WO2007140534A1 - Anticorps vegf-a anti-vegf-b dotés d'une réactivité croisée utilisés comme antagonistes des signaux vegf-a et vegf-b - Google Patents

Anticorps vegf-a anti-vegf-b dotés d'une réactivité croisée utilisés comme antagonistes des signaux vegf-a et vegf-b Download PDF

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WO2007140534A1
WO2007140534A1 PCT/AU2007/000799 AU2007000799W WO2007140534A1 WO 2007140534 A1 WO2007140534 A1 WO 2007140534A1 AU 2007000799 W AU2007000799 W AU 2007000799W WO 2007140534 A1 WO2007140534 A1 WO 2007140534A1
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vegf
antibody
antibodies
heavy
light chain
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PCT/AU2007/000799
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Andrew Nash
Pierre David Scotney
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Csl Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • VEGF-A CROSS-REACTIVE ANTI- VEGF-B ANTIBODIES AS ANTAGONISTS OF VEGF-A AND VEGF-B SIGNALLING
  • the present invention relates generally to anti- VEGF-B antibodies and in particular to anti- VEGF-B antibodies which bind to VEGF-A and which antagonize VEGF-A and VEGF-B signaling.
  • the antibodies of the present invention are useful in the treatment or prevention of VEGF-A- or VEGF-B -mediated diseases or conditions.
  • VEGF vascular endothelial growth factor
  • VEGF-A mRNA is upregulated in many human tumors and VEGF-A appears to be an important angiogenic factor frequently utilized by tumors to switch on blood vessel growth (Dvorak et al, Semin Perinatol 24:75-78, 2000; Ferrara & Alitalo, 1999 supra; Yancopoulos, 2000 supra; Benjamin & Keshet, Proc Natl Acad Sci USA 94:8761-8766, 1997; Ferrara and Davis-Smyth, Endocr. Rev 18:4-25, 1997). VEGF- A also increases vascular permeability, and this is thought to be important for tumor invasion and metastasis (Dvorak et al, Curr Top Microbiol Immunol 237:97-132, 1999).
  • VEGF-A vascular endothelial growth factor-A
  • Bevacizumab a humanized mouse monoclonal antibody that binds to, and inhibits the activity of, VEGF-A.
  • Bevacizumab Avastin has recently been approved by the FDA for the treatment of colorectal cancer.
  • VEGF-A is now recognized as the founding member of a family of structurally related molecules.
  • the 'VEGF family' comprises six members including prototype VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E (Eriksson & Alitalo, Curr Top Microbiol Immunol 237:41-57, 1999).
  • the biological functions of the VEGF family are mediated by the differential activation of at least three structurally homologous tyrosine kinase receptors, VEGFR-I /FIt-I, VEGFR-2/Flk-l/KDR and VEGFR-3/Flt-4.
  • VEGF-A, VEGF-B and PlGF also bind to the non-tyrosine kinase receptors neuropilin-1 and -2 (Soker et al, Cell 92:735-45, 1998; Neufeld et al, Trends Cardiovasc Med. /2:13-19, 2002).
  • the VEGF family can be divided into three subgroups: (1) VEGF-A, which binds to VEGFR-I and VEGFR-2; (2) PlGF and VEGF-B, which bind only to VEGFR-I and; (3) VEGF-C and VEGF-D, which interact with both VEGFR-2 and VEGFR-3 (Ferrara & Alitalo, 1999 supra; Ferrara et al, 2003 supra).
  • VEGFR-2 is the major mediator of VEGF-A associated biological activities such as endothelial cell proliferation, migration and survival, angiogenesis and vascular permeability (Ferrara et al, 2003 supra).
  • VEGF-C and -D also bind to, and activate, VEGFR-3.
  • VEGFR-3 is expressed primarily on lymphatic endothelial cells and VEGF-C and -D are thought to be key regulators of lymphatic angiogenesis [or lymphangiogenesis] (Makinen et al, Nature Medicine 7:199-205, 2001; Skobe et al, Nature Medicine 7:192-8, 2001; Stacker et al, Nature Medicine 7:186-91, 2001).
  • lymphatic angiogenesis or lymphangiogenesis
  • VEGFR-I is expressed on a variety of cell types (Clauss et al, J. Biol. Chem. 277:17629- 17634, 1996; Wang & Keiser, Circ. Res. 55:832-840, 1998; Niida et al, J. Exp. Med. 190:293-29$, 1999) and expression, at least on endothelial cells, is upregulated by hypoxia and a HIF-l ⁇ dependent mechanism (Gerber et al, J. Biol. Chem. 272:23659-23667, 1997).
  • VEGFR-I vascular endothelial growth factor-A
  • VEGF-A binding to VEGFR-I appears not to activate the downstream signals required for key endothelial cell responses such as proliferation and survival (de Vries et al, Science 255:989-991, 1992; Waltenberger et al, J. Biol. Chem. 269:26988- 26995, 1994; Keyt et al, J. Biol. Chem. 277:5638-5646, 1996; Rahimi et al, J. Biol. Chem. 275:16986-16992, 2000).
  • VEGFR-I specific ligand PlGF
  • PlGF VEGFR-I specific ligand
  • VEGFR-1 " ⁇ mice died in utero between days 8.5 and 9.5 and although endothelial cells developed, they failed to organize into vascular channels (Fong et al, Development 726:3015-3025, 1999). Lethality was attributed to excessive angioblast proliferation and this in turn, was attributed to enhanced VEGF-A action (Fong et al, 1999 supra).
  • the observation that mice expressing VEGFR-I lacking the kinase domain were healthy and showed no overt defect in vascular development provided further support for the decoy hypothesis, as the truncated receptor could still bind VEGF-A, but not transmit intracellular signals (Hiratsuka et al, Proc. Natl. Acad. Sci. USA 4:9349-9354, 1998).
  • mice also failed to resolve the confusion surrounding the precise physiological (and pathological) role of VEGFR-I specific ligands and VEGFR-I signaling.
  • VEGF-B "7* mice display no overt defects in vascular development and are healthy and fertile (Bellomo et al, Circ. Res. S6:E29-E35, 2000).
  • the hearts of VEGF-B "7” mice were reduced in size and the response to coronary occlusion and myocardial recovery from ischemia were compromised (Bellomo et al 2000, supra).
  • VEGF-B is essential for the establishment of a fully functional coronary vasculature.
  • a second report describing VEGF-B "7" mice reported only a minor atrial conduction defect (Aase et al, Circulation /04:358-364, 2001).
  • VEGF-B represented a therapeutic target for inhibition of tumor growth and metastasis.
  • VEGF-B had been shown, along with other factors, to be expressed in a variety of tumors (Salven et al, Am J Pathol, 755:103-108, 1998), evidence of upregulation was limited (Li et al, Growth Factors 19:49- 59, 2001) and there were no reports of the efficacy of VEGF-B specific antagonists in xenograft or other relevant animal models.
  • VEGF-B and PlGF
  • PlGF PlGF
  • VEGF-B antibodies to VEGF-B, such as humanized mouse monoclonal antibodies that bind to and inhibit the activity of VEGF-B, are useful in reducing the growth and development of cancer.
  • VEGF-A plays a major role in the progression of angiogenesis and has been implicated in tumor growth.
  • VEGF-B plays a role in the progression of angiogenesis and may play a role in tumor growth.
  • VEGF-A and VEGF-B therefore represent therapeutic targets for the inhibition of angiogenesis and tumor growth.
  • Eriksson et al generated an antibody specific to VEGF-B and VEGF-A in their non-reduced, physiological state, or if the serum contains antibodies specific to VEGF-B and non-specific antibodies to short linear sequences in the VEGF-A protein.
  • Eriksson et al did not go on to isolate and characterise a single, monoclonal antibody that that binds to VEGF-B and VEGF-A.
  • VEGF-B antibodies are identified herein which also bind VEGF-A and which antagonize VEGF-A and VEGF-B signaling.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>l
  • SEQ ID NO:2 sequence identifiers
  • Antibodies raised against VEGF-B are provided which alsobind to VEGF-A.
  • Anti-VEGF-B antibodies which also bind to VEGF-A are referred to herein inter alia as "VEGF-A cross-reactive anti- VEGF-B antibodies”.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are also provided which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are monoclonal antibodies, antigen-binding fragments or antibody-derived polypeptides.
  • VEGF-A cross-reactive anti-VEGF-B antibodies are chimeric antibodies, including humanized antibodies, or human antibodies.
  • VEGF-A cross-reactive anti-VEGF-B antibodies described herein include the murine monoclonal antibodies 2E4, 2E9 and 2El 2 and humanized or chimeric antibodies, including humanized antibodies, derived from the murine monoclonal antibodies 2E4, 2E9 and 2E12. Included herein are VEGF-A cross-reactive anti-VEGF-B antibodies which compete with one or more of the murine monoclonal antibodies 2E4, 2E9 and 2El 2 for binding to VEGF-A and VEGF-B.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies compete with one or more of the murine monoclonal antibodies 2E4, 2E9 and 2El 2 for binding to VEGF-A and VEGF-B and antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies include antibodies which compete with the murine monoclonal antibody 2E4 for binding to VEGF-A and VEGF-B and which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • VEGF-A cross-reactive anti-VEGF-B antibodies described herein bind to human VEGF-A and human VEGF-B. It should be noted, however, that some level of binding to other mammalian forms of VEGF-A and VEGF-B may be desirable in certain circumstances, such as for example, for the purpose of testing antibodies in animal models for their effect on a particular disease and for conducting toxicology studies. Animal species where such antibodies may be desirable include mouse, dog and monkey.
  • a particular group of VEGF-A cross-reactive anti-VEGF-B antibodies are those that bind to human VEGF-A and VEGF-B and which also bind to murine VEGF-A and murine VEGF-B.
  • compositions are also provided herein comprising one or more VEGF-A cross-reactive anti-VEGF-B antibodies either alone or in combination with other agents, such as anticancer agents or angiogenesis-inhibiting agents.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies may be used to treat certain conditions mediated in whole or in part by VEGF-A or VEGF-B.
  • the present invention also contemplates methods for treating these conditions in a subject comprising administering a VEGF-A cross-reactive anti- VEGF-B antibody of the present invention.
  • Conditions to be treated in accordance with the present invention include aberrant angiogenesis, cancers and tumors. Pre-cancerous conditions and dispersed tumors, such as for example, myelomas and lymphomas may also be treated.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are contemplated in the manufacture of a medicament for the treatment of aberrant angiogenesis, cancers and tumors as well as pre-cancerous conditions and dispersed tumors.
  • Figure 1 is a graphical representation demonstrating dose dependant proliferation in response to recombinant !1VEGF-A 11-109 and hVEGF-Bio -1O8 proteins in the h VEGFR- l/EpoR/BaF3 proliferation assay.
  • Figure 2 is a graphical representation of the ability of mAbs raised against !1VEGF-Ai 1-1O9 (12/05-5C9), hVEGF-Bi 67 (2C/99-2H10) and hVEGF-B 10-10 8 (11/05-2E4, 11/05-2E9 & 11/05-2E12) to bind 11VEGF-A 1 1 -109 , JiVEGF-B 10-I08 and hPlGF in an ELISA based assay.
  • Figure 3 is a graphical representation of the ability of mAbs raised against hVEGF -B I0-108 (11/05-2E4, 11/05-2E9 & 11/05-2E12) to bind hVEGF-A n-10 9 and hVEGF-Bi 0- i 08 in Western-blot analysis.
  • Figure 4 is a graphical representation of the ability of the mAb 11/05-2E12 raised against hVEGF- B 10-108 to inhibit the activity of hVEGF-A 11-109 in the hVEGFR-l/EpoR/BaF3 proliferation assay.
  • Figure 5 is a graphical representation of the ability of the mAb 11/05-2E12 raised against hVEGF- B 10-108 to inhibit the activity of hVEGF-B 10-108 in the hVEGFR-l/EpoR/BaF3 proliferation assay.
  • Figure 6 is a graphical representation of the ability of the mAb 11/05-2E4 raised against hVEGF- B 10-I08 to inhibit the activity Of IiVEGF-Ai 1- I 09 in the hVEGFR-2/EpoR/BaF3 proliferation assay.
  • Figure 7 is a graphical representation of the ability of the mAb 11/05-2E9 raised against hVEGF- B 10-108 to inhibit the activity of !1VEGF-A 1 M 09 in the hVEGFR-2/EpoR/BaF3 proliferation assay.
  • Figure 8 is a representation of the single-letter protein sequences for VEGF-A 1 i_io 9 and
  • VEGF-B 10-1 O 8 VEGF-B 10-1 O 8 .
  • Figure 9 is a representation of the variable region nucleotide and translated single-letter amino acid sequences of the heavy chain of the murine monoclonal VEGF-A cross- reactive anti-VEGF-B antibody 2E4.
  • the amino acid sequence corresponds to and was translated from nucleotides 1 to 348 of the nucleotide sequence.
  • Figure 10 is a representation of the variable region nucleotide and translated single-letter amino acid sequences of the light chain of the murine monoclonal VEGF-A cross-reactive anti-VEGF-B antibody 2E4.
  • the amino acid sequence corresponds to and was translated from nucleotides 1 to 321 of the nucleotide sequence.
  • Figure 11 is a representation of the nucleotide and translated single-letter amino acid sequences of the CDRs (complementarity determining regions) of the heavy and light chain of the murine monoclonal VEGF-A cross-reactive anti-VEGF-B antibody 2E4.
  • the subject invention is not limited to specific formulations of components, manufacturing methods, dosage or diagnostic regimes, or the like. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
  • VEGF-B includes a single VEGF-B as well as two or more VEGF-B molecules
  • an antibody includes a single antibody, as well as two or more antibodies
  • reference to “the invention” includes single and multiple aspects of the invention; and so forth.
  • VEGF-A and VEGF-B includes all naturally occurring isoforms of VEGF-A, including VEGF-A 121 , VEGF-A 165 , VEGF-A 189 , and VEGF-A 206 , (Ferrara et. al, J Cell Biochem, 47(3):2 ⁇ 1-218, 1991) and VEGF-B, including VEGF-Bi 67 and VEGF-B 186 (Olofsson et ⁇ /, JBiol Chem. 277:19310-19317, 1996).
  • VEGFR-I and "VEGFR-2" are used herein to refer to the cellular receptors for VEGF-A and VEGF-B.
  • VEGFR-I is a cellular receptor for VEGF-A and VEGF-B.
  • VEGFR-2 is a cellular receptor for VEGF-A but not for VEGF-B.
  • antibody and “antibodies” are used herein to refer to monoclonal antibodies and all the various forms derived from monoclonal antibodies, including but not limited to full antibodies (e.g. having an intact Fc region), antigen-binding fragments, including for example, Fv, Fab, Fab' and F(ab') 2 fragments; and antibody-derived polypeptides produced using recombinant methods such as single chain antibodies, linear antibodies, chimeric antibodies, including humanized antibodies, and complementary determining region (CDR) grafted antibodies.
  • antibody and “antibodies” as used herein also refer to human antibodies produced in transgenic animals or through phage display.
  • monoclonal is used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal” as used herein therefore indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not used to indicate that the antibody was produced by a particular method.
  • monoclonal antibodies contemplated herein may be made by the hybridoma method described by Kohler et al, Nature 256:495, 1975, or may be made by recombinant DNA methods (such as described in U.S. Patent NO: 4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 552:624-628, 1991 or Marks et al, J. MoI. Biol. 222:581-597, 1991.
  • compound used interchangeably herein to refer to a substance that induces a desired pharmacological and/or physiological effect.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active forms thereof, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • references to a "compound”, “active agent”, “pharmacologically active agent”, and “medicament” may include combinations of two or more of such substances, such as for example, two or more antibodies.
  • a “combination” also includes multi-part combinations such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.
  • a multi-part pharmaceutical pack may have two or more antibodies maintained separately or a VEGF-A cross-reactive anti- VEGF-B antibody and an anti-cancer agent or an angiogenesis- inhibiting agent.
  • an effective amount and "therapeutically effective amount” as used herein mean a sufficient amount of a substance, such as a VEGF-A cross-reactive anti- VEGF-B antibody, to provide the desired therapeutic or physiological effect or outcome, such as inhibiting angiogenesis and/or inhibiting growth of cancer including tumor tissue.
  • Undesirable effects e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the exact amount of agent required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount”.
  • an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
  • the ability of a VEGF-A cross-reactive anti- VEGF-B antibody to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • a "pharmaceutically acceptable" carrier and/or diluent is a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, agents used for adjusting tonicity, buffers, chelating agents, and absorption delaying agents and the like.
  • a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that is not biologically or otherwise undesirable.
  • treating and “treatment” as used herein refer to therapeutic treatment and prophylactic or preventative measures.
  • treatment may result in a reduction in severity and/or the frequency of symptoms of cancer, the elimination of symptoms and/or underlying cause of cancer, the prevention of the occurrence of symptoms of cancer and/or their underlying cause and improvement or remediation or amelioration of damage following a cancer.
  • cancer and “tumor” may be used interchangeably and includes pre-cancerous conditions.
  • a "subject” as used herein refers to an animal, such as a mammal and in particular a human who can benefit from the pharmaceutical compositions and methods of the present invention. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical compositions and methods.
  • a subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient as well as subject.
  • the compounds and methods described herein have applications in human medicine and veterinary medicine.
  • Particular animals are humans or laboratory test animals.
  • laboratory test animals include mice, rats, rabbits, guinea pigs, hamsters, cats and dogs.
  • polynucleotide refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in single stranded form, double-stranded form or otherwise.
  • nucleic acid sequence refers to a series of nucleotide bases (also referred to as “nucleotides”) in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.
  • coding sequence or a sequence “encoding” an expression product, such as an mRNA, a polypeptide or a protein, is a nucleotide sequence that, when expressed, results in production of the product.
  • sequence of any nucleic acid may be sequenced by any method known in the art such as by chemical sequencing (Maxam & Gilbert, Proc. Natl. Acad. Sci. USA 74(2):560-564, 1977) or enzymatic sequencing (Sanger et al, Proc. Natl. Acad. Sci. USA 74(12):5463 5467, 1977).
  • Nucleic acids described herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.
  • promoters include promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.
  • IVS internal ribosome entry sites
  • a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell and initiating transcription of a coding sequence.
  • a promoter sequence is generally bounded at its 3 1 terminus by the transcription initiation site and extends upstream in the 5' direction to include the minimum number of bases or elements necessary to initiate transcription at any level.
  • a transcription initiation site as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase may be found within the promoter sequence.
  • the promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter and the SV40 early promoter region.
  • CMV cytomegalovirus
  • a coding sequence is "under the control of, “functionally associated with” or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be trans-RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
  • express and expression mean allowing or causing the information in a gene, RNA or DNA sequence to be converted into a product; for example, producing a protein by activating the cellular functions involved in transcription and translation of a nucleotide sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product” such as RNA (such as mRNA) or a protein (such as a VEGF-A cross-reactive anti-VEGF- B antibody).
  • the expression product itself may also be said to be “expressed” by the cell.
  • vector means the vehicle (such as a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.
  • transfection means the introduction of a nucleic acid into a cell. These terms may refer to the introduction of a nucleic acid encoding a VEGF-A cross- reactive anti- VEGF-B antibody or a fragment thereof into a cell.
  • a host cell that receives the introduced DNA or RNA has been "transformed” and is a “transformant” or a “clone”.
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
  • host cell means any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression of a protein or the replication of a gene.
  • expression system means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are provided.
  • Another embodiment is directed to VEGF-A cross-reactive anti- VEGF-B antibodies which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are in isolated, homogenous or fully or partially purified form.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are monoclonal antibodies, antigen-binding fragments or antibody-derived polypeptides.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies may also be chimeric antibodies, including humanized antibodies, or human antibodies. These include chimeric and humanized antibodies prepared, for example, from murine monoclonal antibodies, and human monoclonal antibodies which may be prepared, for example, using transgenic mice or by phage display.
  • VEGF-A cross-reactive anti-VEGF-B antibodies disclosed herein include the murine monoclonal antibodies 2E4, 2E9, and 2El 2, and more particularly, chimeric antibodies, including humanized antibodies, derived from the murine monoclonal antibodies 2E4, 2E9, and 2E12.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies include complete antibodies (e.g.
  • Fc region having an intact Fc region
  • various forms that can be derived from the antibodies including antigen-binding fragments such as Fv, Fab, Fab' and F(ab') 2 fragments; and antibody-derived polypeptides produced through recombinant methods, such as single chain antibodies, linear antibodies, chimeric antibodies, including humanized antibodies, and complementary determining region (CDR) grafted antibodies.
  • antigen-binding fragments such as Fv, Fab, Fab' and F(ab') 2 fragments
  • antibody-derived polypeptides produced through recombinant methods such as single chain antibodies, linear antibodies, chimeric antibodies, including humanized antibodies, and complementary determining region (CDR) grafted antibodies.
  • CDR complementary determining region
  • VEGF-A cross-reactive anti-VEGF-B antibodies described herein may be prepared by a number of procedures well known in the art, such as the hybridoma method of Kohler et al, 1975 supra, recombinantly as described in U.S. Patent NO: 4,816,567 or using phage antibody libraries as described by Clackson et al, 1991 supra and Marks et al, 1991 supra. See also, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow & Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).
  • the hybridoma method comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with VEGF-B, or an immunogenic part thereof (for example, VEGF- B io-1 o 8 (SEQ ID NO: 1), whereby antibodies directed against VEGF-B or the immunogenic part are generated in said animal.
  • VEGF-B or an immunogenic part thereof
  • Various means for increasing the antigenicity of a particular immunogen such as administering adjuvants, aggregating agents such as alum or conjugated antigens, (which comprise the antigen against which an antibody response is desired and another component), are well known in the art and may be utilized. Immunizations typically involve an initial immunization followed by a series of booster immunizations.
  • VEGF-B vascular endothelial growth factor-B
  • the VEGF-B that may be used in the method described above to immunize animals may be from any mammalian source.
  • the VEGF-B is human VEGF-B, and in another embodiment is human VEGF-B 10-108 (SEQ ID NO: 1).
  • monoclonal antibodies are particularly useful as they can be produced in large quantities, are highly specific and are directed against a single antigenic site. Furthermore, the monoclonal antibody preparations are homogeneous, making them ideal for generating antigen-binding fragments and other engineered antibody derivatives for therapeutic applications. Finally, the immortal nature of hybridoma cells means they can be frozen, thawed, and recultured, thereby providing a renewable source of monoclonal antibodies.
  • polyclonal antibodies are also relatively easily prepared, they are not as useful as monoclonal antibodies as polyclonal antibody preparations typically include different antibodies directed against different antigenic sites and thus are not as suitable for generating antigen-binding fragments and other engineered antibody derivatives for therapeutic applications.
  • hybridoma cell lines generated by the method outlined above, and the anti- VEGF-B monoclonal antibodies produced by them are encompassed herein.
  • the monoclonal antibodies secreted by the hybridoma cell lines may be purified by conventional techniques.
  • the hybridomas or the monoclonal anti- VEGF-B antibodies produced by them may be screened further using, for example, the assays described herein, to identify monoclonal antibodies with particularly desirable properties, such as the ability to bind to VEGF-A, and the ability to antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the hybridoma method described above is used in animals, such as mice, to produce monoclonal antibodies.
  • animals such as mice
  • antibodies derived from animals are generally unsuitable for administration to humans as they may cause an immune response.
  • such antibodies may be modified to become suitable for administration to humans.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies of the present invention may also be produced using recombinant methods (for example, in an E. coli expression system) well known in the art.
  • DNA encoding monoclonal antibodies such as the murine monoclonal antibodies 2E4, 2E9 and 2El 2
  • DNA may be fused to another DNA of interest, or altered (such as by mutagenesis or other conventional techniques) to add, delete, or substitute one or more nucleic acid residues.
  • the DNA may be placed into vectors which are then transfected or transformed into appropriate host cells using methods well known in the art (such as described in U.S. Patent Nos:4,399,216; 4,912,040; 4,740,461 and 4,959,455).
  • the DNA isolated from the hybridoma cell lines may also be modified to change the character of the antibody produced by its expression.
  • chimeric forms of the murine monoclonal antibodies 2E4, 2E9, and 2El 2 may be produced by replacing the nucleotides encoding selected murine heavy and light chain constant domains with nucleotides encoding human heavy and light chain constant domains, such as is described in U.S. Patent No. 4,816,567 and by Morrison et al, Proc.
  • the chimeric antibodies may then be produced in an appropriate cell line, such as a murine myeloma cell line, that has been transfected with modified DNA.
  • an appropriate cell line such as a murine myeloma cell line
  • the heavy and light chain variable regions of the murine monoclonal antibody 2E4 are disclosed in Figures 9 and 10 and in SEQ ID NOs:4 and 6 (amino acid sequences) and 3 and 5 (nucleotide sequences).
  • chimeric VEGF-A cross-reactive anti- VEGF-B antibodies that comprise the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs:4 and 6) fused to non-murine heavy and light chain antibody constant domains.
  • the non-murine heavy and light chain constant domains are human heavy and light chain antibody constant domains.
  • the VEGF-A cross-reactive anti- VEGF-B antibodies herein also include humanized antibodies, which are generally chimeric antibodies which contain a minimal amount of sequence derived from non-human antibodies, such as murine antibodies.
  • humanized antibodies are human antibodies (the recipient antibody) in which the complementarity determining (CDR) region residues have been replaced by CDR region residues from a non-human species (the donor antibody), such as from a mouse, rat, rabbit or non-human primate.
  • CDR complementarity determining
  • the donor antibody such as from a mouse, rat, rabbit or non-human primate.
  • framework region (FR) residues of the human antibody may also be replaced by corresponding non-human residues, or the humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human antibody, and all or substantially all of the FRs are those of a human antibody sequence.
  • the humanized antibody may also optionally comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody.
  • Fc antibody constant region
  • the CDRs of a given antibody may be readily identified, for example using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).
  • the murine monoclonal antibody 2E4 is humanized.
  • the CDRs of the murine monoclonal antibody 2E4 heavy and light chains are disclosed in Figure 11 and in SEQ ID NOs:7 to 12 (amino acid sequences) and 13 to 18 (nucleotide sequences).
  • humanized VEGF-A cross-reactive anti- VEGF-B antibodies that comprise from one to all three of the CDR sequences from the light chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs:7 to 9).
  • humanized VEGF-A cross- reactive anti- VEGF -B antibodies that comprise from one to all three of the CDR sequences from the heavy chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 10 to 12).
  • humanized VEGF-A cross-reactive anti-VEGF-B antibodies comprise one, two, three, four, five or all six CDR sequences from the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 7 to 12).
  • the VEGF-A cross-reactive anti-VEGF-B antibodies also include antigen-binding fragments such as Fv, Fab, Fab' and F(ab') 2 fragments.
  • antigen-binding fragments were generated by the proteolytic digestion of full antibodies (Morimoto et al, Journal of Biochemical and Biophysical Methods 24:107-117,1992; Brennan et al, Science 229:81, 1985).
  • a number of recombinant methods have now been developed for producing antigen-binding fragments of antibodies directly in recombinant host cells.
  • Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al, Bio/Technology 10:163-167, 1992). F(ab') 2 fragments can also be formed using the leucine zipper GCN4 to promote assembly of the F(ab') 2 molecule. Fv, Fab or F(ab') 2 fragments can also be isolated directly from recombinant host cell cultures. A number of recombinant methods have been developed for the production of single chain antibodies including those described in U.S. Patent No. 4,946,778; Bird, Science 242: 423, 1988, Huston et al, Proc. Natl Acad. Sci.
  • Single chain antibodies may be formed by linking heavy (V H ) and light (V L ) chain variable region (Fv region) fragments via an short peptide linker to provide a single polypeptide chain (scFvs).
  • the scFvs may also form dimers or trimers, depending on the length of a peptide linker between the two variable regions (Kortt et al, Protein Engineering 10:423, 1997).
  • Phage display is another well known recombinant method for producing the antigen-binding fragments of the present invention.
  • the antigen-binding fragments and antibody-derived polypeptides contemplated herein may be screened for desired properties using, for example, the assays described herein.
  • the assays described herein provide the means to identify antigen-binding fragments and antibody-derived polypeptides that bind to VEGF-A and to VEGF-B and antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR- 1
  • sequence conservative variants of the nucleic acids are provided which encode the instant antibodies.
  • sequence-conservative variants of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon results in no alteration in the amino acid encoded at that position.
  • Function-conservative variants of the antibodies of the invention are also contemplated herein.
  • “Function-conservative variants” are those in which one or more amino acid residues in a protein have been changed without altering the overall conformation and function of the protein, including, but, by no means, limited to, replacement of an amino acid with one having similar properties. Amino acids with similar properties are well known in the art.
  • polar/hydrophilic amino acids which may be interchangeable include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; nonpolar/hydrophobic amino acids which may be interchangeable include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are included herein which are encoded by nucleic acids also as described herein as well as nucleic acids which hybridize thereto.
  • the nucleic acids hybridize under low stringency conditions, particularly under moderate stringency conditions and particularly under high stringency conditions.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989 supra).
  • Typical low stringency hybridization conditions may be 55 0 C, 5 times SSC, 0.1% w/v SDS, 0.25% w/v milk, and no formamide; or 30% v/v formamide, 5 times SSC and 0.5% w/v SDS.
  • Typical moderate stringency hybridization conditions are similar to the low stringency conditions except the hybridization is carried out in 40% v/v formamide, with 5 or 6 times SSC.
  • High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50% v/v formamide, 5 or 6 times SSC and, optionally, at a higher temperature (e.g., 57 to 68 0 C).
  • SSC 0.15M NaCl and 0.015M Na-citrate.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook et al, 1989 supra).
  • oligonucleotides For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, 1989 supra).
  • nucleic acids comprising nucleotide sequences and polypeptides comprising amino acid sequences which are at least about 70% identical, particularly at least about 80% identical, particularly at least about 90% identical and particularly, at least about 95% identical to the nucleotide and amino acid sequences described herein, when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, and which nucleotide sequences encode polypeptides which have the biological activity of the antibodies encompassed herein.
  • Polypeptides comprising amino acid sequences which are at least about 70% similar, particularly at least about 80% similar, particularly, at least about 90% similar and particularly at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences described herein, when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, and which have the biological activity of the antibodies of the present invention, are also included in the present invention.
  • reference to "at least 70% identity” includes 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% identify.
  • Sequence identity refers to exact matches between the nucleotides or amino acids of two sequences which are being compared.
  • Sequence similarity refers to both exact matches between the amino acids of two polypeptides which are being compared in addition to matches between non-identical, biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.
  • BLAST ALGORITHMS Altschul et al, J. MoI. Biol. 275:403-410, 1990; Altschul et al, Nucleic Acids Res. 25:3389-3402; 1997; Altschul, J. MoI Biol. 219:555-565, 1991.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines may be selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention from host cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
  • IgGl or IgG4 monoclonal antibodies may be derived from an IgM monoclonal antibody, for example, and vice versa.
  • Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody.
  • Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g. DNA encoding the constant region of an antibody of the desired isotype.
  • chimeric VEGF-A cross-reactive anti-VEGF-B antibodies which comprise the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs :4 and 6), or heavy and light chain variable region sequences at least about 70% identical to the heavy and light chain sequences of SEQ ID NOs:4 and 6, fused to non-murine heavy and light chain constant domains.
  • non-murine heavy and light chain constant domains are human heavy and light chain constant domains.
  • humanized VEGF-A cross-reactive anti-VEGF-B antibodies comprise from one to all three of the CDR sequences from the light chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs:7 to 9), or one to three light chain CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs:7 to 9.
  • humanized VEGF-A cross-reactive anti-VEGF-B antibodies comprise from one to all three of the CDR sequences from the heavy chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 10 to 12) or or one to three heavy chain CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 10 to 12.
  • humanized VEGF-A cross-reactive anti-VEGF-B antibodies comprise one, two, three, four, five or all six CDR sequences from the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 7 to 12), or one, two, three, four, five or six heavy and light chain CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs:7 to 12.
  • nucleic acid sequences encoding chimeric VEGF-A cross-reactive anti-VEGF-B antibodies comprise the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 3 and 5), or heavy and light chain variable region sequences that are at least about 70% identical to the heavy and light chain sequences of SEQ ID NOs: 3 and 5, fused to non-murine heavy and light chain constant domains.
  • the non-murine heavy and light chain constant domains are human heavy and light chain constant domains.
  • nucleic acid sequences encoding humanized VEGF-A cross- reactive anti-VEGF-B antibodies are provided which comprise from one to all three of the CDR sequences from the light chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 15), or one to three CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 13 to 15.
  • nucleic acid sequences encoding humanized VEGF-A cross- reactive anti-VEGF-B antibodies comprise from one to all three of the CDR sequences from the heavy chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 16 to 18) or one to three CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 16 to 18.
  • nucleic acid sequences encoding humanized VEGF-A cross-reactive anti-VEGF-B antibodies comprise one, two, three, four, five or all six CDR sequences from the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 18) or one, two, three, four, five or six CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 13 to 18.
  • a method for the production of the VEGF-A cross-reactive anti-VEGF-B antibodies of the present invention, said method comprising cloning nucleic acid sequences encoding the VEGF-A cross-reactive anti-VEGF-B antibodies of the present invention into an appropriate vector, transforming a host cell line with the vector, and culturing the transformed host cell line under conditions suitable for the expression of the VEGF-A cross-reactive anti- VEGF-B antibodies.
  • Vectors available for cloning and expression in host cell lines are well known in the art, and include but are not limited to vectors for cloning and expression in mammalian cell lines, vectors for cloning and expression in bacterial cell lines, vectors for cloning and expression in phage and vectors for cloning and expression insect cell lines.
  • the antibodies can be recovered using standard protein purification methods.
  • a further embodiment is directed to vectors comprising nucleic acid sequences encoding chimeric VEGF-A cross-reactive anti-VEGF-B antibodies that comprise the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs :4 and 6) fused to non-murine heavy and light chain antibody constant domains.
  • vectors comprising nucleic acid sequences encoding chimeric VEGF-A cross-reactive anti-VEGF-B antibodies that comprise the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs:4 and 6), or heavy and light chain variable region sequences at least about 70% identical to the heavy and light chain sequences of SEQ ID NOs :4 and 6, fused to non-murine heavy and light chain constant domains.
  • the non-murine heavy and light chain constant domains are human heavy and light chain constant domains.
  • the vectors comprise nucleic acid sequences encoding humanized VEGF-A cross-reactive anti-VEGF-B antibodies that comprise from one to all three of the CDR sequences from the light chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 15).
  • the vectors comprise nucleic acid sequences encoding humanized VEGF-A cross-reactive anti- VEGF-B antibodies that comprise from one to all three of the CDR sequences from the light chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 15), or one to three CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 13 to 15.
  • the vectors comprise nucleic acid sequences encoding humanized VEGF-A cross-reactive anti- VEGF-B antibodies that comprise from one to all three of the CDR sequences from the heavy chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 16 to 18).
  • vectors comprising nucleic acid sequences encoding humanized VEGF- A cross-reactive anti- VEGF-B antibodies that comprise from one to all three of the CDR sequences from the heavy chain variable region of the murine monoclonal antibody 2E4 (SEQ ID NOs: 16 to 18) or one to three CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 16 to 18.
  • the vectors comprise nucleic acid sequences encoding humanized VEGF-A cross-reactive anti- VEGF-B antibodies that comprise one, two, three, four, five or all six CDR sequences from the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 18).
  • Vectors comprising nucleic acid sequences encoding humanized VEGF-A cross-reactive anti- VEGF -B antibodies are provided which comprise one, two, three, four, five or all six CDR sequences from the heavy and light chain variable regions of the murine monoclonal antibody 2E4 (SEQ ID NOs: 13 to 18) or one, two, three, four, five or six CDR sequences that are at least about 70% identical to the CDR sequences of SEQ ID NOs: 13 to 18.
  • Still a further embodiment is directed to host cell lines transfected or transformed with the subject vectors.
  • Procedures for generating human antibodies in non-human animals have been developed and are well known to those skilled in the art.
  • transgenic mice into which genetic material encoding one or more human immunoglobulin chains has been introduced may be used to produce the subject antibodies.
  • Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos.' 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
  • Phage display techniques for generating human antibodies are well known to those skilled in the art, and include the methods used by companies such as Cambridge Antibody Technology and MorphoSys and which are described in International PCT Publication Nos. WO 1992/01047; WO 1992/20791; WO 1993/06213 and WO 1993/11236.
  • the instant disclosure encompasses human VEGF-A cross-reactive anti- VEGF-B antibodies prepared using transgenic mice or by phage display.
  • human VEGF-A cross-reactive anti- VEGF-B antibodies are provided which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the human VEGF-A cross-reactive anti- VEGF-B antibodies are in isolated, homogenous or fully or partially purified form.
  • the human VEGF-A cross-reactive anti- VEGF-B antibodies may also be monoclonal antibodies, antigen-binding fragments or antibody-derived polypeptides.
  • Antibodies are also contemplated which compete with the VEGF-A cross-reactive anti- VEGF-B antibodies of the present invention for binding to VEGF-A and VEGF-B. Competition for binding to VEGF-A or VEGF-B may be assayed easily in vitro.
  • a VEGF-A cross-reactive anti- VEGF-B antibody of the present invention may be tagged with a specific reporter molecule and assayed in the presence of an untagged test antibody to determine if the untagged test antibody binds the same epitope or an overlapping epitope on VEGF-A and/or VEGF-B.
  • Competition may be determined using an ELISA, for example, in which VEGF-A or VEGF-B is immobilised to a plate and a tagged VEGF-A cross-reactive anti- VEGF-B antibody along with a test antibody is added to the plate. If the untagged antibody competes with the tagged VEGF-A cross-reactive anti- VEGF-B antibody for binding, a decrease in the signal emitted by the tagged antibody will be observed when compared to the control.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are provided which compete with the subject antibodies for binding to VEGF-A and VEGF-B.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are included which compete with the murine monoclonal antibodies 2E4, 2E9 and 2El 2, or with antigen-binding fragments and antibody-derived polypeptides of 2E4, 2E9 or 2El 2, for binding to VEGF-A and VEGF-B.
  • VEGF-A cross-reactive anti-VEGF-B antibodies are provided which compete with the murine monoclonal antibodies 2E4, 2E9 and 2El 2, or with antigen-binding fragments and antibody-derived polypeptides of 2E4, 2E9 or 2El 2, for binding to VEGF-A and VEGF-B, and which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • VEGF-A cross-reactive anti-VEGF-B antibodies which compete with the murine monoclonal antibody 2E4, or with antigen-binding fragments and antibody- derived polypeptides of 2E4, for binding to VEGF-A and VEGF-B.
  • VEGF-A cross-reactive anti-VEGF-B antibodies are provided which compete with the murine monoclonal antibody 2E4, or with antigen-binding fragments and antibody-derived polypeptides of 2E4, for binding to VEGF-A and VEGF-B, and which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR- 1.
  • the antibodies here may exhibit a binding affinity for human VEGF-A and human VEGF- B that is substantially equivalent to or greater than the binding affinity of a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2 for human VEGF-A and human VEGF-B.
  • Antibody affinities may be determined using a biosensor-based approach as described in the Examples section.
  • Antibodies include those which bind human VEGF-A and VEGF-B with a K D value of 1 x 10 "7 M or less; particularly 1 x 10 "8 M or less; particularly, 1 x 10 "9 M or less; and particulary 5 x 10 " M or less as determined by surface plasmon resonance.
  • the antibodies may antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I to substantially the same extent as that observed for a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2.
  • Such antagonist activity may be measured in any suitable assay (e.g. as measured in the VEGFR- l/EpoR/BaF3 proliferation assay described herein or in a reporter gene assay such as that described by Scotney et al, Clin Exp Pharmacol Physiol (Australia), 29(11): 1024- 1029, 2002.
  • Ba/F3 cells are transfected with a chimeric receptor incorporating the intracellular domain of the erythropoietin receptor (EpoR) and the extracellular domain of VEGFR-I or VEGFR-2.
  • EpoR erythropoietin receptor
  • VEGFR-I or VEGFR-2 extracellular domain of VEGFR-I or VEGFR-2.
  • VEGF-A or VEGF-B VEGF-A
  • proliferation of the cells occurs, as measured using an MTS dye reduction assay.
  • VEGF-A cross- reactive anti-VEGF-B antibodies that antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I will inhibit VEGF-A or VEGF-B induced cell proliferation in these assays.
  • VEGF-A cross-reactive anti-VEGF-B antibodies that antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I will inhibit VEGF-A or VEGF-B-mediated activation of the reporter molecule in a reporter gene based assay such as that described by Scotney et al, 2002 supra.
  • the cell based assays herein may be utilised as a basis for screening for modulators of VEGF-A and VEGF-B receptor interaction. While such methods are well known to those skilled in the art, a brief description of the method is provided herein.
  • the method involves subjecting appropriately engineered cells to a signal producing amount of VEGF-A or VEGF-B under conditions where, in the absence of any antagonism of VEGF-A or VEGF-B receptor binding, a signal, for example proliferation or reporter luciferase expression, may be detected.
  • the same experimental procedure is then conducted in the presence of one or more test compounds and the level of signal detected compared with that detected in the absence of a test compound.
  • Test compounds which alter the level of signal detected compared with that detected in the absence of a test compound are then selected for further study.
  • Test compounds may include phage display libraries of antibody variable regions and the like, or panels of monoclonal antibodies against VEGF-A and VEGF-B may be screened across the assay.
  • VEGF-A cross-reactive anti-VEGF-B antibodies in one embodiment bind to human VEGF-A and human VEGF-B.
  • VEGF-A cross-reactive anti-VEGF-B antibodies that bind to and antagonize human and other mammalian forms of VEGF-A and VEGF-B may be desirable and form another embodiment.
  • VEGF-A and VEGF-B Some level of binding with other mammalian forms of VEGF-A and VEGF-B may be desirable in certain circumstances, such as for example, for the purpose of testing antibodies in animal models for their affect on a particular disease and for conducting toxicology studies. Species where binding of an antibody to human VEGF-A and human VEGF-B may be desirable include mice, rats, dogs and monkeys.
  • a particular group of VEGF-A cross-reactive anti- VEGF-B antibodies are those that bind to human VEGF-A and human VEGF-B and which exhibit some level of binding to murine VEGF-A and murine VEGF-B.
  • Such antibodies bind with high affinity to human VEGF-A and human VEGF-B and to VEGF-A and VEGF-B from the other species, and antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • VEGF-A cross-reactive anti- VEGF-B antibodies that bind to VEGF-A and VEGF-B from two or more species and which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • VEGF-A cross-reactive anti- VEGF-B antibodies which bind to human VEGF-A and human VEGF-B and to murine VEGF-A and murine VEGF-B, and which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are in isolated, homogenous or fully or partially purified form.
  • VEGF-A cross-reactive anti-VEGF-B antibodies are monoclonal antibodies, antigen-binding fragments or antibody-derived polypeptides.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are chimeric antibodies, including humanized antibodies, or human antibodies. These include chimeric and humanized antibodies prepared, for example, from murine monoclonal antibodies, and human monoclonal antibodies which may be prepared, for example, using transgenic mice or by phage display.
  • the treatment of VEGF-A and VEGF-B associated diseases and disorders may be by the administration of pharmaceutical compositions comprising the VEGF-A cross-reactive anti- VEGF-B antibodies and one or more pharmaceutically acceptable carriers and/or diluents.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, agents used for adjusting tonicity, buffers, chelating agents, and absorption delaying agents and the like.
  • the use of such media and agents is well known in the art. Except insofar as any conventional media or agent is incompatible with the active form, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants.
  • the prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include agents to adjust tonicity, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. The compositions may also include buffers and chelating agents.
  • Sterile injectable solutions are prepared by incorporating the active form in the required amount in the appropriate solvent and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization.
  • suitable methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of active form plus any additionally desired ingredient.
  • the amount of active form in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • compositions of the present invention are useful in modifying VEGF-A or VEGF-B mediated conditions including but not limited to aberrant angiogenesis, tumors, in particular solid tumors, and any other VEGF-A or VEGF-B mediated diseases or conditions.
  • Pre-cancerous conditions and dispersed tumors such as for example, myelomas and lymphomas may also be treated.
  • the human and humanized antibodies presented herein and in particular humanized forms of the mAbs 2E4, 2E9, and 2El 2 are useful in the treatment of such conditions. Any adverse condition resulting from VEGF-A or VEGF-B interaction with VEGFR-I or VEGFR-2 may be treated by the administration of the antibodies of the present invention such as humanized forms of the mAbs selected from 2E4, 2E9, and 2El 2.
  • VEGF-A cross-reactive anti- VEGF-B antibodies are provided which antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the antibodies herein may be employed in vitro or in vivo to inhibit a biological activity that results from VEGF-A and VEGF-B signaling. Therefore, in an embodiment, the VEGF-A cross-reactive anti- VEGF-B antibodies may be used in therapeutic applications to treat diseases or conditions caused or exacerbated (directly or indirectly) by the signaling of VEGF-A through VEGFR-I, VEGF-A through VEGFR-2 or VEGF-B through VEGFR-I.
  • a therapeutic application involves in vivo administration of a VEGF-A cross-reactive anti-VEGF-B antibody described herein to a mammal in an amount effective to antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies are human or humanized monoclonal antibodies.
  • VEGF-A cross-reactive anti-VEGF-B antibodies may be used to treat diseases or conditions induced by VEGF-A and/or VEGF-B including but not limited to cancer, in particular tumors, more particularly solid tumors.
  • another embodiment contemplates a method for the treatment or prophylaxis of a condition mediated by VEGF-A or VEGF-B such as but not limited to a cancer, said method comprising administering to a subject in need a therapeutically effective amount of a VEGF-A cross-reactive anti-VEGF-B antibody, for example a humanized form of a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2, for a time and under conditions sufficient to antagonize one or more of VEGF-A signaling through VEGFR-I, VEGF-A signaling through VEGFR-2 and VEGF-B signaling through VEGFR-I.
  • a VEGF-A cross-reactive anti-VEGF-B antibody for example a humanized form of a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2
  • the type of tumors contemplated for treatment using the method and compositions disclosed herein include without being limited to breast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumor, hepatoma/cholongio, carcinoma, neuroendocrine tumors, pituitary neoplasm, small round cell tumor, squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors and Wilm's tumor.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the subject VEGF-A cross-reactive anti-
  • VEGF-B antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the invention may be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be by injection, preferably proximal to the site of the target (e.g., tumor). If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day.
  • the subject VEGF-A cross-reactive anti-VEGF-B antibodies are administered to a mammal, particularly a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the VEGF-A cross-reactive anti-VEGF-B antibodies also are suitably administered by intra tumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • the intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors.
  • the appropriate dosage of a VEGF-A cross-reactive anti- VEGF-B antibody depends on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody may be suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 ⁇ g/kg to about 20 mg/kg or more, depending on the factors mentioned above.
  • the treatment is repeated until a desired suppression of disease symptoms occurs. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
  • the subject is a human.
  • veterinary applications are also contemplated for livestock animals as well as companion animals. In such cases it would be necessary to prepare an appropriate antibody designed to avoid an immunogenic response to the antibody by the mammal.
  • a method for treating a VEGF-A or VEGF-B mediated disease or condition in a human subject comprising administering to said subject a therapeutically effective amount of a VEGF-A cross-reactive anti- VEGF-B antibody for a time and under conditions sufficient to produce a desired suppression of the disease or condition.
  • VEGF-A cross-reactive anti-VEGF-B antibody in the manufacture of a medicament in the treatment or prophylaxis of a VEGF-A or VEGF-B mediated disease in a subject.
  • VEGF-A cross-reactive anti- VEGF-B antibody in the manufacture of a medicament in the treatment or prophylaxis of cancer including tumor tissue and pre-cancerous tissue in a subject.
  • the VEGF-A cross-reactive anti- VEGF-B antibody is a monoclonal antibody.
  • the VEGF-A cross-reactive anti- VEGF -B antibodies are in isolated, homogenous or fully or partially purified form.
  • the VEGF-A cross-reactive anti- VEGF-B antibodies are human or humanized monoclonal antibodies suitable for use in human therapeutics. These include humanized antibodies prepared, for example, from murine monoclonal antibodies, and human monoclonal antibodies which may be prepared, for example, using transgenic mice or by phage display.
  • Particular antibodies for use in the above method are those which bind human VEGF-A or VEGF-B with a K D value of 1 x 10 "7 M or less; particularly 1 x 10 "8 M or less; particularly 1 x 10 "9 M or less; and particularly 5 x 10 "10 M or less.
  • a method for ameliorating the effects of VEGF-A or VEGF-B mediated cancer in a human subject comprising administering to said subject an effective amount of a humanized form of a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2 or an humanized or human antibody with equivalent VEGF-A or VEGF-B signaling-blocking activity for a time and under conditions sufficient to reduce the growth or spread of the cancer.
  • a humanized form of a murine monoclonal antibody selected from 2E4, 2E9, and 2El 2 or antibody with equivalent VEGF-A or VEGF-B signaling-blocking activity is also contemplated in the manufacture of a medicament for the treatment or prophylaxis of cancer in a subject.
  • VEGF-A and VEGF-B proteins were produced according to the general methodology previously described for human VEGF-B 1 O-I 0 S (SEQ ID NO:1) (Scotney et al, 2002 supra; Scrofani et al, Protein Science, 9:2018-2025, 2000). This is described below in more detail. Proteins produced using this methodology initially have an additional 16 amino acids at the N-terminus that incorporate a 6xHis tag and a Genenase I cleavage site between the tag and the start of the actual VEGF amino acid sequence. Where desired, these additional amino acids are removed by enzymatic cleavage using the general procedure outlined below.
  • 6xHis.hVEGF-B lo- io8 indicates a protein containing amino acids 10-108 of the mature human VEGF-B with the N-terminal tag and cleavage site.
  • hVEGF-B lo- io 8 indicates the same protein without the N-terminal tag and cleavage site.
  • Other proteins are represented using the same approach.
  • human VEGF-B 10-1O8 with a 6xHis tag and a Genenase I cleavage site (6xHis.hVEGF-Bio-io 8 ) was produced as a recombinant protein by bacterial fermentation following transformation with 6xHis.hVEGF-Bi O- io 8 -pET-15b (pET-15b vector from Novagen USA Cat # 70755-3) into the E. coli BL21-Codon-Plus-[DE3]-RP strain (Stratagene, USA).
  • samples were collected pre- and 1, 2 and 3 hrs post-induction with 1OnM IPTG and assessed by SDS-PAGE and Western blot using an anti-6xHis mAb (murine mAb, QIAGEN USA). Bound mAb was visualized by autoradiography using HRP- conjugated secondary reagent (sheep anti-mouse antibody, Chemicon USA) together with a luminescence substrate (PerkinElmer USA). Following cell lysis 6xHis.hVEGF-Bio.ios was purified from E.
  • the fractions containing predominantly dimeric material were pooled for assaying.
  • Western blot analysis (SDS-PAGE under reducing conditions) was performed on the RP-HPLC fractions using an anti-6xHis mAb. Bound mAb was visualized by autoradiography using a HRP-conjugated secondary reagent together with a luminescence substrate.
  • the N-terminal 6xHis tag was removed from human VEGF-A and -B preparations by enzymatic cleavage as follows. Briefly, 5mg of lyophilised tagged protein was resuspended in a minimum volume of 1 mM acetic acid (500 ⁇ l) and IOOX volume (50 ml) of Genenase I digest solution (5 ⁇ g/ml Genenase I, 100 mM Tris-HCl, 5 mM CaCl 2 , 200 mM NaCl, 0.02% v/v Tween-20) was added. The material was incubated for 24 hrs at 21 0 C with gentle mixing before being dialysed into 0.1 M acetic acid to stop the reaction.
  • Genenase I digest solution 5 ⁇ g/ml Genenase I, 100 mM Tris-HCl, 5 mM CaCl 2 , 200 mM NaCl, 0.02% v/v Tween-20
  • the digested material was then purified by reverse phase chromatography and stored as lyophilised aliquots as previously described (Scotney et al, 2002 supra). Analysis of the activity of purified, refolded hVEGF-A & -B was determined using the VEGFR- l/EpoR/BaF3 cell proliferation assay as described in Example 3. The data shown in Figure 1 demonstrate dose dependant proliferation in response to recombinant hVEGF- A 1 J-1O T and hVEGF-B 10-108 proteins.
  • Monoclonal antibodies were raised against recombinant human VEGF-B t o-ios using standard procedures (see Harlow and Lane, Antibodies - A Laboratory Manual, Cold
  • mice were immunized via the intraperitoneal (i.p) route with 20 ⁇ g of hVEGF-Bio-ios emulsified in Complete
  • CFA Freunds Adjuvant
  • Incomplete Freunds Adjuvant for subsequent immunizations (at least 2, and no less than 3-4 weeks apart).
  • PBS phosphate buffered saline
  • hybridomas secreting mAbs that bound to hVEGF-B were identified by ELISA and, after expansion, cloned by limit dilution on at least two occasions.
  • the anti-VEGF-B mAbs raised against hVEGF-B were tested for cross-reactivity with human VEGF-A using a standard ELISA format.
  • the anti-VEGF-B mAbs were incubated with plate bound hVEGF-A ⁇ -109, hVEGF-B lo- i O 8 and hPIGF at a concentration of 1 ⁇ g/ml and bound mAb was visualized using a HRP-conjugated secondary reagent and TMB substrate (Chemicon International Inc, USA).
  • Results demonstrate that the hVEGF-A specific mAb 12/05-5C9 did not cross react with hVEGF-B and hPIGF and that the hVEGF-B specific mAb 2C/99-2H10 did not cross react with hVEGF-A and hPIGF (see Figure 2).
  • Three anti-VEGF-B mAbs, 11/05-2E4, 11/05-2E9 & 11/05-2E12 were identifed that cross reacted with hVEGF-A, and to a much lesser extent, hPIGF (see Figure 2).
  • the cross-reactivity of the anti-VEGF-B mAbs for VEGF-A was further verified by Western blot analysis.
  • hVEGF-A 11-109 and hVEGF-B 10-108 were run on a reducing SDS-PAGE and transferred to PVDF membrane using standard procedures. Blots were probed with the test mAbs, 11/05-2E4, 11/05-2E9 and 11/05-2E12, or with a negative control mAb (C44) at a concentration of 5 ⁇ g/ml. The bound mAbs were visualized by autoradiography using a HRP-conjugated secondary reagent together with a luminescence substrate. The three test mAbs bound both human VEGF-A 1 I-10 Q and human VEGF-B 10-108 (see Figure 3).
  • Human VEGF-A/B cross-reactive mouse monoclonal antibodies antagonise the activity of VEGF-A & VEGF-B in cell-based assays
  • VEGF receptor 1 VEGF receptor 1
  • VEGFR-2 VEGF receptor 2 proliferation assays.
  • VEGF receptor 1 VEGF receptor 1
  • VEGFR-2 VEGF receptor 2 proliferation assays.
  • EpoR erythropoietin receptor
  • VEGF receptor ligands such as VEGF-A and VEGF-B trigger dimerisation of the chimeric receptor - the subsequent phosphorylation of the cytoplasmic domain of the EpoR leads to the activation of downstream signal transduction molecules and proliferation of the BaF3 cells.
  • This approach has been used to develop proliferation-based biological assays for a number of cytokines and growth factors (for examples see Murayama et al, J. Biol, Chem., 269:591 '6-5980, 1994; Fukada et al, Immunity 5:449-460, 1996; Stacker et al, J. Biol. Chem. 274:34884-34892, 1999).
  • the engineered BaF3 cells are maintained in DMEM (Invitrogen, USA) supplemented with 10% v/v FCS, pencillin (50 units/ml, Invitrogen, USA), streptomycin (50 ⁇ g/ml, Invitrogen, USA) and GlutaMAX-I (2 mM, Invitrogen, USA).
  • VEGFR l/EpoR/BaF3 cells are maintained with the addition of Zeocin (250 ⁇ g/ml, Invitrogen, USA) and hVEGF-Ai 65 (50 ng/ml), and the VEGFR2/EpoR/BaF3 cells are maintained with the addition of mouse IL3 (5 ng/ml, R&D Systems, USA) and G418 (1 mg/ml, Invitrogen, USA).
  • VEGFRl/EpoR/BaF3 or VEGFR2/EpoR/BaF3 cells 6xlO 4 cells/well, flat bottom, 96 well, microtitre plates
  • proliferation was assessed using an MTS dye reduction assay (Mosmann, J Immunol Methods 55:55-63, 1983).
  • Results presented in Figure 4 demonstrate with a dose response that the hVEGF-B raised mAb 11/05-2E12 was able to antagonise the activity of !1VEGF-A 1 j.i O9 in the hVEGFRl/EpoR/BaF3 cell base assay.
  • the same 11/05-2E12 mAb also demonstrates in Figure 5 a dose response partial antagonism of the activity of hVEGF-Bi 0- i 08 in the hVEGFRl/EpoR/BaF3 cell base assay.
  • Results presented in Figure 6 demonstrate with a dose response that the hVEGF-B raised mAb 11/05-2E4 was able to antagonise the activity of hVEGF -A 11-1O g in the hVEGFR2/EpoR/BaF3 cell base assay.
  • the 11/05-2E9 mAb demonstrates in Figure 7 a dose response antagonism of the activity of !1VEGF-A 1 ⁇ 09 in the hVEGFR2/EpoR/BaF3 cell base assay.
  • Biosensor-based approach was used for kinetic analysis of mAb binding to target VEGF-A & -B using a BIAcore (Trademark) 2000 surface plasmon resonance instrument (Biacore AB, Uppsala, Sweden).
  • a biosensor chip was activated for covalent coupling of VEGF-A or -B using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's (Biacore AB, Uppsala, Sweden) instructions.
  • EDC N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • test antibody For kinetics measurements, two-fold serial dilutions of test antibody starting at 313 nM to 2.4 nM were injected in BIAcore (Trademark Biacore, AB, Uppsala, Sweden) buffer (2OmM HEPES, pH 7.8, 0.15M NaCl, 3.4mM EDTA, 0.005% v/v Tween-20) with lmg/ml bovine serum albumin (BSA) at 25°C at a flow rate of 15 ⁇ l/min.
  • BIAcore Trademark Biacore, AB, Uppsala, Sweden
  • BSA bovine serum albumin

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Abstract

La présente invention concerne d'une manière générale des anticorps anti-VEGF-B (facteur de croissance-B des cellules endothéliales vasculaires) et, en particulier, des anticorps anti-VEGF-B qui se lient au VEGF-A (facteur de croissance-A des cellules endothéliales vasculaires) et qui contrarient les signaux déclenchés par le VEGF-A et le VEGF-B. Les anticorps de la présente invention se révèlent utiles dans le traitement ou la prévention de maladies ou de signes cliniques induits par le VEGF-A ou le VEGF-B.
PCT/AU2007/000799 2006-06-08 2007-06-06 Anticorps vegf-a anti-vegf-b dotés d'une réactivité croisée utilisés comme antagonistes des signaux vegf-a et vegf-b WO2007140534A1 (fr)

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Cited By (18)

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AU2009228158B2 (en) * 2008-03-27 2014-02-27 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRbeta and VEGF-A
AU2014202867B2 (en) * 2008-03-27 2016-11-10 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRbeta and VEGF-A
US9708390B2 (en) 2008-03-27 2017-07-18 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRbeta and VEGF-A
US20110177074A1 (en) * 2008-03-27 2011-07-21 Sivakumar Pallavur V Compositions and methods for inhibiting pdgfrbeta and vegf-a
US9441034B2 (en) 2008-03-27 2016-09-13 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRβ and VEGF-A
EP2604279A1 (fr) * 2008-03-27 2013-06-19 ZymoGenetics, Inc. Compositions et procédés pour inhiber PDGFR-bêta et VEGF-A
WO2009120922A3 (fr) * 2008-03-27 2009-12-17 Zymogenetics, Inc. Compositions et procédés pour inhiber pdgfr-bêta et vegf-a
WO2009120922A2 (fr) 2008-03-27 2009-10-01 Zymogenetics, Inc. Compositions et procédés pour inhiber pdgfr-bêta et vegf-a
EP3722310A1 (fr) * 2008-06-25 2020-10-14 Novartis AG Anticorps stables et solubles inhibant le vegf
EP3216803A1 (fr) * 2008-06-25 2017-09-13 ESBATech, an Alcon Biomedical Research Unit LLC Anticorps stables et solubles inhibant le vegf
WO2010005527A1 (fr) 2008-06-30 2010-01-14 Angioblast Systems, Inc. Traitement de maladies oculaires et d’une néovascularisation excessive utilisant un traitement combiné
WO2012172054A1 (fr) * 2011-06-16 2012-12-20 Scil Proteins Gmbh Protéines d'ubiquitine multimères modifiées se liant au vegf-a
US10543270B2 (en) 2013-12-18 2020-01-28 Csl Limited Methods of treating wounds in a diabetic subject
WO2015089585A1 (fr) * 2013-12-18 2015-06-25 Csl Limited Procédé de traitement de plaies
RU2672377C1 (ru) * 2013-12-18 2018-11-14 СиЭсЭл ЛИМИТЕД Способ лечения ран
WO2015166112A1 (fr) * 2014-05-01 2015-11-05 Consejo Nacional De Investigaciones Científicas Y Técnicas (Conicet) Anticorps anti-vegf humain avec affinité de liaison inhabituellement forte pour le vegf-a humain et une réactivité croisée pour le vegf-b humain
CN107108727A (zh) * 2014-05-01 2017-08-29 科学和技术研究全国委员会（科尼特） 对人vegf‑a有异常强的结合亲和力并具有与人vegf‑b的交叉反应性的抗人vegf抗体
US10308943B2 (en) 2016-02-08 2019-06-04 Vitrisa Therapeutics, Inc. Compositions with improved intravitreal half-life and uses thereof

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