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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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  • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
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  • A61P35/00—Antineoplastic agents
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • 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
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/55—Fab or Fab'
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/626—Diabody or triabody
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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  • 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
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  • 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
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  • C07—ORGANIC CHEMISTRY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the present invention is directed to novel antibodies, which comprise single domain binding sites.
• the antibodies can be bivalent or multivalent, and can be bispecif ⁇ c or multispecific. Also disclosed are bispecific and multi specific antibodies that bind to cell surface receptor proteins. When administered to a subject alone or in combination with anti- angiogenic or anti-neoplastic drugs, the antibodies can be used to inhibit tumor growth as well as to inhibit inhibit hyperproliferative diseases.
• Bispecific antibodies are immunoglobulin (Ig)-based molecules that bind to two different epitopes on either the same or distinct antigens. Both laboratory and early clinical studies have demonstrated that BsAb may have significant applications in cancer therapy, for example by targeting tumor cells with cytotoxic agents such as effector cells, radionuclides, drugs and toxins (Weiner et al., 1997, Cancer Immunol. Immunother. 45:190-2.; van Spriel et al., 2000, Immuol. Today 21:391-7; Segal et al., 2000, J. Immunol.
• Methods 248: 1-6. or by simultaneously targeting two different tumor targets (or epitopes) in order to enhance the biological activities of individual antibody therapeutics (Lu et al., 1999, J. Immunol. Methods 230:159-71; Lu et al., 2001, Cancer Res. 61:7002-8.; Lu et al., 2002, J. Immunol. Methods 267:213-26).
• a major obstacle in the development of BsAb-based therapeutics has been the difficulty in producing the materials in sufficient quantity and quality for clinical studies via traditional methods, including the hybrid hybridoma and chemical conjugation (Carter et al., 1995, J. Hematotherapy 4:463-70).
• BsAbs have also been constructed by genetically fusing two single chain Fv (scFv) or Fab fragments with or without the use of flexible linkers (Mallender et al., 1994, J. Biol. Chem. 269:199-206; Mack et al., 1995, Proc. Natl. Acad. Sci. USA.
• the invention provides novel bispecif ⁇ c antibodies which comprise single variable domain (sVD) antigen binding sites.
• the antibodies of the invention can also include antigen binding sites that comprise antibody Fvs in addition to sVD antigen binding sites.
• Antibodies of the invention can be specific for any antigen.
• an antibody of the invention binds to a cell surface antigen, which can be a receptor tyrosine kinase (including, but not limited to PDGFR, VEGFRl, VEGFR2, EGFR).
• a cell surface antigen which can be a receptor tyrosine kinase (including, but not limited to PDGFR, VEGFRl, VEGFR2, EGFR).
• an antibody of the invention binds to a ligand of a cell surface receptor.
• an antibody of the invention has receptor neutralizing activity.
• the invention further provides pharmaceutical compositions of the antibodies.
• the present invention provides methods of inhibiting activation of one or more receptor tyrosine kinases.
• Tumor growth in a mammal can be treated or inhibited by administering to the mammal an effective amount of a present antibody.
• the present antibodies can be coadministered with antibodies that bind to other cell surface antigen (including, e.g., RTKs) or cytokines (including, e.g., RTK ligands).
• the methods also comprise administering to the mammal an anti-neoplastic agent or treatment, including, for example, a chemotherapeutic agent and/or radiation.
• the invention provides a method of treating a non-cancer hyperproliferative disease, e.g., psoriasis, in a mammal.
• FIG. 1 is a schematic diagram depicting examples of single domain antibody-based bispecif ⁇ c antigen binding proteins of the invention.
• the single domain (designatetd V H 2 in the drawing) can be incorporated by fusion to the N-terminus or C- terminus of other antibody domains.
• FIG. 2 depicts expression and purification of mPDGFR ⁇ -specific Fabs.
• the Fabs were expressed in E. coli host HB2151 cells, purified by affinity chromatography, and analyzed by SDS-PAGE.
• Molecular weight markers are in kilodaltons.
• FIG. 3 depicts binding and blocking assays of purified Fabs.
• Various amounts of Fabs were incubated with a fixed amount of mPDGFR ⁇ /Fc in solution. The mixtures were incubated in 96-well plates coated with PDGF-AA followed by anti- human-Fc antibody-HRP conjugate. Bound mPDGFR ⁇ was then quantified by addition of peroxidase substrate, and absorbance was read at A450 nm. Data shown represent the mean ⁇ SD of duplicate samples.
• Figure 4 depicts structures of monovalent and bivalent Fabs of the invention.
• FIG. 5 depicts expression and purification of bivalent mPDGFR ⁇ -specific Fabs.
• the Fabs were expressed in E. coli host HB2151 cells, purified by affinity chromatography, and analyzed by SDS-PAGE.
• Bivalent 1F2-2H Fab is compared with a standard Fab (5C5) under non-reducing conditions.
• Molecular weight markers are in kilodaltons.
• Figure 6 depicts binding and blocking assays of purified antibodies.
• Figure 7 provides a schematic representation of the lF2-based full length IgGs.
• the 1F2 VH is expressed as a fusion protein to CL and/or CH, providing tetravalent (panel A; MJ 1 F-ISO 5 OOO) or bivalent (panels B and C: MFF ⁇ 125,000) antibodies.
• Figure 8 depicts expression and purification of anti-mPDGFRo; antibodies. In the right panel, antibodies were treated with DTT prior the electrophoresis.
• Figure 9 depicts binding and ligand blocking assays for purified antibodies.
• mPDGFRo/Fc Various amounts of antibodies were incubated with a fixed amount of mPDGFRo/Fc and transferred to plates precoated with PDGF-AA. Plates were washed and incubated with an anti-human- Fc antibody-HRP conjugate. Bound mPDGFRo: was then quantified by addition of peroxidase substrate, and absorbance was read at ⁇ 4450 nm. Data represent the mean ⁇ SD of duplicate samples. 2B4 is an antibody directed against mouse VEGFR2 (Flkl).
• FIG. 10 provides a schematic representation of selected lF2-based tetravalent bispecific antibodies that bind to mPDGFRo; and flk-1.
• 1F2 V H is expressed as a fusion to C L
• 2B4 is expressed as a scFv fusion to C H -
• 1F2 V H is expressed as a fusion at the amino terminus of 2B4 V L -
• Figure 11 depicts expression and purification of the 1F2 -based bispecific antibodies.
• the antibodies were expressed in mammalian cells, purified by affinity chromatography, and analyzed by SDS-PAGE. The antibodies were treated with/without (+/-) DTT prior the electrophoresis. Molecular weight markers are in kilodaltons.
• Figure 12 depicts quantitative binding assays of purified anti-mPDGFRo: x anti-Flk-1 bispecific antibodies.
• Figure 13 depicts blocking assays of purified bispecific anti-mPDGFR ⁇ x anti- FIk-I bispecific antibodies.
• various amounts of antibodies were incubated with a fixed amount of receptor CmPDGFRo ⁇ Fc or Flk-1) in solution and transferred to plates precoated with ligand (PDGF-AA or VEGFl 65). The plates were then incubated with an anti-human-Fc antibody-HRP conjugate.
• Bound mPDGFRa was then quantified by addition of peroxidase substrate, and absorbance was read at A450 run. Data shown represent the mean ⁇ SD of duplicate samples.
• Figure 14 depicts inhibition of PDGF and VEGF-stimulated receptor phosphorylation by the bispecific lF2-2B4IgG.
• eEnd.l cells were first incubated with various antibodies at 37°C for 30 min, followed by stimulation with VEGF or PDGF at 37°C for 15 min. After cell lysis, the receptors were immunoprecipitated from the cell lysate supernatant by incubation with an anti-mPDGFR ⁇ . or an anti-mVEGFR2 antibody, followed by ProA/G-sepharose beads. The precipitated receptor proteins were resolved on a 4-12% Nupage Bis-Tris gel and transferred to a polyvinylidene difluoride membrane.
• Phospho- mVEGFR2 and phospho-mPDGFR ⁇ were detected on the blot using an anti-phospho- tyrosine antibody-HRP conjugate.
• Total receptor proteins loaded on the gel were assayed with antibodies to mPDGFR ⁇ or mVEGFR2.
• Figure 15 depicts the amino acid sequences of V H and V L domains of several identified Fab proteins.
• Fabs IElO, IAl 1, 3B2, IClO, 3G7, 1F9, and 1F2 were identified in a screen for Fab-phage that bind to murine PDGFROL Fab 2B4 was identified in a screen for Fab-phage that bind to murine VEGFR2.
• ⁇ frame-shift mutation
• * stop codon resulting from frame-shift mutation @...@: in-frame deletion.
• the invention provides novel antibodies that comprise single variable domain (sVD) antigen binding sites.
• the invention also provides antigen binding proteins which comprise both single domain antigen binding sites and two-domain, Fv-like antigen binding sites.
• Single domain antigen binding sites are similar to immunoglobulin variable domains (e.g., V H or V L ) but are capable of specific binding to antigen in the absence of a second antigen binding domain (e.g., a counterpart V L or V H ). Such single domains may in fact be incapable of stable association with counterpart binding domains.
• single domain antibodies can bind antigen with affinities and avidities similar to those of antibodies that include both V L and V H domains.
• single domain antibodies can block receptor-ligand interactions. Accordingly, antibodies of the invention that comprise single domain binding sites are used for the same purposes as antibodies obtained from, for example, hybridomas, transgenic mice expressing human antibodies, and phage display libraries of Fab or scFv binding domains.
• Antibodies of the invention are immunoglobulin-like in several respects. 1) antibodies of the invention that are IgG-like are heterotetramers, consisting of two of each of two dissimilar polypeptide chains. 2) The dissimilar polypeptide chains associate and may be covalently linked, for example by disulfide bonds in same manner of heavy and light chain constant regions of standard, naturally occurring immunoglobulins. 3) One of the polypeptide chains is capable of stable self association in the manner of naturally occurring immunoglobulin heavy chains. For example, one of the polypeptide chains can include one or more domains corresponding to Q H 2, C H 3, or C H 4 of a naturally occurring immunoglobulin. In a preferred embodiment, an antibody of the invention comprises the constant domain structure of a naturally occurring IgG.
• Antigen-binding proteins of the invention comprise antigen binding sites that are single variable domains (sVDs).
• an sVD binding site substitutes for one variable domain of an IgG-like antibody, and a scFv substitutes for the other variable domain. Examples of such embodiments are depicted in Fig. IA.
• an sVD binding site is grafted to an IgG antibody at the N terminus or C terminus of the IgG heavy or light chain. Examples of such embodiments are depicted in Fig. IB.
• the sVDs can also be incorporated into an immunoglobulin-like antibody at other positions.
• the sVDs can be attached at the C terminus of C H and/or C L constant domains.
• substitutions of s VD binding sites as in Fig. 1 with additions of sVD binding sites to the C terminus of C H and/or C L constant domains, IgG-like antibodies that are multispecific and/or multivalent for a particular antigen can be produced.
• an Fv consists of two domains (e.g., a V H and a V L domain).
• V H and V L domains of the Fv though associated to form an antigen binding site, are not directly linked, but are each joined to linked antibody constant regions.
• the V H and V L domains of the Fv can also be joined with a synthetic linker to form a single chain Fv (scFv).
• Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. Bispecific antibodies (BsAbs) are antibodies which have two different antigen-binding specificities or sites. Where an antigen-binding protein has more than one specificity, the recognized epitopes may be associated with a single antigen or with more than one antigen.
• a natural antibody molecule is composed of two identical heavy chains and two identical light chains. Each light chain is covalently linked to a heavy chain by an interchain disulfide bond. The two heavy chains are further linked to one another by multiple disulfide bonds at the hinge region. The individual chains fold into domains having similar sizes (about 110-125 amino acids) and structures, but different functions.
• the light chain comprises one variable domain (V L ) and one constant domain (C L ).
• the heavy chain comprises one variable domain (V H ) and, depending on the class or isotype of antibody, three or four constant domains (C H I, C H 2, C H 3 and C H 4).
• the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes.
• the portion of an antibody consisting of V L and V H domains is designated "Fv" and constitutes the antigen-binding site.
• a single chain Fv (scFv) is an engineered protein containing a V L domain and a V H domain on one polypeptide chain, wherein the N terminus of one domain and the C terminus of the other domain are joined by a flexible linker.
• Fab refers to the portion of the antibody consisting of V L , V H , C L and CHI domains.
• variable domains show considerable amino acid sequence variablity from one antibody to the next, particularly at the location of the antigen binding site.
• Three regions, called “hypervariable” or “complementarity-determining regions” (CDRs) are found in each of V L and V H -
• Fc is the designation for the portion of an antibody which comprises paired heavy chain constant domains, hi an IgGi antibody, for example, the Fc comprises C H 2 and C H 3 domains.
• the Fc of an IgA or an IgM antibody further comprises a C H 4 domain.
• the Fc is associated with Fc receptor binding, activation of complement-mediated cytotoxicity and antibody-dependent cellular-cytotoxicity.
• complex formation requires Fc constant domains.
• the "hinge” region separates the Fab and Fc portions of the antibody, providing for mobility of Fabs relative to each other and relative to Fc, as well as including multiple disulfide bonds for covalent linkage of the two heavy chains.
• Antibodies of the invention have a combination of desirable features. First, they are essentially homogeneous. By design, m ⁇ spairing of antibody heavy and light chains is greatly reduced or eliminated. For example, some bispecific antibodies make use of two different heavy chains to provide two specificities. Four combinations are possible when such heavy chains are arranged into an IgG type molecule. Two of those consist of mispaired heavy chains such that the product is monospecific. In antibodies of the invention, mispairing is substantially eliminated.
• a second feature of the antibodies of the invention is that they are bivalent for each binding specificity. Many bispecific antibodies are monovalent for each of the antibody binding sites that are comprised. This is significant for antibody function because bivalency allows for cooperativity of binding and a significant increase in binding avidity with resepect to a molecule comprising a single antigen-specific binding site.
• a third advantage of the antibodies of the invention is that one or more heavy chain constant domains which constitute the Fc region (e.g., C H 2 and/or C H 3 for an IgGi molecule) of a natural antibody and which provide other antibody functions are present. Furthermore, the multiple binding domains are separated from the constant domains such that functions provided by the constant domains are not impaired.
• the Fc region e.g., C H 2 and/or C H 3 for an IgGi molecule
• Constant domain functions include binding to certain accessory molecules (e.g., binding to cell surface and soluble Fc receptors, J chain association for IgA and IgM, S protein for IgA), activation of the complement pathway (complement dependent cytoxicity, CDC), recognition of antibody bound to target cells by several different leukocyte populations (antibody-dependent cell- mediated cytoxicity, ADCC) and opsonization (enhancement of phagocytosis). Also, the Fc heavy chain constant domain(s) can confer increased serum half-life.
• accessory molecules e.g., binding to cell surface and soluble Fc receptors, J chain association for IgA and IgM, S protein for IgA
• activation of the complement pathway complement dependent cytoxicity, CDC
• recognition of antibody bound to target cells by several different leukocyte populations antibody-dependent cell- mediated cytoxicity, ADCC
• opsonization enhancement of phagocytosis
• the Fc heavy chain constant domain(s) can confer increased serum half-
• a fourth advantage of proteins of the invention is that there is no requirement for processing in vitro to obtain the complete product. Though rearranged in an artificial manner, each of the domains has a natural character which allows expression in a biological system.
• bispecific antibodies can be expressed in prokaryotic and eukaryotic expression systems.
• the proteins that are produced are substantially bispecific.
• sVD antigen binding sites and Fv region-containing binding sites for use in an antibody of the invention can be obtained by a variety of methods.
• the amino acid sequences of the V H and/or V L portions of a selected binding domain can be obtained from a naturally- occurring antibody or are chosen or be modified, screened, or selected for desired binding characteristics.
• V H and/or V L domains can be obtained directly from a monoclonal antibody which has the desired binding characteristics.
• V H and/or V L domains can be from libraries of V gene sequences from a mammal of choice. Elements of such libraries express random combinations of V H and/or V L domains and are screened with any desired antigen to identify those elements which have desired binding characteristics.
• V H and/or V L domains from a selected non-human source may be incorporated into chimeric antibody which comprises human constant domains.
• V H and/or V L domains from a selected non-human source may be incorporated into chimeric antibody which comprises human constant domains.
• chimeric antibody which comprises human constant domains.
• human constant domains are preferred.
• V domain can be made that is "humanized.”
• Humanized variable domains are constructed in which amino acid sequences which comprise one or more complementarity determining regions (CDRs) of non-human origin are grafted to human framework regions (FRs).
• CDRs complementarity determining regions
• FRs human framework regions
• Variable domains have a high degree of structural homology, allowing easy identification of amino acid residues within variable domains which corresponding to CDRs and FRs. See, e.g., Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest. 5th ed. National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD. Thus, amino acids which participate in antigen binding are easily identified.
• methods have been developed to preserve or to enhance affinity for antigen of humanized binding domains comprising grafted CDRs.
• One way is to include in the recipient variable domain the foreign framework residues which influence the conformation of the CDR regions.
• a second way is to graft the foreign CDRs onto human variable domains with the closest homology to the foreign variable region.
• CDRs are most easily grafted onto different FRs by first amplifying individual FR sequences using overlapping primers which include desired CDR sequences, and joining the resulting gene segments in subsequent amplification reactions. Grafting of a CDR onto a different variable domain can further involve the substitution of amino acid residues which are adjacent to the CDR in the amino acid sequence or packed against the CDR in the folded variable domain structure which affect the conformation of the CDR.
• Humanized variable domains of the invention therefore include human domains which comprise one or more non-human CDRs as well as such domains in which additional substitutions or replacements have been made to preserve or enhance binding characteristics.
• An antibody of the invention may also employ variable domains which have been made less immunogenic by replacing surface-exposed residues so as to make the antibody appear as self to the immune system (Padlan, E. A., 1991, MoI. Immunol. 28, 489- 98). Antibodies have been modified by this process with no loss of affinity (Roguska et al., 1994, Proc. Natl. Acad. ScL USA 91, 969-973). Because the internal packing of amino acid residues in the vicinity of the antigen binding site remains unchanged, affinity is preserved. Substitution of surface-exposed residues according to the invention for the purpose of reduced irnmunogenicity does not mean substitution of CDR residues or adjacent residues which influence binding characteristics.
• variable domains that are essentially human.
• Human binding domains can be obtained from phage display libraries wherein combinations of human heavy and light chain variable domains are displayed on the surface of filamentous phage (See, e.g., McCafferty et al., 1990, Nature 348, 552-54; Aujame et al., 1997, Human Antibodies 8, 155-68). Combinations of variable domains are typically displayed on filamentous phage in the form of Fabs or scFvs. The library is screened for phage bearing combinations of variable domains having desired antigen binding characteristics. Preferred single domain and variable domain combinations display high affinity for a selected antigen and little cross-reactivity to other related antigens.
• human binding domains can be obtained from transgenic animals into which unrearranged human Ig gene segments have been introduced and in which the endogenous mouse Ig genes have been inactivated (reviewed in Br ⁇ ggemann and Taussig, 1997, Curr. Opin. Biotechnol. 8, 455-58).
• Preferred transgenic animals contain very large contiguous Ig gene fragments that are over 1 Mb in size (Mendez et al., 1997, Nature Genet. 15, 146-56) but human Mabs of moderate affinity can be raised from transgenic animals containing smaller gene loci ⁇ See, e.g., Wagner et al., 1994, Eur. J. Immunol. 42, 2672-81; Green et al., 1994, Nature Genet. 7, 13-21).
• the sVD binding sites can be obtained from antigen specific Fv regions (which comprise both V H and V L domains). Often, it can be shown that the binding affinity and specificity of an Fv region is contributed primarily by one of the variable domains. Alternatively, the scFv can be obtained directly.
• Direct sources of sVDs include mammals ⁇ e.g., camelids) that naturally express antibodies containing only V H domain and phage display libraries constructed to express only a single variable domain.
• a human domain antibody phage display library is commercially available from Domantis (Cambridge, UK).
• sVD binding sites specific for PDGFR ⁇ were obtained from an Fab library containing variants in which the V L domain had been spontaneously deleted.
• binding domains of the invention include those for which binding characteristics have been improved by direct mutation or by methods of affinity maturation. Affinity and specificity may be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics ⁇ See, e.g., Yang et al., 1995, J. MoI. Bio. 254, 392-403).
• amino acid residues that are primary determinants of binding of single domain antibodies can be within Kabat defined CDRs, but may include other residues as well, such as, for example, residues that would otherwise be buried in the V H -V L interface of a V H -V L heterodimer.
• CDRs or other residues that determine binding are mutated in a variety of ways. One way is to randomize individual residues or combinations of residues so that in a population of otherwise identical antigen binding sites, all twenty amino acids, or a subset thereof, are found at particular positions. Alternatively, mutations are induced over a range of CDR residues by error prone PCR methods (See, e.g., Hawkins et al., 1992, J. MoI.
• Phage display vectors containing genes encoding binding domains can be propagated in mutator strains of E. coli (See, e.g., Low et al., 1996, J. MoI. Bio. 250, 359-68). These methods of mutagenesis are illustrative of the many methods known to one of skill in the art.
• Each variable domain of the antibodies of the present invention may be a complete immunoglobulin heavy or light chain variable domain, or it may be a functional equivalent or a mutant or derivative of a naturally occurring domain, or a synthetic domain constructed, for example, in vitro using a technique such as one described in WO 93/11236 (Medical Research Council / Griffiths et al.). For instance, it is possible to incorporate domains corresponding to antibody variable domains which are missing one or more amino acids.
• the important characterizing feature is the ability of each variable domain to associate with a complementary variable domain to form an antigen binding site.
• Antigen-binding proteins of the invention have binding sites for any epitope, antigenic site or protein.
• antibodies that are useful for treatment of disease.
• Preferred antibodies neutralize receptor proteins, such as receptors which are involved in angiogenesis and/or oncogenesis.
• Neutralizing a receptor means inactivating the intrinsic kinase activity of the receptor to transduce a signal.
• a reliable assay for receptor neutralization is the inhibition of receptor phosphorylation.
• the present invention is not limited by any particular mechanism of receptor neutralization. Some possible mechanisms include preventing binding of the ligand to the extracellular binding domain of the receptor, and preventing dimerization or oligomerization of receptor. Other mechanisms cannot, however, be ruled out.
• Neutralization of activation of a receptor in a sample of endothelial or non- endothelial cells, such as tumor cells may be performed in vitro or in vivo.
• Neutralizing activation of a receptor in a sample of receptor expressing cells comprises contacting the cells with an antibody of the invention.
• the cells are contacted with the antibody before, simultaneously with, or after, adding VEGF to the cell sample.
• an antibody of the invention is contacted with a receptor by administration to a mammal.
• Methods of administration to a mammal include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.
• Examples of such receptors include, but are not limited to platelet derived growth factor receptor (PDGF-R), VEGF receptors (e.g., VEGFR-2/KDR/Flk-l, VEGFRl/Flt-1, VEGFR3/FH-4), epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR) and the like.
• PDGF-R platelet derived growth factor receptor
• VEGF receptors e.g., VEGFR-2/KDR/Flk-l, VEGFRl/Flt-1, VEGFR3/FH-4
• EGFR epidermal growth factor receptor
• IGFR insulin-like growth factor receptor
• Additional non-limiting examples of receptor tyrosine kinases include Flt-4, HER2/neu, Tek and Tie2.
• FGF fibroblast growth factor
• NGF nerve growth factor
• FGF-R fibroblast growth factor
• NGFR nerve growth factor receptor
• MSP-R macrophage-stimulating protein receptor
• Receptors of interest include human proteins and homologies from other mammals.
• Antibodies of the invention can incorporate Ig antigen binding domains from any source.
• antibodies are known for the above listed receptors and are sources of V H and V L domains for use in antibodies of the present invention.
• scFv variable region binding domains specific for KDR include, for example, V H and V L domains of BMC-ICl 1 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 1 and 2; nucleotide and amino acid sequences of V L : SEQ ID NOS :3 and 4) (see, WO 00/44777), IMC-2C6 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 5 and 6; nucleotide and amino acid sequences of V L : SEQ ID N0S:7 and 8) (see, WO 03/075840), and IMC- 1121 (nucleotide and amino acids sequences of VH: SEQ HD NOS: 5 and 6;
• binding domains specific for FIt-I include 6.12 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 11 and 12; nucleotide and amino acid sequences of V L : SEQ ID N0S:13 and 14) and IMC-18Fl (nucleotide and amino acids sequences of V H : SEQ ID NOS:27 and 28; nucleotide and amino acid sequences of V L : SEQ ID NOS :29 and 30).
• Binding domains specific for EGFR include, for example, ERBITUX (Cetuximab; IMC-C225) (nucleotide and amino acids sequences of V H : SEQ ID NOS: 15 and 16; nucleotide and amino acid sequences of V L : SEQ ID NOS:17 and 18) as disclosed in WO 96/40210 and IMCl 1F8 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 19 and 20; nucleotide and amino acid sequences of V L : SEQ ID NOS:21 and 22).
• binding domain specific for IGFR is IMC-Al 2 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 23 and 24; nucleotide and amino acid sequences of V L : SEQ ID NOS: 25 and 26).
• Antibodies that bind to FGF receptors include, for example, FRl -H7 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 31 and 32; nucleotide and amino acid sequences of V L : SEQ ID NOS: 33 and 34), FRl-Al (nucleotide and amino acids sequences of V H : SEQ ID NOS: 35 and 36; nucleotide and amino acid sequences of V L : SEQ ID NOS: 37 and 38), and FRl -4H (nucleotide and amino acids sequences of V H : SEQ ID NOS: 39 and 40; nucleotide and amino acid sequences of V L : SEQ ID NOS: 41 and 42).
• Antibodies that bind to RON or MSP-R include IMC-41A10 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 43 and 44; nucleotide and amino acid sequences of V L : SEQ ID NOS: 45 and 46) and IMC-41B12 (nucleotide and amino acids sequences of V H : SEQ ID NOS: 47 and 48; nucleotide and amino acid sequences of V L : SEQ ID NOS: 49 and 50).
• Antibodies that bind to PDGFR ⁇ include, for example, 3G3 and 7Gl 1 (Loizos et al., 2005, MoI. Cancer Ther. 4:369).
• binding domains such as the CDR regions
• portions of the above listed binding domains may be incorporated into binding domains used to make the binding proteins described herein.
• a bispecific antigen-binding protein binds to and blocks activation of two different receptor tyrosine kinases involved in angiogenesis.
• the antibody binds to PDGFR and a VEGF receptor such as, for example, VEGFR2/Flk-1/KDR.
• the antibody binds to KDR and FLT-I.
• an antibody of the invention binds to HER2 and EGFR. In yet another preferred embodiment, an antibody of the invention binds to EGFR and IGFR.
• an antigen-binding protein of the invention binds to EGFR and a VEGFR. In a preferred embodiment, the VEGFR is VEGFR2.
• Such an antibody is useful for blocking stimulation of vascular epithelial cells, by blocking signal transduction through both EGFR and VEGFR. This is particularly useful where angiogenesis occurs in response to EGFR ligands, particularly TGFo; secreted by tumor cells.
• Antibodies of the invention can be used to cross-link antigens on target cells with antigens on immune system effector cells. This can be useful, for example, for promoting immune responses directed against cells which have a particular antigens of interest on the cell surface.
• immune system effector cells include antigen specific cells such as T cells which activate cellular immune responses and nonspecific cells such as macrophages, neutrophils and natural killer (NK) cells which mediate cellular immune responses.
• Antibodies of the invention can have a binding site for any cell surface antigen of an immune system effector cell.
• cell surface antigens include, for example, cytokine and lymphokine receptors, Fc receptors, CD3, CD 16, CD28, CD32 and CD64.
• the bispecific antibodies can include binding sites provided by Fvs. Such Fvs may be obtained from antibodies to the aforementioned antigens, from combinatorial libraries, as well as by other methods known in the art.
• the bispecific antibodies which are specific for cytokine and lymphokine receptors can also include binding sites comprising sequences of amino acids that correspond to all or part of the natural ligand for the receptor.
• an bispecific antibody of the invention can have an antigen-binding site which comprises a sequence of amino acids corresponding or IL-2.
• cytokines and lymphokines include, for example, interleukins such as interleukin-4 (IL-4) and interleukin-5 (IL-5), and colony- stimulating factors (CSFs) such as granulocyte-macrophage CSF (GM-CSF), and granulocyte CSF (G-CSF).
• Antibodies of the invention are made by expressing two polypeptide chains, which taken together, comprise at least one single domain antigen binding site.
• the two polypeptide chains each comprise at least one heavy chain constant domain that is capable of dimerization (e.g., C H 2 and/or C H 3).
• Antibodies are conveniently produced in E. coli using DNA constructs which comprise bacterial secretion signal sequences at the start of each polypeptide chain.
• a variety of bacterial signal sequences are known in the art.
• a perferred signal sequence is from the pelB gene o ⁇ Erwinia carotovora.
• the DNA fragments coding for the antibodies can be cloned, e.g., into vectors employing human cytomegalovirus (HCMV) promoters and enhancers for high level expression in mammalian cells, such as, for example, CHO, NSO 3 COS-7, and PER.C6 cells, and cell lines of lymphoid origin such as lymphoma, myeloma, or hybridoma cells.
• HCMV human cytomegalovirus
• a selectable marker is a gene which encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium.
• Typical selectable markers encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli.
• a particularly useful selectable marker confers resistance to methotrexate.
• cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
• Mtx methotrexate
• An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sd. USA 77, 4216.
• the transformed cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding the antibody or antibody fragment.
• an example of a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb et al. (1979) Nature, 282, 39; Kingsman et al. (1979) Gene 1, 141.
• the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones (1977) Genetics 85, 12. The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
• Transformed host cells are cultured by methods known in the art in a liquid medium containing assimilable sources of carbon, e.g. carbohydrates such as glucose or lactose, nitrogen, e.g. amino acids, peptides, proteins or their degradation products such as peptones, ammonium salts or the like, and inorganic salts, e.g. sulfates, phosphates and/or carbonates of sodium, potassium, magnesium and calcium.
• the medium furthermore contains, for example, growth-promoting substances, such as trace elements, for example iron, zinc, manganese and the like.
• Antibodies that bind to growth factor receptors are preferably capable of blocking activation of receptor tyrosine kinase (RTK) activity.
• Tyrosine kinase inhibition can be determined using well-known methods, for example, by measuring the autophosphorylation level of recombinant kinase receptor, and/or phosphorylation of natural or synthetic substrates.
• phosphorylation assays are useful in determining RTK antagonists of the present invention. Phosphorylation can be detected, for example, using an antibody specific for phosphotyrosine in an ELISA assay or on a western blot.
• methods for detection of protein expression can be utilized to determine RTK antagonists, wherein expression of the proteins being measured is mediated by the RTK.
• RTK immunohistochemistry
• FISH fluorescence in situ hybridization
• competitive radioligand binding assays solid matrix blotting techniques, such as Northern and Southern blots, reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA.
• RT-PCR reverse transcriptase polymerase chain reaction
• ELISA e.g., Grandis et al., Cancer, (1996) 78:1284-92; Shimizu et al., Japan J. Cancer Res., (1994) 85:567-71; Sauter et al., Am. J.
• the ability of an antibody to block ligand binding can be measured, for example, by an in vitro competitive assay.
• a ligand of the RTK ⁇ e.g., EGF for EGFR is immobilized, and a binding assay is carried out to determine the effectiveness of the antibody to competitively inhibit binding of the RTK to the immobilized ligand.
• In vivo assays can also be utilized to determine RTK antagonists. For example, receptor tyrosine kinase inhibition can be observed by mitogenic assays using cell lines stimulated with receptor ligand in the presence and absence of inhibitor.
• A431 cells (American Type Culture Collection (ATCC), Rockville, MD) stimulated with EGF can be used to assay EGFR inhibition.
• Another method involves testing for inhibition of growth of EGFR-expressing tumor cells, using for example, human tumor cells injected into a mouse. See U.S. Patent No. 6,365,157 (Rockwell et al.).
• Preferred antibodies of the instant invention have dual specificity and are capable of binding to two different antigens simultaneously.
• the different antigens can be located on different cells or on the same cell.
• Cross-linking of antigen can be shown in vitro, for example by providing a solid surface to which a first antigen has been bound, adding a bispec ⁇ f ⁇ c antibodies specific for the first antigen and a second antigen for which the binding protein is also specific and detecting the presence of bound second antigen.
• Preferred antibodies of the invention are capable of blocking the interaction between two receptors and their respective ligands.
• an antibody specific for KDR and FIt-I inhibits VEGF induced cell migration as well as PlGF induced cell migration.
• Combination of two receptor binding specificities in a bispecific antibodies can be more efficacious in inhibiting cell migration than the individual parent antibodies (see, e.g., Zhu, Z., WO 2004/003211).
• bispecific antibodies can be more potent inhibitors of cellular function.
• VEGF-stimulated cellular functions such as, for example, proliferation of endothelial cells and VEGF- and PlGF-induced migration of human leukemia cells can be more efficiently inhibited by bispecific antibodies, even where affinity for one or both of the two target antigens is reduced.
• An antibody specific (monovalent) for both KDR and FIt-I can be more effective to inhibit VEGF or PlGF induced cell migration than a monospecific scFv directed at either of the target antigens (WO 2004/003211).
• an antibody having dual specificity for both EGFR (or Her2/neu) and IGFR that is capable of binding to both receptors and blocking interaction with their specific ligands is used to neutralizing both EFG- and IGF-stimulated receptor activation and downstream signal transduction. Stimulation of either EGFR or IGFR is observed to result in activation (e.g., phosphorylation) of common downstream signal transduction molecules, including Akt and p44/42, although to different extents. In certain tumor cells, inhibition of EGFR function can be compensated by upregulation of other growth factor receptor signaling pathways, and particularly by IGFR stimulation.
• the antigen-binding proteins are generally useful for treating neoplastic diseases characterized by cell growth or transformation resulting from activation of multiple signal transduction pathways.
• the antibodies of the invention are useful for treatment of a variety of proliferative disorders.
• the present invention provides for treatment of tumors that express and are stimulated through more than one receptor tyrosine kinase. Stimulation through more that one receptor can result in uncontrolled growth that is insensitive to blockage of each receptor alone.
• stimulation of a second receptor can add to the activation observed in response to stimulation through a first receptor.
• the contributions from the individual receptors can be multiplicative. In each of the above instances, significantly improved inhibition of tumor growth is observed in the presence of an antigen-binding protein that blocks both of the receptors.
• the antibodies of the invention are useful for treating diseases in which receptor stimulation is through an EGFR paracrine and/or autocrine loop.
• EGFR expressing tumors are characteristically sensitive to EGF present in their environment, and can further be stimulated by tumor produced EGF or TGF- ⁇ .
• the diseases and conditions that may be treated or prevented by the present methods include, for example, those in which tumor growth is stimulated. The method is therefore effective for treating a solid tumor that is not vascularized, or is not yet substantially vascularized.
• Certain antibodies of the invention are useful for inhibiting angiogenesis associated with a hyperproliferative disease. For example, by blocking tumor associated angiogenesis, tumor growth may be inhibited.
• the antibody binds to a tumor associated RTK and inhibits production of angiogenic ligands (i.e., VEGF) by the tumor, and also binds to a VEGF receptor associated with cells of the vasculature to inhibit proliferation of such cells.
• VEGF angiogenic ligands
• the antibody binds to multiple VEGF receptors, such that VEGF or other ligand of VEGFR (e.g., PlGF) ligand is blocked from binding to more than one type of VEGF receptor.
• Tumors that may be treated include primary tumors and metastatic tumors, as well as refractory tumors.
• Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof.
• Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.
• the tumors may express EGFR or other RTK at normal levels or they may overexpress the RTK at levels, for example, that are at least 10, 100, or 1000 times normal levels.
• tumors that express EGFR and are stimulated by a ligand of EGFR include carcinomas, gliomas, sarcomas, adenocarcinomas, adenosarcomas, and adenomas.
• Such tumors can occur in virtually all parts of the body, including, for example, breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix or liver.
• tumors observed to overexpress EGFR include, but are not limited to, colorectal and head and neck tumors, especially squamous cell carcinoma of the head and neck, brain tumors such as glioblastomas, and tumors of the lung, breast, pancreas, esophagus, bladder, kidney, ovary, cervix, and prostate.
• tumors observed to have constitutively active (i.e., unregulated) receptor tyrosine kinase activity include gliomas, non-small-cell lung carcinomas, ovarian carcinomas and prostate carcinomas.
• tumors include Kaposi's sarcoma, CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.
• RTKs Overexpression of other RTKs can produce similar growth defects. For example, most metastatic bone cancers arise from primary tumors of prostate, breast, or lung. Prostate tumors initially may be hormone dependent, but loss of such dependence coincides with IGFR mediated stimulation of cells that migrate to bone.
• the antibodies are also useful for treating hyperproliferative disesases other than tumors comprising administering to the mammal an effective amount of the antibody of the present invention.
• hyperproliferative disease is defined as a condition caused by excessive growth of non-cancer cells that express a member of the EGFR family or other tyrosine kinase receptors. The excess cells generated by a hyperproliferative disease express the RTK at normal levels or they may overexpress the RTK.
• hyperproliferative disease examples include psoriasis, actinic keratoses, and seborrheic keratoses, warts, keloid scars, and eczema. Also included are hyperproliferative diseases caused by virus infections, such as papilloma virus infection. For example, psoriasis comes in many different variations and degrees of severity.
• psoriasis display characteristics such as pus-like blisters (pustular psoriasis), severe sloughing of the skin (erythrodermic psoriasis), drop-like dots (guttae psoriasis) and smooth inflamed lesions (inverse psoriasis).
• the treatment of all types of psoriasis e. g., psoriasis vulgaris, psoriasis pustulosa, psoriasis erythrodermica, psoriasis arthropathica, parapsoriasis, palmoplantar pustulosis
• psoriasis vulgaris e.g., psoriasis vulgaris, psoriasis pustulosa, psoriasis erythrodermica, psoriasis arthropathica, parapsoriasis, palmoplantar pus
• antibodies can be chemically or biosynthetically conjugated to other agents such as antineoplastic or anti-angiogenic agents for treatment of disease.
• Anti-tumor agents linked to an antibody include any agents which destroy or damage a tumor to which the antibody has bound or in the environment of the cell to which the antibody has bound.
• an anti-tumor agent is a toxic agent such as a chemotherapeutic agent or a radioisotope.
• the chemotherapeutic agents are conjugated to the antibody using conventional methods (See, e.g., Hermentin and Seiler (1988) Behring Inst. Mitt. 82, 197-215), including by peptide and non-peptide linkers.
• Antibodies of the invention can also be linked to detectable signal-producing agents useful in vivo and in vitro for diagnostic purposes.
• the signal producing agent produces a measurable signal which is detectible by external means, usually the measurement of electromagnetic radiation.
• the signal producing agent is an enzyme or chromophore, or emits light by fluorescence, phosphorescence or chemi luminescence.
• Chromophores include dyes which absorb light in the ultraviolet or visible region, and can be substrates or degradation products of enzyme catalyzed reactions.
• the invention further contemplates the use of antibodies with treatment or diagnostic agents incorporated into secondary reagents.
• one member of a binding pair is linked to the antibody of the invention.
• Anti-neoplastic agents for example, are conjugated to second members of such pairs and are thereby directed to the site where the antibody is bound.
• biotin is conjugated to an antibody of the invention, and thereby provides a target for an anti-neoplastic agent or other moiety which is conjugated to avidin or streptavidin.
• biotin or another such moiety is linked to an antibody of the invention and used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin.
• Antibodies can be administered in combination with one or more suitable adjuvants, such as, for example, cytokines (IL-10 and IL-13, for example) or other immune stimulators, such as, but not limited to, chemokine, tumor-associated antigens, and peptides. It should be appreciated, however, that administration of an antibody alone is sufficient to prevent, inhibit, or reduce the progression of the tumor in a therapeutically effective manner.
• suitable adjuvants such as, for example, cytokines (IL-10 and IL-13, for example) or other immune stimulators, such as, but not limited to, chemokine, tumor-associated antigens, and peptides.
• an antibody of the invention that binds to an RTK and blocks ligand binding in combination with another antigen-binding protein that binds to ligand.
• Ligand binding antibodies are well known in the art, and include, e.g., anti-VEGF (Avastin ® ; bevacizumab).
• the antibodies of the invention are also to be used in combined treatment methods by administration with an anti-neoplastic agent such as a chemotherapeutic agent or a radioisotope.
• chemotherapeutic agents include irinotecan (CPT-I l), anthracyclines (e.g. daunomycin and doxorubicin), methotrexate, vindesine, neocarzinostatin, cisplatin, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin and calicheamicin.
• An antibody and an anti-angiogenic or anti-neoplastic agent are admininstered to a patient in amounts effective to inhibit angiogenesis and/or reduce tumor growth.
• the antibodies are also to be administered in combination with other treatment regimes, for example, with treatments such as radiation therapy.
• combination therapies see, e.g., U.S. Patent No. 6,217,866 (Schlessinger et al.) (Anti-EGFR antibodies in combination with anti -neoplastic agents); WO 99/60023 (Waksal et al.) (Anti-EGFR antibodies in combination with radiation).
• any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof.
• the anti-neoplastic agents known in the art or being evaluated can be grouped in to classes based on their target or mode of action.
• alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine.
• anti-metabolites include, but not limited to, doxorubicin, daunorubicin, and paclitaxel, gemcitabine, and topoisomerase inhibitors irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, and topotecan (topoisomerase I) and etoposide (VP- 16) and teniposide (VM-26) (topoisomerase II).
• the source can be either external (external beam radiation therapy — EBRT) or internal (brachytherapy — BT) to the patient being treated.
• EBRT internal beam radiation therapy
• brachytherapy — BT brachytherapy — BT
• Such classifications can be useful for choosing an antineoplastic agent to use. For example, it has been observed that antibodies that bind IGFR may be particularly effective when administered with a topoisomerase inhibitor.
• the dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity tumor being treated and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular dose.
• the antibody is administered before, during, or after commencing therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing the antineoplastic agent therapy.
• the antibody can be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before commencing radiation therapy.
• chemotherapy is administered concurrently with, prior to, or subsequent to antibody therapy.
• any suitable method or route can be used to administer antibodies of the invention, and optionally, to co-administer anti-neoplastic agents, receptor antagonists, or other pharmaceutical composition.
• antineoplastic agent regimens utilized according to the invention include any regimen believed to be optimally suitable for the treatment of a patient's neoplastic condition. Different malignancies can require use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined on a patient to patient basis.
• Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.
• the dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of neoplastic agent, the type and severity tumor being treated and the route of administration of the antineoplastic agent. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.
• antibodies of the invention where used in a mammal for the purpose of prophylaxis or treatment, will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier.
• suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
• Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins.
• the compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.
• kits for inhibiting tumor growth and/or angiogenesis, or treating other disesase comprising a therapeutically effective amount of an antibody of the invention.
• Human or humanized antibodies are preferred.
• the kits can further contain any suitable antagonist of, for example, another growth factor receptor involved in tumorigenesis or angiogenesis (e.g., EGFR, VEGFR-I /FIt-I, VEGFR-2/Flk- 1/KDR, IGFR, PDGFR, NGFR, FGFR, etc, as described above).
• the kits of the present invention can further comprise an anti-neoplastic agent. Examples of suitable anti-neoplastic agents in the context of the present invention have been described herein.
• the kits of the present invention can further comprise an adjuvant; examples have also been described above.
• kits which contain antibodies of the present invention.
• the present receptor binding antibodies thus can be used in vivo and in vitro for investigative, diagnostic, prophylactic, or treatment methods, which are well known in the art.
• investigative, diagnostic, prophylactic, or treatment methods which are well known in the art.
• variations in the principles of invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention. All references mentioned herein are incorporated by reference in their entirety.
• Phage preparations were resuspended in 1 ml of 3% fat-free milk/PBS containing 240 ⁇ g unrelated human IgG and incubated at 37°C for 1 h to block nonspecific binding and binding to the Fc-tag of the mPDGFR ⁇ protein.
• mPDGFRos-Fc-coated Maxisorp Star tubes (Nunc, Rosklide, DenMark) were first blocked with 3% milk/PBS at 37°C for 1 h, and then incubated with the phage preparation at room temperature for 1 h. The tubes were washed 15 times with PBST (PBS containing 0.1% Tween 20) followed by 15 washes with PBS.
• the bound phage was eluted at room temperature for 10 min with ImI of a freshly prepared solution of 100 mM triethylamine (Sigma).
• the eluted phage were incubated with 10 ml of mid-log phase TGl cells at 37°C for 30 min stationary and 30 min shaking.
• the infected TGl cells were pelleted, plated onto three 2 YT AG plates, and incubated overnight at 30 0 C. All of the colonies grown on the plates were scraped into 3—5 ml of 2YTA medium, mixed with glycerol (final concentration: 10%), aliquoted, and stored at -80°C.
• the phage stock 100 ⁇ l
• ELISA screening for antibodies with binding and blocking activities After 2 nd and 3 rd rounds of selection, 190 clones from each round were randomly picked and tested for binding and blocking activities by both phage ELISA and Fab ELISA. Briefly, individual TGl clones recovered after 2 nd and 3 rd rounds of selection were randomly picked and grown at 37 0 C in 96-well plates. To produce phage, the cells were rescued with M13K07 helper phage as described above. To produce soluble Fab, the cells were incubated in 2YTA medium containing ImM of EPTG.
• phage preparations and cell culture supernatants containing soluble Fab were blocked with 1/6 volume of 18% milk/PBS at room temperature for 1 h.
• the blocked phage preparation or cell culture supernatants were then added to 96-well microtiter plates (Nunc) coated with mPDGFRoVFc (1 ⁇ g/ml, 50 ⁇ l, at 4°C overnight), and incubated at room temperature for 1 h. After incubation at room temperature for 1 h, the plates were washed 3 times with PBST.
• phage ELISA For the phage ELISA, the plates were incubated with an anti-M13 phage antibody-HRP conjugate (Amersham Biosciences).
• Fab ELISA the plates were incubated with an anti-human-Fab antibody-HRP conjugate (Jackson ImmunoResearch Laboratory). After three washes, color was developed by addition of TMB peroxidase substrate(KPL, Gaithersburg, MD), and the absorbance was measured at 450 nM using a microplate reader (Molecular Device, Sunnyvale, CA).
• ELISAs for binding to ICl 1 IgG were also performed to eliminate Fc specific antibodies identified as binders in the library screen. More than 77% clones picked after 2 nd selection, and 99% of the recovered clones after 3 rd selection were positive in mPDGFR ⁇ : binding assay, suggesting a high efficiency of the selection process.
• mPDGFR ⁇ -Fc 50 ⁇ of phage preparations or Fab culture supernatants were mixed with a fixed amount of mPDGFR ⁇ -Fc (0.5 ⁇ g/ml) and incubated at RT for 30 min. The mixture was then transferred to 96-well plates precoated with rhPDGF- AA (0.5 /Ag/ml; R&D Systems, Minneapolis, MN) and incubated at RT for 1 h. To quantify bound mPDGFR ⁇ -Fc protein, the plates were then incubated at RT for 1 h with a rabbit anti- human-Fc antibody-HRP conjugate, followed by three washes and addition of the TMS peroxidase substrate.
• the bound mPDGFR ⁇ -Fc protein was quantified by reading the absorbance at 450 nM. Blocking activity was indicated by decreased ELISA signal detected by anti-human-Fc antibody-HRP conjugate. About 4.2% of clones that bound mPDGFR ⁇ showed PDGF-AA blocking activity.
• Nucleotide and amino acids sequences for the Fabs are as follows: IElO V H domain: SEQ ID NOS: 51 and 52; IElO V L domain: SEQ ID NOS: 53 and 54; 1A12 V H domain: SEQ ID NOS: 55 and 56; 1A12 V L domain: SEQ ID NOS: 57 and 58; 3B2 V H domain: SEQ ID NOS: 59 and 60; 3B2 V L domain: SEQ ID NOS: 61 and 62; IClO V H domain: SEQ ID NOS: 63 and 64; IClO V L domain: SEQ ID NOS: 65 and 66; 3G7 V H domain: SEQ ID NOS: 67 and 68; 3G7 V L domain nucleotide sequence: SEQ ID NO: 69; 3G7 V L domain: QAW; 1F9 V H domain: SEQ ID NOS: 70 and 71; 1F9 V L domain: SEQ ID NOS: 72 and 73; 1
• the Fab fragments of six clones were expressed in E. coli HB2151 host cells, and purified by affinity chromatography Protein G column. Phagemids of the individual selected clones were used to transform a nonsupressor E.coli host HB2151. Expression of the Fab fragments in HB2151 was induced by culturing the cells in 2YTA medium containing 1 mM of IPTG at 30°C. A periplasm ⁇ c extract of the cells was prepared as described by Lu (x). The soluble Fab protein was purified using a Protein G column followirip the manufacturer's protocol (Amersham Biosciences).
• the purified antibodies were electrophoresed in NuP AGETM 4-12% Bis-Tris gel (Invitrogen) and visualized by staining with the solution of SimplyBlueTM SafeStain (Invitrogen).
• the binding kinetics of various anti-mPDGFRa antibodies were determined by surface plasmon resonance using a BIAcore 3000 biosensor and evaluated using the program BIA Evaluation 2.0 (Biacore, Inc., Uppsala, Sweden).
• the affinity constant, Kd was calculated from the ratio of dissociation rate (&off)/association rate (&on). Reported values represent the mean ⁇ S.E. from at least two determinations for Fabs and three determinations for IgG. (Table 2). Consistent with binding assays, sVD antibody 1F2 has much higher affinity (KJ) of about 0.5 nM, more than 23-fold higher than the selected normal Fab, IElO.
• 1F2-2H Fab is an engineered divalent Fab containing a second identical VH domain expressed as a fusion to CL.
• the divalent Fab was expressed in E. coli and purified by Protein G chromatography. SDS-PAGE analysis of the purified 1F2-2H Fab demonstrated one single protein band of approximately 50 kD, similar to that of the standard Fab fragment (Fig. 5).
• the affinity of the divalent Fab is 79.5 pM, compared to 418 pM for the monovalent 1F2 Fab, indicating a 5.2-fold enhancement (Table 2).
• VH- ⁇ DFc and VL-pLC ⁇ The heavy chain and light chain expression vectors (VH- ⁇ DFc and VL-pLC ⁇ ) were then co-transfected into COS-7 cells for transient expression as previously described.
• Cell culture media were collected at 48 and 96 hours after transfection, and pooled.
• Antibodies were purified from the supernatant of cell cultures by affinity chromatography affinity chromatography using Protein- A columns following the manufacture's protocol (Amersham Biosciences). After function of IgG was confirmed, expressed pairs of VH and VL genes were cloned into a single expression vectors and transfected into COS-7 cells. Antibody purification was as described above.
• IgG like constructs were produced from 1F2 Ig which contains only one VH (see Fig. 7), including tetravalent 1F2-2H IgG with four V H s (A), and divalent 1F2 with 1F2 V H expressed as a fusion either to C H or C L (B and C).
• the plasmids for expressing the light and heavy chains were used to co-transfect COS-7 cells for transient expression. Expression levels of antibodies were monitored by ELISA using the supernatants of cell cultures. Expression of 1F2-2H was consistently less than both divalent 1F2 antibodies, about one-fourth of divalent 1F2 antibodies.
• the antibodies from the supernatant of cell cultures were purified by affinity chromatography using a Protein A column.
• V H and V L genes of IElO were first cloned into pDFc and pLC/c, respectively.
• the expressed V H / V L pair was cloned into a single expression vector which was then used to transfect COS-7 cells followed by antibody purification.
• Binding and blocking activities of full length anti-mPDGFRa antibodies All three purified 1F2 variant antibodies, 1F2-2H IgG, 1F2-CH/CL and 1F2-CL/CH, and antibody IElO were compared for their binding affinity to mPDGFRo: by ELISA. Various amount of antibodies were incubated in the ELISA plate coated with mPDGFRo/Fc, mPDGFR ⁇ -bound antibodies were then detected by an anti-human- ⁇ antibody HRP conjugate.
• Quantitative blocking assays were also performed to evaluate the anti- mPDGFRor antibodies. As described above, various amounts of antibodies were incubated with a fixed amount of mPDGFR ⁇ /Fc fusion in solution for 30 min, and the mixtures were then transferred to 96-well plates coated with rhPDGF-AA and incubated for 1 h. Bound mPDGFR ⁇ was then quantified by quantifying bound anti-human-Fc-Ab.
• the divalent 1F2-CH/CL and 1F2-CL/CH were better blockers than 1F2-2H IgG and IElO IgG: the IC50 values were 3.2, 2.7, 17, and 9.6 nM, for 1F2-CH/CL,1F2- CL/CH, 1F2-2H IgG and IElO IgG, respectively (Fig. 9B).
• the binding affinities of 2B4 Fab and IgG to mVEGFR2 are 6.7 ⁇ 3.0 nM and 0.39 ⁇ 0.1 nM, respectively.
• 2B4 IgG blockes VEGFR2/VEGFi 6 5 interaction with an IC50 value of approximately 3.5 nM (Fig. 13B).
• the scFv2B4- CH expressing vector was then paired with the 1F2VH-CL expressing vector as described above by co-transfection of COS-7 cells for transient expression.
• the V H gene of 1 F2 and the V L of 2B4 were first assembled using overlapping PCR.
• the COOH terminus of 1F2 V H was linked to the amino terminus of 2B4 V L via a 5-amino acid linker from 5' end of C H -
• the 1F2 VH-2B4 VL encoding gene was then cloned into vector pLCK via Hind III/BsiW I sites for expression of 1F2 VH-2B4 VL - CL-fusion protein.
• the 2B4 V H gene was cloned into pDFc vector as described above for V H expression.
• the plasmids for expressing light and heavy chains paired as depicted in Fig. 1OB, were used to co-transfect COS-7 cells for transient expression.
• the Ig expression constructs were combined into a single expression vector which was then used transfect COS-7 cells for transient expression of designed IgG. Antibodies were purified from the supernatant of cell cultures as described above.
• the 1F2/SCFV2B4 and 1F2-2B4 antibodies were produced by transfection and transient expression in COS-7 cells (100-200ml cell cultures). Antibodies were purified from cell culture supernatants affinity chromatography using Protein A columns.
• the upper band correlates with a scFv-heavy chain fusion protein (MW -62,500).
• the upper band of 1F2-2B4 corresponds to the heavy chain of a standard IgG (2B4) and the lower band is observed at a position expected for a VH-light chain fusion (MW -37,500).
• the antigen binding efficiency of the domain-based bispecific antibodies was determined on immobilized mPDGFRa and FIk-I .
• ELISA showed that the bispecific antibody, 1F2/SCFV2B4 and 1F2-2B4 bound to mPDGFR ⁇ and FIk-I, but not as efficiently as the parental antibody 1F2 and 2B4 (Fig. 12).
• the binding of 1F2-2B4 antibody was slightly more efficient compared with 1F2/SCFV2B4.
• the FIk-I- specific antibody 2B4 did not bind to mPDGFR ⁇ : (Fig. 12A), nor did the mPDGFR ⁇ -specific antibody 1F2 bind to FIk-I (Fig. 12B).
• the binding kinetics of the bispecific antibodies to mPDGFR ⁇ and FIk-I were determined by surface plasmon resonance using a BIAcore instrument (Table 3). Consistent with the observations from ELISA, the 1F2-2B4 antibody has greater affinity to both mPDGFR ⁇ ; and FIk-I in comparison with 1F2/SCFV2B4 antibody.
• Fig. 13 shows that both bispecific antibodies inhibit mPDGFR ⁇ : from binding to immobilized PDGF-AA (Fig. 13A) with estimated IC50 of 9.9 nM and 25.3 nM for 1F2/SCFV2B4 and 1F2-2B4, respectively.
• the antibodies also block FIk-I from binding to immobilized VEGFj ⁇ s (Fig. 13B) with IC50 value of 9.5 nM and 19.5 nM, respectively.
• 2B4 had no effects on mPDGFR ⁇ /PDGF-AA interaction
• 1F2 had no effects on Flk-l/VEGFi65 interaction.
• MFI mean fluorescence intensity
• eEnd.l cells were plated onto 6 cm dishes and grown to 70-80% confluence, after which the cells were washed twice in PBS and cultured overnight in serum free medium. The cells were first incubated with various antibodies at 37°C for 30 min, followed by stimulation with VEGF or PDGF-AA at 37°C for 15 min.
• the cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na 3 VO 4, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, and 1 ⁇ g/ml aprotinin) for 1 h, followed by centrifugation of the lysate at 12, 000 rpm for 10 min at 4°C.
• lysis buffer 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na 3 VO 4, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, and 1 ⁇ g/ml a
• Receptors were immunoprecipitated from the cell lysate supernatant using anti-mPDGFR ⁇ (eBioscience, San Diego, CA) or anti-mVEGFR2 antibody (Santa Cruz Biotech, Santa Cruz, CA), followed by the addition of 20 ⁇ l of ProA/G- sepharose beads (Santa Cruz Biotech).
• the precipitated receptor proteins were resolved on a 4-12% Nupage Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane.
• Phospho-mVEGFR2 and phospho-mPDGFR ⁇ were detected on the blot using an anti-phospho-tyrosine antibody-HRP conjugate (Santa Cruz Biotech).
• Total receptor proteins loaded on the gel were assayed with antibodies to mPDGFR ⁇ or mVEGFR2 (both from Santa Cruz Biotech).
• the BsAb inhibited both PDGF and VEGF-stimulated phosphorylation of mPDGFR ⁇ . and mVEGFR2 receptors, whereas monospecific parent antibodies only blocked the activation of a single receptor stimulated by its cognate ligand.
• the anti-EGFR antibody, C225 did not have any effect on ligand-stimulated activation of either receptor.

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