WO2010002862A2 - Fibroblast growth factor receptor 3 (fgfr3) binding proteins - Google Patents

Fibroblast growth factor receptor 3 (fgfr3) binding proteins Download PDF

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Publication number
WO2010002862A2
WO2010002862A2 PCT/US2009/049211 US2009049211W WO2010002862A2 WO 2010002862 A2 WO2010002862 A2 WO 2010002862A2 US 2009049211 W US2009049211 W US 2009049211W WO 2010002862 A2 WO2010002862 A2 WO 2010002862A2
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Prior art keywords
seq
variable region
chain variable
heavy chain
light chain
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PCT/US2009/049211
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French (fr)
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WO2010002862A3 (en
Inventor
Zhigang Weng
William M. Winston, Jr.
Lyne Breault
Kristan Meetze
Solly Weiler
Jeno Gyuris
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Aveo Pharmaceuticals, Inc.
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Priority to EP09774313A priority Critical patent/EP2313435A4/en
Publication of WO2010002862A2 publication Critical patent/WO2010002862A2/en
Publication of WO2010002862A3 publication Critical patent/WO2010002862A3/en

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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
    • C07K16/28—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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

Definitions

  • the field of the invention is molecular biology, immunology and oncology. More particularly, the field is antibody-based binding proteins that bind human fibroblast growth factor receptor 3 (FGFR3).
  • FGFR3 human fibroblast growth factor receptor 3
  • Fibroblast Growth Factor Receptor 3 is one member of a family of receptor tyrosine kinases (FGFRl, FGFR2, FGFR3, FGFR4) that binds fibroblast growth factors (FGFs) (Keegan et al. (1991) PROC. NATL. ACAD. SCI. USA 88: 1095-1099).
  • FGFs fibroblast growth factors
  • FGF receptors are characterized as having three extracellular immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain.
  • FGF ligand binding induces FGF receptor dimerization and tyrosine autophosphorylation resulting in cell proliferation, differentiation, and migration (Gomez-Roman et al. (2005) CLIN. CANCER RES. 11:459-65; Chang et al. (2005) BLOOD 106:353-6; Eswarakumar et al. (2005) CYTOKINE GROWTH FACTOR REV. 16(2) : 139-49) .
  • the IHb splice variant has high affinity for FGFl (acidic FGF) ligand and lower affinity for FGF8 (androgen-induced growth factor) and FGF9 (glial activating factor) (Chellaiah et al. (1999) J. BlOL. CHEM. 274:34785-94; Gomez-Roman et al. (2005) supra).
  • the IIIc splice variant is characterized as a promiscuous receptor binding numerous FGF ligands including FGFl, FGF2, FGF4, FGF8, FGF9, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, and FGF23 (Chellaiah et al. (1994) J. BIOL.
  • the FGFR3-FGF signaling pathway plays a role in the differentiation of adipocytes, chondrocytes and neurons, wound healing, angiogenesis, embryo development, and malignancies (Keegan et al. (1991) supra).
  • FGFR3 Activating mutations of FGFR3 have been associated with cancer and skeletal disorders including dwarfism, achondroplasia, and hypochondroplasia (Gomez-Roman et al. (2005) supra; Delezoide et al. (1997) HUMAN MOL. GENETICS 6:1899-1906).
  • Certain FGFR3 antibodies are known. See, e.g., U.S. 2005/0147612 (Yayon).
  • Antibodies are multimeric proteins that contain four polypeptide chains (Figure 1). Two of the polypeptide chains are called heavy chains (H chains), and two of the polypeptide chains are called light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds.
  • a light chain consists of one variable region (V L in Figure 1) and one constant region (C L in Figure 1).
  • the heavy chain consists of one variable region (V H in Figure 1) and at least three constant regions (CH 1 , CH 2 and CH 3 in Figure 1). The variable regions determine the specificity of the antibody.
  • Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs relatively conserved framework regions
  • the invention is based, in part, upon the discovery of a family of binding proteins that specifically bind human FGFR3.
  • the binding proteins contain FGFR3 binding sites based on the CDRs of a family of antibodies that specifically bind FGFR3.
  • the binding proteins can be used as diagnostic and therapeutic agents.
  • the binding proteins are engineered, e.g., humanized, to reduce or eliminate an immune response when administered to a human patient.
  • the binding proteins prevent or inhibit the activation of (i.e., neutralize) human FGFR3.
  • the binding proteins prevent FGFR3 from binding to a ligand, e.g., FGFl, thereby neutralizing FGFR3 activation.
  • the binding proteins can be used to inhibit the proliferation of tumor cells or stimulate the proliferation of chondrocytes.
  • the binding proteins can inhibit or reduce tumor growth in the mammal.
  • Figure 1 (prior art) is a schematic representation of a typical antibody.
  • Figure 2 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin heavy chain variable region of antibodies 15D8, 27H2, 2G4, 4E7 (7D12), and 20B4. The amino acid sequences for each antibody are aligned against one another, and CDR 1 , CDR 2 , and CDR 3 are identified in boxes. The unboxed sequences represent framework (FR) sequences.
  • Figure 3 is a schematic diagram showing the CDR 1 , CDR 2 , and CDR 3 sequences for each of the immunoglobulin heavy chain variable region sequences in Figure 2. For antibody 15D8, three alternative CDR 2 sequences are shown (15D8, 15D8-2, and 15D8-3).
  • Figure 4 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin light chain variable region of antibodies 15D8, 27H2, 2G4, 4E7 (7D12), and 20B4.
  • the amino acid sequences for each antibody are aligned against one another, and CDR 1 , CDR 2 , and CDR 3 are identified in boxes.
  • the unboxed sequences represent framework (FR) sequences.
  • Figure 5 is a schematic diagram showing the CDR 1 , CDR 2 , and CDR 3 sequences for each of the immunoglobulin light chain variable region sequences in Figure 4.
  • Figure 6 is a graph summarizing results from an experiment to measure neutralization activity of negative control IgGl (A) and anti-FGFR3 monoclonal antibodies 15D8 (X), 27H2 (+), 2G4 ( ⁇ ), 4E7 (T), 7D12 (0), and 20B4 (•) to inhibit FGFR3 binding to FGFl.
  • Figure 7 is a graph summarizing results from an experiment to measure neutralization activity of negative control IgGl Fab (D) and anti-FGFR3 Fab fragments 15D8 (0), 27H2 (•), 2G4 (*), 4E7 ( ⁇ ), and 7D12 ( A) to inhibit FGFR3 binding to FGFl.
  • Figure 8 is a graph summarizing results from an experiment to measure anti- proliferation activity of negative control (murine IgGl) ( ⁇ ) and anti-FGFR3 monoclonal antibodies 15D8 (T), 27H2 ( ⁇ ), 2G4 (•), and 20B4 ( A) in FDCP-FGFR3 IIIc-109 cells.
  • Figure 9 is a graph summarizing results from an experiment to measure tumor inhibitory activity of a murine IgG control at 20 mg/kg ( ⁇ ) and anti-FGFR3 antibody 15D8 in a OPM-2 xenograft tumor model (antibody 15D8 at 5 mg/kg ( ⁇ ); antibody 15D8 at 10 mg/kg (A); and antibody 15D8 at 20 mg/kg (•)).
  • Figure 10 is a graph summarizing results from an experiment to measure tumor inhibitory activity of a murine IgG control at 1 mg/kg ( ⁇ ) and anti-FGFR3 antibodies dosed in an OPM-2 xenograft tumor model (murine antibody 15D8 at 1 mg/kg (A); murine antibody 4E7 at 1 mg/kg (X); murine antibody 27H2 at 1 mg/kg (o); and murine antibody 2G4 at 1 mg/kg ( ⁇ )).
  • murine antibody 15D8 at 1 mg/kg (A)
  • murine antibody 4E7 at 1 mg/kg
  • X murine antibody 27H2 at 1 mg/kg (o)
  • murine antibody 2G4 at 1 mg/kg ( ⁇ )
  • the invention is based, in part, upon the discovery of a family of binding proteins that specifically bind and neutralize the activity of human FGFR3.
  • the binding proteins can be used in a variety of diagnostic and therapeutic applications.
  • the binding proteins are based upon the antigen binding sites of certain monoclonal antibodies that have been selected for their ability to bind and neutralize the activity of FGFR3.
  • the binding proteins contain immunoglobulin variable region CDR sequences that define a binding site for FGFR3.
  • the binding proteins can be engineered to minimize or eliminate an immune response when administered to a human patient.
  • the binding proteins are fused or conjugated to other moieties, such as detectable labels (e.g., radiolabels) or effector molecules (e.g., other proteins or small molecule therapeutics).
  • detectable labels e.g., radiolabels
  • effector molecules e.g., other proteins or small molecule therapeutics.
  • the binding protein comprises (i) an immunoglobulin heavy chain variable region comprising the structure CDR HI -CDR H2 -CDR H3 and (ii) an immunoglobulin light chain variable region comprising three complementarity determining regions (CDRs), wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human FGFR3.
  • CDR HI comprises the amino acid sequence X 1 Tyr Asn Met Tyr (SEQ ID NO: 81), wherein amino acid X 1 is Asp or Ser.
  • CDR H2 comprises the amino acid sequence Tyr He Asp Pro Tyr Asn GIy GIy Thr X 2 X 3 Asn X 4 X 5 Phe X 6 GIy (SEQ ID NO: 82), wherein amino acid X 2 is Arg or Ser, amino acid X 3 is Asp or Tyr, amino acid X 4 is GIn or Pro, amino acid X 5 is a Lys or Ser, and amino acid X 6 is Lys or GIn.
  • CDR H3 comprises the amino acid sequence X 7 X 8 GIy X 9 X 10 X 11 X 12 Xi 3 Phe X 14 Tyr (SEQ ID NO: 89), wherein amino acid X 7 is GIu or Ser, amino acid X 8 is GIy or Leu, amino acid X 9 is Asn or a peptide bond, amino acid X 10 is Tyr or a peptide bond, amino acid X 11 is GIu or a peptide bond, amino acid X 12 is Ala or Pro, amino acid X 13 is Trp or Asp, and amino acid X 14 is Ala or Asp.
  • the binding protein comprises (i) an immunoglobulin light chain variable region comprising the structure CDR L1 -CDR L2 -CDR L3 and (ii) an immunoglobulin heavy chain variable region comprising three CDRs, wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human FGFR3.
  • CDR L1 comprises the amino acid sequence Ser Ala Ser Ser Ser VaI X 15 Tyr Met X 16 (SEQ ID NO: 83), wherein amino acid X 15 is Ser or Asn, and X 16 is Tyr or His.
  • CDR L2 comprises the amino acid sequence X 17 Thr Ser X 18 Leu Ala Ser (SEQ ID NO: 84), wherein the amino acid X 17 is Leu or Asp, and the amino acid X 18 is Asn, Lys, or Tyr.
  • CDR L3 comprises the amino acid sequence GIn GIn Trp X 19 Ser X 2 o Pro Leu Thr (SEQ ID NO: 85), wherein the amino acid X 19 is Ser or Asn, and the amino acid X 2 o is Asn or Tyr.
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising the structure CDR H1 -CDR H2 -CDR H3 , wherein (i) CDR H1 comprises the amino acid sequence Ser Tyr Asn Met Tyr (SEQ ID NO: 17), (ii) CDR H2 comprises the amino acid sequence Tyr He Asp Pro Tyr Asn GIy GIy Thr X 1 X 2 Asn X 3 X 4 Phe X 5 GIy (SEQ ID NO: 86), wherein amino acid X 1 is Arg or Ser, amino acid X 2 is Asp or Tyr, amino acid X 3 is GIn or Pro, amino acid X 4 is Lys or Ser, and amino acid X 5 is Lys or GIn, and (iii) CDR H3 comprises the amino acid sequence GIu GIy GIy Asn Tyr GIu Ala Trp Phe Ala Tyr (SEQ ID NO: 19), and an immunoglobulin light chain variable region comprising the structure CDR L
  • the binding protein can comprise both the immunoglobulin light chain and the immunoglobulin heavy chain sequences or the fragments thereof, described above.
  • the binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site.
  • the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed with framework regions (FR).
  • the framework regions optionally can be humanized or fully human.
  • the binding protein comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDR H1 -CDR H2 -CDR H3 and (b) immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR3.
  • the CDR H1 comprises a sequence selected from the group consisting of SEQ ID NO: 17 (15D8, 27H2, 4E7(7D12), 2G4) and SEQ ID NO: 29 (20B4).
  • the CDR H2 comprises a sequence selected from the group consisting of SEQ ID NO: 18 (15D8, 20B4), SEQ ID NO: 20 (15D8-2), SEQ ID NO: 21 (15D8-3), SEQ ID NO: 25 (27H2, 4E7(7D12)), and SEQ ID NO: 28 (2G4).
  • the CDR H3 comprises a sequence selected from the group consisting of SEQ ID NO: 19 (15D8, 27H2, 4E7(7D12), 2G4) and SEQ ID NO: 30 (20B4).
  • SEQ ID NO: 29 (20B4) means SEQ ID NO: 29 comes from antibody 20B4.
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDR H i comprising the sequence of SEQ ID NO: 17 (15D8), a CDR H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), SEQ ID NO: 20 (15D8-2), and SEQ ID NO: 21 (15D8-3), and a CDR H3 comprising the sequence of SEQ ID NO: 19 (15D8).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDR H1 comprising the sequence of SEQ ID NO: 17 (15D8), a CDR H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), and a CDR H3 comprising the sequence of SEQ ID NO: 19 (15D8).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 17 (27H2, 4E7(7D12)), a CDR H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25 (27H2, 4E7(7D12)), and a CDR H3 comprising the sequence of SEQ ID NO: 19 (27H2, 4E7(7D12)).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 17 (2G4), a CDR H2 comprising the sequence of SEQ ID NO: 28 (2G4), and a CDR H3 comprising the sequence of SEQ ID NO: 19 (2G4).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 29 (20B4), a CDR H2 comprising the sequence of SEQ ID NO: 18 (20B4), and a CDR H3 comprising the sequence of SEQ ID NO: 30 (20B4).
  • the CDRm, CDR H2 , and CDR H3 sequences are interposed between human or humanized immunoglobulin FRs.
  • the binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
  • the binding protein comprises (a) an immunoglobulin light chain variable region comprising the structure CDR LI -CDR L2 -CDR L3 , and (b) an immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region together define a single binding site for binding human FGFR3.
  • the CDR L1 comprises a sequence selected from the group consisting of SEQ ID NO: 22 (15D8, 27H2, 2G4, 4E7(7D12)) and SEQ ID NO: 31 (20B4);
  • the CDR L2 comprises a sequence selected from the group consisting of SEQ ID NO: 23 (15D8), SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 32 (20B4);
  • the CDR L3 comprises a sequence selected from the group consisting of SEQ ID NO: 24 (15D8), SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 33 (20B4),.
  • the binding protein comprises an immunoglobulin light chain variable region comprising: a CDR LI comprising the sequence of SEQ ID NO: 22 (15D8); a CDR L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23 (15D8); and a CDR L3 comprising the sequence of SEQ ID NO: 24 (15D8).
  • the binding protein comprises an immunoglobulin light chain variable region comprising: a CDR LI comprising the sequence of SEQ ID NO: 22 (27H2, 2G4, 4E7(7D12)); a CDR L2 comprising the sequence of SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)); and a CDR L3 comprising the sequence of SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)).
  • the binding protein comprises an immunoglobulin light chain variable region comprising: a CDR LI comprising the sequence of SEQ ID NO: 31 (20B4); a CDR L2 comprising the sequence of SEQ ID NO: 32 (20B4); and a CDR L3 comprising the sequence of SEQ ID NO: 33 (20B4).
  • the CDR L1 , CDR L2 , and CDR L3 sequences are interposed between human or humanized immunoglobulin FRs.
  • the binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
  • the binding protein comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDR HI -CDR H2 -CDR H3 and (b) an immunoglobulin light chain variable region comprising the structure CDR LI -CDR L2 - CDR L3 , wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR3.
  • the CDR H i comprises SEQ ID NO: 17 (15D8, 27H2, 2G4, 4E7(7D12)); a CDR H2 is selected from the group consisting of SEQ ID NO: 18 (15D8), SEQ ID NO: 20 (15D8-2), SEQ ID NO: 21 (15D8-3), SEQ ID NO: 25 (27H2, 4E7(7D12)), and SEQ ID NO: 28 (2G4); and the CDR H3 comprises SEQ ID NO: 19 (15D8, 27H2, 2G4, 4E7(7D12)).
  • the CDR L1 comprises SEQ ID NO: 22 (15D8, 27H2, 2G4, 4E7(7D12)); a CDR L2 is selected from the group consisting SEQ ID NO: 23 (15D8) and SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)); and a CDR L3 is selected from the group consisting SEQ ID NO: 24 (15D8) and SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)).
  • the binding protein comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (15D8), SEQ ID NO: 6 (27H2), SEQ ID NO: 10 (2G4), SEQ ID NO: 12 (4E7(7D12)), and SEQ ID NO: 14 (20B4), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (15D8), SEQ ID NO: 8 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 16 (20B4).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 2 (15D8), and an immunoglobulin light chain variable region comprising SEQ ID NO: 4 (15D8).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 6 (27H2), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (27H2).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 10 (2G4), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (2G4).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 12 (4E7(7D12)), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (4E7(7D12)).
  • the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 14 (20B4), and an immunoglobulin light chain variable region comprising SEQ ID NO: 16 (20B4).
  • the binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
  • the binding protein comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 39 (15D8), SEQ ID NO: 43 (27H2), SEQ ID NO: 47 (2G4), SEQ ID NO: 51 (4E7(7D12)), and SEQ ID NO: 55 (20B4), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 41 (15D8), SEQ ID NO: 45 (27H2), SEQ ID NO: 49 (2G4), SEQ ID NO: 53 (4E7(7D12)) and SEQ ID NO: 57 (20B4).
  • the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 39 (15D8), and an immunoglobulin light chain comprising SEQ ID NO: 41 (15D8).
  • the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 43 (27H2), and an immunoglobulin light chain comprising SEQ ID NO: 45 (27H2).
  • the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 47 (2G4), and an immunoglobulin light chain comprising SEQ ID NO: 49 (2G4).
  • the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 51 (4E7(7D12)), and an immunoglobulin light chain comprising SEQ ID NO: 53 (4E7(7D12)).
  • the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 55 (20B4), and an immunoglobulin light chain comprising SEQ ID NO: 57 (20B4).
  • Each of the binding proteins described above can be an intact antibody, e.g., a monoclonal antibody.
  • the binding protein can be an antigen binding fragment of an antibody, or can be a biosynthetic antibody binding site.
  • Antibody fragments include Fab, Fab', (Fab') 2 and Fv fragments. Techniques for making antibody fragments are known in the art.
  • Biosynthetic antibody binding sites are known in the art, e.g., single Fv and sFv molecules. See, e.g., U.S. Patent No. 5,476,786.
  • Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, e.g., antibodies or antibody fragments that bind at least two different antigens.
  • bispecific binding proteins can bind human FGFR3 and another antigen of interest.
  • Methods for making bispecific antibodies are known in art. Such methods include fusing hybridomas or by linking Fab' fragments. See, e.g., Songsivilai et ⁇ l. (1990) CLIN. EXP. IMMUNOL. 79: 315-325; Kostelny et ⁇ l. (1992) J. IMMUNOL. 148: 1547- 1553.
  • an isolated binding protein binds human FGFR3 with a K D of 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or lower, wherein the K D values are determined by surface plasmon resonance methods under the conditions described in Example
  • an isolated binding protein binds human FGFR3 with a K D of 200 pM, 150 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, or lower, wherein the K D values are determined by a kinetic exclusion assay (See, e.g., Darling and Brault (2004) ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES 2: 647-657) under the conditions described in Example 7.
  • the binding proteins inhibit hFGFR3 from binding to FGFl.
  • the binding proteins can have an IC 50 (concentration at 50% of maximum inhibition) of about 10, 11, 12, 13, 14, 15, 16, 17 or 18 nM, when assayed using the protocol described in Example 4.
  • binding proteins of the invention are known in the art.
  • DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized using the sequence information provided herein.
  • Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired binding proteins. Production of defined gene constructs is within routine skill in the art.
  • sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or PCR techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells.
  • the nucleic acids encoding the desired binding proteins can be introduced (ligated) into expression vectors, which can be introduced into a host cell through conventional transfection or transformation techniques.
  • exemplary host cells include E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce immunoglobulin protein.
  • Transfected host cells are grown under conditions that permit the host cells to express the genes of interest, e.g., genes that encode the immunoglobulin light or heavy chain variable regions.
  • a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a signal sequence, e.g., a sequence encoding fragment B of protein A (FB).
  • a suitable bacterial promoter e.g., Trp or Tac
  • a signal sequence e.g., a sequence encoding fragment B of protein A (FB).
  • FB sequence encoding fragment B of protein A
  • the expressed fusion protein accumulates in refractile or inclusion bodies in the bacterial cytoplasm, and may be harvested after disruption of the cells by French press or sonication.
  • the refractile bodies then are solubilized, and the proteins refolded and cleaved by methods already established for other recombinant proteins.
  • the engineered gene is to be expressed in eukaryotic host cells, e.g., myeloma cells or CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, immunoglobulin enhancers, and various introns.
  • This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed.
  • the gene construct can be transfected into myeloma cells or CHO cells using established transfection protocols.
  • Such transfected cells can express V L or V H fragments, V L -V H heterodimers, V H -V L or V L -V H single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a protein domain having another function (e.g., cytotoxicity).
  • the binding proteins can be modified to optimize performance, depending upon the intended use of the binding proteins. For example, when the binding protein is being used as a therapeutic agent, the binding protein can be modified to reduce its immunogenicity in a human patient. Alternatively or in addition, the binding protein can be fused or chemically conjugated to another protein or peptide, e.g., a growth factor, cytokine, or cytotoxin.
  • another protein or peptide e.g., a growth factor, cytokine, or cytotoxin.
  • the binding proteins When the binding proteins are to be administered to a human, the binding proteins preferably are engineered ("humanized") to reduce or eliminate antigenicity in humans. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non-humanized binding protein from which it was derived.
  • chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison, et al. (1984) PROC. NAT. ACAD. SCI. 81: 6851-6855, Neuberger et al. (1984) NATURE 312: 604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).
  • CDR grafting the CDRs of the light and heavy chain variable regions are grafted into frameworks from another species.
  • murine CDRs can be grafted into human FR sequences.
  • the CDRs of the light and heavy chain variable regions of an anti-FGFR3 antibody are grafted into human FRs or consensus human FRs.
  • consensus human FRs FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described in U.S. Patent Nos.
  • human immunogenicity is reduced or eliminated by an alternative form of grafting.
  • human CDR sequences are chosen from a set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al. (2002) J. IMMUNOL 169:1119-1125.
  • ACTIVMABTM technology Vaccinex, Inc., Rochester, NY
  • ACTIVMABTM technology Vaccinex, Inc., Rochester, NY
  • High levels of combinatorial diversity of immunoglobulin heavy and light chains are said to be produced. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).
  • HUMAN ENGINEERINGTM HETM
  • US XOMA LLC
  • Any suitable approach including any of the above approaches, can be used to reduce or eliminate human immunogenicity of a binding protein of the invention.
  • Binding proteins of the invention can be conjugated to, or fused with, other molecules, depending upon their intended use. For example, if the binding protein is going to be used as a therapeutic, then the binding protein can be conjugated with another agent, for example, an effector molecule that modulates or otherwise promotes the therapy. A small molecule drug, a radiolabel or toxin, then, the agent can be chemically coupled to the binding protein using standard in vitro coupling chemistries. If the effector molecule is a protein or peptide, the binding protein can be chemically coupled to the effector using in vitro coupling chemistries or can be coupled to the effector as a fusion protein. Fusion proteins can be constructed and expressed using the techniques similar to those discussed in section II.
  • Binding proteins of the invention can be used as a research agent, diagnostic agent or therapeutic agent.
  • binding proteins of the invention prevent or inhibit the activation of FGFR3, they can be used in therapeutic applications.
  • certain binding proteins of the invention are useful in the prevention or treatment of hyperproliferative diseases or disorders, e.g., various forms of cancer and skeletal disorders.
  • the binding proteins can be used to inhibit or reduce the proliferation of cancer cells.
  • the cancer cells are exposed to a therapeutically effective amount of the binding protein so as to inhibit or reduce proliferation of the cancer cell.
  • the binding proteins inhibit cancer cell proliferation by at least 50%, 60%, 70%, 80%, 90%, or 95%.
  • the binding protein is used to inhibit or reduce proliferation of a tumor cell wherein the binding protein inhibits binding of hFGFR3 to an FGF ligand, e.g., FGFl.
  • the binding protein can be used in a method to inhibit tumor growth in a mammal, e.g., a human patient.
  • the method comprises administering to the mammal a therapeutically effective amount of the binding protein.
  • Cancers associated with FGFR3 activation include bladder cancer, cervical cancer, oral squamous cell cancer, non- small cell lung cancer, breast cancer, lymphoma, and multiple myeloma.
  • Exemplary skeletal disorders that are associated with FGFR3 activation include achondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TD I and TD II), and craniosynostosis syndromes.
  • treat, "treating” and “treatment” mean the treatment of a disease in a mammal, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.
  • a therapeutically effective amount of active component will be in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg.
  • the amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the binding protein, the pharmaceutical formulation, and the route of administration.
  • the initial dosage may be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue level. Alternatively, the initial dosage may be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment.
  • Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg.
  • Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.
  • a preferred route of administration is parenteral, e.g., intravenous infusion.
  • Formulation of monoclonal antibody-based drugs is within ordinary skill in the art.
  • the binding protein e.g., monoclonal antibody
  • the binding protein e.g., monoclonal antibody
  • the binding protein e.g., monoclonal antibody
  • binding proteins may be administered either alone or in combination with other pharmaceutically active ingredients, e.g., a chemotherapeutic drug.
  • the other active ingredients e.g., immunomodulators, can be administered together with the binding protein, before or after the binding protein.
  • the binding proteins preferably are combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means buffers, carriers, and excipients, that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the carrier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • compositions containing binding proteins of the invention can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration.
  • routes of administration are intravenous (IV), intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration.
  • IV infusion A preferred route of administration for monoclonal antibodies is IV infusion.
  • Useful formulations can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
  • Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene-diamine-tetra-acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include aqueous solutions (where water soluble) or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
  • Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted prior to or following lyophilization and reconstitution. Once the pharmaceutical composition has been formulated, it can be stored, for example, in vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • the binding proteins When the binding proteins are used for diagnostic purposes, either in vitro or in vivo, the binding proteins typically are labeled either directly or indirectly with a detectable moiety.
  • the detectable moiety can be any moiety that produces, either directly or indirectly, a detectable signal.
  • the detectable moiety can be a radioisotope, e.g., 3 H, 14 C, 32 P, 35 S, 125 I or
  • a fluorescent or chemiluminescent compound e.g., fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase
  • a spin label e.g., a colored particle, e.g., a latex particle or gold particle.
  • the binding protein can be conjugated to the detectable moiety by any suitable method. See, e.g., Hunter et ⁇ l. (1962) NATURE 144: 945; David et ⁇ l. ⁇ 191 A) BIOCHEMISTRY 13: 1014; Pain et ⁇ l. (1981) J.
  • binding proteins can be employed in immunoassay techniques.
  • Exemplary immunoassays include sandwich immunoassays, competitive immunoassays, and immunohistochemical procedures.
  • two antibodies that bind an analyte or antigen of interest are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with a detectable moiety.
  • the antigen binds to both the immobilized antibody and the labeled antibody, to form a "sandwich" immune complex on the surface of the support.
  • the complexed protein is detected by washing away non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support's surface.
  • the antibody free in solution can be detected by a third antibody labeled with a detectable moiety which binds the free antibody.
  • a detectable moiety which binds the free antibody.
  • Labeled binding proteins are useful as in vivo imaging agents, whereby the binding proteins can target the imaging agents to tissues of interest.
  • An exemplary remotely detectable moiety for in vivo imaging is the radioactive atom Technetium "99 " 1 ( 99m Tc), a gamma emitter with a half- life of about six hours.
  • Non-radioactive moieties useful in in vivo imaging include nitroxide spin labels, lanthanide and transition metal ions, all of which induce proton relaxation in situ.
  • the complexed radioactive moieties may be used in radioimmunotherapy protocols to destroy the targeted cell. Suitable isotopes for radioimmunotherapy include the radioactive atoms 90 Yt, 131 I and 111 In.
  • This Example describes the production of a number of anti-hFGFR3 monoclonal antibodies.
  • Immunizations, fusions, and primary screens were conducted at Maine Biotechnology Services Inc. following the Repetitive Immunization Multiple Sites (RIMMS) protocol.
  • Five AJ mice and five Balb/c mice were immunized with recombinant human FGFR3 IHb (R&D Systems, Catalog No. 1264-FR-050) and FGFR3 IIIc (R&D Systems, Catalog No. 766-FR-050) where the Fc fragment was removed from each by Factor Xa protease cleavage.
  • Example 2 Sequence Analysis of anti-hFGFR3 Monoclonal Antibodies
  • the light-chain isotype and heavy chain isotype of each monoclonal antibody was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit according the manufacturer's instructions (Roche Applied Science). [0098] All antibodies were determined to be Kappa immunoglobulin light chain and IgGl immunoglobulin heavy chain.
  • variable regions of the Kappa and Heavy (IgGl) immunoglobulin chains were amplified by PCR (Polymerase Chain Reaction) using the Expand High-Fidelity PCR System (Roche Applied Science) according the manufacturer's instructions.
  • Heavy chain variable regions were amplified with the GeneRacerTM 5' Primer, 5'-cgactggagcacgaggacactga-3' (SEQ ID NO: 58) (Invitrogen), and a 3' IgGl Constant Region specific primer, either 5' tatgcaaggcttacaaccaca 3' (SEQ ID NO: 59) or 5' gccagtggatagacagatgggggtgtcg 3' (SEQ ID NO: 60).
  • Kappa chain variable regions were amplified with the GeneRacerTM 5' Primer and a 3' Kappa Constant Region specific primer, either 5' ctcattcctgttgaagctcttgacaat 3' (SEQ ID NO: 61) or 5' cgactgaggcacctccagatgtt 3' (SEQ ID NO: 62).
  • PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquick Gel Purification kit according to the manufacturer's instructions (Qiagen). The PCR products were subsequently cloned into the pCR2.1 TOPO plasmid using the topoisomerase based cloning kit TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) according to the manufacturer's instructions (Invitrogen) and transformed into DH5-alpha bacteria through standard molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using M 13 Forward (5'
  • Monoclonal antibodies 4E7 and 7D12 have identical heavy chain sequences and identical light chain sequences.
  • Table 1 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
  • each variable sequence above is combined with its respective constant region.
  • a complete heavy chain comprises a heavy variable sequence followed by the murine IgGl heavy chain constant sequence and a complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence.
  • variable region sequences described herein can be ligated to each of a number of other constant region sequences known to those skilled in the art to produce active full length immunoglobulin heavy and light chains.
  • Protein Sequence Defining the Full Length 27H2 Heavy Chain Sequence (27H2 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 43) 1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtry
  • Protein Sequence Defining the Full Length 27H2 Light Chain Sequence (27H2 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 45) 1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasgvpar
  • (20B4 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 54) i gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
  • Table 2 provides a concordance chart showing the correspondence between the full length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.
  • the antibodies were captured in an individual flow cell at a flow rate of 10 ⁇ l/min. Injection time was varied for each antibody to yield approximately 40-50 RU of antibody captured for each cycle. Buffer or rhFGFR3 IHb Fc (R&D Systems, Catalog No. 1264-FR- 050) or rhFGFR3 IIIc Fc (R&D Systems, Catalog No. 766-FR-050) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 300 sec at 60 ⁇ l/min. The dissociation phase was monitored for 30 minutes.
  • the surface was then regenerated with two 60-seconds injection of 10 mM Glycine- HCl, pH 1.7 (made from Glycine pH 1.5 (Biacore, Catalog No. BR- 1003-54) and pH 2.0 (Biacore, Catalog No. BR- 1003-55)) at a flow rate of 60 ⁇ l/min.
  • rhFGFR3 Fc concentrations tested were 0.62 nM to 40 nM.
  • Kinetic parameters were determined using the kinetic function of the BIAevalutation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, k a (association rate constant), k ⁇ j (dissociation rate constant) and K D (equilibrium dissociation constant) were determined. Kinetic values of the monoclonal antibodies at 25 0 C are summarized in Table 3.
  • Example 1 The antibodies produced in Example 1 were characterized for their ability to inhibit recombinant hFGFR3 IHb binding to FGFl (also known as FGF acidic).
  • the antibodies were tested by ECL (Electrochemiluminescence) assay for inhibition of hFGFR3 IHB binding to FGF-I.
  • MA2400 96- well high binding plates (Meso Scale Discovery, Catalog No. L15XB-6) were coated with 25 ⁇ l of 0.8 ⁇ g/mL FGF-I (R&D Systems, Catalog No. 232-FA-025) in PBS (Invitrogen, Catalog No. 14040-133) for 1 hour at room temperature with agitation. The plates then were washed 3 times with PBS and blocked with 200 ⁇ l of PBS containing 5% BSA (Sera Care Life Sciences, Catalog No.
  • AP-4510-80 and 5 ⁇ g/mL heparin (Sigma, Catalog No. H4784) for 1 hour at room temperature.
  • the antibodies (concentration range: 0.029-30 ⁇ g/mL) were incubated for 1 hour at room temperature with 1.7 ⁇ g/mL rhFGFR3 HIb Fc (R&D Systems, Catalog No. 1264-FR-050) and 5 ⁇ g/mL heparin. After washing the plates 3 times with PBS, 25 ⁇ l of the antibody- receptor mixture was added to the plates for another hour at room temperature with agitation.
  • the plates were washed three times with PBS and incubated with 25 ⁇ l of 1 ⁇ g/mL ST-anti-human IgG antibody (Meso Scale Discovery, Catalog No. R32AJ-1) for 1 hour at room temperature with agitation. The plates then were washed 3 times with PBS, and 150 ⁇ l of IX read buffer (Meso Scale Discovery, Catalog No. R92TC-1) was added to each well before the plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery) instrument.
  • Example 5 Anti-Proliferative Activity of Anti-hFGFR3 Antibodies
  • the antibodies produced in Example 1 were characterized for their ability to inhibit FGFl dependent proliferation of cells.
  • FDCP-I cells mouse bone marrow cells obtained from German Collection of Microorganisms and Cell Cultures
  • plasmids encoding human FGFR3 IHb, FGFR3 IHc, or a mutant variant G380R (an activating mutation associated with the skeletal disorder, achondroplasia (Webster and Donoghue (1996) EMBO J. 15:520-527) by electroporation and selected with G418 (600 ⁇ g/mL).
  • G418 600 ⁇ g/mL
  • FDCP-FGFR3 IHb #122, FDCP-FGFR3 IIIc #109, FGFR3 IIIc G380R #1 exhibited FGF-I induced proliferation in the absence of IL3.
  • hybridoma supernatants containing FGFR3 antibodies were added to FDCP-FGFR3 IHb #122 or FDCP-FGFR3 IIIc #109 cells cultured in basic growth medium (70% ISCOVE' s Modified Dulbecco's Medium (Invitrogen, Catalog No. 12440-053), 20% horse serum (Invitrogen, Catalog No.
  • FGFR3 antibodies were added to the cells along with FGFl (8ng/mL) and heparin (50 ⁇ g/mL).
  • the cells were cultured in basic growth medium (70% ISCOVE' s, 20% horse serum and 10% WEHI-culture medium (90% Iscove's MDM + 10% FBS + 2 mM L-glutamine + 0.0025 mM mercaptoethanol)) in a 96-well plate (70,000 cells/ well).
  • the final concentration of FGFl and heparin used in the assay is 8 ng/mL and 5 ⁇ g/mL, respectively.
  • MTT assay was conducted one to three days post FGFl stimulation.
  • OPM-2 xenograft model The ability of murine monoclonal antibodies of the invention to inhibit tumor growth was tested in an OPM-2 xenograft model.
  • OPM-2 cells were grown in culture at 37°C in an atmosphere containing 5% CO 2 , using RMPI medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). Cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice (Taconic Labs) with 5 x 10 6 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237).
  • Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width x width x length/2. When tumors reached approximately 150 mm 3 , the mice were randomized into 4 groups of 10 mice each.
  • This Example describes the humanization of the murine antibody designated 15D8, and the characterization of the resulting humanized antibody.
  • the humanized anti-FGFR3 antibody was designed using the SUPERHUMANIZATIONTM method (Arana Therapeutics Ltd. and Hwang, W.Y. et al. (2005) METHODS 36:35-42). Certain framework residues were converted to murine 15D8 residues to improve the antibody's affinity toward FGFR3, and the antibody's activity in inhibiting the biological activity of FGFR3, or both.
  • the designed amino acid sequences were converted to codon-optimized DNA sequences, including (in the following order): 5' HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, humanized variable region, human IgGl or Kappa constant region, stop codon, and a 3' EcoRI restriction site.
  • Chimeric (murine variable region and human constant region) 15D8 heavy (human IgGl) and light (human Kappa) chains were also constructed.
  • the murine variable regions were fused to the human constant region using overlap extension PCR, including (in the following order): 5' HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, mouse variable region, human IgGl or Kappa constant region, stop codon, and 3' EcoRI restriction site.
  • the humanized and chimeric IgGl heavy chains were subcloned into pEE6.4 (Lonza Biologies) via HindIII and EcoRI sites.
  • the humanized and chimeric Kappa light chains were subcloned into pEE14.4 (Lonza Biologies) via HindIII and EcoRI sites.
  • Chimeric 15D8 Full Length Chimeric 15D8 Heavy Chain (Mouse Variable Region and Human IgGl Constant Region) (SEQ ID NO: 66) plus Full Length Chimeric 15D8 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 68)
  • Humanized 15D8 Full Length Humanized 15D8 Heavy Chain (Humanized Variable Region and Human IgGl Constant Region) (SEQ ID NO: 74) plus Full Length Humanized 15D8 Light Chain (Humanized Variable Region and Human Kappa Constant Region) (SEQ ID NO: 76) [0182]
  • the nucleic acid sequences encoding and the polypeptide sequences defining the chimeric and humanized antibodies are summarized below (amino terminal signal sequences are not shown).
  • CDR sequences Kabat definition) are shown in bold/underlined in the amino acid sequences.
  • Nucleic Acid Sequence Defining the Full Length Humanized 15D8 (Hul5D8) Heavy Chain (Humanized Variable Region and Human IgGl Constant Region) (SEQ ID NO: 73) 1 gaggtccaac tggtgcaatc tggggctgag gtcaagaaac ccggggaatc tctcaaaatt
  • Table 8 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
  • the KD was determined. Unbound anti-FGFR3 antibody was detected by allowing the anti-FGFR3 antibody/FGFR3 IIIc Fc solution to flow through the FGFR3 IIIc Fc PMMA beads. The anti- FGFR3 antibody captured by these beads was then detected with Cy5-conjugated anti-human secondary antibody 0.3 ug/ml (Jackson ImmunoResearch) or Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) 0.5ug/ml in PBS BSA 1 mg/ml. The detected signal for captured anti-FGFR3 antibody is directly proportional to the remaining free binding sites, thus allowing KD determination.

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Abstract

Monoclonal antibodies that bind and inhibit activation of fibroblast growth factor receptor 3 (FGFR3) are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation of FGFR3.

Description

FIBROBLAST GROWTH FACTOR RECEPTOR 3 (FGFR3) BINDING PROTEINS
RELATED APPLICATIONS
[0001] This application claims the benefit and priority of U.S. Provisional Application No. 61/077,278, filed July 1, 2008, the entire contents of which are incorporated herein by reference.
FIELD OF INVENTION [0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field is antibody-based binding proteins that bind human fibroblast growth factor receptor 3 (FGFR3).
BACKGROUND
[0003] Fibroblast Growth Factor Receptor 3 (FGFR3) is one member of a family of receptor tyrosine kinases (FGFRl, FGFR2, FGFR3, FGFR4) that binds fibroblast growth factors (FGFs) (Keegan et al. (1991) PROC. NATL. ACAD. SCI. USA 88: 1095-1099). FGF receptors are characterized as having three extracellular immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. FGF ligand binding induces FGF receptor dimerization and tyrosine autophosphorylation resulting in cell proliferation, differentiation, and migration (Gomez-Roman et al. (2005) CLIN. CANCER RES. 11:459-65; Chang et al. (2005) BLOOD 106:353-6; Eswarakumar et al. (2005) CYTOKINE GROWTH FACTOR REV. 16(2) : 139-49) .
[0004] Alternative splicing of the FGFR3 transcript results in two isoforms, IHb and IHc. The FGFR3 isoforms are differentially expressed with epithelial cells expressing predominantly the IHb isoform, whereas fibroblast cells express a mixture of IHb and IIIc transcripts (Scotet et al. (1995) BiocHiM. BIOPHYS. ACTA 1264:238-42). In addition, the IHb and IIIc splice variants differ in their specificity for FGF ligand. The IHb splice variant has high affinity for FGFl (acidic FGF) ligand and lower affinity for FGF8 (androgen-induced growth factor) and FGF9 (glial activating factor) (Chellaiah et al. (1999) J. BlOL. CHEM. 274:34785-94; Gomez-Roman et al. (2005) supra). The IIIc splice variant is characterized as a promiscuous receptor binding numerous FGF ligands including FGFl, FGF2, FGF4, FGF8, FGF9, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, and FGF23 (Chellaiah et al. (1994) J. BIOL. CHEM. 269:11620-7; Gomez-Roman et al. (2005) supra; Ornitz et al. (1996) J. BIOL. CHEM. 271(25): 15292-7; Lee et al. (2000) J. BIOL. CHEM. 275(43):33679-87). [0005] The FGFR3-FGF signaling pathway plays a role in the differentiation of adipocytes, chondrocytes and neurons, wound healing, angiogenesis, embryo development, and malignancies (Keegan et al. (1991) supra). Activating mutations of FGFR3 have been associated with cancer and skeletal disorders including dwarfism, achondroplasia, and hypochondroplasia (Gomez-Roman et al. (2005) supra; Delezoide et al. (1997) HUMAN MOL. GENETICS 6:1899-1906). Certain FGFR3 antibodies are known. See, e.g., U.S. 2005/0147612 (Yayon).
[0006] Antibodies are multimeric proteins that contain four polypeptide chains (Figure 1). Two of the polypeptide chains are called heavy chains (H chains), and two of the polypeptide chains are called light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL in Figure 1) and one constant region (CL in Figure 1). The heavy chain consists of one variable region (VH in Figure 1) and at least three constant regions (CH1, CH2 and CH3 in Figure 1). The variable regions determine the specificity of the antibody. [0007] Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity.
[0008] Although certain anti-FGFR3 antibodies are known in the art, there is still a need for additional FGFR3 modulators that can be used as therapeutic and diagnostic agents.
SUMMARY OF THE INVENTION
[0009] The invention is based, in part, upon the discovery of a family of binding proteins that specifically bind human FGFR3. The binding proteins contain FGFR3 binding sites based on the CDRs of a family of antibodies that specifically bind FGFR3. The binding proteins can be used as diagnostic and therapeutic agents. When used as a therapeutic agent, the binding proteins are engineered, e.g., humanized, to reduce or eliminate an immune response when administered to a human patient.
[0010] The binding proteins prevent or inhibit the activation of (i.e., neutralize) human FGFR3. In some embodiments, the binding proteins prevent FGFR3 from binding to a ligand, e.g., FGFl, thereby neutralizing FGFR3 activation. The binding proteins can be used to inhibit the proliferation of tumor cells or stimulate the proliferation of chondrocytes. Furthermore, when administered to a mammal, the binding proteins can inhibit or reduce tumor growth in the mammal.
[0011] These and other aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims. As used herein, "including" means without limitation, and examples cited are non-limiting.
DESCRIPTION OF THE DRAWINGS
[0012] The invention can be more completely understood with reference to the following drawings.
[0013] Figure 1 (prior art) is a schematic representation of a typical antibody. [0014] Figure 2 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin heavy chain variable region of antibodies 15D8, 27H2, 2G4, 4E7 (7D12), and 20B4. The amino acid sequences for each antibody are aligned against one another, and CDR1, CDR2, and CDR3 are identified in boxes. The unboxed sequences represent framework (FR) sequences. [0015] Figure 3 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin heavy chain variable region sequences in Figure 2. For antibody 15D8, three alternative CDR2 sequences are shown (15D8, 15D8-2, and 15D8-3).
[0016] Figure 4 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin light chain variable region of antibodies 15D8, 27H2, 2G4, 4E7 (7D12), and 20B4. The amino acid sequences for each antibody are aligned against one another, and CDR1, CDR2, and CDR3 are identified in boxes. The unboxed sequences represent framework (FR) sequences.
[0017] Figure 5 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin light chain variable region sequences in Figure 4. [0018] Figure 6 is a graph summarizing results from an experiment to measure neutralization activity of negative control IgGl (A) and anti-FGFR3 monoclonal antibodies 15D8 (X), 27H2 (+), 2G4 (■), 4E7 (T), 7D12 (0), and 20B4 (•) to inhibit FGFR3 binding to FGFl. [0019] Figure 7 is a graph summarizing results from an experiment to measure neutralization activity of negative control IgGl Fab (D) and anti-FGFR3 Fab fragments 15D8 (0), 27H2 (•), 2G4 (*), 4E7 (■), and 7D12 ( A) to inhibit FGFR3 binding to FGFl.
[0020] Figure 8 is a graph summarizing results from an experiment to measure anti- proliferation activity of negative control (murine IgGl) (■) and anti-FGFR3 monoclonal antibodies 15D8 (T), 27H2 (♦), 2G4 (•), and 20B4 ( A) in FDCP-FGFR3 IIIc-109 cells.
[0021] Figure 9 is a graph summarizing results from an experiment to measure tumor inhibitory activity of a murine IgG control at 20 mg/kg (♦) and anti-FGFR3 antibody 15D8 in a OPM-2 xenograft tumor model (antibody 15D8 at 5 mg/kg (■); antibody 15D8 at 10 mg/kg (A); and antibody 15D8 at 20 mg/kg (•)). [0022] Figure 10 is a graph summarizing results from an experiment to measure tumor inhibitory activity of a murine IgG control at 1 mg/kg (♦) and anti-FGFR3 antibodies dosed in an OPM-2 xenograft tumor model (murine antibody 15D8 at 1 mg/kg (A); murine antibody 4E7 at 1 mg/kg (X); murine antibody 27H2 at 1 mg/kg (o); and murine antibody 2G4 at 1 mg/kg (Δ)). DETAILED DESCRIPTION
[0023] The invention is based, in part, upon the discovery of a family of binding proteins that specifically bind and neutralize the activity of human FGFR3. The binding proteins can be used in a variety of diagnostic and therapeutic applications. The binding proteins are based upon the antigen binding sites of certain monoclonal antibodies that have been selected for their ability to bind and neutralize the activity of FGFR3. The binding proteins contain immunoglobulin variable region CDR sequences that define a binding site for FGFR3.
[0024] In view of the neutralizing activity of these antibodies, they are useful for modulating the growth and/or proliferation of certain cancer cells. When used as a therapeutic agent, the binding proteins can be engineered to minimize or eliminate an immune response when administered to a human patient. In some embodiments of the invention, the binding proteins are fused or conjugated to other moieties, such as detectable labels (e.g., radiolabels) or effector molecules (e.g., other proteins or small molecule therapeutics). Various features and aspects of the invention are discussed in more detail below.
I - Binding Proteins That Bind FGFR3 [0025] In certain embodiments of the invention, the binding protein comprises (i) an immunoglobulin heavy chain variable region comprising the structure CDRHI-CDRH2-CDRH3 and (ii) an immunoglobulin light chain variable region comprising three complementarity determining regions (CDRs), wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human FGFR3. CDRHI comprises the amino acid sequence X1 Tyr Asn Met Tyr (SEQ ID NO: 81), wherein amino acid X1 is Asp or Ser. CDRH2 comprises the amino acid sequence Tyr He Asp Pro Tyr Asn GIy GIy Thr X2 X3 Asn X4 X5 Phe X6 GIy (SEQ ID NO: 82), wherein amino acid X2 is Arg or Ser, amino acid X3 is Asp or Tyr, amino acid X4 is GIn or Pro, amino acid X5 is a Lys or Ser, and amino acid X6 is Lys or GIn. CDRH3 comprises the amino acid sequence X7 X8 GIy X9 X10 X11 X12 Xi3 Phe X14 Tyr (SEQ ID NO: 89), wherein amino acid X7 is GIu or Ser, amino acid X8 is GIy or Leu, amino acid X9 is Asn or a peptide bond, amino acid X10 is Tyr or a peptide bond, amino acid X11 is GIu or a peptide bond, amino acid X12 is Ala or Pro, amino acid X13 is Trp or Asp, and amino acid X14 is Ala or Asp.
[0026] In some embodiments of the invention, the binding protein comprises (i) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3 and (ii) an immunoglobulin heavy chain variable region comprising three CDRs, wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human FGFR3. CDRL1 comprises the amino acid sequence Ser Ala Ser Ser Ser VaI X15 Tyr Met X16 (SEQ ID NO: 83), wherein amino acid X15 is Ser or Asn, and X16 is Tyr or His. CDRL2 comprises the amino acid sequence X17 Thr Ser X18 Leu Ala Ser (SEQ ID NO: 84), wherein the amino acid X17 is Leu or Asp, and the amino acid X18 is Asn, Lys, or Tyr. CDRL3 comprises the amino acid sequence GIn GIn Trp X19 Ser X2o Pro Leu Thr (SEQ ID NO: 85), wherein the amino acid X19 is Ser or Asn, and the amino acid X2o is Asn or Tyr. [0027] In some embodiments, the binding protein comprises an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, wherein (i) CDRH1 comprises the amino acid sequence Ser Tyr Asn Met Tyr (SEQ ID NO: 17), (ii) CDRH2 comprises the amino acid sequence Tyr He Asp Pro Tyr Asn GIy GIy Thr X1 X2 Asn X3 X4 Phe X5 GIy (SEQ ID NO: 86), wherein amino acid X1 is Arg or Ser, amino acid X2 is Asp or Tyr, amino acid X3 is GIn or Pro, amino acid X4 is Lys or Ser, and amino acid X5 is Lys or GIn, and (iii) CDRH3 comprises the amino acid sequence GIu GIy GIy Asn Tyr GIu Ala Trp Phe Ala Tyr (SEQ ID NO: 19), and an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, wherein (i) CDRL1 comprises the amino acid sequence Ser Ala Ser Ser Ser VaI Ser Tyr Met Tyr (SEQ ID NO: 22), (ii) CDRL2 comprises the amino acid sequence Leu Thr Ser X6 Leu Ala Ser (SEQ ID NO: 87), wherein the amino acid X6 is Asn or Tyr, and (iii) CDRL3 comprises the amino acid sequence GIn GIn Trp Ser Ser X7 Pro Leu Thr (SEQ ID NO: 88), wherein the amino acid X7 is Asn or Tyr.
[0028] The binding protein can comprise both the immunoglobulin light chain and the immunoglobulin heavy chain sequences or the fragments thereof, described above. The binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site.
[0029] In some embodiments, the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed with framework regions (FR). The framework regions optionally can be humanized or fully human.
[0030] In some embodiments of the invention, the binding protein comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR3. The CDRH1 comprises a sequence selected from the group consisting of SEQ ID NO: 17 (15D8, 27H2, 4E7(7D12), 2G4) and SEQ ID NO: 29 (20B4). The CDRH2 comprises a sequence selected from the group consisting of SEQ ID NO: 18 (15D8, 20B4), SEQ ID NO: 20 (15D8-2), SEQ ID NO: 21 (15D8-3), SEQ ID NO: 25 (27H2, 4E7(7D12)), and SEQ ID NO: 28 (2G4). The CDRH3 comprises a sequence selected from the group consisting of SEQ ID NO: 19 (15D8, 27H2, 4E7(7D12), 2G4) and SEQ ID NO: 30 (20B4). Throughout the specification a particular SEQ ID NO. is followed in parentheses by the antibody that was the origin of that sequence. For example, "SEQ ID NO: 29 (20B4)" means SEQ ID NO: 29 comes from antibody 20B4.
[0031] In certain embodiments, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRHi comprising the sequence of SEQ ID NO: 17 (15D8), a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), SEQ ID NO: 20 (15D8-2), and SEQ ID NO: 21 (15D8-3), and a CDRH3 comprising the sequence of SEQ ID NO: 19 (15D8). In a preferred embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ ID NO: 17 (15D8), a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), and a CDRH3 comprising the sequence of SEQ ID NO: 19 (15D8).
[0032] In some embodiments, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 17 (27H2, 4E7(7D12)), a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25 (27H2, 4E7(7D12)), and a CDRH3 comprising the sequence of SEQ ID NO: 19 (27H2, 4E7(7D12)).
[0033] In some embodiments, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 17 (2G4), a CDRH2 comprising the sequence of SEQ ID NO: 28 (2G4), and a CDRH3 comprising the sequence of SEQ ID NO: 19 (2G4).
[0034] In one embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRm comprising the sequence of SEQ ID NO: 29 (20B4), a CDRH2 comprising the sequence of SEQ ID NO: 18 (20B4), and a CDRH3 comprising the sequence of SEQ ID NO: 30 (20B4). [0035] Preferably, the CDRm, CDRH2, and CDRH3 sequences are interposed between human or humanized immunoglobulin FRs. The binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
[0036] In some embodiments, the binding protein comprises (a) an immunoglobulin light chain variable region comprising the structure CDRLI-CDRL2-CDRL3, and (b) an immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region together define a single binding site for binding human FGFR3. The CDRL1 comprises a sequence selected from the group consisting of SEQ ID NO: 22 (15D8, 27H2, 2G4, 4E7(7D12)) and SEQ ID NO: 31 (20B4); the CDRL2 comprises a sequence selected from the group consisting of SEQ ID NO: 23 (15D8), SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 32 (20B4); and the CDRL3 comprises a sequence selected from the group consisting of SEQ ID NO: 24 (15D8), SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 33 (20B4),.
[0037] In some embodiments, the binding protein comprises an immunoglobulin light chain variable region comprising: a CDRLI comprising the sequence of SEQ ID NO: 22 (15D8); a CDRL2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23 (15D8); and a CDRL3 comprising the sequence of SEQ ID NO: 24 (15D8).
[0038] In one embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising: a CDRLI comprising the sequence of SEQ ID NO: 22 (27H2, 2G4, 4E7(7D12)); a CDRL2 comprising the sequence of SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)); and a CDRL3 comprising the sequence of SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)).
[0039] In one embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising: a CDRLI comprising the sequence of SEQ ID NO: 31 (20B4); a CDRL2 comprising the sequence of SEQ ID NO: 32 (20B4); and a CDRL3 comprising the sequence of SEQ ID NO: 33 (20B4). [0040] Preferably, the CDRL1, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FRs. The binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
[0041] In some embodiments of the invention, the binding protein comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRHI-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region comprising the structure CDRLI -CDRL2- CDRL3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR3. The CDRHi comprises SEQ ID NO: 17 (15D8, 27H2, 2G4, 4E7(7D12)); a CDRH2 is selected from the group consisting of SEQ ID NO: 18 (15D8), SEQ ID NO: 20 (15D8-2), SEQ ID NO: 21 (15D8-3), SEQ ID NO: 25 (27H2, 4E7(7D12)), and SEQ ID NO: 28 (2G4); and the CDRH3 comprises SEQ ID NO: 19 (15D8, 27H2, 2G4, 4E7(7D12)). The CDRL1 comprises SEQ ID NO: 22 (15D8, 27H2, 2G4, 4E7(7D12)); a CDRL2 is selected from the group consisting SEQ ID NO: 23 (15D8) and SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)); and a CDRL3 is selected from the group consisting SEQ ID NO: 24 (15D8) and SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)).
[0042] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (15D8), SEQ ID NO: 6 (27H2), SEQ ID NO: 10 (2G4), SEQ ID NO: 12 (4E7(7D12)), and SEQ ID NO: 14 (20B4), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (15D8), SEQ ID NO: 8 (27H2, 2G4, 4E7(7D12)), and SEQ ID NO: 16 (20B4). [0043] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 2 (15D8), and an immunoglobulin light chain variable region comprising SEQ ID NO: 4 (15D8).
[0044] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 6 (27H2), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (27H2).
[0045] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 10 (2G4), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (2G4).
[0046] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 12 (4E7(7D12)), and an immunoglobulin light chain variable region comprising SEQ ID NO: 8 (4E7(7D12)).
[0047] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 14 (20B4), and an immunoglobulin light chain variable region comprising SEQ ID NO: 16 (20B4). [0048] In each of the foregoing embodiments, the binding protein can be an intact antibody, an antigen-binding antibody fragment, or a biosynthetic antibody site.
[0049] In other embodiments, the binding protein comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 39 (15D8), SEQ ID NO: 43 (27H2), SEQ ID NO: 47 (2G4), SEQ ID NO: 51 (4E7(7D12)), and SEQ ID NO: 55 (20B4), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 41 (15D8), SEQ ID NO: 45 (27H2), SEQ ID NO: 49 (2G4), SEQ ID NO: 53 (4E7(7D12)) and SEQ ID NO: 57 (20B4).
[0050] In another embodiment, the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 39 (15D8), and an immunoglobulin light chain comprising SEQ ID NO: 41 (15D8).
[0051] In another embodiment, the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 43 (27H2), and an immunoglobulin light chain comprising SEQ ID NO: 45 (27H2).
[0052] In another embodiment, the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 47 (2G4), and an immunoglobulin light chain comprising SEQ ID NO: 49 (2G4).
[0053] In another embodiment, the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 51 (4E7(7D12)), and an immunoglobulin light chain comprising SEQ ID NO: 53 (4E7(7D12)). [0054] In another embodiment, the binding protein comprises an immunoglobulin heavy chain comprising SEQ ID NO: 55 (20B4), and an immunoglobulin light chain comprising SEQ ID NO: 57 (20B4).
[0055] Each of the binding proteins described above can be an intact antibody, e.g., a monoclonal antibody. Alternatively, the binding protein can be an antigen binding fragment of an antibody, or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab', (Fab')2 and Fv fragments. Techniques for making antibody fragments are known in the art. Biosynthetic antibody binding sites are known in the art, e.g., single Fv and sFv molecules. See, e.g., U.S. Patent No. 5,476,786. Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, e.g., antibodies or antibody fragments that bind at least two different antigens. For example, bispecific binding proteins can bind human FGFR3 and another antigen of interest. Methods for making bispecific antibodies are known in art. Such methods include fusing hybridomas or by linking Fab' fragments. See, e.g., Songsivilai et αl. (1990) CLIN. EXP. IMMUNOL. 79: 315-325; Kostelny et αl. (1992) J. IMMUNOL. 148: 1547- 1553. [0056] In some embodiments of the invention, an isolated binding protein binds human FGFR3 with a KD of 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or lower, wherein the KD values are determined by surface plasmon resonance methods under the conditions described in Example
3. [0057] In some embodiments of the invention, an isolated binding protein binds human FGFR3 with a KD of 200 pM, 150 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, or lower, wherein the KD values are determined by a kinetic exclusion assay (See, e.g., Darling and Brault (2004) ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES 2: 647-657) under the conditions described in Example 7. [0058] In some embodiments, the binding proteins inhibit hFGFR3 from binding to FGFl. For example, the binding proteins can have an IC50 (concentration at 50% of maximum inhibition) of about 10, 11, 12, 13, 14, 15, 16, 17 or 18 nM, when assayed using the protocol described in Example 4.
II - Production of Binding Proteins [0059] Methods for producing binding proteins of the invention are known in the art. For example, DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired binding proteins. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or PCR techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells.
[0060] The nucleic acids encoding the desired binding proteins can be introduced (ligated) into expression vectors, which can be introduced into a host cell through conventional transfection or transformation techniques. Exemplary host cells include E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce immunoglobulin protein. Transfected host cells are grown under conditions that permit the host cells to express the genes of interest, e.g., genes that encode the immunoglobulin light or heavy chain variable regions.
[0061] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a signal sequence, e.g., a sequence encoding fragment B of protein A (FB). The expressed fusion protein accumulates in refractile or inclusion bodies in the bacterial cytoplasm, and may be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods already established for other recombinant proteins.
[0062] If the engineered gene is to be expressed in eukaryotic host cells, e.g., myeloma cells or CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, immunoglobulin enhancers, and various introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be transfected into myeloma cells or CHO cells using established transfection protocols. Such transfected cells can express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a protein domain having another function (e.g., cytotoxicity). III - Modifications to the Binding Proteins
[0063] The binding proteins can be modified to optimize performance, depending upon the intended use of the binding proteins. For example, when the binding protein is being used as a therapeutic agent, the binding protein can be modified to reduce its immunogenicity in a human patient. Alternatively or in addition, the binding protein can be fused or chemically conjugated to another protein or peptide, e.g., a growth factor, cytokine, or cytotoxin.
[0064] Various techniques for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the binding proteins are to be administered to a human, the binding proteins preferably are engineered ("humanized") to reduce or eliminate antigenicity in humans. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non-humanized binding protein from which it was derived. [0065] In one humanization approach, chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison, et al. (1984) PROC. NAT. ACAD. SCI. 81: 6851-6855, Neuberger et al. (1984) NATURE 312: 604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).
[0066] In another approach, known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted into frameworks from another species. For example, murine CDRs can be grafted into human FR sequences. In some embodiments of the invention, the CDRs of the light and heavy chain variable regions of an anti-FGFR3 antibody are grafted into human FRs or consensus human FRs. In order to create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described in U.S. Patent Nos. 7,022,500 (Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297 (Carter); 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones et al. (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT 430: 92-94.
[0067] In an approach called "superhumanization," human immunogenicity is reduced or eliminated by an alternative form of grafting. In superhumanization, human CDR sequences are chosen from a set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al. (2002) J. IMMUNOL 169:1119-1125.
[0068] Other methods to reduce immunogenicity include "reshaping," "hyperchimerization," and "veneering/resurfacing." See, e.g., Vaswami et al. (1998) ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81: 105; Roguska et al. (1996) PROT. ENGINEER 9: 895- 904; and U.S. Patent No. 6,072,035 (Hardman). In the veneering/resurfacing approach, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues more frequently found at the same positions in a human antibody. This type of antibody resurfacing is described, e.g., in U.S. Patent No. 5,639,641 (Pedersen).
[0069] Another approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which involves a vaccinia virus-based vector to express antibodies in mammalian cells. High levels of combinatorial diversity of immunoglobulin heavy and light chains are said to be produced. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).
[0070] Another approach for converting a mouse antibody into a form suitable for use in humans is technology practiced commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to produce an "epitope focused" library for antibody selection.
[0071] Another approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERING™ (HE™) technology, which is practiced commercially by XOMA (US) LLC. See, e.g., International Application Publication No. WO 93/11794 and U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619.
[0072] Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of a binding protein of the invention.
[0073] Binding proteins of the invention can be conjugated to, or fused with, other molecules, depending upon their intended use. For example, if the binding protein is going to be used as a therapeutic, then the binding protein can be conjugated with another agent, for example, an effector molecule that modulates or otherwise promotes the therapy. A small molecule drug, a radiolabel or toxin, then, the agent can be chemically coupled to the binding protein using standard in vitro coupling chemistries. If the effector molecule is a protein or peptide, the binding protein can be chemically coupled to the effector using in vitro coupling chemistries or can be coupled to the effector as a fusion protein. Fusion proteins can be constructed and expressed using the techniques similar to those discussed in section II.
IV - Use of Binding Proteins
[0074] Binding proteins of the invention can be used as a research agent, diagnostic agent or therapeutic agent.
(1) Therapeutic Applications
[0075] Because the binding proteins of the invention prevent or inhibit the activation of FGFR3, they can be used in therapeutic applications. For example, certain binding proteins of the invention are useful in the prevention or treatment of hyperproliferative diseases or disorders, e.g., various forms of cancer and skeletal disorders. [0076] The binding proteins can be used to inhibit or reduce the proliferation of cancer cells. In such an approach, the cancer cells are exposed to a therapeutically effective amount of the binding protein so as to inhibit or reduce proliferation of the cancer cell. In some embodiments, the binding proteins inhibit cancer cell proliferation by at least 50%, 60%, 70%, 80%, 90%, or 95%.
[0077] In some embodiments, the binding protein is used to inhibit or reduce proliferation of a tumor cell wherein the binding protein inhibits binding of hFGFR3 to an FGF ligand, e.g., FGFl.
[0078] The binding protein can be used in a method to inhibit tumor growth in a mammal, e.g., a human patient. The method comprises administering to the mammal a therapeutically effective amount of the binding protein.
[0079] Cancers associated with FGFR3 activation include bladder cancer, cervical cancer, oral squamous cell cancer, non- small cell lung cancer, breast cancer, lymphoma, and multiple myeloma. [0080] Exemplary skeletal disorders that are associated with FGFR3 activation include achondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TD I and TD II), and craniosynostosis syndromes.
[0081] As used herein, "treat, "treating" and "treatment" mean the treatment of a disease in a mammal, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.
[0082] Generally, a therapeutically effective amount of active component will be in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the binding protein, the pharmaceutical formulation, and the route of administration. The initial dosage may be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue level. Alternatively, the initial dosage may be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments of the invention, the binding protein, e.g., monoclonal antibody, is lyophilized and reconstituted in buffered saline at the time of administration.
[0083] The binding proteins may be administered either alone or in combination with other pharmaceutically active ingredients, e.g., a chemotherapeutic drug. The other active ingredients, e.g., immunomodulators, can be administered together with the binding protein, before or after the binding protein.
[0084] For therapeutic use, the binding proteins preferably are combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients, that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
[0085] Pharmaceutical compositions containing binding proteins of the invention can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration. A preferred route of administration for monoclonal antibodies is IV infusion. Useful formulations can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
[0086] Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene-diamine-tetra-acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0087] In general, pharmaceutical compositions suitable for injection include aqueous solutions (where water soluble) or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. [0088] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted prior to or following lyophilization and reconstitution. Once the pharmaceutical composition has been formulated, it can be stored, for example, in vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
(2) Diagnostic Applications
[0089] When the binding proteins are used for diagnostic purposes, either in vitro or in vivo, the binding proteins typically are labeled either directly or indirectly with a detectable moiety. The detectable moiety can be any moiety that produces, either directly or indirectly, a detectable signal. The detectable moiety can be a radioisotope, e.g., 3H, 14C, 32P, 35S, 125I or
131I; a fluorescent or chemiluminescent compound, e.g., fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; a spin label; or a colored particle, e.g., a latex particle or gold particle. The binding protein can be conjugated to the detectable moiety by any suitable method. See, e.g., Hunter et αl. (1962) NATURE 144: 945; David et αl. {191 A) BIOCHEMISTRY 13: 1014; Pain et αl. (1981) J. IMMUNOL. METH. 40: 219; and Nygren (1982) J. HISTOCHEM. AND CYTOCHEM. 30: 407. [0090] The binding proteins can be employed in immunoassay techniques. Exemplary immunoassays include sandwich immunoassays, competitive immunoassays, and immunohistochemical procedures.
[0091] In a sandwich immunoassay, two antibodies that bind an analyte or antigen of interest are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with a detectable moiety. When a sample containing the antigen is introduced into this system, the antigen binds to both the immobilized antibody and the labeled antibody, to form a "sandwich" immune complex on the surface of the support. The complexed protein is detected by washing away non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support's surface. Alternatively, the antibody free in solution can be detected by a third antibody labeled with a detectable moiety which binds the free antibody. See, e.g., Butt, ed., (1984) PRACTICAL IMMUNOLOGY, Marcel Dekker, New York; Harlow et al. eds. (1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor Laboratory; and Diamandis et al., eds. (1996) IMMUNOASSAY, Academic Press, Boston.
[0092] Labeled binding proteins are useful as in vivo imaging agents, whereby the binding proteins can target the imaging agents to tissues of interest. An exemplary remotely detectable moiety for in vivo imaging is the radioactive atom Technetium"99"1 (99mTc), a gamma emitter with a half- life of about six hours. Non-radioactive moieties useful in in vivo imaging include nitroxide spin labels, lanthanide and transition metal ions, all of which induce proton relaxation in situ. In addition to immunoimaging, the complexed radioactive moieties may be used in radioimmunotherapy protocols to destroy the targeted cell. Suitable isotopes for radioimmunotherapy include the radioactive atoms 90Yt, 131I and 111In.
EXAMPLES [0093] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
Example 1 - Production of Anti-hFGFR3 Monoclonal Antibodies
[0094] This Example describes the production of a number of anti-hFGFR3 monoclonal antibodies. [0095] Immunizations, fusions, and primary screens were conducted at Maine Biotechnology Services Inc. following the Repetitive Immunization Multiple Sites (RIMMS) protocol. Five AJ mice and five Balb/c mice were immunized with recombinant human FGFR3 IHb (R&D Systems, Catalog No. 1264-FR-050) and FGFR3 IIIc (R&D Systems, Catalog No. 766-FR-050) where the Fc fragment was removed from each by Factor Xa protease cleavage. Two Balb/c mice with sera displaying highest anti-FGFR3 activity by Enzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the appropriate mice were harvested. B -cells then were harvested and fused with a myeloma line. Fusion products were serially diluted onto forty 96-well plates to near clonality. Three thousand seven hundred and sixty- three supernatants from the resulting fusions were screened for their binding to recombinant human FGFR3 IHb and IIIc by ELISA. Three hundred fifty-six supernatants identified to contain antibodies to FGFR3 were further characterized by in vitro biochemical and cell-based assays as discussed in the following examples. A panel of hybridomas was selected and the hybridomas were subcloned and expanded. Hybridoma cell lines were transferred to BioXCell (formerly Bio-Express) for antibody expression and purification by affinity chromatography on Protein G resin under standard conditions.
Example 2 - Sequence Analysis of anti-hFGFR3 Monoclonal Antibodies [0096] This Example describes isotype and sequence analysis of the anti-FGFR3 monoclonal antibodies produced in Example 1. a. Determination of FGFR3 Murine Monoclonal Antibody Isotypes
[0097] The light-chain isotype and heavy chain isotype of each monoclonal antibody was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit according the manufacturer's instructions (Roche Applied Science). [0098] All antibodies were determined to be Kappa immunoglobulin light chain and IgGl immunoglobulin heavy chain.
Ix Determination of Nucleotide Sequences Encoding Immunoglobulin Heavy and Light Chain Variable Regions
[0099] The heavy and light chain variable regions of the mouse monoclonal antibodies were sequenced using 5' RACE. Total RNA was extracted from each monoclonal hybridoma cells line using the RNeasy Miniprep kit according to the manufacturer's instructions (Qiagen). Full-length first strand cDNA containing 5' ends was generated using the GeneRacer™ Kit according to the manufacturer's instructions (Invitrogen) using random primers for the purpose of 5' RACE (Rapid Amplification of cDNA Ends). [0100] The variable regions of the Kappa and Heavy (IgGl) immunoglobulin chains were amplified by PCR (Polymerase Chain Reaction) using the Expand High-Fidelity PCR System (Roche Applied Science) according the manufacturer's instructions. Heavy chain variable regions were amplified with the GeneRacer™ 5' Primer, 5'-cgactggagcacgaggacactga-3' (SEQ ID NO: 58) (Invitrogen), and a 3' IgGl Constant Region specific primer, either 5' tatgcaaggcttacaaccaca 3' (SEQ ID NO: 59) or 5' gccagtggatagacagatgggggtgtcg 3' (SEQ ID NO: 60). Kappa chain variable regions were amplified with the GeneRacer™ 5' Primer and a 3' Kappa Constant Region specific primer, either 5' ctcattcctgttgaagctcttgacaat 3' (SEQ ID NO: 61) or 5' cgactgaggcacctccagatgtt 3' (SEQ ID NO: 62).
[0101] Individual PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquick Gel Purification kit according to the manufacturer's instructions (Qiagen). The PCR products were subsequently cloned into the pCR2.1 TOPO plasmid using the topoisomerase based cloning kit TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) according to the manufacturer's instructions (Invitrogen) and transformed into DH5-alpha bacteria through standard molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using M 13 Forward (5'
GTAAAACGACGGCCAGT 3') (SEQ ID NO: 63) and M13 Reverse primers (5' CAGGAAACAGCTATGACC 3') (SEQ ID NO: 64) by Agencourt Bioscience using standard dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using Vector NTI software (Invitrogen) and the IMGT/V-Quest web server to identify and confirm variable region sequences.
[0102] The nucleic acid sequences encoding and the protein sequences defining each of the immunoglobulin heavy chain and light chain variable regions are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences are shown in bold and are underlined in the amino acid sequences. [0103] Nucleic Acid Sequence Encoding the 15D8 Heavy Chain Variable Region (SEQ ID NO: 1)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc 121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca [0104] Protein Sequence Defining the 15D8 Heavy Chain Variable Region (SEQ ID NO: 2)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtsy 61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
[0105] Nucleic Acid Sequence Encoding the 15D8 Kappa Chain Variable Region (SEQ ID NO: 3)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tcctacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgctcacgtt cggtgctgga
301 accaagctgg agctgaaa
[0106] Protein Sequence Defining the 15D8 Kappa Chain Variable Region (SEQ ID NO: 4)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt sylasgvpar 61 fsgsgsgtsy sltissmeae daatyycqqw ssypltfgag tklelk
[0107] Nucleic Acid Sequence Encoding the 27H2 Heavy Chain Variable Region (SEQ ID
NO: 5)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc 121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac
181 aaccagaagt tcaagggcaa ggccacaatg actgttgaca agtcctccag cacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
[0108] Protein Sequence Defining the 27H2 Heavy Chain Variable Region (SEQ ID NO: 6)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtry 61 nqkfkgkatm tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
[0109] Nucleic Acid Sequence Encoding the 27H2 Kappa Chain Variable Region (SEQ ID NO: 7)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg
301 accaagctgg agctgaaa [0110] Protein Sequence Defining the 27H2 Kappa Chain Variable Region (SEQ ID NO: 8)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasqvpar 61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfqaq tklelk [0111] Nucleic Acid Sequence Encoding the 2G4 Heavy Chain Variable Region (SEQ ID NO: 9)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcaca agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagggac 181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag tacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
[0112] Protein Sequence Defining the 2G4 Heavy Chain Variable Region (SEQ ID NO: 10) 1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtrd
61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
[0113] Nucleic Acid Sequence Encoding the 2G4 Kappa Chain Variable Region (SEQ ID
NO: 7)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga 121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg 301 accaagctgg agctgaaa
[0114] Protein Sequence Defining the 2G4 Kappa Chain Variable Region (SEQ ID NO: 8)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasqvpar 61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelk [0115] Nucleic Acid Sequence Encoding the 4E7 (7D12) Heavy Chain Variable Region (SEQ ID NO: 11)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac 181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
[0116] Protein Sequence Defining the 4E7 (7D12) Heavy Chain Variable Region (SEQ ID NO: 12)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtry 61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
[0117] Nucleic Acid Sequence Encoding the 4E7 (7D12) Kappa Chain Variable Region (SEQ ID NO: 7)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga 121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg 301 accaagctgg agctgaaa
[0118] Protein Sequence Defining the 4E7 (7D12) Kappa Chain Variable Region (SEQ ID NO: 8)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasgvpar
61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelk
[0119] Nucleic Acid Sequence Encoding the 20B4 Heavy Chain Variable Region (SEQ ID NO: 13)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta ctcactcact gactacaaca tgtactgggt gaagcagagc 121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagatcgttg
301 ggacctgatt ttgactactg gggccaaggc accactctca cagtctcctc a [0120] Protein Sequence Defining the 20B4 Heavy Chain Variable Region (SEQ ID NO:
14)
1 eiqlqqsgpe lvkpgasvkv sckasgyslt dynmywvkqs hgkslewigy idpynggtsy
61 nqkfkgkatl tvdkssstaf mhlnsltsed savyycarsl gpdfdywgqg ttltvss [0121] Nucleic Acid Sequence Encoding the 20B4 Kappa Chain Variable Region (SEQ ID NO: 15)
1 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaaat tacatgcact ggtaccagca gaagtcaggc
121 acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatactgcca cttattactg tcaacagtgg aatagtaacc cactcacgtt cggtgcgggg
301 accaagctgg agctgaaa
[0122] Protein Sequence Defining the 20B4 Kappa Chain Variable Region (SEQ ID NO: 16)
1 qivltqspai msaspgekvt mtcsasssvn ymhwyqqksg tspkrwiydt sklasgvpar
61 fsgsgsgtsy sltissmeae dtatyycqqw nsnpltfgag tklelk
[0123] Nucleic Acid Sequence Encoding the Murine IgGl Heavy Chain Constant Region Determined for 15D8, 20B4, 27H2, 2G4, and 4E7 (7D12) (SEQ ID NO: 34)
1 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
61 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
121 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac
181 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 241 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc
361 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
421 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag
481 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 541 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc
601 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 721 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 781 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 841 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 901 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 961 tctcctggta aa
[0124] Protein Sequence Defining the Murine IgGl Heavy Chain Constant Region Determined for 15D8, 20B4, 27H2, 2G4, and 4E7 (7D12) (SEQ ID NO: 35) 1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd
61 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif
121 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv
181 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv
241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 301 tcsvlheglh nhhtekslsh spgk
[0125] Nucleic Acid Sequence Encoding the Murine Kappa Light Chain Constant Region Determined for 15D8, 20B4, 27H2, 2G4, and 4E7 (7D12) (SEQ ID NO: 36)
1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 61 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 121 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 181 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 241 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 301 agcttcaaca ggaatgagtg t
[0126] Protein Sequence Defining the Murine Kappa Light Chain Constant Region Determined for 15D8, 20B4, 27H2, 2G4, and 4E7 (7D12) (SEQ ID NO: 37)
1 radaaptvsi fppsseqlts ggasvvcfln nfypkdmvk wkidgserqn gvlnswtdqd 61 skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec
[0127] The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 1 are aligned in Figure 2. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. The sequences defining Complementary Determining Region (CDR) sequences (Kabat definition), CDR1, CDR2, and CDR3, are identified by boxes. Figure 3 shows an alignment of the separate CDR1, CDR2, and CDR3 sequences for each of the antibodies.
[0128] The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies produced in Example 1 are aligned in Figure 4. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. The sequences defining CDR1, CDR2, and CDR3 are identified by boxes. Figure 5 shows an alignment of the separate CDR1, CDR2, and CDR3 sequences for each of the antibodies.
[0129] Monoclonal antibodies 4E7 and 7D12 have identical heavy chain sequences and identical light chain sequences. [0130] Table 1 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
Table 1
Figure imgf000027_0001
[0131] To create the complete heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by the murine IgGl heavy chain constant sequence and a complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence.
[0132] The following sequences represent the actual or contemplated full length heavy and light chain sequences (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. The variable region sequences described herein can be ligated to each of a number of other constant region sequences known to those skilled in the art to produce active full length immunoglobulin heavy and light chains.
[0133] Nucleic Acid Sequence Encoding the Full Length 15D8 Heavy Chain Sequence
(15D8 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 38)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
361 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
421 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
481 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac
541 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc
601 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
661 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc
721 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
781 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag
841 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 901 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 961 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg
1021 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 1081 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 1141 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 1201 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 1261 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 1321 tctcctggta aa [0134] Protein Sequence Defining the Full Length 15D8 Heavy Chain Sequence (15D8 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 39)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtsy
61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd
181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif
241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv
301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv
361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf
421 tcsvlheglh nhhtekslsh spgk
[0135] Nucleic Acid Sequence Encoding the Full Length 15D8 Light Chain Sequence (15D8 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 40)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tcctacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgctcacgtt cggtgctgga
301 accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc
361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac
481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg
541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca
601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt [0136] Protein Sequence Defining the Full Length 15D8 Light Chain Sequence (15D8 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 41)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt sylasgvpar
61 fsgsgsgtsy sltissmeae daatyycqqw ssypltfgag tklelkrada aptvsifpps
121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl
181 tkdeyerhns ytceathkts tspivksfnr nee
[0137] Nucleic Acid Sequence Encoding the Full Length 27H2 Heavy Chain Sequence
(27H2 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 42)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac
181 aaccagaagt tcaagggcaa ggccacaatg actgttgaca agtcctccag cacagcctac
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
361 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
421 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
481 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac
541 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc
601 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
661 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 721 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
781 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag
841 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc
901 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 961 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg
1021 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc
1081 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg
1141 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct
1201 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 1261 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac
1321 tctcctggta aa
[0138] Protein Sequence Defining the Full Length 27H2 Heavy Chain Sequence (27H2 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 43) 1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtry
61 nqkfkgkatm tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd
181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif
241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv 301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv
361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 421 tcsvlheglh nhhtekslsh spgk
[0139] Nucleic Acid Sequence Encoding the Full Length 27H2 Light Chain Sequence (27H2 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 44)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg
301 accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc
361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac
481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca
601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt
[0140] Protein Sequence Defining the Full Length 27H2 Light Chain Sequence (27H2 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 45) 1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasgvpar
61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps
121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl
181 tkdeyerhns ytceathkts tspivksfnr nee
[0141] Nucleic Acid Sequence Encoding the Full Length 2G4 Heavy Chain Sequence (2G4 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 46)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcaca agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagggac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag tacagcctac 241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
361 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
421 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
481 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 541 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc
601 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
661 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc
721 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 781 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag
841 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc
901 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc
961 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg
1021 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 1081 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg
1141 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct
1201 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc
1261 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac
1321 tctcctggta aa
[0142] Protein Sequence Defining the Full Length 2G4 Heavy Chain Sequence (2G4 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 47)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtrd
61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa 121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd
181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif
241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv
301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv
361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 421 tcsvlheglh nhhtekslsh spgk
[0143] Nucleic Acid Sequence Encoding the Full Length 2G4 Light Chain Sequence (2G4 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 48)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg
301 accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac
481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg
541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca
601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt
[0144] Protein Sequence Defining the Full Length 2G4 Light Chain Sequence (2G4 Kappa
Chain Variable Region and Constant Region) (SEQ ID NO: 49)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasgvpar
61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps 121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl
181 tkdeyerhns ytceathkts tspivksfnr nee
[0145] Nucleic Acid Sequence Encoding the Full Length 4E7 (7D12) Heavy Chain
Sequence (4E7 (7D12) Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 50)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
361 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac
421 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
481 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac
541 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc
601 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg
661 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc
721 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
781 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag
841 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc
901 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc
961 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg
1021 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc
1081 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg
1141 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct
1201 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc
1261 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac
1321 tctcctggta aa
[0146] Protein Sequence Defining the Full Length 4E7 (7D12) Heavy Chain Sequence (4E7 (7D12) Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 51)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtry
61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa
121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd
181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif
241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv
301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv
361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf
421 tcsvlheglh nhhtekslsh spgk
[0147] Nucleic Acid Sequence Encoding the Full Length 4E7 (7D12)Light Chain Sequence (4E7 (7D12) Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 52)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga
121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg
301 accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc
361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac
481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg
541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca
601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt
[0148] Protein Sequence Defining the Full Length 4E7 (7D12) Light Chain Sequence (4E7 (7D12) Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 53)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt snlasgvpar
61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps
121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl
181 tkdeyerhns ytceathkts tspivksfnr nee [0149] Nucleic Acid Sequence Encoding the Full Length 20B4 Heavy Chain Sequence
(20B4 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 54) i gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta ctcactcact gactacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc
241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagatcgttg
301 ggacctgatt ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg
361 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg
421 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct
481 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact
541 ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
601 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt
661 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag
721 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc
781 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca
841 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt
901 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca
961 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca
1021 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc
1081 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag
1141 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc
1201 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct
1261 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt
1321 aaa
[0150] Protein Sequence Defining the Full Length 20B4 Heavy Chain Sequence (20B4 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 55)
1 eiqlqqsgpe lvkpgasvkv sckasgyslt dynmywvkqs hgkslewigy idpynggtsy
61 nqkfkgkatl tvdkssstaf mhlnsltsed savyycarsl gpdfdywgqg ttltvssakt
121 tppsvyplap gsaaqtnsmv tlgclvkgyf pepvtvtwns gslssgvhtf pavlqsdlyt
181 lsssvtvpss twpsetvtcn vahpasstkv dkkivprdcg ckpcictvpe vssvfifppk
241 pkdvltitlt pkvtcvvvdi skddpevqfs wfvddvevht aqtqpreeqf nstfrsvsel
301 pimhqdwlng kefkcrvnsa afpapiekti sktkgrpkap qvytipppke qmakdkvslt
361 cmitdffped ltvewqwngq paenykntqp imdtdgsyfv ysklnvqksn weagntftcs
421 vlheglhnhh tekslshspg k
[0151] Nucleic Acid Sequence Encoding the Full Length 20B4 Light Chain Sequence (20B4 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 56)
1 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc
61 atgacctgca gtgccagctc aagtgtaaat tacatgcact ggtaccagca gaagtcaggc
121 acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc
181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa
241 gatactgcca cttattactg tcaacagtgg aatagtaacc cactcacgtt cggtgcgggg
301 accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc
361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac
481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg
541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca
601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt [0152] Protein Sequence Defining the Full Length 20B4 Light Chain Sequence (20B4 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 57)
1 qivltqspai msaspgekvt mtcsasssvn ymhwyqqksg tspkrwiydt sklasgvpar 61 fsgsgsgtsy sltissmeae dtatyycqqw nsnpltfgag tklelkrada aptvsifpps 121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl 181 tkdeyerhns ytceathkts tspivksfnr nee
[0153] For convenience, Table 2 provides a concordance chart showing the correspondence between the full length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.
Table 2
Figure imgf000033_0001
Example 3 - Binding Affinities of Anti-FGFR3 Monoclonal Antibodies
[0154] The binding affinities and kinetics of interaction of the monoclonal antibodies (15D8, 27H2, 2G4, and 4E7(7D12)) produced in Example 1 against recombinant human FGFR3 (HIb and IIIc isoforms) Fc fusion protein (rhFGFR3 IHb Fc or rhFGFR3 IIIc Fc) were measured by surface plasmon resonance using a Biacore™ TlOO (Biacore) instrument.
[0155] Rabbit anti-mouse immunoglobulins (Biacore, Catalog No. BR- 1005- 14) were immobilized on carboxymethylated dextran CM4 sensor chips (Biacore, Catalog No. BR- 1005- 34) by amine coupling (Biacore, Catalog No. BR- 1000-50) using a standard coupling protocol according to manufacturer's instructions. The analyses were performed at 250C and 370C using PBS (Invitrogen, Catalog No. 14040-133) containing 0.05% surfactant P20 (Biacore, Catalog No. BR- 1000-54) as running buffer.
[0156] The antibodies were captured in an individual flow cell at a flow rate of 10 μl/min. Injection time was varied for each antibody to yield approximately 40-50 RU of antibody captured for each cycle. Buffer or rhFGFR3 IHb Fc (R&D Systems, Catalog No. 1264-FR- 050) or rhFGFR3 IIIc Fc (R&D Systems, Catalog No. 766-FR-050) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 300 sec at 60 μl/min. The dissociation phase was monitored for 30 minutes. The surface was then regenerated with two 60-seconds injection of 10 mM Glycine- HCl, pH 1.7 (made from Glycine pH 1.5 (Biacore, Catalog No. BR- 1003-54) and pH 2.0 (Biacore, Catalog No. BR- 1003-55)) at a flow rate of 60 μl/min. rhFGFR3 Fc concentrations tested were 0.62 nM to 40 nM.
[0157] Kinetic parameters were determined using the kinetic function of the BIAevalutation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), k<j (dissociation rate constant) and KD (equilibrium dissociation constant) were determined. Kinetic values of the monoclonal antibodies at 250C are summarized in Table 3.
Table 3
Figure imgf000034_0001
Standard deviation not calculated when n < 3
[0158] Kinetic values of the monoclonal antibodies at 370C are summarized in Table 4. Table 4
Figure imgf000035_0001
Example 4 - Neutralization Activity of Anti-hFGFR3 Antibodies
[0159] The antibodies produced in Example 1 were characterized for their ability to inhibit recombinant hFGFR3 IHb binding to FGFl (also known as FGF acidic).
[0160] The antibodies were tested by ECL (Electrochemiluminescence) assay for inhibition of hFGFR3 IHB binding to FGF-I. MA2400 96- well high binding plates (Meso Scale Discovery, Catalog No. L15XB-6) were coated with 25 μl of 0.8 μg/mL FGF-I (R&D Systems, Catalog No. 232-FA-025) in PBS (Invitrogen, Catalog No. 14040-133) for 1 hour at room temperature with agitation. The plates then were washed 3 times with PBS and blocked with 200 μl of PBS containing 5% BSA (Sera Care Life Sciences, Catalog No. AP-4510-80) and 5 μg/mL heparin (Sigma, Catalog No. H4784) for 1 hour at room temperature. The antibodies (concentration range: 0.029-30 μg/mL) were incubated for 1 hour at room temperature with 1.7 μg/mL rhFGFR3 HIb Fc (R&D Systems, Catalog No. 1264-FR-050) and 5 μg/mL heparin. After washing the plates 3 times with PBS, 25 μl of the antibody- receptor mixture was added to the plates for another hour at room temperature with agitation. The plates were washed three times with PBS and incubated with 25 μl of 1 μg/mL ST-anti-human IgG antibody (Meso Scale Discovery, Catalog No. R32AJ-1) for 1 hour at room temperature with agitation. The plates then were washed 3 times with PBS, and 150 μl of IX read buffer (Meso Scale Discovery, Catalog No. R92TC-1) was added to each well before the plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery) instrument.
[0161] The interaction of FGFl with FGFR3 was inhibited by 4E7, 7D12, 15D8, 27H2, 2G4, and 20B4 IgGl as shown in Figure 6. The interaction of FGFl with FGFR3 was also inhibited by Fab fragments as shown in Figure 7 (20B4 not shown). [0162] The IC50 and maximum percent inhibition values for the murine anti-human FGFR3 antibodies (IgGl) and Fab fragments (Fab) were calculated and are summarized in Table 5.
Table 5
Figure imgf000036_0001
* Standard deviation not calculated when n < 3
[0163] The results demonstrate that all the antibodies (i.e., 15D8, 27H2, 2G4, 4E7, 7D12) except for 20B4 efficiently neutralize hFGFR3 binding to FGFl. The 2G4 Fab fragment neutralized hFGFR3 binding to FGFl better than the 2G4 IgGl antibody.
Example 5 - Anti-Proliferative Activity of Anti-hFGFR3 Antibodies [0164] In this Example, the antibodies produced in Example 1 were characterized for their ability to inhibit FGFl dependent proliferation of cells.
[0165] FDCP-I cells (mouse bone marrow cells obtained from German Collection of Microorganisms and Cell Cultures) were transfected with plasmids encoding human FGFR3 IHb, FGFR3 IHc, or a mutant variant G380R (an activating mutation associated with the skeletal disorder, achondroplasia (Webster and Donoghue (1996) EMBO J. 15:520-527) by electroporation and selected with G418 (600 μg/mL). Single clones were isolated and tested for their FGFl -dependent proliferation in the absence of IL3 containing WEHI-conditioned medium. FDCP-FGFR3 IHb #122, FDCP-FGFR3 IIIc #109, FGFR3 IIIc G380R #1 exhibited FGF-I induced proliferation in the absence of IL3. [0166] To screen for antagonistic FGFR3 antibodies, hybridoma supernatants containing FGFR3 antibodies were added to FDCP-FGFR3 IHb #122 or FDCP-FGFR3 IIIc #109 cells cultured in basic growth medium (70% ISCOVE' s Modified Dulbecco's Medium (Invitrogen, Catalog No. 12440-053), 20% horse serum (Invitrogen, Catalog No. 26050-088) and 10% WEHI-culture medium (90% ISCOVE' s MDM + 10% FBS (Invitrogen, Catalog No. 10438- 026) + 2 mM L-glutamine (Invitrogen, Catalog No. 25030-081) + 0.0025 mM mercaptoethanol (Invitrogen, Catalog No. 21985-023))) at a 1:1 ratio (volume) in a 96-well plate (70,000 cells/ well) in the absence or presence of FGFl (8ng/mL) and heparin (5 μg/mL). MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were conducted two to three days post FGFl stimulation. Top antagonistic antibodies were selected for further characterization.
[0167] To test the effect of FGFR3 antibodies on the proliferation of various FGFR3-driven FDCP cells, varying amounts of antibodies were added to the cells along with FGFl (8ng/mL) and heparin (50 μg/mL). The cells were cultured in basic growth medium (70% ISCOVE' s, 20% horse serum and 10% WEHI-culture medium (90% Iscove's MDM + 10% FBS + 2 mM L-glutamine + 0.0025 mM mercaptoethanol)) in a 96-well plate (70,000 cells/ well). The final concentration of FGFl and heparin used in the assay is 8 ng/mL and 5 μg/mL, respectively. MTT assay was conducted one to three days post FGFl stimulation.
[0168] An example of the dose dependent inhibition of FDCP-FGFR3c cell proliferation by murine anti-human FGFR3 antibodies is shown in Figure 8. Inhibition data of FDCP-FGFR3 cell line proliferation with monoclonal antibodies (15D8, 27H2, 4E7, 2G4, and 20B4) are summarized in Table 6.
Table 6
Figure imgf000037_0001
[0169] The results in Table 6 demonstrate that all the antibodies (i.e., 15D8, 27H2, 4E7, 2G4) except for 20B4 strongly inhibited FGFl induced proliferation in FDCP-FGFR3 IHb and FDCP-FGFR3 IIIc cell lines. Inhibition by 20B4 was maximally 40% and an IC50 value was not calculated. The antibodies also have an inhibitory effect on FGFR3 IIIc G380R, a mutant variant that is correlated with the skeletal disorder, achondroplasia.
Example 6 - Tumor Inhibition in OPM-2 Xenograft Model
[0170] The ability of murine monoclonal antibodies of the invention to inhibit tumor growth was tested in an OPM-2 xenograft model. OPM-2 cells were grown in culture at 37°C in an atmosphere containing 5% CO2, using RMPI medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). Cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice (Taconic Labs) with 5 x 106 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237).
[0171] Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width x width x length/2. When tumors reached approximately 150 mm3, the mice were randomized into 4 groups of 10 mice each.
[0172] Each group (10 mice each) received one of the following treatments: murine IgG control at 20 mg/kg, or 15D8 at 5, 10 or 20 mg/kg. Treatment was given intra-peritoneal twice weekly for 2 weeks. Each 15D8 treatment group demonstrated similar tumor growth inhibition of 70% (p<0.001) as shown in Figure 9. All 15D8 treatments were well-tolerated with no significant body weight loss.
[0173] A study was performed to compare four of the murine antibodies. Each group (10 mice each) received one of the following treatments: murine IgG, 15D8, 4E7, 27H2, or 2G4, each dosed at 1 mg/kg. As can be seen in Figure 10, the four murine antibodies demonstrated similar efficacy at approximately 40% tumor growth inhibition in this model. [0174] Thus, these results demonstrate that treatment with the murine 15D8, 4E7, 27H2, and 2G4 antibodies slows tumor development.
Example 7 - Humanization of Anti-FGFR3 Antibodies a. Construction of Humanized and Chimeric Anti-FGFR3 Antibodies
[0175] This Example describes the humanization of the murine antibody designated 15D8, and the characterization of the resulting humanized antibody. The humanized anti-FGFR3 antibody was designed using the SUPERHUMANIZATION™ method (Arana Therapeutics Ltd. and Hwang, W.Y. et al. (2005) METHODS 36:35-42). Certain framework residues were converted to murine 15D8 residues to improve the antibody's affinity toward FGFR3, and the antibody's activity in inhibiting the biological activity of FGFR3, or both. The designed amino acid sequences were converted to codon-optimized DNA sequences, including (in the following order): 5' HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, humanized variable region, human IgGl or Kappa constant region, stop codon, and a 3' EcoRI restriction site.
[0176] Chimeric (murine variable region and human constant region) 15D8 heavy (human IgGl) and light (human Kappa) chains were also constructed. The murine variable regions were fused to the human constant region using overlap extension PCR, including (in the following order): 5' HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, mouse variable region, human IgGl or Kappa constant region, stop codon, and 3' EcoRI restriction site.
[0177] The humanized and chimeric IgGl heavy chains were subcloned into pEE6.4 (Lonza Biologies) via HindIII and EcoRI sites. The humanized and chimeric Kappa light chains were subcloned into pEE14.4 (Lonza Biologies) via HindIII and EcoRI sites.
[0178] Humanized antibody chains or chimeric antibody chains were transiently transfected into 293T cells to produce antibody for purification and subsequent in vitro analysis. Binding of the chimeric and humanized antibodies to human FGFR3 was measured as described below. The results are summarized in Table 9. Additionally, the chimeric and humanized antibodies were tested for inhibition of FGF-stimulated proliferation of FDCP-FGFR3b cells (as described in Example 5). The results are summarized in Table 10.
[0179] Each of the possible combinations of immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth in Table 7A. Table 7A
Figure imgf000039_0001
[0180] Each of the possible combinations of immunoglobulin heavy chains and immunoglobulin light chains are set forth in Table 7B.
Table 7B
Figure imgf000040_0001
[0181] The antibody constructs containing the full length chimeric or humanized immunoglobulin heavy and light chains are designated below:
Chimeric 15D8 = Full Length Chimeric 15D8 Heavy Chain (Mouse Variable Region and Human IgGl Constant Region) (SEQ ID NO: 66) plus Full Length Chimeric 15D8 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 68)
Humanized 15D8 = Full Length Humanized 15D8 Heavy Chain (Humanized Variable Region and Human IgGl Constant Region) (SEQ ID NO: 74) plus Full Length Humanized 15D8 Light Chain (Humanized Variable Region and Human Kappa Constant Region) (SEQ ID NO: 76) [0182] The nucleic acid sequences encoding and the polypeptide sequences defining the chimeric and humanized antibodies are summarized below (amino terminal signal sequences are not shown). CDR sequences (Kabat definition) are shown in bold/underlined in the amino acid sequences.
[0183] Nucleic Acid Sequence Encoding the Full Length Chimeric 15D8 Heavy Chain (Mouse Variable Region and Human IgGl Constant Region) (SEQ ID NO: 65)
1 gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
61 tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactgggt gaagcagagc
121 catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac
181 aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 241 atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg
301 ggtaactacg aggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
361 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
421 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
481 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 541 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
601 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc
661 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
721 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 781 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
841 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
901 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
961 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa gaccatctcc
1021 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1081 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1141 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1201 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1261 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1321 cagaagagcc tctccctgtc tccgggtaaa
[0184] Protein Sequence Defining the Full Length Chimeric 15D8 Heavy Chain (Mouse Variable Region and Human IgGl Constant Region) (SEQ ID NO: 66)
1 eiqlqqsgpe lvkpgasvkv sckasgyaft synmywvkqs hgkslewigy idpynggtsy
61 nqkfkgkatl tvdkssstay mhlnsltsed savyycareg gnyeawfayw gqgtlvtvsa 121 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss
181 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg
241 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn
301 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree
361 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421 qqgnvfscsv mhealhnhyt qkslslspgk
[0185] Nucleic Acid Sequence Encoding the Full Length Chimeric 15D8 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 67)
1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccaaga 121 tcctccccca aaccctggat ttatctcaca tcctacctgg cttctggagt ccctgctcgc 181 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgctcacgtt cggtgctgga 301 accaagctgg agctgaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 361 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 421 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 481 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 541 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 601 agctcgcccg tcacaaagag cttcaacagg ggagagtgt
[0186] Protein Sequence Defining the Full Length Chimeric 15D8 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 68)
1 qivltqspal msaspgekvt mtcsasssvs ymywyqqkpr sspkpwiylt sylasgvpar 61 fsgsgsgtsy sltissmeae daatyycqqw ssypltfgag tklelkrtva apsvfifpps 121 deqlksgtas vvcllnnfyp reakvqwkvd nalqsgnsqe svteqdskds tyslsstltl 181 skadyekhkv yacevthqgl sspvtksfnr gee [0187] Nucleic Acid Sequence Encoding Humanized 15D8 (Hul5D8) Heavy Chain Variable Region (SEQ ID NO: 69)
1 gaggtccaac tggtgcaatc tggggctgag gtcaagaaac ccggggaatc tctcaaaatt
61 tcatgcaaag gttctggtta cagtttcacc tcatataaca tgtactgggt taggcagatg 121 cctggtaaag gcttggagtg gatggggtac attgatccct ataacggcgg cactagttac
181 aatcagaagt tcaagggcaa ggccacattg actgttgaca agtccatctc aactgcttac
241 ctgcaatggt cctctctcaa agccagcgac actgctatgt actactgcgc aagggaggga
301 ggcaattacg aggcttggtt cgcttattgg ggacaaggca ctcttgtcac cgtctcctca
[0188] Protein Sequence Defining Humanized 15D8 (Hul5D8) Heavy Chain Variable Region (SEQ ID NO: 70)
1 evqlvqsgae vkkpgeslki sckgsgysft synmywvrqm pgkglewmgy_ idpynggtsy
61 nqkfkgkatl tvdksistay lqwsslkasd tamyycareg gnyeawfayw gqgtlvtvss
[0189] Nucleic Acid Sequence Encoding Humanized 15D8 (Hul5D8) Light Chain Variable Region (SEQ ID NO: 71)
1 gatatccaac tcacccagtc cccttcatcc ctgtctgcat cagtcgggga cagagtgaca
61 attacttgtt ccgccagctc tagtgtctca tacatgtatt ggtttcagca aaagccagga
121 aaagctccca aacccctgat ctatctgacc agctatctgg caagcggcgt gccttctcgg 181 ttcagtggat cagggtccgg tacagacttt accctgacta ttagcagtct gcaaccagag
241 gacttcgcca cttattactg ccaacagtgg agttcatatc ccctgacttt tggcggaggg
301 accaaggtcg agatcaag
[0190] Protein Sequence Defining Humanized 15D8 (Hul5D8) Light Chain Variable Region (SEQ ID NO: 72) 1 diqltqspss lsasvgdrvt itcsasssvs ymywfqqkpg kapkpliyl^ sylasgvpsr 61 fsgsgsgtdf tltisslqpe dfatyycqqw ssypltfggg tkveik
[0191] Nucleic Acid Sequence Defining the Full Length Humanized 15D8 (Hul5D8) Heavy Chain (Humanized Variable Region and Human IgGl Constant Region) (SEQ ID NO: 73) 1 gaggtccaac tggtgcaatc tggggctgag gtcaagaaac ccggggaatc tctcaaaatt
61 tcatgcaaag gttctggtta cagtttcacc tcatataaca tgtactgggt taggcagatg
121 cctggtaaag gcttggagtg gatggggtac attgatccct ataacggcgg cactagttac
181 aatcagaagt tcaagggcaa ggccacattg actgttgaca agtccatctc aactgcttac
241 ctgcaatggt cctctctcaa agccagcgac actgctatgt actactgcgc aagggaggga 301 ggcaattacg aggcttggtt cgcttattgg ggacaaggca ctcttgtcac cgtctcctca
361 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg
421 ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc
481 tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct
541 ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601 tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc
661 aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt
721 cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc
781 gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg
841 tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901 agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa
961 gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt
1021 aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa
1081 atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141 gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201 ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261 cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321 cagaagtcac tgagcctgag cccagggaag
[0192] Protein Sequence Defining the Full Length Humanized 15D8 (Hul5D8) Heavy Chain (Humanized Variable Region and Human IgGl Constant Region) (SEQ ID NO: 74)
1 evqlvqsgae vkkpgeslki sckgsgysft synmywvrqm pgkglewmgy_ idpynggtsy 61 nqkfkgkatl tvdksistay lqwsslkasd tamyycareg gnyeawfayw gqgtlvtvss
121 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss
181 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg
241 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn
301 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw
421 qqgnvfscsv mhealhnhyt qkslslspgk
[0193] Nucleic Acid Sequence Encoding the Full Length Humanized 15D8 (Hul5D8) Light Chain (Humanized Variable Region and Human Kappa Constant Region) (SEQ ID NO: 75)
1 gatatccaac tcacccagtc cccttcatcc ctgtctgcat cagtcgggga cagagtgaca
61 attacttgtt ccgccagctc tagtgtctca tacatgtatt ggtttcagca aaagccagga
121 aaagctccca aacccctgat ctatctgacc agctatctgg caagcggcgt gccttctcgg
181 ttcagtggat cagggtccgg tacagacttt accctgacta ttagcagtct gcaaccagag 241 gacttcgcca cttattactg ccaacagtgg agttcatatc ccctgacttt tggcggaggg 301 accaaggtcg agatcaagcg cacagtcgcc gctccctccg tgttcatctt tccaccaagt 361 gatgagcaac tgaagtctgg tactgcttca gtcgtgtgtc tgctgaacaa tttctaccct 421 cgagaagcca aagtccaatg gaaggtagac aacgcactgc agtccggcaa tagccaagaa 481 tcagttaccg aacaggattc aaaggacagt acatattccc tgagcagcac tctgaccctg 541 tcaaaggccg attacgagaa acacaaggtc tatgcttgcg aagtgacaca tcagggactg 601 tccagcccag tgacaaaatc ttttaaccgt ggggagtgt
[0194] Protein Sequence Defining the Full Length Humanized 15D8 (Hul5D8) Light
Chain (Humanized Variable Region and Human Kappa Constant Region) (SEQ ID NO: 76)
1 diqltqspss lsasvgdrvt itcsasssvs ymywfqqkpg kapkpliyl^ sylasgvpsr 61 fsgsgsgtdf tltisslqpe dfatyycqqw ssypltfggg tkveikrtva apsvfifpps 121 deqlksgtas vvcllnnfyp reakvqwkvd nalqsgnsqe svteqdskds tyslsstltl 181 skadyekhkv yacevthqgl sspvtksfnr gee
[0195] Nucleic Acid Sequence Encoding Human IgGl Heavy Chain Constant Region (SEQ ID NO: 77)
1 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
61 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
121 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 181 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
241 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc
301 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
361 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
421 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 481 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 541 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 601 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa gaccatctcc 661 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 721 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 781 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 841 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 901 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 961 cagaagagcc tctccctgtc tccgggtaaa
[0196] Protein Sequence Defining Human IgGl Heavy Chain Constant Region (SEQ ID NO: 78)
1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss
61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg
121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn
181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree
241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffIy skltvdksrw
301 qqgnvfscsv mhealhnhyt qkslslspgk
[0197] Nucleic Acid Sequence Encoding Human Kappa Chain Constant Region (SEQ ID NO: 79)
1 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct
61 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag
121 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac
181 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag
241 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag
301 agcttcaaca ggggagagtg t
[0198] Protein Sequence Defining Human Kappa Chain Constant Region (SEQ ID NO: 80)
1 rtvaapsvfi fppsdeqlks gtasvvcllnn fypreakvq wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec
[0199] For convenience, Table 8 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.
Table 8
Figure imgf000044_0001
Figure imgf000045_0001
Ix Binding Affinities of Humanized and Chimeric Anti-FGFR3 Monoclonal Antibodies
[0200] The binding affinities of monoclonal 15D8, chimeric 15D8, and humanized 15D8 antibodies for recombinant human FGFR3 IIIc Fc fusion protein was measured using a kinetic exclusion assay, KinExA® technology (Sapidyne Instruments, Inc.). First, beads were prepared for the purpose of detecting anti-FGFR3 antibody that is unbound to FGFR3 IIIc Fc. This was done by adding 1 ml recombinant human FGFR3IIIc Fc (R&D Systems, Inc.) 10 ug/ml in PBS to 200 mg polymethyl methacrylate (PMMA) hard beads. The suspension was mixed and rotated for two hours at room temperature. Next, the mixture was centrifuged and supernatant was discarded. The bead pellet was rinsed once with 1 ml BSA 10 mg/ml in PBS by incubation for 1 hour at room temperature with rotation. The beads were resuspended in 27 ml PBS with 0.02% NaN3. Next, a fixed concentration of anti-FGFR3 antibody (0.5 nM) was incubated in solution with a series of FGFR3 IIIc Fc concentrations (started with 50 nM (in PBS BSA (1 mg/ml)) and serially diluted 1:2 in PBS BSA(I mg/ml) to obtain 50 to 0.0122 nM FGFR3 III Fc) at room temperature for at least 4 hours to allow equilibrium to be reached. By measuring the amount of anti-FGFR3 antibody that is not bound to FGFR3 IIIc Fc, the KD was determined. Unbound anti-FGFR3 antibody was detected by allowing the anti-FGFR3 antibody/FGFR3 IIIc Fc solution to flow through the FGFR3 IIIc Fc PMMA beads. The anti- FGFR3 antibody captured by these beads was then detected with Cy5-conjugated anti-human secondary antibody 0.3 ug/ml (Jackson ImmunoResearch) or Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) 0.5ug/ml in PBS BSA 1 mg/ml. The detected signal for captured anti-FGFR3 antibody is directly proportional to the remaining free binding sites, thus allowing KD determination. The experiments were repeated, varying the concentrations of anti-FGFR3 antibody or FGFR3 III Fc used in solution, and the KD was calculated with the KinExA® software using n-curve analysis. The resulting data are shown in Table 9. These data demonstrated that 15D8, chimeric 15D8, and humanized 15D8 strongly bind FGFR3 with nearly equal affinity.
Table 9
Figure imgf000046_0001
α Antiproliferative Activity of Humanized and Chimeric Anti-FGFR3 Monoclonal Antibodies
[0201] The chimeric and humanized 15D8 antibodies were tested for inhibition of FGFl- induced proliferation of FDCP-FGFR3 IHb #122, as described in Example 5. Inhibition data are summarized in Table 10.
Table 10
Figure imgf000046_0002
[0202] The results in Table 10 demonstrate that chimeric 15D8 and humanized 15D8 strongly inhibited FGFl -induced proliferation in FDCP-FGFR3 IHb cells with nearly equal potency.
INCORPORATION BY REFERNCE
[0203] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. EQUIVALENTS
[0204] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
[0205] What is claimed is :

Claims

CLAIMS 1. An isolated binding protein that binds human fibroblast growth factor receptor 3 (FGFR3) comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region selected from the group consisting of: (a) (i) an immunoglobulin heavy chain variable region comprising a CDRH i comprising the sequence SEQ ID NO: 17 (15D8), a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), and a CDRH3 comprising the sequence SEQ ID NO: 19 (15D8); and (ii) an immunoglobulin light chain variable region comprising a CDRu comprising the sequence SEQ ID NO: 22 (15D8), a CDRL2 comprising the sequence SEQ ID NO: 23 (15D8), and a CDRL3 comprising the sequence SEQ ID NO: 24 (15D8); (b) (i) an immunoglobulin heavy chain variable region comprising a CDRHi comprising the sequence SEQ ID NO: 17 (27H2, 4E7(7D12)), a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25 (27H2, 4E7(7D12)), and a CDRH3 comprising the sequence SEQ ID NO: 19 (27H2, 4E7(7D12)); and (ii) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence SEQ ID NO: 22 (27H2, 4E7(7D12)), a CDRL2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26 (27H2, 4E7(7D12)), and a CDRL3 comprising the sequence SEQ ID NO: 27 (27H2, 4E7(7D12)); (c) (i) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence SEQ ID NO: 17 (2G4), a CDRH2 comprising the sequence SEQ ID NO: 28 (2G4), and a CDRH3 comprising the sequence SEQ ID NO: 19 (2G4); and (ii) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence SEQ ID NO: 22 (2G4), a CDRL2 comprising the sequence SEQ ID NO: 26 (2G4), and a CDRL3 comprising the sequence SEQ ID NO: 27 (2G4); and (d) (i) an immunoglobulin heavy chain variable region comprising a CDRHI comprising the sequence SEQ ID NO: 29 (20B4), a CDRH2 comprising the sequence SEQ ID NO: 18 (20B4), and a CDRH3 comprising the sequence SEQ ID NO: 30 (20B4); and (ii) an immunoglobulin light chain variable region comprising a CDRu comprising the sequence SEQ ID NO: 31 (20B4), a CDRL2 comprising the sequence SEQ ID NO: 32 (20B4), and a CDRL3 comprising the sequence SEQ ID NO: 33 (20B4).
2. An isolated binding protein that binds human fibroblast growth factor receptor 3 (FGFR3) comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region selected from the group consisting of: (a) (i) an immunoglobulin heavy chain variable region comprising a CDRH i comprising the sequence SEQ ID NO: 17 (15D8, 27H2, 2G4, 4E7(7D12)); a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 (15D8), SEQ ID NO: 25 (27H2, 4E7(7D12)), and SEQ ID NO: 28 (2G4); and a CDRH3 comprising the sequence SEQ ID NO: 19 (15D8, 27H2, 2G4, 4E7(7D12)); and (ii) an immunoglobulin light chain variable region comprising a CDRu comprising the sequence SEQ ID NO: 22 (15D8, 27H2, 2G4, 4E7(7D12)); a CDRL2 comprising an amino acid sequence selected from the group consisting SEQ ID NO: 23 (15D8) and SEQ ID NO: 26 (27H2, 2G4, 4E7(7D12)); and a CDRL3 comprising an amino acid sequence selected from the group consisting SEQ ID NO: 24 (15D8) and SEQ ID NO: 27 (27H2, 2G4, 4E7(7D12)).
3. The binding protein of claim 1 or 2, wherein the CDR sequences are interposed between human and humanized framework sequences.
4. The binding protein of claim 1 or 2, wherein the binding protein is a monoclonal antibody or antigen binding protein fragment thereof.
5. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region of claim 1 or 2.
6. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region of claim 1 or 2.
7. An expression vector containing the nucleic acid of claim 5.
8. An expression vector containing the nucleic acid of claim 6.
9. A host cell comprising at least one expression vector that expresses a protein of claim 1 or 2.
10. A method of producing a binding protein, the method comprising: (a) growing the host cell of claim 9 under conditions so that the host cell expresses the immunoglobulin heavy chain variable region; and (b) harvesting the immunoglobulin heavy chain variable region.
11. The method of claim 10, wherein, after step (b), the immunoglobulin heavy chain variable region is covalently linked to an immunoglobulin light chain variable region, so that the heavy and light chain variable regions together bind human FGFR3.
12. A method of producing a binding protein, the method comprising: (a) growing the host cell of claim 9 under conditions so that the host cell expresses the immunoglobulin light chain variable region; and (b) harvesting the immunoglobulin light chain variable region.
13. The method of claim 12, wherein, after step (b), the immunoglobulin light chain variable region is covalently linked to an immunoglobulin heavy chain variable region, so that the light and heavy chain variable regions together bind human FGFR3.
14. An isolated binding protein that binds human fibroblast growth factor receptor 3 (FGFR3) comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region selected from the group consisting of: (a) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (15D8), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (15D8); (b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 (27H2), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 (27H2); (c) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 (2G4), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 (2G4); (d) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (4E7(7D12)), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 (4E7(7D12)); (e) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (20B4), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (20B4); and (f) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 70 (Hul5D8), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 72 (Hul5D8).
15. The binding protein of claim 14, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 (15D8), and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 (15D8).
16. The binding protein of claim 14, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 (27H2), and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 8 (27H2).
17. The binding protein of claim 14, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70 (Hul5D8), and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 72 (Hul5D8).
18. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region of claim 14.
19. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region of claim 14.
20. An expression vector containing the nucleic acid of claim 18.
21. An expression vector containing the nucleic acid of claim 19.
22. A host cell comprising at least one expression vector that expresses a protein of claim 14.
23. A method of producing a binding protein, the method comprising: (a) growing the host cell of claim 22 under conditions so that the host cell expresses the immunoglobulin heavy chain variable region; and (b) harvesting the immunoglobulin heavy chain variable region.
24. The method of claim 23, wherein, after step (b), the immunoglobulin heavy chain variable region is covalently linked to an immunoglobulin light chain variable region, so that the heavy and light chain variable regions together bind human FGFR3.
25. A method of producing a binding protein, the method comprising: (a) growing the host cell of claim 22 under conditions so that the host cell expresses the immunoglobulin light chain variable region; and (b) harvesting the immunoglobulin light chain variable region.
26. The method of claim 25, wherein, after step (b), the immunoglobulin light chain variable region is covalently linked to an immunoglobulin heavy chain variable region, so that the light and heavy chain variable regions together bind human FGFR3.
27. An isolated binding protein that binds human fibroblast growth factor receptor 3 (FGFR3) comprising an immunoglobulin heavy chain and an immunoglobulin light chain selected from the group consisting of: (a) an immunoglobulin heavy chain of SEQ ID NO: 39 (15D8), and an immunoglobulin light chain of SEQ ID NO: 41 (15D8); (b) an immunoglobulin heavy chain of SEQ ID NO: 43 (27H2) , and an immunoglobulin light chain of SEQ ID NO: 45 (27H2); (c) an immunoglobulin heavy chain of SEQ ID NO: 47 (2G4), and an immunoglobulin light chain of SEQ ID NO: 49 (2G4); (d) an immunoglobulin heavy chain of SEQ ID NO: 51 (4E7(7D12)), and an immunoglobulin light chain of SEQ ID NO: 53 (4E7(7D12)); (e) an immunoglobulin heavy chain of SEQ ID NO: 55 (20B4), and an immunoglobulin light chain of SEQ ID NO: 57 (20B4); (f) an immunoglobulin heavy chain of SEQ ID NO: 66 (Chimeric 15D8), and an immunoglobulin light chain of SEQ ID NO: 68 (Chimeric 15D8); and (g) an immunoglobulin heavy chain of SEQ ID NO: 74 (Hul5D8), and an immunoglobulin light chain of SEQ ID NO: 76 (Hul5D8).
28. The binding protein of claim 27, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 39 (15D8), and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 41 (15D8).
29. The binding protein of claim 27, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 43 (27H2), and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 45 (27H2).
30. The binding protein of claim 27, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 66 (Chimeric 15D8), and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 68 (Chimeric 15D8).
31. The binding protein of claim 27, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 74 (Hul5D8), and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 76 (Hul5D8).
32. The binding protein of claim 14 or 27, wherein the binding protein is a monoclonal antibody or an antigen binding fragment thereof.
33. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain of claim 27.
34. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain of claim 27.
35. An expression vector containing the nucleic acid of claim 33.
36. An expression vector containing the nucleic acid of claim 34.
37. A host cell comprising at least one expression vector that expresses a protein of claim 27.
38. A method of producing a binding protein, the method comprising: (a) growing a host cell of claim 37 comprising one expression vector that expresses an immunoglobulin heavy chain and a second expression vector that expresses an immunoglobulin light chain under conditions so that the host cell expresses both immunoglobulin heavy chain and immunoglobulin light chain, and (b) harvesting an antibody comprising an immunoglobulin heavy chain and an immunoglobulin light chain capable of binding human FGFR3.
39. The binding protein of claim 1, 2, 14, or 27, wherein the binding protein has a KD of 4 nM or lower as measured by surface plasmon resonance.
40. The binding protein of claim 1, 2, 14, or 27, wherein the binding protein has a KD of 100 pM or lower as measured by a kinetic exclusion assay.
41. A method of inhibiting or reducing proliferation of a tumor cell comprising exposing the cell to an effective amount of the binding protein of claim 1, 2, 14, or 27 to inhibit or reduce proliferation of the tumor cell.
42. A method of inhibiting or reducing tumor growth in a mammal, the method comprising exposing the mammal to an effective amount of the binding protein of claim 1, 2, 14, or 27 to inhibit or reduce proliferation of the tumor.
43. A method of treating cancer in a mammal, the method comprising administering an effective amount of the binding protein of claim 1, 2, 14, or 27 to a mammal in need thereof.
44. The method of claim 43, wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, oral squamous cell cancer, non- small cell lung cancer, breast cancer, lymphoma, and multiple myeloma.
45. The method of claim 43, wherein the mammal is a human.
46. A method of treating a cell proliferative disorder, the method comprising administering an effective amount of the binding protein of claim 1, 2, 14, or 27 to a subject in need thereof.
47. The method of claim 46, wherein the cell proliferative disorder is a cancer or a skeletal disorder.
48. The method of claim 47, wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, oral squamous cell cancer, non- small cell lung cancer, breast cancer, lymphoma, and multiple myeloma.
49. The method of claim 47, wherein the skeletal disorder is selected from the group consisting of achondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia, and craniosynostosis syndromes.
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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010111367A1 (en) 2009-03-25 2010-09-30 Genentech, Inc. Anti-fgfr3 antibodies and methods using same
WO2013033008A2 (en) 2011-08-26 2013-03-07 Merrimack Pharmaceuticals, Inc. Tandem fc bispecific antibodies
WO2013087725A1 (en) 2011-12-12 2013-06-20 Institut National De La Sante Et De La Recherche Medicale (Inserm) Antagonist of the fibroblast growth factor receptor 3 (fgfr3) for use in the treatment or the prevention of skeletal disorders linked with abnormal activation of fgfr3
JP2014514314A (en) * 2011-04-20 2014-06-19 ゲンマブ エー／エス Bispecific antibodies against HER2 and CD3
JP2014517823A (en) * 2011-04-20 2014-07-24 ゲンマブ エー／エス Bispecific antibody against HER2
US8828385B2 (en) 2001-06-20 2014-09-09 Fibron Limited Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
WO2014138449A1 (en) 2013-03-06 2014-09-12 Merrimack Pharmaceuticals, Inc. Anti-c-met tandem fc bispecific antibodies
WO2014138364A2 (en) 2013-03-06 2014-09-12 Genentech, Inc. Methods of treating and preventing cancer drug resistance
WO2015191986A1 (en) 2014-06-13 2015-12-17 Genentech, Inc. Methods of treating and preventing cancer drug resistance
WO2016139227A1 (en) 2015-03-03 2016-09-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Fgfr3 antagonists
WO2018185050A1 (en) 2017-04-03 2018-10-11 Covagen Ag Fgfr3 binding molecules
JP2018184478A (en) * 2011-10-25 2018-11-22 プロシーナ バイオサイエンシーズ リミテッド Antibody formulation and method
US10208120B2 (en) 2014-11-05 2019-02-19 Genentech, Inc. Anti-FGFR2/3 antibodies and methods using same
US10294289B2 (en) 2016-07-07 2019-05-21 Therachon Sas Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof
US10724014B2 (en) 2013-01-16 2020-07-28 Institut National De La Sante Et De La Recherche Medicale Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
US11046771B2 (en) 2010-05-27 2021-06-29 Genmab A/S Monoclonal antibodies against HER2
CN114716548A (en) * 2021-01-05 2022-07-08 (株)爱恩德生物 anti-FGFR 3 antibodies and uses thereof
US11505611B2 (en) 2020-08-21 2022-11-22 Genzyme Corporation FGFR3 antibodies and methods of use
WO2024152014A1 (en) 2023-01-13 2024-07-18 Regeneron Pharmaceuticals, Inc. Fgfr3 binding molecules and methods of use thereof

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060034845A1 (en) * 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
EP1797084A1 (en) * 2004-09-20 2007-06-20 4Sc Ag NOVEL HETEROCYCLIC NF-kB INHIBITORS
PL2173379T3 (en) 2007-07-02 2016-02-29 Oncomed Pharm Inc Compositions and methods for treating and diagnosing cancer
AR073770A1 (en) * 2008-10-20 2010-12-01 Imclone Llc ISOLATED ANTIBODY THAT LINKS SPECIFICALLY WITH, AND INDUCES THE DEGRADATION OF THE RECEPTOR-3 OF THE HUMAN FIBROBLAST GROWTH FACTOR (FGFR-3), FGFR-3 HUMAN LINK FRAGMENT OF THE SAME, PHARMACEUTICAL COMPOSITION AND PRODUCT COMPOSITION
EP2710042A2 (en) 2011-05-16 2014-03-26 Fabion Pharmaceuticals, Inc. Multi-specific fab fusion proteins and methods of use
WO2013012747A1 (en) 2011-07-15 2013-01-24 Oncomed Pharmaceuticals, Inc. Rspo binding agents and uses thereof
SG11201401518TA (en) 2011-10-28 2014-05-29 Teva Pharmaceuticals Australia Pty Ltd Polypeptide constructs and uses thereof
WO2014012007A2 (en) 2012-07-13 2014-01-16 Oncomed Pharmaceuticals, Inc. Rspo3 binding agents and uses thereof
CN104662044B (en) 2012-08-24 2018-10-30 加利福尼亚大学董事会 For treating ROR1 cancers and inhibiting the antibody and vaccine that shift
MX369550B (en) * 2012-09-27 2019-11-12 Chugai Pharmaceutical Co Ltd Fgfr3 fusion gene and pharmaceutical drug targeting same.
US11117975B2 (en) 2013-04-29 2021-09-14 Teva Pharmaceuticals Australia Pty Ltd Anti-CD38 antibodies and fusions to attenuated interferon alpha-2B
PT2992013T (en) 2013-04-29 2020-03-05 Teva Pharmaceuticals Australia Pty Ltd Anti-cd38 antibodies and fusions to attenuated interferon alpha-2b
US10780182B2 (en) 2014-04-25 2020-09-22 The Trustees Of The University Of Pennsylvania Methods and compositions for treating metastatic breast cancer and other cancers in the brain
UA119352C2 (en) 2014-05-01 2019-06-10 Тева Фармасьютикалз Острейліа Пті Лтд Combination of lenalidomide or pomalidomide and cd38 antibody-attenuated interferon-alpha constructs, and the use thereof
DK3142750T3 (en) 2014-05-13 2020-09-14 Univ Pennsylvania COMPOSITIONS INCLUDING AAV EXPRESSING DUAL ANTIBODY CONSTRUCTIONS AND USES THEREOF
EP3180027A4 (en) * 2014-08-15 2018-01-10 Oncomed Pharmaceuticals, Inc. Rspo1 binding agents and uses thereof
EP3193935A4 (en) 2014-09-16 2018-03-21 Oncomed Pharmaceuticals, Inc. Treatment of fibrotic diseases
CA2965414C (en) 2014-10-29 2024-01-09 Teva Pharmaceuticals Australia Pty Ltd Interferon .alpha.2.beta. variants
US10870701B2 (en) 2016-03-15 2020-12-22 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
CN109069602B (en) * 2016-04-14 2022-11-22 刘扶东 Inhibition of SCUBE2, a novel VEGFR2 co-receptor, inhibition of tumor angiogenesis
IL299099A (en) 2016-06-27 2023-02-01 Univ California Cancer treatment combinations
US11359014B2 (en) 2017-05-16 2022-06-14 Alector Llc Anti-siglec-5 antibodies and methods of use thereof
US20210024936A1 (en) * 2018-04-09 2021-01-28 Board Of Regents, The University Of Texas System Therapeutic targeting of oncogenes using exosomes
EA202190183A1 (en) * 2018-07-27 2021-05-18 Алектор Ллс ANTIBODIES TO SIGLEC-5 AND METHODS OF THEIR APPLICATION
WO2023113806A1 (en) 2021-12-16 2023-06-22 Affinia Therapeutics, Inc. Recombinant aav for treatment of neural disease
EP4323531A1 (en) 2021-04-12 2024-02-21 Affinia Therapeutics Inc. Recombinant aav for treatment of neural disease
EP4329885A1 (en) 2021-04-27 2024-03-06 Generation Bio Co. Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
US20240158515A1 (en) * 2022-11-14 2024-05-16 Regeneron Pharmaceuticals, Inc. Anti-fgfr3 antibodies and antigen-binding fragments and methods of use thereof

Family Cites Families (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0481000T3 (en) 1989-07-06 1999-11-15 Univ California Receptors for fibroblast growth factors
US20010029293A1 (en) * 1992-01-27 2001-10-11 Icos Corporation Icam-related protein
US7381803B1 (en) 1992-03-27 2008-06-03 Pdl Biopharma, Inc. Humanized antibodies against CD3
AU6446194A (en) 1993-03-17 1994-10-11 Whittier Institute For Diabetes And Endocrinology, The Monoclonal antibodies specific for fibroblast growth factor receptors, immunotoxins, and use thereof
AU2466895A (en) 1995-04-28 1996-11-18 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5948658A (en) 1996-06-25 1999-09-07 The Trustees Of Columbia University In The City Of New York Anti-cocaine catalytic antibody
WO1998040488A1 (en) 1997-03-12 1998-09-17 Smithkline Beecham Corporation Anti-alphabeta3 humanized monoclonal antibodies
CA2325346A1 (en) 1998-04-03 1999-10-14 Chugai Seiyaku Kabushiki Kaisha Humanized antibody against human tissue factor (tf) and process for constructing humanized antibody
US20030206912A1 (en) 1998-06-12 2003-11-06 Yeda Research And Development Co., Ltd. FGFR3 as a marker for mesenchymal skeletal progenitor cells
MXPA01008098A (en) 1999-02-12 2002-10-23 Inst Genetics Llc Humanized immunoglobulin reactive with b7 molecules and methods of treatment therewith.
EP1208231B2 (en) 1999-05-05 2009-12-30 Institut Curie Means for detecting and treating pathologies linked to fgfr3
US20100293669A2 (en) * 1999-05-06 2010-11-18 Jingdong Liu Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement
AU1410801A (en) 1999-11-17 2001-05-30 Compugen Ltd. Variants of alternative splicing
AU2001240020B9 (en) * 2000-03-01 2008-12-04 Medimmune, Llc High potency recombinant antibodies and method for producing them
US20040043930A1 (en) 2000-02-08 2004-03-04 Anderson David W. Novel proteins and nucleic acids encoding same
US6630584B1 (en) 2000-03-16 2003-10-07 Ramot At Tel-Aviv University Ltd. Single chain antibody against mutant p53
PL358215A1 (en) 2000-03-24 2004-08-09 Micromet Ag Multifunctional polypeptides comprising a binding site to an epitope of the nkg2d receptor complex
WO2002015846A2 (en) 2000-08-21 2002-02-28 Smithkline Beecham Corporation Anti-rank ligand monoclonal antibodies useful in treatment of rank ligand mediated disorders
US20020111302A1 (en) 2000-11-30 2002-08-15 Y. Tom Tang Novel nucleic acids and polypeptides
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
WO2002087618A1 (en) 2001-04-27 2002-11-07 Takeda Chemical Industries, Ltd. Preventive/therapeutic method for cancer
EP1423428B2 (en) 2001-06-20 2012-11-14 Fibron Ltd. Antibodies that block fgfr3 activation, methods of screening for and uses thereof
CA2459219A1 (en) 2001-09-17 2003-03-27 Protein Design Labs, Inc. Methods of diagnosis of cancer compositions and methods of screening for modulators of cancer
US20060194265A1 (en) 2001-10-23 2006-08-31 Morris David W Novel therapeutic targets in cancer
US20070237770A1 (en) 2001-11-30 2007-10-11 Albert Lai Novel compositions and methods in cancer
ES2363765T3 (en) 2002-01-31 2011-08-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. FGFR AGONISTS.
EP1478667B1 (en) * 2002-02-26 2010-09-08 SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. Anti-human tenascin monoclonal antibody
WO2003083046A2 (en) 2002-03-08 2003-10-09 Curagen Corporation Novel proteins and nucleic acids encoding same
WO2004009805A1 (en) 2002-07-19 2004-01-29 Japan Science And Technology Agency Novel process for producing antibody enzyme, novel antibody enzyme and utilization thereof
WO2004022095A1 (en) 2002-09-04 2004-03-18 Abtech Anti-idiotypic antibodies as vegf or fgf agonists for bone therapy
JP2004121026A (en) 2002-09-30 2004-04-22 Univ Nihon Genetic engineering method for producing antibody molecule to glycosyltransferase
EP1565491B1 (en) 2002-11-20 2010-03-31 Cancer Research Technology Limited Antibodies binding to human magic roundabout (mr), polypeptides and uses thereof for inhibition of angiogenesis
US20040253606A1 (en) 2002-11-26 2004-12-16 Protein Design Labs, Inc. Methods of detecting soft tissue sarcoma, compositions and methods of screening for soft tissue sarcoma modulators
AU2003287918A1 (en) 2002-12-20 2004-07-14 Enkam Pharmaceuticals A/S Method of modulation of interaction between receptor and ligand
WO2004074506A2 (en) 2003-02-13 2004-09-02 Mergen Ltd Polynucleotide sequences and corresponding encoded polypeptides of particular secreted and membrane-bound proteins overexpressed in certain cancers
ES2330634T3 (en) 2003-03-26 2009-12-14 Progenika Biopharma, S.A. IN VITRO METHOD TO DETECT TRANSITIONAL CARCINOMA OF BLADDER.
IL156495A0 (en) 2003-06-17 2004-01-04 Prochon Biotech Ltd Use of fgfr3 antagonists for treating t cell mediated diseases
WO2005007800A2 (en) 2003-07-18 2005-01-27 Mochida Pharm Co Ltd Monoclonal antibody against platelet membrane glycoprotein vi
WO2005066211A2 (en) * 2003-12-19 2005-07-21 Five Prime Therapeutics, Inc. Fibroblast growth factor receptors 1, 2, 3, and 4 as targets for therapeutic intervention
DE60319240D1 (en) 2003-12-19 2008-04-03 Charite Universitaetsmedizin Use of ligands of CD52 antigen for the treatment of solid tumors and bone cancers
CA2558758C (en) 2004-02-24 2015-06-23 Allergan, Inc. Botulinum toxin screening assays
WO2005094446A2 (en) * 2004-03-23 2005-10-13 Eli Lilly And Company Anti-myostatin antibodies
WO2005115363A2 (en) 2004-05-25 2005-12-08 Yale University Method for treating skeletal disorders resulting from fgfr malfunction
US20090175866A1 (en) 2004-11-04 2009-07-09 Avner Yayon Treatment of b-cell malignancies
EP1659175A1 (en) 2004-11-18 2006-05-24 Institut Curie Alterations in seborrheic keratoses and their applications
JP2008535853A (en) 2005-04-07 2008-09-04 ノバルティス ヴァクシンズ アンド ダイアグノスティクス インコーポレイテッド Cancer-related genes
SI1912675T1 (en) 2005-07-25 2014-07-31 Emergent Product Development Seattle, Llc B-cell reduction using cd37-specific and cd20-specific binding molecules
US7498142B2 (en) 2006-01-31 2009-03-03 Yeda Research And Development Co., Ltd. Methods of identifying combinations of antibodies with an improved anti-tumor activity and compositions and methods using the antibodies
NZ573819A (en) * 2006-06-02 2011-09-30 Aveo Pharmaceuticals Inc Hepatocyte growth factor (hgf) binding proteins
US8101721B2 (en) 2006-06-15 2012-01-24 Fibron Ltd. Antibodies blocking fibroblast growth factor receptor activation and methods of use thereof
US20100092457A1 (en) * 2006-08-14 2010-04-15 Forerunner Pharma Research Co., Ltd. Diagnosis and Treatment of Cancer Using Anti-Desmoglein-3 Antibodies
FR2909092B1 (en) 2006-11-24 2012-10-19 Pf Medicament NEW ANTI-PROLIFERATION ANTIBODIES

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP2313435A4 *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8828385B2 (en) 2001-06-20 2014-09-09 Fibron Limited Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
US11401333B2 (en) 2009-03-25 2022-08-02 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
US8410250B2 (en) 2009-03-25 2013-04-02 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
EP3702371A1 (en) 2009-03-25 2020-09-02 Genentech, Inc. Anti-fgfr3 antibodies and methods using same
EP2679600A1 (en) 2009-03-25 2014-01-01 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
US8710189B2 (en) 2009-03-25 2014-04-29 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
US9499623B2 (en) 2009-03-25 2016-11-22 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
US10287356B2 (en) 2009-03-25 2019-05-14 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
WO2010111367A1 (en) 2009-03-25 2010-09-30 Genentech, Inc. Anti-fgfr3 antibodies and methods using same
US9161977B2 (en) 2009-03-25 2015-10-20 F. Hoffmann-La Roche Ag Anti-FGFR3 antibodies and methods using same
US10000571B2 (en) 2009-03-25 2018-06-19 Genentech, Inc. Anti-FGFR3 antibodies and methods using same
US11046771B2 (en) 2010-05-27 2021-06-29 Genmab A/S Monoclonal antibodies against HER2
US11091553B2 (en) 2010-05-27 2021-08-17 Genmab A/S Monoclonal antibodies against HER2
JP2014517823A (en) * 2011-04-20 2014-07-24 ゲンマブ エー／エス Bispecific antibody against HER2
JP2017197516A (en) * 2011-04-20 2017-11-02 ゲンマブ エー／エス Bispecific antibody against her2 and cd3
JP2014514314A (en) * 2011-04-20 2014-06-19 ゲンマブ エー／エス Bispecific antibodies against HER2 and CD3
US11578141B2 (en) 2011-04-20 2023-02-14 Genmab A/S Bispecific antibodies against HER2 and CD3
WO2013033008A2 (en) 2011-08-26 2013-03-07 Merrimack Pharmaceuticals, Inc. Tandem fc bispecific antibodies
JP2018184478A (en) * 2011-10-25 2018-11-22 プロシーナ バイオサイエンシーズ リミテッド Antibody formulation and method
US10517830B2 (en) 2011-10-25 2019-12-31 Prothena Biosciences Limited Antibody formulations and methods
WO2013087725A1 (en) 2011-12-12 2013-06-20 Institut National De La Sante Et De La Recherche Medicale (Inserm) Antagonist of the fibroblast growth factor receptor 3 (fgfr3) for use in the treatment or the prevention of skeletal disorders linked with abnormal activation of fgfr3
US10724014B2 (en) 2013-01-16 2020-07-28 Institut National De La Sante Et De La Recherche Medicale Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
US11814654B2 (en) 2013-01-16 2023-11-14 Institut National De La Sante Et De La Recherche Medicale Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
US11702642B2 (en) 2013-01-16 2023-07-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
WO2014138364A2 (en) 2013-03-06 2014-09-12 Genentech, Inc. Methods of treating and preventing cancer drug resistance
WO2014138449A1 (en) 2013-03-06 2014-09-12 Merrimack Pharmaceuticals, Inc. Anti-c-met tandem fc bispecific antibodies
US9458245B2 (en) 2013-03-06 2016-10-04 Merrimack Pharmaceuticals, Inc. ANTI-C-MET tandem Fc bispecific antibodies
WO2015191986A1 (en) 2014-06-13 2015-12-17 Genentech, Inc. Methods of treating and preventing cancer drug resistance
US10208120B2 (en) 2014-11-05 2019-02-19 Genentech, Inc. Anti-FGFR2/3 antibodies and methods using same
WO2016139227A1 (en) 2015-03-03 2016-09-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Fgfr3 antagonists
US11021528B2 (en) 2016-07-07 2021-06-01 INSERM (Institut National de la Santé et de la Recherche Médicale Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof
US10294289B2 (en) 2016-07-07 2019-05-21 Therachon Sas Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof
US11697678B2 (en) 2016-07-07 2023-07-11 Pfizer Inc. Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof
US11351267B2 (en) 2017-04-03 2022-06-07 Cilag Gmbh International FGFR3 binding molecules
US10722589B2 (en) 2017-04-03 2020-07-28 Covagen Ag FGFR3 binding molecules
WO2018185050A1 (en) 2017-04-03 2018-10-11 Covagen Ag Fgfr3 binding molecules
US11505611B2 (en) 2020-08-21 2022-11-22 Genzyme Corporation FGFR3 antibodies and methods of use
CN114716548A (en) * 2021-01-05 2022-07-08 (株)爱恩德生物 anti-FGFR 3 antibodies and uses thereof
WO2022149837A1 (en) * 2021-01-05 2022-07-14 (주)에임드바이오 Anti-fgfr3 antibody and use thereof
WO2024152014A1 (en) 2023-01-13 2024-07-18 Regeneron Pharmaceuticals, Inc. Fgfr3 binding molecules and methods of use thereof

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EP2313435A2 (en) 2011-04-27

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