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XOMA Technology Ltd., Patent - December 17,2009

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XOMA Technology Ltd., Patent - December 17,2009

  1. 1. llIIlIlllIlIlIllIlIllIllIllIIlllIIllIlIlllIIlIIlllIlllIIlllIlIllIIlIlll US 20090311257A1 119) United States 112) Patent Application Publication 110) No.:US 2009/0311257 Pub. A1 Masat et al. (43)Pub.Date: Dec. 17, 2009 154) HUMAN ANTIBODIES SPECIFIC FOR Related U.S. Application Data GASTRIN MATERIALS AND METHODS 160) Provisional application No. 60/784,501, filed on Mar. 175) Inventors: Linda Masat, Walnut Creek, CA 20, 2006. 1US); Marina Roell, Concord, CA 1US) Publication Classification 151) Int. Cl. Correspondence Address: 261K 39/395 12006.01) MARSHALL, GERSTEIN 4 BORUN LLP C07K 16/00 12006.01) 233 SOUTH WACKER DRIVE, 6300 SEARS C07H 21/04 12006.01) TOWER C12N 15/63 12006.01) CHICAGO, IL 60606-6357 (US) C12N 5/00 12006.01) C12P 21/04 12006.01) 173) Assignee: XOMA Technology Ltd., Hamilton 1BM) 152) U.S. Cl..... ... 424/139.1; 530/3873; 536/23.53; 435/320.1; 435/325; 435/69.6 121) Appl. No.: 12/293,890 157) ABSTRACT 122) PCT Filed: Dec. 13, 2006 The present invention relates to materials and methods for 186) PCT No.: PC T/US2006/047S40 human antibodies specific for the peptide hormone gastrin and uses of these antibodies in the treatment of subjects ) 371 1c)11), having cancer and other conditions or disorders related to 12), 14) Date: Jun. 3, 2009 gastrin expression.
  2. 2. Patent Application Publication Dec. 17, 2009 Sheet 1 of 7 US 2009/0311257 A1 A K Qo p HR C «4 Kl A tA A Cfl +I R lg C9 O C4 AA A a A n a C LA ~l Ch M M H «4 MH H M «4 «3 5 C5
  3. 3. Patent Application Publication Dec. 17, 2009 Sheet 2 of 7 US 2009/0311257 A1 Hi UO U Cll CCI K K O V co g g Q LA Wn A Cl CQO 'Ol A U' C CQ M Ul H H Mn U U U 4o Q Q CQLn C4 CCI O C4,m CO .U U U r- ltl P4 Q II A a A A 0 J CX M FCI C4 g 00 ~, 0 Co o O+ rI Q Ca Z Ca ICL U3 A ©'R O' R Rg O' R ©'R ~U F4 Co CQ gg 03 Cll CJO ce 4 CII 4 Co cM Cfi M MN CO Ql I,Q g a.~l rK & K CI P4 K 5 ga Fc O~r~
  4. 4. Patent Application Publication Dec. 17, 2009 Sheet 3 of 7 US 2009/0311257 A1 M 0I AM A a Ol A 0I A 8 A 0 A 0 .hl g M C4 C5 M M 0 M I M C9 Cll C4 M I-I I. I 8 g g F M CII C 8 CC IVI mg ~~ -R 0 &r 0 Ro g R. oR 0 C4 oR oR. 8A A Cll 8 ACQ A CO' H H HQ H Ol OI5 Ol U ol 'a gL Ol Cll Cll 2 I5 OI I 8 I O0 O Ql C4 CM. IIC 'A 0I C4 C40I C4a n n nR nK n0 n nk nQ
  5. 5. Patent Application Publication Dec. 17, 2009 Sheet 4 of 7 US 2009/0311257 A1
  6. 6. Patent Application Publication Dec. 17, 2009 Sheet 5 of 7 US 2009/0311257 A1 Figure 5 s XPA067 = 1.00 =: 0.47 nM XPA067.06 = 0.17 =' 0.04 nM C XPA067,18 = 0.20 =: 0.08 nNl 0,1.
  7. 7. Patent Application Publication Dec. 17, 2009 Sheet 6 of 7 US 2009/0311257 A1 Figure 6 Famotidine model 6 4) 6 K 3 F(KLH) G+F(XPA067.006) G+F(XPA067) G+F(KLH) Treatment
  8. 8. Patent Application Publication Dec. 17, 2009 Sheet 7 of 7 US 2009/0311257 A1 Figure 7 Telenzepine mode'I tD To CL 1 T(KLH) 6+T(XPA067.006) G+T(XPA067) Treatment
  9. 9. US 2009/0311257 A1 Dec. 17, 2009 HUMAN ANTIBODIES SPECIFIC FOR gastrin receptors has been demonstrated in primary tumors GASTRIN MATERIALS AND METHODS taken from cancer patients. Since some tumor types appear to produce and secrete their own gastrin, gastrin can act to stimulate tumor growth via autocrine and paracrine pathways [0001] This application claims the priority benefit of U.S. as well as via an endocrine pathway. Several studies pub- Provisional PatentApplication No. 60/784,501, filed Mar. 20, lished in the literature have demonstrated that tumors taken 2006, hereby incorporated by reference. from cancer patients both produce gastrin and express high levels of gastrin receptors (Schmitz et al., Eur J Clin Invest FIELD OF THE INVENTION 31:812-20. 2001; Finley et al., Cancer Res 53:2919-26. 1993; Weinberg et al., J Clin Invest 100:597-603, 1997; Caplin et [0002] This invention relates to materials and methods for al., Br J Surg 87:1035-40, 2000). human antibodies specific for the peptide hormone gastrin [0007] The therapeutic approach of disrupting the gastrin- and uses of these antibodies in the treatment of subjects mediated mitogenesis of cancer cells has been tried in the having cancer and other conditions or disorders related to clinic using small molecule antagonists to the gastrin recep- gastrin expression. tor. Several small molecule antagonists for gastrin receptors have been tested in clinical trials for oncology indications. BACKGROUND OF THE INVENTION [0008] Antibodies represent a powerful approach to neu- tralize therapeutic targets due to their high degree of speci- [0003] Gastrin is a peptide hormone that signals through ficity and affinity. Monoclonal antibodies specific for murine the G-protein coupled receptor (GPCR)CCK2R, and has a variety of effects including stimulation of gastric epithelial gastrin peptides have been disclosed in U.S. Pat. Nos. 6,861, 510 and 5,688,506. However, these antibodies do not possess cell proliferation and acid secretion by parietal cells (Yassin R R, Peptides 20:885-98, 1999). It has also been characterized the desired specificity for human gastrin asneeded for clinical as a factor in the progression of gastric cancers and presents a therapy. potential target for therapies that neutralize its function [0009] Thus there remains a need in the art to develop specific antibodies against human gastrin to use in the treat- (Smith et al. Gut 47:820-24, 2000). ment of cancers and other conditions or disorders associated [0004] Gastrin is a hormone produced in the digestive tract with gastrin expression. of many species, including humans. Normal adults produce gastrin in only one cell type the G cells which line the SUMMARY OF THE INVENTION gastric mucosa in the antral portion of the stomach (Ganong, Review of medical physiology. Norwalk, Conn., Appleton & [0010] The materials and methods of the present invention Lange, 1995). Food intake stimulates the G cells to produce fulfill the aforementioned and other related needs in the art. gastrin. Specifically, distension of the lumen of the stomach [0011] In one embodiment the invention provides antigen- or the presence of peptides and amino acids in the stomach binding compounds, including functional fragments, having stimulate gastrin secretion. There is also a neural pathway for the amino acid sequencesset forth in SEQ ID NOs: 1-12 and gastrin release as the sight or smell of food may stimulate 23-33. In a related embodiment, an aforementioned antigen (through the Vagus nerve) release of gastrin. binding compound is selected from the group consisting of a [0005] Once secreted, gastrin has a range of activities on fully assembled tetrameric antibody, a polyclonal antibody, a the digestive tract. The primary roles of gastrin in a normal monoclonal antibody including a HUMAN ENGI- adult are to stimulate acid production by the parietal cells of NEEREDâ„¢ antibody; a humanized antibody; a human anti- the stomach and to act as a trophic factor for cells lining the body; a chimeric antibody; a multispecific antibody, an anti- gastrointestinal tract. Gastrin also serves other secondary body fragment, Fab, F(ab')~; Fv; scFv or single-chain roles in the digestive tract such as stimulating pepsin and antibody fragment; a diabody; triabody, tetrabody, minibody, pancreatic enzyme release, and gall bladder contraction and linear antibody; chelating recombinant antibody, a tribody or small intestine motility. Gastrin is produced in a precursor bibody, an intrabody, a nanobody,a small modular immunop- form of 101 amino acids called pre-pro-gastrin. This protein harmaceutical (SMIP), a binding-domain immunoglobulin goes through a series of cleavage steps to generate several fusion protein, a camelized antibody, a V~~ containing anti- different proteins of varying length (Mulholland et al., Sur- body, or a variant or derivative of any one of these antibodies, gery 103:135-47, 1988). Additional post-translational steps that comprise one or more CDR sequencesof the invention include glycine addition and amidation. Gastrin may be and exhibit the desired biological activity. The antigen bind- expressed as pre-pro-gastrin, pregastrin, gastrin 34 (G34, ing compounds of the invention preferably retain binding having 34 amino acids), gastrin 17 (G17, having 18 amino affinity of at least 10, 10, 10 M or higher asmeasured by acids) and gastrin 14 (G14, having 14 amino acids). Gastrin surface plasmon resonance. (G34) stimulates stomach acid secretion and has a trophic [0012] In one aspect, the antibodies of the invention com- effect on gastrointestinal tract (G.I) mucosa. Glycine-Ex- prise the antibodies. XPA061, XPA063, XPA065, XPA067 tended Gastrin (Gly-G17) and amidated Gastrin (G17-NH2) and XPA081 set out in amino acid sequences SEQ ID NO: also stimulate stomach acid secretion and exhibit a trophic 1-10. It is further contemplated that the antibodies may com- effect on G.I. mucosa. prise all or part of the antibodies set out in the above amino [0006] Gastrin functions in healthy adults are limited to acid sequences.In one embodiment, the antibodies comprise preparing the gastrointestinal tract for the process of digest- at least one of CDRI, CDR2, or CDR3 of the heavy chain of ing ingested food. However, much recent research has impli- SEQ ID NOs: I, 3, 5, 7, 9 and 11, or at least one of CDRI, cated gastrin as a growth factor for some types of cancer CDR2 or CDR3 of the light chain of SEQ ID NOs: 2, 4, 6, 8, (Baldwin et al., Gut 42:581-4, 1998; Smith et al., Aliment 10 and 12. Pharmacal Ther 14:1231-47, 2000) including pancreatic, [0013] In another embodiment of the invention, variants of gastric, and colorectal carcinoma. Expression of gastrin and the aforementioned antibody are provided, comprising a vari-
  10. 10. US 2009/0311257 A1 Dec. 17, 2009 able heavy chain amino acid sequencewhich is at least 60, 65, [0020] Antibodies comprising any one of the heavy chains 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% described above may further comprise a light chain, prefer- homologous to the amino acid sequence set forth in SEQ ID ably a light chain that binds to target antigen, and most pref- NOs: I, 3, 5, 7, 9 and 11. In a related embodiment, the erably a light chain comprising light chain CDR sequencesof antibody comprises a variable light chain amino acid the invention described below. sequencewhich is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, [0021] Another aspect of the invention provides an anti- 94, 95, 96, 97, 98, or 99% homologous to the amino acid body that binds target antigen comprising a light chain that sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10 and 12. comprises any one, two, and/or three of the light chain CDR [0014] One aspect of the invention provides an antibody sequencesof the invention described below. that binds target antigen comprising a heavy chain that com- [0022] Preferably the light chain comprises an amino acid prises any one, two, and/or three of the heavy chain CDR sequence identified as a light chain CDR3 sequence. Such a sequencesof the invention described below. "light chain CDR3 sequence" includes an amino acid [0015] Preferably the heavy chain comprises an amino acid sequenceidentified as a light chain CDR3 sequencein FIG. 2 sequenceidentified as a heavy chain CDR3 sequence.Such a and within SEQ ID NOs: 2, 4, 6, 8 10 and 12. Alternatively, "heavy chain CDR3 sequence" includes an amino acid the light chain CDR3 sequence comprises an amino acid sequenceidentified as a heavy chain CDR3 sequence set out sequencethat contains one or more amino acid changes com- in FIG. 1 or FIG. 3 and SEQ ID NOs: I, 3, 5, 7, 9, 11 and pared to any light chain CDR3 amino acid sequenceidentified 23-33. Alternatively, the heavy chain CDR3 sequence com- in FIG. 2, i.e. a substitution, insertion or deletion. Preferable prises an amino acid sequence that contains one or more substitutions include a substitution to an amino acid at the amino acid changes compared to any heavy chain CDR3 corresponding position within another light chain CDR3 of amino acid sequence identified in FIG. 1 or FIG. 3, i.e. a FIG. 2. Alternatively, the light chain CDR3 sequence may substitution, insertion or deletion. Preferable substitutions comprise a consensus amino acid sequence of light chain include a substitution to an amino acid at the corresponding CDR3 shown in FIG. 2. position within another heavy chain CDR3 of FIG. 1 or FIG. [0023] The light chain comprising a light chain CDR3 3. Alternatively, the heavy chain CDR3 sequencemay com- sequenceof the invention described above may further com- prise a consensusamino acid sequenceof heavy chain CDR3 prise a "light chain CDRI sequence" of the invention, which shown in FIG. 1 or FIG. 3. includes any of the amino acid sequencesidentified as a light [0016] The heavy chain comprising a heavy chain CDR3 chain CDRI in FIG. 2, amino acid sequencesthat contain one sequenceof the invention described above may further com- or more amino acid changes compared to any light chain prise a "heavy chain CDRI sequence" of the invention, which CDRI identified in FIG. 2, preferably a substitution to an includes any of the amino acid sequencesidentified as a heavy amino acid at the corresponding position within another light chain CDRI in FIG. 1 or FIG. 3, amino acid sequencesthat chain CDRI of FIG. 2, or a consensussequenceof light chain contain one or more amino acid changes compared to any CDRI shown in FIG. 2. heavy chain CDRI identified in FIG. 1 or FIG. 3, preferably [0024] Alternatively, the light chain comprising a light a substitution to an amino acid at the corresponding position chain CDR3 sequenceof the invention described above may within another heavy chain CDRI of FIG. 1, or a consensus further comprise a "light chain CDR2 sequence" of the inven- sequence of heavy chain CDRI shown in FIG. 1 or FIG. 3. tion, which includes any of the amino acid sequencesidenti- [0017] Alternatively, the heavy chain comprising a heavy fied as a light chain CDR2 in FIG. 2, amino acid sequences chain CDR3 sequenceof the invention described above may that contain one or more amino acid changescompared to any further comprise a "heavy chain CDR2 sequence" of the light chain CDR2 identified in FIG. 2, preferably a substitu- invention, which includes any of the amino acid sequences tion to an amino acid at the corresponding position within identified as a heavy chain CDR2 in FIG. 1 or FIG. 3, amino another light chain CDR2 of FIG. 2, or a consensussequence acid sequencesthat contain one or more amino acid changes of light chain CDR2 shown in FIG. 2. compared to any heavy chain CDR2 identified in FIG. 1 or [0025] The light chain comprising a light chain CDR3 FIG. 3, preferably a substitution to an amino acid at the sequenceof the invention described above may also comprise corresponding position within another heavy chain CDR2 of both (a) a light chain CDRI sequence of the invention FIG. 1 or FIG. 3, or a consensus sequence of heavy chain described above and (b) a light chain CDR2 sequenceof the CDR2 shown in FIG. 1 or FIG. 3. invention described above. [0018] The heavy chain comprising a heavy chain CDR3 [0026] Antibodies comprising any one of the light chains sequenceof the invention described above may also comprise described above may further comprise a heavy chain, prefer- both (a) a heavy chain CDRI sequence of the invention ably a heavy chain that binds to target antigen, and most described above and (b) a heavy chain CDR2 sequenceof the preferably a heavy chain comprising heavy chain CDR invention described above. sequencesof the invention described above. [0019] Any of the heavy chain CDR sequencesdescribed [0027] In yet another embodiment, the antibody or antigen- above may also include amino acids added to either end of the binding compound comprises a constant region and one or CDRs. Preparation of variants and derivatives of antibodies more heavy and light chain variable framework regions of a and antigen-binding compounds of the invention, including human antibody sequence.In a related embodiment, the anti- affinity maturation or preparation of variants or derivatives body comprises a modified or unmodified constant region of containing amino acid analogs, is described in further detail a human IgGI, IgG2, IgG3 or IgG4. In an exemplary embodi- below. Exemplary variants include those containing a conser- ment, the constant region is optionally modified to enhance or vative or non-conservative substitution of a corresponding decreasecertain properties. For example, modifications to the amino acid within the amino acid sequence,or a replacement constant region, particularly the hinge or CH2 region, may of an amino acid with a corresponding amino acid of a human increase or decreaseeffector function, including ADCC and/ antibody sequence. or CDC activity. In other embodiments, an IgG2 constant
  11. 11. US 2009/0311257 A1 Dec. 17, 2009 region is modified to decrease antibody-antigen aggregate [0038] Yet another aspect of the invention provides non- formation. In the case of IgG4, modifications to the constant immunoglobulin-like recombinant polypeptides or other region, particularly the hinge region, may reduce the forma- compounds that comprise any of the heavy chain or light tion of half-antibodies. chain CDR sequences of the invention described above, or [0028] In exemplary embodiments, the antibody of the any combinations of these CDR sequences. For example, invention is derived from, based on, or contains part of the such compounds may comprise a CDR sequenceof the inven- human antibody consensus sequence, human germline tion as a single copy or in multiple copies in, for example, a sequence,human consensusgermline sequence,or any one of tandemly repeated or multivalent configuration. Such com- the human antibody sequences in Kabat, NCBI Ig Blast pounds may further comprise other CDR sequencesin single (http: //www.ncbi.nlm.nih.gov/igblast/showGermline.cgi), or multiple copies. Such compounds may also include non- which enables searching all Ig sequences in the database, peptidyl linkages. including germline sequences (maintained by the National [0039] In still another embodiment of the invention, an Center for Biotechnology Information); Kabat Database isolated nucleic acid is provided comprising a nucleic acid http: //www.bioinf.org.uk/abs/sexiest.html, Martin, A.C.R. sequenceencoding the aforementioned antibody. In a related "Accessing the Kabat Antibody SequenceDatabase by Com- embodiment, the isolated nucleic acid comprises a heavy puter" Proteins: Structure, Function and Genetics, 25 (1996), chain nucleic acid sequencewhich is at least 60, 65, 70, 75, 130-133; 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to [0029] ImMunoGeneTics database (Montpellier France) the heavy chain nucleotide sequenceset forth in SEQ ID NO: (http: //imgt.cines.fr/), Lefranc, M.-P. et al., Nucleic Acids 13, 15, 17, 19 and 21. In yet another related embodiment, the Research, 27, 209-212, 1999; isolated nucleic acid comprises a light chain nucleic acid [0030] V-Base, Tomlinson, I. M., Williams, S. C., Ignatov- sequencewhich is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, ich, O., Corbett, S. J. & Winter, G. (1996) VBASE Sequence 94, 95, 96, 97, 98, or 99% identical to the light chain nucle- Directory (Medical Research Council Centre for Protein otide sequenceset forth in SEQ ID NOs: 14, 16, 18, 20 and 22. Engineering, Cambridge, UK); [0040] In another embodiment, a vector comprising the [0031] Zurich University (http: //www.biochem.unizh.ch/ aforementioned isolated nucleic acid is provided. In a related embodiment, the aforementioned vector is provided wherein antibody the isolated nucleic acid is operably linked to a regulatory [0032] /Sequences/index.html), Burmester, et al., Selec- control sequence. In still another embodiment, a host cell is tion, characterization and X-ray structure of anti-ampicillin provided comprising the aforementioned vector. single chain Fv fragments from phage-displayed murine anti- [0041] Numerous methods are contemplated in the present body libraries. J. Mol. Biol., 309 (2001) 671-685; invention. For example, a method of producing an aforemen- [0033] The Therapeutic Antibody Human Homology tioned antibody is provided comprising culturing the afore- Project (TAHHP) (http: //www.path.cam.ac.uk/-mrc7/hu- mentioned host cell such that the isolated nucleic acid is manisation/TAHHP.html), "Reshaping antibodies for expressed to produce the antibody. In a related embodiment, therapy" Edward G. Routledge, Scott D. Gorman and Mike the method further comprises the step of recovering the anti- Clark, in Protein Engineering of Antibody Molecules for body from the host cell culture. In a related embodiment, an Prophylactic and Therapeutic Applications in Man pp. 13-44 isolated antibody produced by the aforementioned method is (1993), Academic Titles, Nottingham, England; provided. [0034] Humanization by design (http: //people.cryst.bbk. [0042] A further aspect of the invention addressesthe por- ac.uk/-ubcg07s/), Bendig, M. M., Kettleborough, C. A., tions of the compounds of the invention that do not bind the Jones,S. T., Maeda, H. and Saldanha, J. (1993), "The humani- target antigen but instead are responsible for other functions, sation of mouse monoclonal antibodies by CDR-grafting: such as circulating half-life, direct cytotoxic effect, detectable Examples with anti-viral and anti-tumour cell antibodies", in labeling, or activation of the recipient's endogenous comple- Monoclonal Antibodies 2: Applications in Clinical Oncology ment cascadeor endogenous cellular cytotoxicity. Antibodies ed. A. A. Epenetos, pp 119-140, Chapman & Hall Medical of the invention may comprise all or a portion of the constant Publishers; Leger, O. J. P. and Saldanha, J. W. (2000), "Prepa- region and may be of any isotype, including IgA (e.g., IgAI or ration of recombinant antibodies from immune rodent IgA2), IgD, IgE, IgG (e.g. IgGI, IgG2, IgG3 or IgG4), or spleens and the design of their humanization by CDR graft- IgM. In addition to, or instead of, comprising a constant ing", in Monoclonal Antibodies: A Practical Approach eds. P. region, antigen-binding compounds of the invention may Shepherd and C. Dean, pp 25-69, Oxford University Press; include an epitope tag, a salvage receptor epitope, a label [0035] Antibody Resources (http: //www.antibodyre- moiety for diagnostic or purification purposes, or a cytotoxic source.corn/educational. html), Antibody Engineering (by moiety such as a radionuclide or toxin. TT Wu), Humana Press. [0043] In another embodiment of the invention, a pharma- [0036] In yet another embodiment of the invention, the ceutical composition is provided comprising any one of the aforementioned antibody has an affinity Kd of at least 10[ ] aforementioned antibodies and a pharmaceutically suitable M. In a related embodiment, the antibody has an affinity Kd of carrier, excipient or diluent. Preferably the antibodies and at least 10[ ]M. compounds of the invention are administered in a therapeu- [0037] In one aspect, the invention specifically contem- tically effective amount, i.e., an amount sufficient to amelio- plates sterile compositions of isolated monoclonal antibody rate a clinical sign or symptom of a condition or disorder that binds to gastrin with an affinity Kd ranging from about associated with the target protein expression, to a subject in 10 M to 10 M, or about 10 M to 10 M, or 10 M to need of such treatment. In a related embodiment, the phar- 10 " M; in a relatedaspect, inventioncontemplates the the maceutical composition further comprises a secondtherapeu- use of such compositions to treat disorders associated with tic agent. In yet another related embodiment, the pharmaceu- gastrin expression. tical composition is provided wherein the second therapeutic
  12. 12. US 2009/0311257 A1 Dec. 17, 2009 agent is a growth factor, a cytokine, a chemotherapeutic [0054] FIG. 3 shows the heavy chain amino acid sequences agent, or a radiotherapeutic agent. In another embodiment the of the XPA.067 affinity matured antibodies (SEQ ID NOs: second therapeutic agent is another antibody. 23-33). CDRs are underlined. [0044] In another embodiment of the invention, the afore- [0055] FIG. 4 is a comparison of the heavy chain CDR mentioned methods are provided wherein the subject is a regions of the originating XPA067 antibody and the affinity mammal. In a related embodiment, the mammal is human. matured antibodies. [0045] In another embodiment, the aforementioned meth- [0056] FIG. 5 illustrates the improved gastrin neutraliza- ods are provided wherein the antibody inhibits the interaction tion capacity of reformatted, affinity matured antibodies between the target and a binding partner. In yet another XPA067.06 and XPA067.18 compared to parent antibody embodiment, the aforementioned methods are provided XPA067. wherein the antibody is administered at a dosebetween about [0057] FIG. 6 shows the neutralization of anti-gastrin anti- 2 pg/kg to 50 mg/kg, 0.1 mg/kg to 30 mg/kg, or 0.1 mg/kg to bodies in a gastric pH famotidine mouse model. F: Fainoti- 10 mg/kg. dine, G: h-G17 (human gastrln), XPA067: parental a-gastrin mAb, XPA067.06: affinity mature u-gastrin mAb. [0046] In another embodiment of the invention, the use of [0058] FIG. 7 shows the neutralization of anti-gastrin anti- an antibody of the invention is contemplated in the manufac- bodies in a gastric pH telenzepine mouse model. T: Telen- ture of a medicament for preventing or reducing a condition zepine, G: h-G17 (human gastrln), XPA067: parental u-gas- or disorder associated with target protein expression, as trin mAb, XPA067.06: affinity mature u-gastrln mAb. defined herein. [0047] In any of the aforementioned uses, the medicament DETAILED DESCRIPTION OF THE INVENTION is coordinated with treatment using a second therapeutic agent. [0059] The present invention addressesa need in the art to [0048] In another embodiment of the invention, the use of a develop therapeutics to treat conditions or disorders associ- synergistic combination of an antibody of the invention for ated with target antigen expression. The present invention preparation of a medicament for treating a patient exhibiting provides molecules or agents that interact with the target to symptoms of a condition or disorder disclosed herein wherein eliminate signaling through binding partners of the target. the medicament is coordinated with treatment using a second [0060] In order that the invention may be more completely therapeutic agent is contemplated. In a related embodiment, understood, several definitions are set forth. the second therapeutic agent is a chemokine, a cytokine, a [0061] As used herein, "target" or "target antigen" refers to growth factor, a chemotherapeutic agent, a radiotherapeutic the gastrin peptide hormone. Gastrin may be the 34 amino agent, or radiation therapy. acid gastrin peptide or may be a shorterversion of the peptide, such as the 17 amino acid or 14 amino acid variant of gastrin. [0049] Embodiments of any of the aforementioneduses are [0062] In a preferred embodiment the gastrin is human contemplated wherein the amount of antibody in the medica- gastrin. ment is at a dose effective to reduce the dosage of second [0063] As used herein, the "desired biological activity" of therapeutic agent required to achieve a therapeutic effect. an anti-target antibody is the ability to bind to gastrin and [0050] The amount of antibody in any of the aforemen- inhibit its functional effects. tioned medicaments may be at a dosebetween about 2 pg/kg [0064] As used herein, a "condition" or "disorder associ- to 50 mg/kg body weight. In a related embodiment, the ated with target expression" is a condition or disorder in amount of antibody in the medicament is at a dose between which target activity is detrimental and includes diseasesand about 0.1 mg/kg to 30 mg/kg body weight. In still another other disorders in which high levels o ftarget have been shown embodiment, the amount of antibody in the medicament is at to be or are suspected of being either responsible for the a dose between about 0.1 mg/kg to 10 mg/kg body weight. pathophysiology of the disorder or a factor that contributes to [0051] Kits are also contemplated by the present invention. a worsening of the disorder, as well as diseases and other In one embodiment, a kit comprises a therapeutically effec- disorders in which high levels of target expression are asso- tive amount of a composition of the invention, packaged in a ciated with undesirable clinical signs or symptoms. Such container, such as a vial or bottle, and further comprising a disorders may be evidenced, for example, by an increase in label attached to or packaged with the container, the label the levels of target secreted and/or on the cell surface or describing the contents of the container and providing indi- increased signalling inthe affected cells ortissues ofa subject cations and/or instructions regarding use of the contents of the suffering from the disorder. The increase in target levels may container to prevent or reduce a condition or disorder associ- be detected, for example, using an target specific antibody as ated with target protein expression. described above. [0065] Exemplary conditions or disorders associated with BRIEF DESCRIPTION OF THE DRAWINGS target expression include cancers, such as pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, and [0052] FIG. 1 shows the heavy chain amino acid sequences small lung cell carcinoma, as well as gastric ulcer, duodenal of anti-gastrin antibodies XPA061, XPA063, XPA065, ulcer, other ulcers or conditions associated with H. Pylori, XPA067, XPA081 and a consensus sequence. CDRs are gastroesophageal reflux disease, autoimmune gastritis, atro- underlined and Chothia numbering for all sequences is phic body gastritis, Zollinger-Ellison syndrome associated included beneath the consensus sequence. with tumor of the pancreas (gastrinoma), and inflammatory [0053] FIG. 2 shows the light chain amino acid sequences bowel disease. of anti-gastrin antibodies XPA061, XPA063, XPA065, [0066] An "immunoglobulin" or "native antibody" is a tet- XPA067, XPA081 and a consensus sequence. CDRs are rameric glycoprotein. In a naturally-occurring immunoglo- underlined and Chothia numbering for all sequences is bulin, each tetramer is composed of two identical pairs of included beneath the consensus sequence. polypeptide chains, each pair having one "light" (about 25
  13. 13. US 2009/0311257 A1 Dec. 17, 2009 kDa) and one "heavy" chain (about 50-70 kDa). The amino- complementarity determining region (CDR) of said antibody terminal portion of each chain includes a variable region of light chain variable domain. The light chain variable region about 100 to 110 or more amino acids primarily responsible may contain one, two, or three CDRs of said antibody light for antigen recognition. The carboxy-terminal portion of each chain, which may be either a kappa or lambda light chain chain defines a constant region primarily responsible for depending on the antibody. effector function. Human light chains are classified as kappa [0073] The term "antibody" is used in the broadest sense (K) and lambda P.) light chains. Heavy chains are classified as and includes fully assembled antibodies, monoclonal anti- mu (p), delta (A), gamma (7), alpha (u), and epsilon (e), and bodies, polyclonal antibodies, multispecific antibodies (e.g., define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, bispecific antibodies), antibody fragments that can bind anti- respectively. Within light and heavy chains, the variable and gen (e.g., Fab', F'(ab)~, Fv, single chain antibodies, diabod- constant regions arej oined by a "I" region of about 12 or more ies), and recombinant peptides comprising the forgoing as amino acids, with the heavy chain also including a "D" region long as they exhibit the desired biological activity. Antigen- of about 10 more amino acids. See generally, Fundamental binding portions may be produced by recombinant DNA Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,N.Y. techniques or by enzymatic or chemical cleavage of intact (1989)) (incorporated by reference in its entirety for all pur- antibodies. Antibody fragments or antigen-binding portions poses). The variable regions of each light/heavy chain pair include, inter alia, Fab, Fab', F(ab')~, Fv, domain antibody form the antibody binding site such that an intact immuno- (dAb), complementarity determining region (CDR) frag- globulin has two binding sites. ments, single-chain antibodies (scFv), single chain antibody [0067] Each heavy chain has at one end a variable domain fragments, chimeric antibodies, diabodies, triabodies, tetra- (V~) followed by a number of constant domains. Each light bodies, minibody, linear antibody; chelating recombinant chain has a variable domain at one end (Vz) and a constant antibody, a tribody or bibody, an intrabody, a nanobody, a domain at its other end; the constant domain of the light chain small modular immunopharmaceutical (SMIP), a antigen- is aligned with the first constant domain of the heavy chain, binding-domain immunoglobulin fusion protein, a camelized and the light chain variable domain is aligned with the vari- antibody, a V~~ containing antibody, or a variant or a deriva- able domain of the heavy chain. Particular amino acid resi- tive thereof, and polypeptides that contain at least a portion of dues are believed to form an interface between the light and an immunoglobulin that is sufficient to confer specific antigen heavy chain variable domains (Chothia et al., J. Mol. Biol. binding to the polypeptide, such as a CDR sequence,as long 196:901-917, 1987). as the antibody retains the desired biological activity. [0068] Immunoglobulin variable domains exhibit the same [0074] "Monoclonal antibody" refers to an antibody general structure of relatively conserved framework regions obtained from a population of substantially homogeneous (FR) joined by three hypervariable regions or CDRs. From antibodies, i.e., the individual antibodies comprising the ¹erminus to C-terminus, both light and heavy chains com- population are identical except for possible naturally occur- prise the domains FRI, CDRI, FR2, CDR2, FR3, CDR3 and ring mutations that may be present in minor amounts. FR4. The assignment of amino acids to each domain is in [0075] "Antibody variant" as used herein refers to an anti- accordance with the definitions of Kabat Sequences of Pro- body polypeptide sequence that contains at least one amino teins of Immunological Interest (National Institutes of acid substitution, deletion, or insertion in the variable region Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, of the natural antibody variable region domains. Variants may (J. Mol. Biol. 196:901-917, 1987); Chothia et al., (Nature be substantially homologous or substantially identical to the 342: 878-883, 1989). unmodified antibody. [0069] The hypervariable region of an antibody refers to the [0076] A "chimeric antibody," as used herein, refers to an CDR amino acid residues of an antibody which are respon- antibody containing sequence derived from two different sible for antigen-binding. The hypervariable region com- antibodies (see,e.g., U.S. Pat. No. 4,816,567) which typically prises amino acid residues from a CDR [i.e., residues 24-34 originate from different species. Most typically, chimeric (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable antibodies comprise human and rodent antibody fragments, domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the generally human constant and mouse variable regions. heavy chain variable domain as described by Kabat et al., [0077] A "neutralizing antibody" is an antibody molecule Sequences Proteins of Immunological Interest, 5' Ed. of which is able to eliminate or significantly reduce an effector Public Health Service, National Institutes of Health, function of a target antigen to which it binds. Accordingly, a Bethesda, Md. (1991)] and/or those residues from a hyper- "neutralizing" anti-target antibody is capable of eliminating variable loop (i.e., residues 26-32 (Ll), 50-52 (L2) and 91-96 or significantly reducing an effector function, such as enzyme (L3) in the light chain variable domain and 26-32 (HI), 53-55 activity, ligand binding, or intracellular signaling. (H2) and 96-101 (H3) in the heavy chain variable domain as [007S] An "isolated" antibody is one that has been identi- described by [Chothia et al., J. Mol. Biol. 196: 901-917 fied and separated and recovered from a component of its (1987)]. natural environment. Contaminant components of its natural [0070] Framework or FR residues are those variable environment are materials that would interfere with diagnos- domain residues other than the hypervariable region residues. tic or therapeutic uses for the antibody, and may include [0071] "Heavy chain variable region" as used herein refers enzymes, hormones, and other proteinaceous or non-pro- to the region of the antibody molecule comprising at least one teinaceous solutes. In preferred embodiments, the antibody complementarity determining region (CDR) of said antibody will be purified (I) to greater than 95% by weight of antibody heavy chain variable domain. The heavy chain variable region as determined by the Lowry method, and most preferably may contain one, two, or three CDRs of said antibody heavy more than 99% by weight, (2) to a degree sufficient to obtain chain. at least 15 residues of ¹erminal or internal amino acid [0072] "Light chain variable region" as used herein refers sequenceby use of a spinning cup sequenator,or (3) to homo- to the region of an antibody molecule, comprising at least one geneity by SDS-PAGE under reducing or nonreducing con-
  14. 14. US 2009/0311257 A1 Dec. 17, 2009 ditions using Coomassie blue or, preferably, silver stain. Iso- ornithine, which do not normally occur in human proteins. lated antibody includes the antibody in situ within Derivatives retain the binding properties of underivatized recombinant cells since at least one component of the anti- molecules of the invention. body's natural environment will not be present. Ordinarily, [00S3] "Detectable moiety" or a "label" refers to a compo- however, isolated antibody will be prepared by at least one sition detectable by spectroscopic, photochemical, biochemi- purification step. cal, immunochemical, or chemical means. For example, use- [0079] As used herein, an antibody that "specifically binds" ful labels include P, S, fluorescent dyes, electron-dense is "target specific", is "specific for" target or is "immunore- reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for active" with the target antigen refers to an antibody or anti- which antisera or monoclonal antibodies are available, or body substance of the invention that binds the target antigen nucleic acid molecules with a sequencecomplementary to a with greater affinity than with similar antigens. In one aspect, target. The detectable moiety often generates a measurable the target-binding polypeptides of the invention, or frag- signal, such as a radioactive, chromogenic, or fluorescent ments, variants, or derivatives thereof, will bind with a greater signal, that can be used to quantitate the amount of bound affinity to human target as compared to its binding affinity to detectable moiety in a sample. target of other, i.e., non-human, species,but binding polypep- [00S4] The term "therapeutically effective amount" is used tides that recognize and bind orthologs of the target are within herein to indicate the amount of target-specific composition the scope of the invention. of the invention that is effective to ameliorate or lessen symp- [00SO] For example, a polypeptide that is an antibody or toms or signs of diseaseassociatedwith target protein expres- fragment thereof "specific for" its cognate antigen indicates sion. that the variable regions of the antibodies recognize and bind [00S5] The present invention provides a target-specific the polypeptide of interest with a detectable preference (i.e., antibody, which may comprise those exemplary sequences able to distinguish the polypeptide of interest from other set out in FIGS. 1 and 2, fragments, variants and derivatives known polypeptides of the same family, by virtue of measur- thereof, pharmaceutical formulations including a target-spe- able differences in binding affinity, despite the possible exist- cific antibody recited above, methods of preparing the phar- ence of localized sequence identity, homology, or similarity maceutical formulations, and methods of treating patients between family members). It will be understood that specific with the pharmaceutical formulations and compounds. antibodies may also interact with other proteins (for example, [00S6] Depending on the amino acid sequenceof the con- S. aureus protein A or other antibodies in ELISA techniques) stant domain of their heavy chains, immunoglobulins can be through interactions with sequences outside the variable assigned to different classes, IgA, IgD, IgE, IgG and IgM, region of the antibodies, and in particular, in the constant which may be further divided into subclassesor isotypes, e.g. region of the molecule. Screening assaysto determine bind- IgGI, IgG2, IgG3, IgG4, IgAI and IgA2. The subunit struc- ing specificity of an antibody for use in the methods of the tures and three-dimensional configurations of different invention are well known and routinely practiced in the art. classes of immunoglobulins are well known. Different iso- For a comprehensive discussion of such assays,seeHarlow et types have different effector functions; for example, IgGI and al. (Eds), Antibodies A I aborato~ Manual; Cold Spring Har- IgG3 isotypes have ADCC activity. An antibody of the inven- bor Laboratory, Cold Spring Harbor, N.Y. (1988), Chapter 6. tion, if it comprises a constantdomain, may be of any of these Antibodies for use in the invention can be produced using any subclassesor isotypes. method known in the art. [00S7] The antibodies of the present invention may exhibit [00S1] The term "epitope" refers to that portion of any binding affinity to antigen of a Ka of greater than or equal to molecule capable of being recognized by and bound by a about 10 M ', greaterthan or equal to about 10 M ', or selective binding agent at one or more of the antigen binding greater thanor equalto about10 M ', or greaterthanor equal regions. Epitopes usually consist of chemically active surface to about 10 M ', or greaterthan or equalto about 10 M ', groupings of molecules, such as, amino acids or carbohydrate 10' M ',10"M 'or10' M '.Suchaffinitiesmaybereadily side chains, and have specific three-dimensional structural determined using conventional techniques, such as by equi- characteristics as well as specific charge characteristics. librium dialysis; by using the BIAcore 2000 instrument, using Epitopes as used herein may be contiguous or non-contigu- general procedures outlined by the manufacturer; by radio- ous. Moreover, epitopes may be mimetic (mimotopes) in that immunoassay using ' I labeledtargetantigen;or by another they comprise a threedimensional structure that is identical to method known to the skilled artisan. The affinity data may be the epitope used to generate the peptibody, yet comprise none analyzed, for example, by the method of Scatchard et al., (Ann or only some of the amino acid residues found in the target N.Y. Acad. Sci., 51:660, 1949). that were used to stimulate the peptibody immune response. As used herein, a mimotope is not considered a different Antibody Polypeptides of the Invention antigen from the epitope bound by the selective binding agent; the selective binding agent recognizes the same three- [00SS] The present invention encompasses amino acid dimensional structure of the epitope and mimotope. molecules encoding target specific antibodies. In exemplary [00S2] The term "derivative" when used in connection with embodiments, a target specific antibody of the invention can antibody substancesand polypeptides of the invention refers comprise a human kappa (K) or a human lambda P.) light to polypeptides chemically modified by such techniques as chain or an amino acid sequence derived therefrom, or a ubiquitination, conjugation to therapeutic or diagnostic human heavy chain or a sequencederived therefrom, or both agents, labeling (e.g., with radionuclides or various heavy and light chains together in a single chain, dimeric, enzymes), covalent polymer attachment such as pegylation tetrameric or other form. In some embodiments, a heavy (derivatization with polyethylene glycol) and insertion or chain and a light chain of a target specific immunoglobulin substitution by chemical synthesis of amino acids such as are different amino acid molecules. In other embodiments,
  15. 15. US 2009/0311257 A1 Dec. 17, 2009 the sameamino acid molecule contains a heavychain variable which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, region and a light chain variable region of a target specific 96, 97, 98, or 99% homologous to any of the amino acid antibody. sequencesset forth herein. [0089] In some embodiments, the amino acid sequence of [0095] It is further contemplated that the CDR of the anti- the human anti-target antibody comprises one or more CDRs body heavy and light chains comprise variant amino acid sequenceswhich may improve antibody binding affinity and of the amino acid sequenceof the light chain variable region are derived through, for example, affinity maturation. In one (V~) of antibodies XPA061, XPA063, XPA065, XPA067, and aspect it is contemplated that an antibody of the invention XPA081 set out in FIG. 2 or variants thereof. In some embodi- comprises a heavy chain CDR2 sequencehaving about 35% ments, the V~ comprises the amino acid sequence from the identity to a CDR2 of a parent antibody sequence set out in beginning of the CDRI to the end of the CDR3 of the light SEQ ID NOs: I, 3, 5, 7, 9, 11 or 23-33. In a related aspect it chain of any one of the foregoing antibodies. is contemplated that an antibody of the invention comprises a [0090] In one embodiment, the target specific antibody heavy chain CDR3 sequencehaving about 50% identity to a comprises a light chain CDRI, CDR2 or CDR3, each of CDR3 of a parent antibody sequenceset out in SEQ ID NOs: which are independently selected from the CDRI, CDR2 and I, 3, 5, 7, 9, 11 or 23-33. CDR3 regions of an antibody having a light chain variable region comprising the amino acid sequenceof the V~ region Antibody Nucleic Acids of the Invention set out in SEQ ID NOs: 2, 4, 6, 8, 10 and 12, or encoded by a [0096] The present invention also encompasses nucleic nucleic acid molecule encoding the V~ region set out in SEQ ID NOs: 14, 16, 18, 20 and 22. In one aspect the light chain acid molecules encoding target specific antibodies. In some embodiments, different nucleic acid molecules encode a CDRI is from approximately residues 24-34, CDR2 is from approximately residues 50-56 and CDR3 extends from heavy chain variable region and a light chain variable region of a target specific antibody. In other embodiments, the same approximately residues 89-97, according to Chothia number- nucleic acid molecule encodesa heavy chain and a light chain ing. A polypeptide of the target specific antibody may com- variable regions of a target specific antibody. prise the CDRI, CDR2 and CDR3 regions of an antibody [0097] In one embodiment, the nucleic acid encodes a tar- comprising the amino acid sequenceof the V~ region selected from the group consisting of XPA.061XPA063, XPA065, get specific antibody of the invention. XPA067, and XPA081. [0098] In one aspect, a nucleic acid molecule of the inven- tion comprises a nucleotide sequence that encodes the V~ [0091] In some embodiments, the human target specific amino acid sequence of antibodies XPA061, XPA063, antibody comprises one or more CDRs of the amino acid XPA065, XPA067, and XPA081 set out in SEQ ID NOs: 2, 4, sequenceof the heavy chain variable region (V~) of antibody 6, 8 and 10 or a portion thereof. In a related aspect, the V~ XPA.061, XPA063, XPA065, XPA067, and XPA081 set out amino acid sequenceis a consensussequenceset out in SEQ in FIG. 1 or FIG. 3 or variants thereof. In some embodiments, ID NO: 12. In some embodiments, the nucleic acid encodes the V~ comprises the amino acid sequence from the begin- the amino acid sequence of the light chain CDRs of said ning of the CDRI to the end of the CDR3 of any one of the antibody. In some embodiments, said portion is a contiguous heavy chain of the foregoing antibodies. portion comprising CDRI-CDR3. In one embodiment, said [0092] In one embodiment, the target specific antibody portion comprises at least one, two or three of a light chain comprises a heavy chain CDRI, CDR2 or CDR3, each of CDRI, CDR2, or CDR3 region. which are independently selected from the CDRI, CDR2 and [0099] In a related aspect, the nucleic acid molecule com- CDR3 regions of an antibody having a heavy chain variable prises a nucleotide sequence that encodes the light chain region comprising the amino acid sequenceof the V~ region amino acid sequenceof one of SEQ ID NOs: 2, 4, 6, 8, 10 and set out in SEQ ID NOs: I, 3, 5, 7, 9 and 11, or encoded by a 12 or a portion thereof. In one embodiment, the nucleic acid nucleic acid molecule encoding the V~ region set out in SEQ molecule comprises the light chain nucleotide sequence of ID NO: 13, 15, 17, 19 and 21. It is further contemplated that any one of SEQ ID NOs: 14, 16, 18, and 22 or a portion a target specific antibody comprises a heavy chain CDRI, thereof. CDR2 or CDR3, each of which are independently selected [0100] In some embodiments, the nucleic acid molecule from the CDRI, CDR2 and CDR3 regions of an antibody encodes a V~ amino acid sequencethat is at least 60, 65, 70, having a heavy chain variable region comprising the amino 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 97, 98 or 99% identical acid sequenceof the V~ region set out in SEQ ID NOs: 23-33. to a V~ amino acid sequence set out in SEQ ID NOs: 2, 4, 6, In one aspect the heavy chain CDRs are located according to 8, 10 and 12. Nucleic acid molecules of the invention include Chlothia numbering set out in FIG. 1: CDRI is from approxi- nucleic acids that hybridize under highly stringent condi- mately residues 31-35, CDR2 is from approximately residues tions, such as those described herein, to a nucleic acid 50-65 and CDR3 extends from approximately residues sequenceencoding the light chain variable region amino acid 95-102. A polypeptide of the target specific antibody may sequenceof SEQ IDNOs: 2,4, 6, 8, 10 and 12, orthathas the comprise the CDRI, CDR2 and CDR3 regions of an antibody light chain variable region nucleic acid sequence of SEQ ID comprising the amino acid sequenceo fthe V~ region selected NOs: 14, 16, 18, 20 and 22. from the group consisting of XPA061, XPA063, XPA065, [0101] It is further contemplated that a nucleic acid mol- XPA067, and XPA081. ecule of the invention comprises a nucleotide sequence that [0093] In another embodiment, the antibody comprises a encodesthe V~ amino acid sequenceof any one of antibodies light chain as disclosed above and a heavy chain as disclosed XPA061, XPA063, XPA065, XPA067, and XPA081, or a above. portion thereof. In some embodiments, the nucleic acid [0094] It is contemplated that a variant of the antibody encodesthe amino acid sequenceof the heavy chain CDRs of sequencerefers to amino acid sequences,comprising a vari- said antibody. In some embodiments, said portion is a con- able heavy chain or a variable light chain amino acid sequence tiguous portion comprising heavy chain CDRI-CDR3. In one
  16. 16. US 2009/0311257 A1 Dec. 17, 2009 embodiment, said portion comprises at least one, two or three sites.At 7-14 days post-booster injection, the animals are bled of a heavy chain CDRI, CDR2, or CDR3 region. and the serum is assayed for antibody titer. Animals are [0102] In a related aspect, the nucleic acid molecule com- boosted until the titer plateaus. Preferably, the animal is prises a nucleotide sequence that encodes the heavy chain boosted with the conjugate of the same antigen, but conju- amino acid sequenceof one of heavy chain of SEQ ID NOs: gated to a different protein and/or through a different cross- I, 3, 5, 7, 9 and 11 or a portion thereof. In one embodiment, linking reagent. Conjugates also can be made in recombinant the nucleic acid molecule comprises the heavy chain nucle- cell culture as protein fusions. Also, aggregating agents such otide sequenceof SEQ ID NO: 13, 15, 17, 19 and 21 a portion as alum are suitably used to enhance the immune response. thereof. [0103] In some embodiments, the nucleic acid molecule Monoclonal Antibodies encodes a V~ amino acid sequencethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a [0109] Monoclonal antibody refers to an antibody obtained V~ amino acid sequence XPA061, XPA063, XPA065, from a population of substantially homogeneous antibodies. XPA067, and XPA081 set out in SEQ ID NOs: I, 3, 5, 7 and Monoclonal antibodies are generally highly specific, and may 9. In a related aspect, the V~ amino acid sequence is a con- be directed against a single antigenic site, in contrast to con- sensus sequence set out in SEQ ID NO: 11. Nucleic acid ventional (polyclonal) antibody preparations that typically molecules of the invention include nucleic acids that hybrid- include different antibodies directed against different deter- ize under highly stringent conditions, such as those described minants (epitopes). In addition to their specificity, the mono- above, to a nucleic acid sequence encoding the heavy chain clonal antibodies are advantageous in that they are synthe- variable region amino acid sequenceof SEQ ID NOs: I, 3, 5, sized by the homogeneous culture, uncontaminated by other 7, 9 and 11, or that has the heavy chain variable region nucleic immunoglobulins with different specificities and characteris- acid sequenceof any one of SEQ ID NO: 13, 15, 17, 19 and tics. 21. [0110] Monoclonal antibodies to be used in accordance [0104] It is further contemplated that the nucleic acids of with the present invention may be made by the hybridoma the invention encode a full-length light chain or heavy chain method first described by Kohler et al., (Nature, 256:495-7, of an antibody selected from XPA061, XPA063, XPA065, 1975), or may be made by recombinant DNA methods (see, XPA067, and XPA081 wherein a full-length light chain or e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies full-length heavy chain comprises a light chain constant may also be isolated from phage antibody libraries using the region or a heavy chain constant region, respectively. techniques described in, for example, Clackson et al., (Nature [0105] In one aspect, the full length light chain antibody 352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581- comprises the sequencesset out in SEQ ID NOs: 2, 4, 6, 8, 10 597, 1991). and 12. It is further contemplated that the nucleotide encoding [0111] In the hybridoma method, a mouse or other appro- the full-length light chain encodes the sequences SEQ ID priate host animal, such as a hamster or macaque monkey, is NOs: 2, 4, 6, 8, 10 and 12, and comprises the nucleotides immunized as herein described to elicit lymphocytes that sequence set forth in SEQ ID NOs: 14, 16, 18, 20 and 22. produce or are capable of producing antibodies that will spe- [0106] In one aspect, the full length heavy chain antibody cifically bind to the protein used for immunization. Alterna- comprises the sequencesin any one of SEQ ID NOs: I, 3, 5, tively, lymphocytes may be immunized in vitro. Lympho- 7, 9 and 11. It is further contemplated that the nucleotide cytes then are fused with myeloma cells using a suitable encoding the full-length heavy chain encodes the sequences fusing agent, such as polyethylene glycol, to form a hybri- heavy chain of SEQ ID NOs: I, 3, 5, 7, 9 and 11 and comprises doma cell (Goding, Monoclonal Antibodies: Principles and the nucleotides sequenceset forth in any one of SEQ ID NO: Practice, pp. 59-103 (Academic Press, 1986)). 13, 15, 17, 19 and 21. [0112] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains Polyclonal Antibodies one or more substancesthat inhibit the growth or survival of [0107] Polyclonal antibodies are preferably raised in ani- the unfused, parental myeloma cells. For example, if the mals by multiple subcutaneous (sc) or intraperitoneal (ip) parental myeloma cells lack the enzyme hypoxanthine gua- injections of the relevant antigen and an adjuvant. An nine phosphoribosyl transferase (HGPRT or HPRT), the cul- improved antibody responsemay be obtained by conjugating ture medium for the hybridomas typically will include hypox- the relevant antigen to a protein that is immunogenic in the anthine, aminopterin, and thymidine (HAT medium), which species to be immunized, e.g., keyhole limpet hemocyanin, substancesprevent the growth of HGPRT-deficient cells. serum albumin, bovine thyroglobulin, or soybean trypsin [0113] Preferred myeloma cells are those that fuse efft- inhibitor using a bifunctional or derivatizing agent, for ciently, support stable high-level production of antibody by example, maleimidobenzoyl sulfosuccinimide ester (conju- the selected antibody-producing cells, and are sensitive to a gation through cysteine residues), N-hydroxysuccinimide medium. Human myeloma and mouse-human heteromy- (through lysine residues), glutaraldehyde, succinic anhydride cloma cell lines also have been described for the production or other agents known in the art. of human monoclonal antibodies (Kozbor, J. Immunol., 133: [0108] Animals are immunized against the antigen, immu- 3001 (1984); Brodeur et al., Monoclonal Antibody Produc- nogenic conjugates, or derivatives by combining, e.g., 100 pg tion Techniques and Applications, pp. 51-63 (Marcel Dekker, or 5 pg of the protein or conjugate (for rabbits or mice, Inc., New York, 1987)). Exemplary murine myeloma lines respectively) with 3 volumes of Freund's: complete adjuvant include those derived from MOP-21 and M.C.-II mouse and injecting the solution intradermally at multiple sites. One tumors available from the Salk Institute Cell Distribution month later, the animals are boostedwith 'ts to (fraction Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 ('bio) theoriginal amountof peptideor conjugate Freund's ) in cells available from the American Type Culture Collection, complete adjuvant by subcutaneous injection at multiple Rockville, Md. USA.
  17. 17. US 2009/0311257 A1 Dec. 17, 2009 [0114] Culture medium in which hybridoma cells are grow- [0119] Additional antibody fragment include a domain ing is assayed for production of monoclonal antibodies antibody (dAb) fragment (Ward et al., Nature 341:544-546, directed against the antigen. Preferably, the binding specific- 1989) which consists of a Vu domain. Diabodies are bivalent ity of monoclonal antibodies produced by hybridoma cells is antibodies in which Vu and Vz domains are expressed on a determined by immunoprecipitation or by an in vitro binding single polypeptide chain, but using a linker that is too short to assay, such as radioimmunoassay (RIA) or enzyme-linked allow for pairing between the two domains on the samechain, immunoabsorbent assay (ELISA). The binding affinity of the thereby forcing the domains to pair with complementary monoclonal antibody can, for example, be determined by domains of another chain and creating two antigen binding Scatchard analysis (Munson et al., Anal. Biochem., 107:220 sites (see e.g., EP 404,097; WO 93/11161; Holliger et al., (1980)). Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak et [0115] After hybridoma cells are identified that produce al., Structure 2:1121-1123, 1994). Diabodies can be bispe- antibodies of the desired specificity, affinity, and/or activity, cific or monospecific. the clones may be subcloned by limiting dilution procedures [0120] Functional heavy-chain antibodies devoid of light and grown by standard methods (Goding, Monoclonal Anti- chains are naturally occurring in nurse sharks (Greenberg et bodies: Principles and Practice, pp. 59-103 (Academic Press, al., Nature 374:168-73, 1995), wobbegong sharks Outtall et 1986)). Suitable culture media for this purpose include, for al., MolImmunol. 38:313-26, 2001) and Camelidae (Hamers- example, DMEM or RPMI-1640 medium. In addition, the Casterman et al., Nature 363: 446-8, 1993; Nguyen et al., J. hybridoma cells may be grown in vivo as ascites tumors in an Mol. Biol. 275: 413, 1998), such as camels, dromedaries, animal. The monoclonal antibodies secretedby the subclones alpacas and llamas. The antigen-binding site is reduced to a are suitably separatedfrom the culture medium, ascites fluid, single domain, the Vuu domain, in these animals. These anti- or serum by conventional immunoglobulin purification pro- bodies form antigen-binding regions using only heavy chain cedures such as, for example, protein A-Sepharose, hydroxy- variable region, i.e., these functional antibodies are lapatite chromatography, gel electrophoresis, dialysis; or homodimers of heavy chains only having the structure H~L~ affinity chromatography. (referred to as "heavy-chain antibodies" or "HCAbs"). Cam- [0116] It is further contemplated that antibodies of the elidVuureportedly recombines with IgG2 and IgG3 constant invention may be used as smaller antigen binding fragments regions that contain hinge, CH2, and CH3 domains and lack of the antibody well-known in the art and described herein. a CHI domain (Hamers-Casterman et al., supra). For example, llama IgGI is a conventional (H~L~) antibody iso- Antibody Fragments type in which Vu recombines with a constant region that contains hinge, CHI, CH2 and CH3 domains, whereas the [0117] Antibody fragments comprise a portion of an intact llama IgG2 and IgG3 are heavy chain-only isotypes that lack full length antibody, preferably an antigen binding or variable region of the intact antibody. Examples of antibody fragments CH I domains and that contain no light chains. Camelid Vuu domains have been found to bind to antigen with high affinity include Fab, Fab', F(ab')~, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001) and possesshigh stability in solution (Ewert et al., Biochemistry multispecific antibody fragments such as bispecfic, trispe- 41:3628-36, 2002). Classical sonly fragments are difficult cific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies); minibody; chelating recombinant antibody; tribodies or to produce in soluble form, but improvements in solubility and specific binding can be obtained when framework resi- bibodies; intrabodies; nanobodies; small modular immunop- harmaceuticals (SMIP), binding-domain immunoglobulin dues are altered to be more VH~like. (See, e.g., Reichman, et al., I Immunol Methods 1999, 231:25-38.) Methods for gen- fusion proteins; camelized antibodies; Vuu containing anti- bodies; and other polypeptides formed from antibody frag- erating antibodies having camelid heavy chains are described ments. in, for example, in U.S. Patent Publication Nos. 20050136049 and 20050037421. [0118] Papain digestion of antibodies produces two identi- cal antigen-binding fragments, called "Fab" fragments, [0121] Because the variable domain of the heavy-chain- monovalent fragments consisting of the Vz, Vu, Cz and Cu antibodies is the smallest fully functional antigen-binding domains each with a single antigen-binding site, and a fragment with a molecular mass of only 15 kDa, this entity is residual "Fc" fragment, whose name reflects its ability to referred to as a nanobody (Cortez-Retamozo et al., Cancer crystallize readily. Pepsin treatment yields a F(ab')~fragment, Research 64:2853-57, 2004). A nanobody library may be a bivalent fragment comprising two Fab fragments linked by generated from an immunized dromedary as described in a disulfide bridge at the hinge region, that has two "Single- Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, chain Fv" or "scFv" antibody fragments comprise the Vu and 2001) or using recombinant methods as described in Vz domains of antibody, wherein these domains are present in [0122] Production of bispecific Fab-scFv ("bibody") and a single polypeptide chain. Preferably, the Fv polypeptide trispecific Fab-(scFv)(2) ("trlbody") are described in further comprises a polypeptide linker between the Vu and Vz Schoonjans et al. (J Immunol. 165:7050-57, 2000) and domains that enables the Fv to form the desired structure for Willems et al. (J Chromatogr B Analyt Technol Biomed Life antigen binding, resulting in a single-chain antibody (scFv), Sci. 786:161-76, 2003). For bibodies or tribodies, a scFv in which a Vz and Vu region are paired to form a monovalent molecule is fused to one or both of the VL-CL (L) and VH- molecule via a synthetic linker that enables them to be made CH, (Fd) chains, e.g., to produce a tribody two scFvs are as a single protein chain (Bird et al., Science 242:423-426, fused to C-term of Fab while in a bibody one scFv is fused to 1988, and Huston et al., Proc. Natl. Acad. Sci. USA. 85:5879- C-term of Fab. 5883, 1988). For a review of sFv see Pluckthun, in The Phar- [0123] A "minibody" consisting of scFv fused to CH3 via a macology of Monoclonal Antibodies, vol. 113, Rosenburg peptide linker (hingeless) or via an IgG hinge has been and Moore eds., Springer-Verlag, New York, pp. 269-315 described in Olafsen, et al., Protein Eng Des Sel. 2004 April; (1994). An Fd fragment consists of the Vu and Cul domains. 17(4):315-23.
  18. 18. US 2009/0311257 A1 Dec. 17, 2009 10 [0124] Intrabodies are single chain antibodies which dem- ing the yield of the heterodimer over other unwanted end- onstrate intracellular expression and can manipulate intrac- products such as homodimers. See WO96/27011 published ellular protein function (Biocca, et al., EMBO J. 9:101-108, Sep. 6, 1996. 1990; Colby et al., Proc Natl Acad Sci U SA. 101:17616-21, [0130] Bispecific antibodies include cross-linked or "het- 2004). Intrabodies, which comprise cell signal sequences eroconjugate" antibodies. For example, one of the antibodies which retain the antibody contruct in intracellular regions, in the heteroconjugate can be coupled to avidin, the other to may be produced as described in Mhashilkar et al (EMBO J biotin. Heteroconjugate antibodies may be made using any 14:1542-51, 1995) and Wheeler et al. (FASEB J. 17:1733-5. convenient cross-linking methods. Suitable cross-linking 2003). Transbodies are cell-permeable antibodies in which a agents are well known in the art, and are disclosed in U.S. Pat. protein transduction domains (PTD) is fused with single chain variable fragment (scFv) antibodies Heng et al., (Med. No. 4,676,980, along with a number of cross-linking tech- Hypotheses. 64:1105-8, 2005). niques. [0125] Further contemplated are antibodies that are SMIPs [0131] Techniques for generating bispecific antibodies or binding domain immunoglobulin fusion proteins specific from antibody fragments have also been described in the for target protein. These constructs are single-chain polypep- literature. For example, bispecific antibodies can be prepared tides comprising antigen binding domains fused to immuno- using chemical linkage. Brennan et al., (Science 229:81-83, globulin domains necessary to carry out antibody effector 1985) describe a procedure wherein intact antibodies are functions. See e.g., WO03/041600, U.S. Patent publication proteolytically cleaved to generate F(ab')~ fragments. These 20030133939 and US Patent Publication 20030118592. fragments are reduced in the presence of the dithiol complex- [0126] One or more CDRs may be incorporated into a mol- ing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' frag- ecule either covalently or noncovalently to make it an immu- noadhesin. An immunoadhesin may incorporate the CDR(s) ments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then as part of a larger polypeptide chain, may covalently link the reconverted to the Fab'-thiol by reduction with mercaptoet- CDR(s) to another polypeptide chain, or may incorporate the hylamine and is mixed with an equimolar amount of the other CDR(s) noncovalently. The CDRs permit the immunoad- Fab'-TNB derivative to form the bispecific antibody. The hesin to specifically bind to a particular antigen of interest. bispecific antibodies produced can be used as agents for the [0127] Thus, a variety of compositions comprising one, selective immobilization of enzymes. In yet a further embodi- two, and/or three CDRs of a heavy chain variable region or a ment, Fab'-SH fragments directly recovered from E. cali can light chain variable region of an antibody may be generated be chemically coupled in vitro to form bispecific antibodies. by techniques known in the art. (Shalaby et al., J. Exp. Med. 175:217-225 (1992)) [0132] Shalaby et al., J. Exp. Med. 175:217-225 (1992) Multispecific Antibodies describe the production of a fully humanized bispecific anti- [0128] In some embodiments, it may be desirable to gen- body F(ab')~ molecule. Each Fab' fragment was separately erate multispecific (e.g. bispecific) anti-target antibody of the secreted from E. cali and subjected to directed chemical invention having binding specificities for at least two differ- coupling in vitro to form the bispeefic antibody. The bispe- ent epitopes of the same or different molecules. Exemplary cific antibody thus formed was able to bind to cells overex- bi specificantibodies may bind to two different epitopes o fthe pressing the HER2 receptor and normal human T cells, as target molecule. Alternatively, a target-specific antibody arm well as trigger the lytic activity of human cytotoxic lympho- may be combined with an arm which binds to a cell surface cytes against human breast tumor targets. molecule, such as a T-cell receptor molecule (e.g., CD2 or [0133] Various techniques for making and isolating bispe- CD3), or Fc receptors for igG (FcyR), such as FcyRI (CD64), cific antibody fragments directly from recombinant cell cul- FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular ture have also been described. For example, bispecific anti- defense mechanisms to the target. Bispecific antibodies may bodies have been produced using leucine zippers. (Kostelny also be used to localize cytotoxic agents to cells which et al., J. Immunol. 148:1547-1553, 1992). The leucine zipper express or take up the target. These antibodies possess a peptides from the Fos and Jun proteins were linked to the Fab' target-binding arm and an arm which binds the cytotoxic portions of two different antibodies by gene fusion. The anti- agent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricinA body homodimers were reduced at the hinge region to form chain, methotrexate or radioactive isotope hapten). Bispecific monomers and then re-oxidized to form the antibody het- antibodies can be prepared as full length antibodies or anti- erodimers. This method can also be utilized for the produc- body fragments (e.g., F(ab')2 bispecific antibodies). tion of antibody homodimers. The "diabody" technology [0129] According to another approach for making bispe- described by Hollinger et al. (Proc. Natl. Acad. Sci. USA cific antibodies, the interface between a pair of antibody 90: 6444-48, 1993) has provided an alternative mechanism for molecules can be engineered to maximize the percentage of making bispecific antibody fragments. heterodimers which are recovered from recombinant cell cul- [0134] The fragments comprise a heavy chain variable ture. The preferred interface comprises at least a part of the region (Vu) connected to a light-chain variable region (Vz) by Cu3 domain of an antibody constant domain. In this method, a linker which is too short to allow pairing between the two one or more small amino acid side chains from the interface of domains on the same chain. Accordingly, the Vu and Vz the first antibody molecule are replaced with larger side domains of one fragment are forced to pair with the comple- chains (e.g., tyrosine or tryptophan). Compensatory "cavi- mentary Vz and Vu domains of another fragment, thereby ties" of identical or similar size to the large side chain(s) are forming two antigen-binding sites. Another strategy for mak- created on the interface of the second antibody molecule by ing bispecific antibody fragments by the use of single-chain replacing large amino acid side chains with smaller ones (e.g., Fv (sFv) dimers has also been reported. See Gruber et al., J. alanine or threonine). This provides a mechanism for increas- Immunol. 152: 5368 (1994).

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