ization. Antibodies XPA0122, XPA0121, XPA0120,XPA0119, XPA0118, XPA0123, PD-71, and XPA092 wereidentified as good neutralizers in a second screening round.Antibodies that showed over 70'zo neutralization of calciumflux were chosen for further study.[0370] Candidates that neutralize at this threshold werefurther characterized at a lower concentration, e.g., 2x molarexcess, to further differentiate the neutralization potencies ofthe antibodies. Antibody XPA061 exhibited approximately70'zo neutralization when used at 2x molar excess, whileantibody XPA0120 showed approximately 50'zo inhibition,XPA0121 and XPA088 both showed approximately 40'zoinhibition, XPA0118 and XPA064 showed slightly greaterthan 30'zo inhibition, and XPA0116, XPA0123 and XPA0119demonstrated between 20 and 30'zo inhibition. The other anti-bodies gave under 20'zo neutralization.[0371] These top candidates that showed a high degree ofneutralization at lower concentrations were further analyzedin cell based functional assays and were selected for re-forming of antibody fragments to IgG as described below.

Example 4

ELISA Measurement of Neutralization of ERKI/2Phosphorylation Using Antibodies to GPCRs

[0372] Gastrin binding to CCK2R leads to activation ofERKI/2 (pERKI/2) in CCK2R expressing cells. In order toevaluate the effect of gastrin antibodies on downstreameffects of gastrin-CCK2R binding, activation of ERKI/2 wasmeasured in the presence of anti-gastrin antibodies.[0373] Cells were seeded in microtiter plates in completegrowth medium for 24 hours at 37' C. followed by 24 hourincubation in serum- free media to reduce basal levels of intra-cellular signaling. Cells were incubated with gastrin ~poten-tial neutralizing anti-gastrin antibodies for 5 minutes at 37' C.and immediately washed with ice cold PB S be fore addition o fstandard lysis buffer containing detergents, chelators, andvarious protease and phosphatase inhibitors to generate thecell lysates. The levels of phosphorylated ERKI/2 (pERKI/2) were measured using standard ELISA (ke. DUOSET® ICPhospho-ERKI/ERK2, R&D Systems, Inc. or FACEâ„¢ERKI/2, Active Motif). Results of pERKI/2 assay usingselect antibodies are shown in Table 2.

Example 5

Measurement of Anti-Proliferative/Apoptotic Activ-ity of Neutralizing Antibodies to GPCRs

[0374] To examine the effects of anti-gastrin antibodies onthe growth and proliferation of CCK2R expressing cells,growth inhibition or cellular cytoxicity as a consequence ofantibody neutralization of gastrin is measured using well-

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known techniques in the art. Such techniques include stainingwith viability dyes to monitor metabolically active cells (ke.MTT, XTT, and WST-I assays), quantitating DNA synthesismonitoring incorporation of modified nucleotides (e.g.,Brdu) or radiolabeled nucleotides (e.g., H-thymidine), orquantitating total cell numbers using DNA interacting dyes(e.g., CYQUANTâ„¢, Molecular Probes).[0375] Induction of apoptosis as a consequence of antibodyneutralization of gastrin is measured using well-known tech-niques in the art including monitoring increased extracellularexposure of phosphatidylserine (PS) as an early apoptoticmarker using labeled Annexin V, activation of cellularcaspase-3 using labeled substrates (e.g., DEVD-APC, R & DSystems, Inc.), or DNA fragmentation as measured by anincrease in a sub-GO/Gl population in cell cycle analysis (e.g,BrdU Flow Kit, BD Biosciences).

Example 6

Converting Antibody Candidates Identified by PhageDisplay to Whole IgG

[0376] To convert the lead candidate binders from the initialscreen to antibodies comprising antibody heavy and lightchain constant regions, the variable regions of both heavy andlight chains binders were cloned into a proprietary mamma-lian expression vector (WO 2004/033693) encoding for eitherthe kappa (K) >lambda p.) and gamma-2 (72) constant regiongenes.[0377] Antibodies were transiently expressed in 293E cellsas described in Handa et al (2004 American Society of CancerBiology Poster ¹1937). Supernatant of trans fected cells washarvested at day 6 of culture and IgG was quantified. IgGyield from the top five candidates in 4 mls of culture, rangedfrom 32-60 pg/ml (32, 53, 54, 58 and 60 pg/ml), with total IgGin the culture ranging from 128 to 240 pg.[0378] These results show that high affinity scFv or Fabidentified by phage display techniques may be recombinantlyconjugated to immunoglobulin contstant regions in order togenerate high-affinity whole human antibodies.

Example 7

Analysis by Neutralization Assay of Re-FormattedIgG Antibodies

[0379] Whole human gastrin-specific IgG was isolatedfrom cell supernatants and analyzed by calcium flux neutral-ization assay as described previously.[0380] Re-formed whole IgG anti-gastrin antibodies weretested at IC» (03 nM) and IC75 (1.7 nM) values as calculatedfrom an earlier dose response experiment with murine mono-clonal antibody Mu mAb2. Mu mAb2 and XPA067 were alsotested at 67 nM as a control for maximum neutralization inthis experiment.[0381] Analysis of reformatted XPA067, XPA08,XPA0121, and XPA016 antibodies showed that XPA067 andMu mAb2 at 67 nM exhibited approximately 85'zo and 95'zoneutralization, respectively. At 1.7 nM, Mu mAb2 andXPA067 each showed approximately 55'zo calcium flux neu-tralization, XPA088 and XPA011 each exhibited approxi-mately 30'zo inhibiton, and XPAO121 exhibited approxi-mately 10'zo neutralization. At 0.3 nM, all antibodiesdemonstrated 10'zo or less neutralization.[0382] Lead antibody selection was then based on two cri-teria: (I) calcium flux IC50 value and (2) KD measurement. A

more qualitative pERKI/2 dose response was also performedto confirm neutralization of gastrin-induced signaling in adifferent signaling pathway. Dose response studies indicatethat at 0.01 nM neutralization is 100'zo for XPA067, XPA061and murine antibodies Mu mAB2 and Mu mAb4. IC50, K~and pERK IC50 are shown in Table 2.

TABLE 2

Ca FluxIC60 (nM1

KD pERK(nM1 IC60 (nM1Antibody

3.96.29.9

14.2

XPA067Mu mAb2XPA061Mu mAb4

0.66 + 0.130.28 + 0.030.42 + 0.110.49 ~ 0.36

0.76 + 0.380.63 + 0.291.34+ 0.21

2. 63

[0383] Results from the calcium flux dose response experi-ments and affinity measurements indicate that antibodiesderived from human phage display libraries and off-rate cri-teria to select high affinity binding antibodies leads to gen-eration of human antibodies equivalent to murine hybridomaderived antibodies in potency and ability to neutralize cal-cium flux.

Example 8

Affinity Maturation of Anti-Gastrin IgG Antibodies

Example 9

Screening of Affinity Matured Ab Using theDELFIA® Competition Assay

[0386] Individual scFv obtained from the affinity-basedselection of libraries of antibodies from CDR randomization

[0384] Affinity maturation was carried out for XPA067 asfollows to optimize affinity. Libraries of antibodies were pro-duced where random mutagenesis of the following regionswas carried out; V~ and Vz CDR3, V~ CDRI, V~ CDR2 andVz CDRI. The libraries were constructed using standardmolecular biology techniques as described in Clackson andLowman, Phage Display A Practical Approach (OxfordUniversity Press 2004).[0385] Each CDR3 was randomized in two blocks of 6amino acids in order to cover the entire CDR, producinglibraries H3B I (N terminal block of 6 aa VH CDR3), H3B2(C terminal block of 6 aa in VH CDR3), L3B I (N terminalblock of 6 aa in VL CDR3) and L3B2 (C terminal block of 6aa in VL CDR3). V~ CDRI was randomized including thevernier residue at position 30 (Kabat, E.A. et al, Sequences o fProteins of Immunological Interest. 4' Edition, US Depart-ment of Health and Human Services. 1987, and Kabat, E. A.et al. (1991) Sequences of Proteins of Immunological Inter-est, 5th Edition. US Department of Health and Human Ser-vices, Public Service, NIH, Washington) to produce libraryHl. V~ CDR2 was randomized at residues 50, 52, 53, 54, 56and 58, to produce library H2. Vz CDRI was randomized atresidues 27A, 27B, 27C, 29, 31 and 32 to produce library Ll.Affinity-based selections were then performed on five(H3BI, H3B2, L3BI, L3B2, H2) of the seven libraries,whereby the concentration of target antigen was reduced oversuccessive rounds of selection (Clackson and Lowman,Phage Display A Practical Approach, Oxford UniversityPress, 2004). At each stage of the optimization process, scFvthat were able to inhibit the binding of clone XPA067 IgG I tothe target antigen were identified and assessed as described.

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Example 10

Determination of Binding Affinity of Matured Abs

[03SS] Affinities of the parental (XPA067) and maturedantibody fragments were determined by surface plasmonresonance using a BIACORE® 2000. Affinity constants werecalculated from the kinetic rate constants for antibody bind-ing to biotinylated peptide immobilized on a Streptavidinsensor chip.[03S9] A very low density of peptide was immobilized inorder to avoid bivalent binding by the antibodies to the sensorchip. Antibodies were tested in triplicate at six concentrationsof three-fold serial dilutions. Data were analyzed using BiaE-val Software.[0390] The gastrin-binding affinities of four matured anti-body fragments are shown in Table 3.

TABLE 3

Approximate AffinityAffinity Matured Ab

XPA067.18XPA067.06XPA067.09XPA067.11XPA067

40-80 pM20 pM

180 pM(500 pM

5 nM

[0391] These results indicate that the affinity maturationprocess success fully generates antibdoes with greater antigenaffinity that the starting parent antibody.

Example 11

Experimental Models Useful in Measuring AntibodyEfficacy

[0392] To evaluate the efficacy of anti-gastrin antibodies asa therapeutic in conditions or diseases in which gastrin activ-ity is detrimental, experimental animal modles that areaccepted models for human disease are used as a readout.Usefulexperimental models include, but are not limited to,those described below.[0393] Pancreatic Cancer[0394] In one embodiment, an orthotopic model is used inwhich PAN I cells (human pancreatic cancer) are injected intothe tail of the pancreas of 5-7 nude mice (104 cells in 0.1 mL).Rabbit anti-gastrin G17 (antiserum is administered daily, iv(or ip) at 150 mg protein/mouse. Rabbits vaccinated with G17

of Ab clone XPA067, were tested for the ability to inhibit thebinding of gastrin to the parent XPA067 IgG antibody. Themicroplate based competitive screening DELFIA® assay(Perkin Elmer) was performed according to protocols pro-vided by manufacturer. ScFv contained in periplasmicextracts of individual clones were assayed at different dilu-tions to reflect 20 lo, 10 ' and 5'zo periplasmic extract contentin the sample assay. ScFv that at the highest dilution inhibitedmore than 80'zo the binding of gastrin to XPA067 IgG werefurther characterized to isolate the lead affinity matured anti-body.[03S7] The top 11 scFv were converted to scFv-Fc asdescribed in Example 5 and used for analytical and functionalranking ofAb as described in Examples 2, 6, and 9. The aminoacid sequences of the 11 affinity matured antibodies is set outin FIG. 3 and SEQ ID NOs: 23-33. FIG. 4 is a comparison ofthe CDR regions of the originating XPA067 antibody and theaffinity matured antibodies.

will raise antibodies capable of binding human gastrin forms.Thus, rabbit G17 antisera contains gastrin-neutralizing anti-bodies. In this model both monotherapy with the compositionof theinvention and combination therapy antibody substanceplus the a second therapeutic such as gemcitibine(GEMZAR®) are evaluated. In combination therapy experi-ments gemcitibine is given on day I, 3, and 6 at 4 mg/kg.Studies are terminated when the tumor burden affected clini-cal condition of mice (weight loss, ascites and cachexia).[0395] Gastric Cancer[0396] In one embodiment, an intraperitoneal tumor modelusing cell lines: MGLVAI and ST16 isolated from humangastric tumors is used (Watson et al., Gut 45:812-7 (1999)).Cells are injected into the peritoneal cavity of SCID mice(n=10) and gastrin-specific antibody of the invention isadministered daily, iv (serum ABC of 7.5x10-9M). The studyis terminated when tumor burden affected clinical conditionof mice (weight loss, ascites and cachexia).[0397] Colorectal Cancer[039S] In one embodiment, APSLV cells (Sx10 ) areinjected into abdominal wall muscle layer of SCID mice(Watson et al., Int J Cancer 61:233-40 (1995)). Atnibody ofthe invention is administered daily, iv (serum ABC of 3.75x10-9M). Mice are terminated on day 28 and lung metastasis ismeasured.[0399] Evidence that certain G.I. tumors produce gastrinand are mitogenic to gastrin can be found in several areas: invitro cell line studies, studies performed with human tumortissue, and in vivo animal models. It is expected that treatmentof certain G.I. tumors with gastrin-neutralizing Abs of theinvention will have clinical benefit if the G.I. tumors prolif-erate in response to gastrin exposure and/or produce gastrinwhich stimulates them to through autocrine and paracrinepathways to proliferate.[0400] Experimental Autoimmune Gastritis[0401] Experimental models of gastritis can be induced inBALB/c mice by immunization with the gastric H/K-ATPase(Alderuccio et al., Autoimmunity 25:167-175 (1997)); Scarffet al., Immunology 92:91-98 (1997)) or by a variety ofmanipulations that result in transient lyrnphopenia (Gleesonet al., Immunol Rev 149:97-125 (1996)), or by thymectomiz-ing mice (Barrett et al., Eur J Immunol 25:238-244 (1995)).Further, a spontaneous mutation in mice that induces autoim-mune gastritis characterized by autoantibodies to the gastricH/K-ATPase (Alderuccio et al., Am J of Pathol. 153:1311-1318 (1998)).[0402] Duodenal Ulcers/HPyiori Infection[0403] Experimental models of duodenal ulcers induced byethanol gavage in Sprague-Dawley rats are described in Kran-tis et al., (Dig Dis Sci. 38:722-9 (1993)). Infection ofHPyioriand induction of ulcers is described in Ross et al., (Am JPathol. 141:721-7 (1992)), which describes induction andmonitoring of H. pylori induced ulcers in Sprague Dawleyrats, and in Mahler et al., (Helicobacter. 10:332-44 (2005)),which describes H. pylori induced ulcers in the hispid cottonrat. Other animals models useful to evaluate H. pylori infec-tion are available and known in the art. See e.g., a mousemodel is described in Day et al., (Dig Dis Sci. 46:1943-51(2001)), and a Mongolian gerbil model is described inSawada et al., (J. Gastroenterol. 34 Suppl 11:55-60 (1999)).

Example 12

Affinity and Efficacy of Reformatted Antibody InVitro

[0404] Affinity matured antibodies were reformatted tocomprise an IgG tail as described in Example 6 and the

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affinity of these reformatted antibodies to bind gastrin and toneutralize the effects of gastrin in vitro were measured.[0405] Antibody affinity was measured as described previ-ously. The reformatted, affinity matured antibodies showedimproved affinities compared to the parent antibody. SeeTable 4.

TABLE 4

Sample Id Approximate Affinity

17 pM31 pM6 nM

XPA067.18XPA067.06XPA067

Example 13

Efficacy of Reformatted Antibody In Vivo

[0409] Monitoring pH changes can serve as a pharmaco-logical end point to measure the in vivo efficacy of the thera-peutic anti-gastrin antibody. Previous studies have demon-strated that agents such as famotidine, an Hn-receptorantagonist that inhibits stomach acid production, and telen-zepine, a muscarinic Ml-receptor antagonist that inhibitsgastric acid secretion, can increase the gastric pH in CD-1mice. A biologically active anti-gastrin antibody should alsoneutralize the function of endogenous gastrin and increase thegastric pH level.[0410] Human gastrin interacts with mouse receptors andhas effect on gastric acid secretion in vivo. Therefore, a gas-tric pH model was modified to assess in vivo gastrin neutral-ization activity of anti-gastrin antibodies by introducinghuman gastrin (h-G17) into CD-1 mice and administeringanti-human gastrin antibodies. Thus, the neutralization of theexogenous human gastrin by the anti-gastrin antibody couldbe detected by measuring gastric pH.[0411] To evaluate the in vivo efficacy of the anti-gastrinmAbs, the effect of target neutralization was measured usinggastric pH as the readout. On study day — 2 (48 hours beforegastric fluid collection), CD-1 mice (12-14 weeks old) weretreated with anti-gastrin mAbs (20 mg/kg body wt) or anti-

[0406] To determine the efficacy of gastrin neutralizationby these antibodies in vitro, an in vitro calcium flux assay wasperformed as described in Example 3. Briefly, flow cytomet-ric measurement of intracellular calcium flux was carried outin a CCK2R overexpressing cell line (AGS-TR).[0407] Reformatted antibodies XPA067, XPA067.06 andXPA067.18 were assayed for their ability to neutralize gastrinactivation of calcium flux in these cells. FIG. 5 shows theresults of the calcium flux assay. Parent antibody XPA067demonstrated approximately 50'ro gastrin neutralization at aconcentration of approximately I nM. Affinity matured anti-body XPA067.06 reached 50'ro neutralization at about 0.17nM and affinity matured antibody XPA067.18 showed 50'roneutralization at about 0.2 nM.[0408] These results demonstrate that the affinity maturedantibodies exhibit greater affinity for gastrin and thereforeshow increased neutralization at a lower concentration ofantibody compared to the parent antibody. This suggests thata lower dosage of a higher affinity antibody would be requiredto effectuate neutralization of gastrin in vivo. However, theparent antibody was able to achieve 90'ro neutralization atabout 10 nM and, therefore, may be suitable for some thera-peutic applications.

KLH IgGI antibody control (20 mg/kg body wt) by intrap-eritoneal injection. Mice were given NAPA-NECTAR™water gel instead of solid Rodent Chow. On study day — I (24hours before gastric fluid collection), mice were fasted over-night with free access to water. On study day 0 (the day ofgastric fluid collection), human gastrin h-G17 (I mg/kg bodywt; Sigma) or PBS (pH 7.4) was injected subcutaneously.Twenty minutes after h-G17 or PBS injection, a H2R antago-nist (famotidine; 30 mg/kg body wt; Sigma), a muscarinic Mlreceptor antagonist (telenzepine; 30 mg/kg body wt; Sigma)or PBS was administered intravenously into each mouse. Thestomach was removed one hour later. Approximately 50 pL o fgastric fluid was collected, and the pH was directly measuredby using a pH meter (model D-51; Horiba Ltd., Kyoto, Japan)with micro electrode (model 9669-IOD; Horiba Ltd.).

[0412] FIG. 6 shows that administration of famotidinealone neutralizes endogenous mouse gastric acid and raisesthe pH levels in the stomach to approximately pH 5.5, whileadministration of exogenous human gastrin and famotidinereduced the stomach pH levels to approximately pH 2. Thus,at the doses given famotidine cannot neutralize the effect ofexogenous gastrin. Administration of anti-gastrin antibodyXPA067, human gastrin and famatodine demonstrated aslight increase in stomach pH, to pH 2.5. However, adminis-tering the affinity matured anti-gastrin antibody XPA067.06instead of XPA067 can further increase the pH in the stomachto approximately pH 4.

[0413] FIG. 7 demonstrates that administration of telen-zepine alone neutralizes endogenous mouse gastric acid andraises the pH levels in the stomach to approximately pH 3,while administration of exogenous human gastrin and telen-zepine reduced the stomach pH levels to approximately pH13. Administration of anti-gastrin antibody XPA067, humangastrin and telenzepine demonstrated a slight increase instomach pH, to pH 1.5. However, administering the affinitymatured anti-gastrin antibody XPA067.06 instead of XPA067can further increase the pH in the stomach to approximatelypH 2.

[0414] These results demonstrate that normal levels of gas-tric acid secretion are decreased by administration of theselective H2R antagonist, famotidine, or the muscarinic Mlreceptor antagonist, telenzepine. In addition, these effects canbe abolished by pre-introducing exogenous human gastrin toCD-1 mice. The anti-gastrin mabs neutralizes exogenoushuman gastrin in vivo. Elevation of gastric pH was observedafter administration of the neutralizing anti-gastrin antibod-ies. The in vivo data also indicates the affinity-matured anti-body, XPA067.06, is a more potent gastrin-neutralizing agentthan the parental antibody, XPA067.

[0415] The data herein show that a monoclonal antibodywith affinity of about 10 M was able to achieve 90'ro neu-tralization of gastrin-induced calcium flux activity in vitro ata concentration of about 10 nM, and has minimal influence onthe acid-stimulating effects of gastrin in vivo in two animalmodels. Monoclonal antibodies with affinity of about 10 'M achieved 90'ro neutralization of gastrin in vitro at a con-centration of 0.17 or 0.2 nM, and produced marked neutral-ization of gastrin's acid-stimulating effects in vivo in the sameanimal models. The relatively modest 5-fold improvement inneutralization upon a 3 log increase in affinity indicates thatantibodies of much higher affinity (ke. with an affinity valueof less than 10 ' or 10 " M) may not provide significantlybetter neutralization.

US 2009/0311257 A1 Dec. 17, 2009

37

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 33

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Lys Val Ser Cys Lys Ile Ser Gly Gly Thr Phe Ser Ser His20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile35 40 45

Gly Arg Ile Ile Pro Ser Leu Asp Leu Asp Asn Ser Ala Pro Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Asn Thr Ala Tyr65 70 75 80

Met Glu Leu Arg Asn Leu Arg Pro Glu Asp Thr Ala Phe Tyr Tyr Cys85 90 95

Thr Gly Asp Pro Leu Tyr Asn Trp Thr Gly Ala His Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 2

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Asp Ile Asn Val Gly Thr20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Ser Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Arg Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys85 90 95

Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Val100 105 110

[0416] The relatively modest 5-fold improvement in neu-tralization upon a 3 log increase in affinity indicates thatantibodies of much higher affinity (ke. with an affinity valueof less than 10 ' or 10 " M) provide better neutralizationand enhance pharmacological efiicacy.

<210> SEQ ID NO 1<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

<210> SEQ ID NO 2<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

[0417] Numerous modifications and variations in theinvention as set forth in the above illustrative examples areexpected to occur to those skilled in the art. Consequentlyonly such limitations as appear in the appended claims shouldbe placed on the invention.

US 2009/0311257 A1 Dec. 17, 2009

38

-continued

Thr Val Leu115

<210> SEQ ID NO 3<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 3

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Ser Leu Asp Leu Asp Asn Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Lys Leu Ser Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 4<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 4

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Thr20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys85 90 95

Thr Val Leu115

<210> SEQ ID NO 5<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence

Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu100 105 110

US 2009/0311257 A1 Dec. 17, 2009

39

-continued

<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 5

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Thr Leu Asp Leu Ala Asn Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 6<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 6

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Thr1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Ser20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Arg Pro Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Val65 70 75 80

Leu Phe Ile Ser Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys85 90 95

Thr Val Leu115

<210> SEQ ID NO 7<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 7

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Met Ile Trp His Asn Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu100 105 110

US 2009/0311257 A1 Dec. 17, 2009

40

-continued

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Thr Leu Asp Leu Ala Asn Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 8<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 8

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Thr20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys85 90 95

Thr Val Leu115

<210> SEQ ID NO 9<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Ser Leu Asp Leu Asp Asn Ser Ala Gln Lys Phe50 55 60

Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu100 105 110

US 2009/0311257 A1 Dec. 17, 2009

41

-continued

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Lys Leu Ser Asn Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Gln Trp Ser Gly Ser Tyr Trp Gly Lys Gly100 105 110

Thr Leu Val Thr Val Ser Ser115

<210> SEQ ID NO 10<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 10

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Asp Ile Asn Val Gly Thr20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Gly Asp Gly Ala Asp Tyr Tyr Cys85 90 95

Thr Val Leu115

<210> SEQ ID NO 11<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 11

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Ser Leu Asp Leu Asp Asn Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Ser Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly

Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu100 105 110

US 2009/0311257 A1 Dec. 17, 2009

42

-continued

100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 12<211> LENGTH: 115<212> TYPE: PRT<213> ORGANISM: Arti<220> FEATURE:<223> OTHER INFORMAT

ficial sequence

ION: Synthetic peptide

<400> SEQUENCE: 12

Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Thr20 25 30

Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys85 90 95

Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu100 105 110

Thr Val Leu115

<210> SEQ ID NO 13<211> LENGTH: 357<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 13

caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

120tcctgcaaaa tttctggagg caccttcagc agtcatgcta tcggctgggt gcgacaggcc

cctggacaag ggcttgagtg gataggtagg atcatcccga gccttgatct cgataactcc 180

240gcaccgaagt tccaggacag agtcacgatt accgcggaca aattcacgaa cacagcctac

300atggagctga ggaacctgag acctgaggac acggccttct attactgtac gggagacccc

357ctctataatt ggactggggc ccactggggc cgggggacaa tggtcaccgt ctcgagt

<210> SEQ ID NO 14<211> LENGTH: 346<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 14

60ccaggctgtg ctgactcagc cgtcttccct ctctgcatct cctggagcat cagccagtct

120cacctgcacc ttgcgcagtg acatcaatgt tggtacctac aggatatact ggtaccagca

180gaagtcaggg agtcctcccc agtatctcct gagatacaga tcagactcag ataagcagca

US 2009/0311257 A1 Dec. 17, 2009

43

-continued

346cagcagcgct tgggtgttcg gcggagggac caaggtcacc gtccta

<210> SEQ ID NO 15<211> LENGTH: 357<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 15

gaagtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgacggtc 60

120tcctgcaagg cctctggagg cacgttcgac aagtatgcta tcggctgggt gcgacaggcc

cctggacaag ggcttgagtg gatgggaagg atcatcccta gccttgattt agacaactcc 180

240gcacagaagt tccaggacag agtcacgatt accgcggaca aatttacgac cacagtctac

300atgaagctga gcaacctgag acctgaggac acggccatgt actactgtac gcgagacccc

357ctctataact ggagtgggtc ctactggggc agagggacaa tggtcaccgt ctcgagt

<210> SEQ ID NO 16<211> LENGTH: 345<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 16

60caggctgtgc tgactcagcc gtcttccctc tctgcatctc ctggagcatc agccagtctc

acctgcacct tgcgcagtgg catcaatgtt ggtacctaca ggatatactg gtaccagcag 120

180aagccaggga gtcctcccca gtatctcctg aggtacaaat cagactcaga taagcagcag

240ggctctggag tccccagccg cttctctgga tccaaagatg cttcggccaa tgcagggatt

300ttactcatct ctgggctcca gtctgaggat gaggctgact attactgtat gatttggcac

agcagcgcgt gggtgttcgg cggagggacc aagctgaccg tccta 345

<210> SEQ ID NO 17<211> LENGTH: 357<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 17

gaggtccagc tggtacagtc tggagctgag gtgaagaagc ctgggtcctc ggtgacggtc 60

120tcctgcaagg cctctggagg caccttcgac aagtatgcta tcggctgggt gcggcaggcc

180cctggacaag ggcttgagtg gatgggaagg atcatcccta cccttgattt agcaaactcc

240gcacagaagt tccaggacag agtcacgatt accgcggaca aattcacgac cacagtctac

300atggagctga ccaacctgag acctgaggac acggccatgt actactgtac gagagacccc

ctctataact ggagtgggtc ctactggggc cgagggacaa tggtcaccgt ctcgagt 357

<210> SEQ ID NO 18<211> LENGTH: 345

gggctctgga gtccccagcc gcttctctgg atccaaagat gcttcggcca atgcagggat 240

tttactcatc tctgggctcc agtctgagga tgaggctgac tattactgta tgatttggca 300

US 2009/0311257 A1 Dec. 17, 2009

44

-continued

<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 18

60caggctgtgc tgactcagcc gtcttccctc tctgcatctc ctggaacgtc agccagtctc

120acctgcacct tgcgcagtgg catcaatgtc ggtagttaca ggatatactg gtaccagcag

180

240

300ttattcatct ctgggctcca gtctgacgat gaggctgact attactgtat gatttggcac

aacagcgctt gggtgttcgg cggagggacc aagctgaccg tccta 345

<210> SEQ ID NO 19<211> LENGTH: 357<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 19

gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgacggtc 60

120tcctgcaagg cctctggagg caccttcgac aagtatgcta tcggctgggt gcggcaggcc

cctggacaag ggcttgagtg gatgggaagg atcatcccta cccttgattt agcaaactcc 180

240gcacagaagt tccaggacag agtcacgatt accgcggaca aattcacgac cacagtctac

300atggagctga ccaacctgag acctgaggac acggccatgt actactgtac gagagacccc

357ctctataact ggagtgggtc ctactggggc cgagggacaa tggtcaccgt ctcgagt

<210> SEQ ID NO 20<211> LENGTH: 345<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 20

60caggctgtgc tgactcagcc gtcttccctc tctgcatctc ctggagcatc agccagtctc

acctgcacct tacgcagtgg catcaatgtt ggtacctaca ggatatactg gtaccagcag 120

180aagccaggga gtcctcccca gtatctcctg aggtacaaat cagactcaga taagcagcag

240ggctctggag tccccagccg cttctctgga tccaaagatg cttcggccaa tgcagggatt

300ttactcatct ctgggctcca gtctgaggat gaggctgact attactgtat gatttggcac

agcagcgctt gggtgttcgg cggagggacc aagctgaccg tccta 345

<210> SEQ ID NO 21<211> LENGTH: 357<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 21

60gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgacggtc

tcctgcaagg cctctggagg tacgttcgac aagtatgcta tcggctgggt gcgacaggcc 120

aggccaggga gtcctcccca gtatctcctg aggtataaat cagattcaga taagcagcag

ggttctggag tccccagccg cttctctgga tccaaagatg cttcggccaa tgcaggggtt

US 2009/0311257 A1 Dec. 17, 2009

45

-continued

180cctggacaag ggcttgagtg gatgggaagg atcatccctt cccttgattt agacaactcc

240

300atgaagctga gcaacctgag acctgaggac acggccatat actactgtac gcgagacccc

357ctctatcagt ggagtgggtc ctactggggc aaaggaaccc tggtcaccgt ctcgagt

<210> SEQ ID NO 22<211> LENGTH: 345<212> TYPE: DNA<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 22

60caggctgtgc tgactcagcc gtcttccctc tctgcatctc ctggagcatc agccagtctc

acctgcacct tgcgcagtga catcaatgtt ggtacctaca ggatatactg gtaccagcag 120

180aagccaggga gtcctcccca gtatctcctg aggtacaaat cagactcaga taagcagcag

240ggctctggag tccccagccg cttctctgga tccaaagatg cttcggccaa tgcaggaatt

300ttactcatct ctgggctcca gtctggggat ggggctgact actactgtat gatttggcac

agcagcgctt gggtgttcgg cggagggacc aagctgaccg tccta 345

<210> SEQ ID NO 23<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 23

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Thr Ile Ile Pro Ser Leu Asp Arg Ala Ile Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 24<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 24

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

gcacagaagt tccaggacag agtcacgatt accgcggaca aatttacgac cacagtctac

US 2009/0311257 A1 Dec. 17, 2009

46

-continued

10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Gly Thr Ile Ile Pro Ser Leu Asp Arg Ala Val Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 25<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 25

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Thr Ile Ile Pro Asp Glu Asp Arg Ala Ile Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 26<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 26

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

US 2009/0311257 A1 Dec. 17, 2009

47

-continued

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 27<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 27

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Thr Ile Ile Pro Glu Leu Asp Arg Ala Ile Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 28<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 28

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Ala Ile Asn Pro Ser Glu Asp Gln Ala Ile Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Gly Ser Ile Asn Pro Ser Ala Asp Arg Ala Ile Ser Ala Gln Lys Phe50 55 60

US 2009/0311257 A1 Dec. 17, 2009

48

-continued

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 29<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 29

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Thr Ile Met Pro Ser Leu Asp Arg Ala Ile Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 30<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 30

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Ala Ile Asn Pro Ala Val Asp Leu Ala Arg Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

US 2009/0311257 A1 Dec. 17, 2009

49

-continued

<210> SEQ ID NO 31<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 31

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Met Ile Ile Pro Ser Leu Asp Arg Ala Lys Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Pro Leu Tyr Asn Trp Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 32<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Lys Tyr20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Thr Leu Asp Leu Ala Asn Ser Ala Gln Lys Phe50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr Asn Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys85 90 95

Thr Arg Asp Gly Pro Val Gly Met Ser Gly Ser Tyr Trp Gly Arg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

<210> SEQ ID NO 33<211> LENGTH: 119<212> TYPE: PRT<213> ORGANISM: Artificial sequence<220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide

<400> SEQUENCE: 33

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15

US 2009/0311257 A1 Dec. 17, 2009

50

-continued

Glu Val Gln Leu Val Gln Ser Gly P.la Glu Val Lys Lys Pro Gly Ser1 5 10 15

Ser Val Thr Val Ser Cys Lys P.la Ser Gly Gly Thr Phe P.sp Lys Tyr20 25 30

P.la Ile Gly Trp Val P.rg Gln P.la Pro Gly Gln Gly Leu Glu Trp Met35 40 45

Gly P.rg Ile Ile Pro Thr Leu P.sp Leu P.la P.sn Ser P.la Gln Lys Phe50 55 60

Gln P.sp Prg Val Thr Ile Thr P.la P.sp Lys Phe Thr Thr Thr Val Tyr65 70 75 80

Met Glu Leu Thr P.sn Leu P.rg Pro Glu P.sp Thr P.la Met Tyr Tyr Cys85 90 95

Thr P.rg P.sp Gly Pro Thr Ile Glu Ser Gly Ser Tyr Trp Gly P.rg Gly100 105 110

Thr Met Val Thr Val Ser Ser115

1-3. (canceled)4. An isolated antibody that binds gastrin with an acuity kd

of 10 M or less that comprises:(a) a heavy chain CDRI amino acid sequence set forth in

FIG. 1, FIG. 3 or SEQ ID NOs: I, 3, 5, 7, 9, 11 or 23-33,a variant thereof in which one or two amino acids havebeen changed, or a consensus sequence thereof;

(b) a heavy chain CDR2 amino acid sequence set forth inFIG. 1, FIG. 3 or SEQ ID NOs: I, 3, 5, 7, 9, 11 or 23-33,or a variant thereof in which one or two amino acids havebeen changed or a consensus sequence thereof; and

(c) a heavy chain CDR3 amino acid sequence set forth inFIG. 1, FIG. 3 or SEQ ID NOs: I, 3, 5, 7, 9, 11 or 23-33a variant thereof in which one or two amino acids havebeen changed, or a consensus sequence thereof.

5.-7. (canceled)S. The antibody of claim 4 wherein one or more of said

heavy chain CDRI, CDR2 or CDR3 amino acid sequences isa consensus sequence displayed in FIG. 1.

9. The antibody of claim 4 wherein(a) an amino acid in a heavy chain CDRI amino acid

sequence is replaced with an amino acid from a corre-sponding position within a different heavy chain CDRIamino acid sequence set forth in FIG. 1 or FIG. 3; or

(b) an amino acid in a heavy chain CDR2 amino acidsequence is replaced with an amino acid from a corre-sponding position within a different heavy chain CDR2amino acid sequence set forth in FIG. 1 or FIG. 3; or

(c) an amino acid in a heavy chain CDR3 amino acidsequence is replaced with an amino acid from a corre-sponding position within a different heavy chain CDR3amino acid sequence set forth in FIG. 1 or FIG. 3.

10. (canceled)11. The antibody of claim 4 comprising a CDR2 amino

acid consensus sequence XIXPXXDXAXSAQKFQD,wherein X is any amino acid.

12. (canceled)13. The antibody of claim 4 comprising a CDR3 amino

acid consensus sequence DXXXXXSGSY, wherein X is anyamino acid.

14. The antibody of claim 4 that comprises an amino acidsequence at least 65'ro identical to a heavy chain variableregion amino acid sequence set forth in FIG. 1 or FIG. 3.

15. The antibody of claim 14 that comprises an amino acidsequence at least 85'ro identical to a heavy chain variableregion amino acid sequence set forth in FIG. 1 or FIG. 3.

16. The antibody of claim 14 that comprises an amino acidsequence at least 95'ro identical to a heavy chain variableregion amino acid sequence set forth in FIG. 1 or FIG. 3.

17. The antibody of claim 14 that comprises a heavy chainvariable region amino acid sequence set forth in FIG. 1 orFIG. 3.

1S. The antibody of claim 4 in which one or more heavychain framework amino acids have been replaced with corre-sponding amino acid(s) from another human antibody aminoacid sequence.

19. (canceled)

20. The antibody of claim 4 further comprising a humanheavy chain constant region attached to said heavy chainvariable region.

21.-22. (canceled)

23. The antibody of claim 4 that further comprises

(a) a light chain CDRI amino acid sequence set forth inFIG. 2, SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variantthereof in which one or two amino acids have beenchanged;

(b) a light chain CDR2 amino acid sequence set forth inFIG. 2, SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variantthereof in which one or two amino acids have beenchanged; and

(c) a light chain CDR3 amino acid sequence set forth inFIG. 2, SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variantthereof in which one or two amino acids have beenchanged.

24.-25. (canceled)

26. An isolated antibody that binds gastrin with an acuitykd of 10 M or less that comprises:

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Dec. 17, 2009

(a) a light chain CDRI amino acid sequence set forth inFIG. 2, SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variantthereof in which one or two amino acids have beenchanged;

(b) a light chain CDR2 amino acid sequence set forth inFIG. 2 that is from the same, SEQ ID NOs: 2, 4, 6, 8, 10or 12, or a variant thereof in which one or two aminoacids have been changed; and

(c) a light chain CDR3 amino acid sequence set forth inFIG. 2, SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variantthereof in which one or two amino acids have beenchanged.

27.-29. (canceled)30. The antibody of claim 26 wherein one or more of said

light chain CDRI, CDR2 or CDR3 amino acid sequences is aconsensus sequence displayed in FIG. 2.

31. The antibody of claim 26 wherein(a) an amino acid in a light chain CDRI amino acid

sequence is replaced with an amino acid from a corre-sponding position within a different light chain CDRIamino acid sequence set forth in FIG. 2; or

(b) an amino acid in a light chain CDR2 amino acidsequence is replaced with an amino acid from a corre-sponding position within a different light chain CDR2amino acid sequence set forth in FIG. 2; or

(c) an amino acid in a light chain CDR3 amino acidsequence is replaced with an amino acid from a corre-sponding position within a different light chain CDR3amino acid sequence set forth in FIG. 2.

32.-33. (canceled)34. The antibody of claim 26 that comprises an amino acid

sequence at least 65% identical to a light chain variable regionamino acid sequence set forth in FIG. 2.

35. The antibody of claim 34 that comprises an amino acidsequence at least 85% identical to a light chain variable regionamino acid sequence set forth in FIG. 2.

36. The antibody of claim 34 that comprises an amino acidsequence at least 95% identical to a light chain variable regionamino acid sequence set forth in FIG. 2.

37. The antibody of claim 34 that comprises a light chainvariable region amino acid sequence set forth in FIG. 2.

3S. The antibody of claim 26 in which one or more lightchain framework amino acids have been replaced with corre-sponding amino acid(s) from another human antibody aminoacid sequence.

39. (canceled)40. The antibody of claim 26 further comprising a human

light chain constant region attached to said light chain vari-able region.

41. (canceled)

42. An antibody of claim 4 or 26 that exhibits at least 70%neutralization of calcium flux in an assay using AGS-TRcells, wherein the tested antibody is at a concentration in 100molar excess over gastrin, and wherein gastrin is at the EC50concentration (I nM).

43. The antibody of claim 4 that comprises at least 5 of theCDRs of any of XPA061, XPA063, XPA065, XPA067,XPA081, XPA067.06, XPA067.18.

44. The antibody of claim 4 or 26 which is a single chainantibody.

45.-46. (canceled)47. The antibody claim 4 or 26 that is conjugated to another

diagnostic or therapeutic agent.4S. An isolated nucleic acid molecule comprising a nucle-

otide sequence that encodes the heavy chain or light chain ofclaim 4 or 26.

49. An expression vector comprising the nucleic acid mol-ecule of claim 4S operably linked to a regulatory controlsequence.

50. A host cell comprising the vector of claim 49.51. A method of using the host cell of claim 50 to produce

an antibody, comprising culturing the host cell of claim 50under suitable conditions and recovering said antibody.

52. An antibody produced by the method of claim 51.53. (canceled)54. A pharmaceutical composition comprising the anti-

body of claim 52 and a pharmaceutically acceptable carrier.55. A kit comprising an antibody of claim 52 and instruc-

tions for use.56. A method for treating a condition or disorder associated

with gastrin expression comprising the step of administeringto a subject in need a therapeutically effective amount of thepharmaceutical composition of claim 54.

57. (canceled)

5S. The method of claim 56 wherein the antibody is admin-istered in conjunction with a second therapeutic agent.

59. (canceled)

60. An isolated monoclonal antibody that binds gastrinwith an afftnity kd of 10 M or less.

61. The antibody of claim 4 wherein the heavy chain CDR2and CDR3 amino acid sequences are from the same heavychain variable region as the CDRI amino acid sequence.

62. The antibody of claim 26 wherein the light chain CDR2and CDR3 amino acid sequences are from the same lightchain variable region as the CDRI amino acid sequence.