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WO2014105849A1 - Anticorps spécifiques contre le fgfr4 et méthodes d'utilisation - Google Patents

Anticorps spécifiques contre le fgfr4 et méthodes d'utilisation Download PDF

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WO2014105849A1
WO2014105849A1 PCT/US2013/077552 US2013077552W WO2014105849A1 WO 2014105849 A1 WO2014105849 A1 WO 2014105849A1 US 2013077552 W US2013077552 W US 2013077552W WO 2014105849 A1 WO2014105849 A1 WO 2014105849A1
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seq
cancer
antibody
amino acid
acid sequence
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Vinay Bhaskar
Resi B. Gerstner
Kristen MICHELSON
Hassan ISSAFRAS
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Xoma US LLC
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Xoma US LLC
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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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates, in general, to materials and methods for antibodies specific for fibroblast growth factor receptor 4 (FGFR4), and uses of these antibodies in the treatment of subjects having cancer, or any other disease, condition or disorder related to FGFR4-mediated signaling.
  • FGFR4 fibroblast growth factor receptor 4
  • FGFs fibroblast growth factors
  • FGFRs high affinity fibroblast growth factors
  • Irregular FGFR signaling through either altered expression, mutation, constitutive activation, translocation, or truncation, has been associated with a variety of cancer types (Turner, et al. Nature Reviews. Cancer 10, 116-29 (2010)).
  • FGFR4 requires FGF 19 for its proliferative activity (M. H. Xie et al, Cytokine 11, 729-35 (1999)).
  • FGFR4 overexpression has been associated with a variety of human cancers, including hepatocellular (Desnoyers et al, Oncogene 27, 85-97 (2008)), prostate, breast, and lung cancer.
  • FGFR4 promotes stroma-induced epithelial-to-mesenchymal transition in colorectal cancer (Liu, et al, Cancer Res. 73, 5926-35 (2013)) and FGFR4 blockade exerts antitumorigenic effects in rhabdomyosarcomas (Crose et al., Clin.
  • FGFR4 inhibition has been shown to reduce Bcl-xl expression and increase apoptosis rate following doxorubicin induction of cancer cell lines (Roidl et al., Clin. Cancer Res. 15, 2058-66 (2009)).
  • Several genetic alterations have been reported for FGFR4, the most prominent of which is a polymorphism at G388R associated with a significantly higher risk of cancer, reduced survival, and a more aggressive phenotype (Bange et al, Survival 62, 840 - 847 (2002); Wang, et al, Neoplasia 10, 847-856 (2008); Xu et al, Eur. J.
  • the present disclosure provides methods and compositions for the treatment of conditions, diseases or disorders associated with FGFR4 signaling/dysregulation.
  • the disclosure provides antibodies that bind FGFR4. It was discovered herein that the disclosed FGFR4-specific antibodies unexpectedly modulate FGFR4 signaling and are capable of neutralizing FGF-19 induced phosphorylation of intracellular proteins, as well as inhibiting tumor growth in vivo.
  • the subject antibodies are contemplated for treatment of tumors associated with FGFR4 signaling, as well as other conditions, diseases or disorders associated with FGFR4 signaling.
  • the present disclosure provides an isolated antibody that specifically binds to FGFR4 and results in greater inhibition of FGF19-induced ERK 1/2 phosphorylation as compared to an antibody that includes variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO: 7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO: 9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 11 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO: 7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence
  • an antibody according to the first embodiment competes for specific binding to FGFR4 with an antibody that includes: a heavy chain complementary determining region 1 (HCDRl) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:5; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO:5; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97-107 of SEQ ID NO:5; a light chain complementary determining region 1 (LCDRl) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:6; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 51-53 of SEQ ID NO:6; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 90-100 of SEQ ID NO:6.
  • HCDRl heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 having the amino
  • the antibody that competes for specific binding to FGFR4 as described above optionally includes: a heavy chain complementary determining region 1 (HCDRl) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:5; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO:5; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97-107 of SEQ ID NO:5; a light chain complementary determining region 1 (LCDRl) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:6; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 51-53 of SEQ ID NO:6; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 90-100 of SEQ ID NO:6.
  • HCDRl heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 having the amino acid sequence of
  • an antibody according to the first embodiment comprises a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:5.
  • the antibody comprises a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:6.
  • the antibody comprises heavy and light chain polypeptides comprising variable regions comprising the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO:6, respectively.
  • An antibody according to the first embodiment may be an antibody fragment, such as an Fv, scFv, Fab, F(ab')2, or Fab'.
  • a subject conjugate includes any of the antibodies described above with respect to the first embodiment, and a therapeutic agent.
  • the therapeutic agent is a toxin.
  • pharamaceutical formulations e.g., sterile pharamaceutical formulations.
  • a subject formulation includes: any of the antibodies described above with respect to the first embodiment, or a conjugate as described above; and a pharmaceutically acceptable carrier.
  • Methods for treating a disease, condition or disorder associated with FGFR4 signaling are also provided by the present disclosure.
  • the methods include the step of administering to a subject in need thereof a therapeutically effective amount of any of the antibodies described above with respect to the first embodiment, a conjugate as described above, or a pharmaceutical composition as described above.
  • the methods find use in treating a wide variety of diseases, conditions, or disorders associated with FGFR4 signaling.
  • the disease, condition or disorder is selected from cancer, irritable bowel syndrome (IBS), coronary artery disease, and gallstone disease.
  • the disease, condition or disorder is cancer
  • the cancer is optionally selected from hepatocellular (liver) cancer, breast cancer, prostate cancer, ovarian cancer, thyroid cancer, gastric cancer, pituitary cancer, pancreatic cancer, colorectal cancer, stomach cancer, adrenocortical (adrenal gland) cancer, glioma, lung cancer, bladder cancer, head and neck cancer, oral cancer, uterine cancer, cervical cancer, brain cancer, neuroma (nerve tissue) cancer, kidney cancer, bladder cancer, skin cancer, endocrine cancer, neuroendocrine cancer, testis cancer, soft tissue sarcoma, bone sarcoma, Hodgkin's lymphoma, pituitary adenomas, breast fibroadenomas, Cushing's disease, acoustic neuromas, and leukemia.
  • the subject method is a method of treating hepatocellular (liver) cancer.
  • the present disclosure also provides an isolated antibody that specifically binds to FGFR4, where the antibody binds to an epitope of FGFR4 that is different from an epitope bound by an antibody comprising variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO:7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO:9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 11 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • the antibody that binds to a different epitope as described above competes for specific binding to FGFR4 with an antibody that includes: a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO: l; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO: l; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97- 115 of SEQ ID NO : 1 ; a light chain complementary determining region 1 (LCDRl ) having the amino acid sequence of amino acids 26-34 of SEQ ID NO:2; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 52-58 of SEQ ID NO:2; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 97-106 of SEQ ID NO:2.
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 HC
  • the antibody described above that competes for specific binding to FGFR4 with an antibody that includes the heavy and light chain complementary determining regions present in SEQ ID NOs: 1 and 2, respectively, includes the following: a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO: l; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO: l; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97-115 of SEQ ID NO: l; a light chain complementary determining region 1 (LCDRl) having the amino acid sequence of amino acids 26-34 of SEQ ID NO:2; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 52-58 of SEQ ID NO:2; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 97-106 of SEQ ID NO:2.
  • HCDR1 heavy chain complementary
  • the antibody that binds to a different epitope as described above includes a heavy chain polypeptide including a variable region that includes the amino acid sequence set forth in SEQ ID NO: 1.
  • the antibody that binds to a different epitope as described above includes a light chain polypeptide including a variable region that includes the amino acid sequence set forth in SEQ ID NO:2.
  • the antibody includes a heavy chain polypeptide including a variable region that includes the amino acid sequence set forth in SEQ ID NO: l and a light chain polypeptide including a variable region that includes the amino acid sequence set forth in SEQ ID NO:2.
  • the antibody that binds to a different epitope as described above may be an antibody fragment, such as an Fv, scFv, Fab, F(ab')2, or Fab'.
  • a subject conjugate includes an antibody that binds to a different epitope as described above, and a therapeutic agent.
  • the therapeutic agent is a toxin.
  • a subject formulation includes: an antibody that binds to a different epitope as described above, or a conjugate that includes such an antibody as described above; and a pharmaceutically acceptable carrier.
  • the methods include the step of administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to a different epitope as described above, or a conjugate or pharmaceutical formulation including the same.
  • the methods find use in treating a wide variety of diseases, conditions, or disorders associated with FGFR4 signaling.
  • the disease, condition or disorder is selected from cancer, irritable bowel syndrome (IBS), coronary artery disease, and gallstone disease.
  • the disease, condition or disorder is cancer, and the cancer is optionally selected from
  • the subject method is a method of treating hepatocellular (liver) cancer.
  • a subject antibody includes a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:5; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO:5; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97-107 of SEQ ID NO:5; a light chain complementary determining region 1 (LCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:6; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 51-53 of SEQ ID NO:6; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 90-100 of SEQ ID NO:6.
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 having the amino acid sequence of amino acids 97-107 of SEQ ID NO:5
  • the antibody includes a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:5, a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:6, or a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 5 and a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:6.
  • the present disclosure provides an antibody that specifically binds to FGFR4 and includes: a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO:3; a heavy chain
  • HCDR2 complementary determining region 2
  • HCDR3 heavy chain complementary determining region 3
  • LCDR1 light chain complementary determining region 1
  • LCDR2 light chain complementary determining region 2
  • LCDR3 light chain complementary determining region 3
  • the antibody includes a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:3, a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:4, or a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:3 and a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:4.
  • an antibody that competes for specific binding to FGFR4 with an antibody that includes: a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO: l; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO: l; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97- 115 of SEQ ID NO : 1 ; a light chain complementary determining region 1 (LCDR1) having the amino acid sequence of amino acids 26-34 of SEQ ID NO:2; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 52-58 of SEQ ID NO:2; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 97-106 of SEQ ID NO:2.
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 having the amino acid
  • the antibody that competes for specific binding as described in the preceding paragraph includes: a heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of amino acids 26-33 of SEQ ID NO: l; a heavy chain complementary determining region 2 (HCDR2) having the amino acid sequence of amino acids 51-58 of SEQ ID NO: l; a heavy chain complementary determining region 3 (HCDR3) having the amino acid sequence of amino acids 97- 115 of SEQ ID NO : 1 ; a light chain complementary determining region 1 (LCDR1) having the amino acid sequence of amino acids 26-34 of SEQ ID NO:2; a light chain complementary determining region 2 (LCDR2) having the amino acid sequence of amino acids 52-58 of SEQ ID NO:2; and a light chain complementary determining region 3 (LCDR3) having the amino acid sequence of amino acids 97-106 of SEQ ID NO:2.
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • HCDR3 having the amino acid sequence of
  • the antibody includes a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: l, a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:2, or a heavy chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain polypeptide comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:2.
  • any of the above-described isolated antibodies may be antibody fragments, such as an Fv, scFv, Fab, F(ab')2, or Fab' fragment.
  • conjugates of the isolated antibodies described above are also provided.
  • the conjugate may include an antibody as set forth above, and a therapeutic agent.
  • the therapeutic agent is a toxin.
  • compositions e.g., sterile pharmaceutical compositions
  • any of the isolated antibodies described above, or any of the conjugates set forth above, are also provided.
  • the methods include the step of administering to a subject in need thereof a therapeutically effective amount of any isolated antibody as set forth above, or a conjugate or pharmaceutical formulation including the same.
  • the methods find use in treating a wide variety of diseases, conditions, or disorders associated with FGFR4 signaling.
  • the disease, condition or disorder is selected from cancer, irritable bowel syndrome (IBS), coronary artery disease, and gallstone disease.
  • the disease, condition or disorder is cancer
  • the cancer is optionally selected from hepatocellular (liver) cancer, breast cancer, prostate cancer, ovarian cancer, thyroid cancer, gastric cancer, pituitary cancer, pancreatic cancer, colorectal cancer, stomach cancer, adrenocortical (adrenal gland) cancer, glioma, lung cancer, bladder cancer, head and neck cancer, oral cancer, uterine cancer, cervical cancer, brain cancer, neuroma (nerve tissue) cancer, kidney cancer, bladder cancer, skin cancer, endocrine cancer, neuroendocrine cancer, testis cancer, soft tissue sarcoma, bone sarcoma, Hodgkin's lymphoma, pituitary adenomas, breast fibroadenomas, Cushing's disease, acoustic neuromas, and leukemia.
  • the subject method is a method of treating hepatocellular (liver) cancer.
  • each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein.
  • each of these types of embodiments is a non- limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination.
  • Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are
  • FIG. 1 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure bind specifically to FGFR4.
  • the binding of antibodies XPA.48.056, XPA.48.117, and XPA.48.148 was evaluated by measuring binding to CHO-hFGFR4 cells (FIG. 1, Panel A); HepG2 (FIG. 1, Panel B); and HuH7 cells (FIG. 1, Panel C) by FACS.
  • FIG. 2 shows data indicating that an example of an anti-FGFR4 antibody of the present disclosure (XPA.48.56) inhibits FGF19 binding to FGFR4 as monitored by SPR.
  • FIG. 3 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure do not bind to other members of the FGFR family (FGFRl-3). Binding was assessed by SPR using recombinant extracellular domains of FGFRl-3 and anti-FGFR4 antibodies XPA.48.117 (FIG. 3, Panel A), XPA.48.148 (FIG. 3, Panel B) and XPA.48.056 (FIG. 3, Panel C).
  • FIG. 4 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure are capable of neutralizing FGF-19 induced phosphorylation of
  • Each of the three example antibodies were able to neutralize pERK (FIG. 4, Panel A) and pGSK-3P (FIG. 4, Panel B) in the presence of a sub-maximal concentration of rfiFGF-19 as measured using electrochemiluminescence.
  • FIG. 5 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure are capable of mediating antibody-dependent cellular cytotoxicity in HepG2 cells (FIG. 5, Panel A), HuH-7 cells (FIG. 5, Panel B) and HCT116-FGFR4 cells (FIG. 5, Panel C). Percent specific cytotoxicity was calculated using 1 or 10 ⁇ g/mL antibody.
  • FIG. 6 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure are capable of binding to mouse FGFR4 in addition to human FGFR4.
  • FIG. 6 Panel A shows dose-dependent binding of XPA.48.056, XPA.48.117, and
  • FIG. 6 Panel B shows FACS binding of 50 nM XPA.48.056, XPA.48.117, and XPA.48.148 to HEK293E cells transiently transfected with mouse FGFR4 cDNA. Data was normalized to the degree of signal measured for untransfected cells.
  • FIG. 7 shows data indicating that three examples of anti-FGFR4 antibodies of the present disclosure are capable of inhibiting tumor growth in the HuH-7 xenograft model. Antibodies were administered at 30 mg/kg/week for a total of 4 weeks (15 mice per group).
  • FIG. 8 shows an amino acid sequence alignment of examples of anti-FGFR4 antibodies of the present disclosure, and comparator anti-FGFR4 antibodies.
  • BM1 heavy chain SEQ ID NO:7
  • BM2 heavy chain SEQ ID NO:9
  • BM3 heavy chain SEQ ID NO: 8.
  • XPA.48.117 heavy chain SEQ ID NO:5
  • XPA.48.148 heavy chain SEQ ID NO:3
  • BM1 light chain SEQ ID NO: 8
  • BM2 light chain SEQ ID NO: 10
  • BM3 light chain SEQ ID NO: 12
  • BM4 light chain SEQ ID NO: 14
  • XPA.48.056 light chain SEQ ID NO:2
  • the present disclosure provides therapeutics to treat diseases, conditions or disorders associated with FGFR4 signaling, for example, cancer, irritable bowel syndrome, coronary artery disease, and gallstone disease.
  • the present disclosure provides molecules or agents that interact with FGFR4 and inhibit one or more of its functional effects, such as for example, signaling through the FGFR4 (e.g., signal transduction (e.g., phosphorylation of intracellular proteins) resulting from the binding of an FGFR4 ligand (e.g., FGF19 or any other FGFR4 ligand) to FGFR4.
  • the compositions disclosed can modulate cell proliferation and tumor growth, thereby providing, in one aspect, a method to treat cancer by affecting a cell population that directly or indirectly affects growth of the tumor.
  • FGFR4 refers to fibroblast growth factor receptor 4 protein, including wild-type FGFR4 or an FGFR4 variant, which variant includes but is not limited to, FGFR4 having a single nucleotide polymorphism (SNP) or mutation (e.g., a cancer-causing SNP or mutation, e.g., a G338R substitution, a D756N substitution, an S778R substitution, etc.), an FGFR4 splice variant, or any other FGFR4 variant).
  • SNP single nucleotide polymorphism
  • mutation e.g., a cancer-causing SNP or mutation, e.g., a G338R substitution, a D756N substitution, an S778R substitution, etc.
  • the "desired biological activity" of an anti-FGFR4 antibody is the ability to bind to FGFR4 and inhibit one or more of its functional effects (e.g., FGFR4- mediated signal transduction).
  • a disease, condition or disorder associated with "FGFR4 signaling” is a disease, condition or disorder in which FGFR4 activity is detrimental and includes diseases, conditions and disorders in which high levels of FGFR4 and/or high levels of FGFR4 activity have been shown to be or are suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder, as well as diseases and disorders in which high levels of FGFR4 expression or activity are associated with undesirable clinical signs or symptoms.
  • Such disorders may be evidenced, for example, by an increase in the levels of FGFR4 on the cell surface and/or increased signaling (e.g., dysregulation) in the affected cells or tissues of a subject suffering from the disorder.
  • An increase in FGFR4 levels may be detected, for example, using an FGFR4-specific antibody (e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody) as described herein.
  • an FGFR4-specific antibody e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • Examples of diseases, conditions or disorders associated with FGFR4 signaling that can be treated with an antibody substance that binds FGFR4 include cancers, such as hepatocellular (liver) cancer, breast cancer, prostate cancer, ovarian cancer, thyroid cancer, gastric cancer, pituitary cancer, pancreatic cancer, colorectal cancer, stomach cancer, adrenocortical (adrenal gland) cancer, glioma, lung cancer, bladder cancer, head and neck cancer, oral cancer, uterine cancer, cervical cancer, brain cancer, neuroma (nerve tissue) cancer, kidney cancer, bladder cancer, skin cancer, endocrine cancer, neuroendocrine cancer, testis cancer, soft tissue sarcoma, bone sarcoma, Hodgkin's lymphoma, pituitary adenomas, breast fibroadenomas, Cushing's disease, acoustic neuromas, and leukemia.
  • cancers such as hepatocellular (liver) cancer, breast cancer, prostate cancer
  • non-cancer diseases, conditions or disorders associated with FGFR4 signaling include irritable bowel syndrome (IBS), coronary artery disease, gallstone disease and any other disease, condition or disorder associated with FGFR4 signaling.
  • IBS irritable bowel syndrome
  • coronary artery disease CAD
  • gallstone disease CAD
  • an "immunoglobulin” or “native antibody” is a tetrameric glycoprotein.
  • each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa ( ⁇ ) and lambda ( ⁇ ) light chains.
  • Heavy chains are classified as mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), alpha (a), and epsilon ( ⁇ ), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).
  • Immunoglobulin variable domains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions or CDRs. From N- terminus to C -terminus, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, (J. Mol. Biol. 196:901-917, 1987); Chothia et al, (Nature 342:878-883, 1989).
  • the hypervariable region of an antibody refers to the CDR amino acid residues of an antibody responsible for antigen-binding .
  • the hypervariable region may include amino acid residues from a CDR as described by Kabat et al, Sequences of Proteins of
  • CDRs have also been identified and numbered according to ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The
  • Framework or FR residues are those variable domain residues other than the hypervariable region residues.
  • Heavy chain variable region refers to the region of the antibody molecule comprising at least one complementarity determining region (CDR) of said antibody heavy chain variable domain.
  • the heavy chain variable region may contain one, two, or three CDRs of said antibody heavy chain.
  • Light chain variable region refers to the region of an antibody molecule, comprising at least one complementarity determining region (CDR) of said antibody light chain variable domain.
  • the light chain variable region may contain one, two, or three CDR of said antibody light chain, which may be either a kappa or lambda light chain depending on the antibody.
  • antibody is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies (e.g. humanized monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen ("antigen-binding fragments" or "antigen- binding portions", e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and
  • immunoglobulin or "tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region.
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antibody fragments or antigen- binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), a antigen- binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as aone, two, three, four, five or six CDR sequences, as long as the antibody retains the desired biological activity.
  • dAb domain antibody
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Antibody variant refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the reference antibody variable region domains. Variants may be substantially homologous or substantially identical to the unmodified antibody.
  • a "chimeric antibody,” as used herein, refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and rodent antibody fragments, generally human constant and mouse variable regions.
  • a “neutralizing antibody” is an antibody molecule which is able to eliminate or significantly reduce a biological function of a target antigen to which it binds. Accordingly, a “neutralizing" anti-target antibody is capable of eliminating or significantly reducing a biological function of FGFR4, such as signal tranduction.
  • an "isolated" antibody is one that has been separated from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and can be more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • an antibody that "specifically binds to FGFR4" is “FGFR4 specific”, is “specific for FGFR4" or is “immunoreactive” with FGFR4 refers to an antibody or antibody substance that binds the FGFR4 with greater affinity than with similar antigens.
  • the FGFR4-binding polypeptides, or fragments, variants, or derivatives thereof will bind with a greater affinity to human FGFR4 as compared to its binding affinity to FGFR4 of other, i.e., non-human (e.g., mouse), species, but binding polypeptides that recognize and bind orthologs of FGFR4 are within the scope provided.
  • a polypeptide that is an antibody or fragment thereof "specific for" its cognate antigen indicates that the variable regions of the antibodies recognize and bind the polypeptide of interest with a detectable preference (i.e., able to distinguish the polypeptide of interest from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members).
  • detectable preference i.e., able to distinguish the polypeptide of interest from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members.
  • specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule.
  • epitope refers to that portion of any molecule capable of being recognized by and bound by a selective binding agent at one or more of the antigen binding regions.
  • Epitopes usually consist of chemically active surface groupings of molecules, such as, amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes as used herein may be contiguous or non-contiguous.
  • epitopes may be mimetic (mimotopes) in that they comprise a three dimensional structure that is identical to the epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in the target (FGFR4) that were used to stimulate the antibody immune response.
  • FGFR4 target
  • a mimotope is not considered a different antigen from the epitope bound by the selective binding agent; the selective binding agent recognizes the same three-dimensional structure of the epitope and mimotope.
  • derivative when used in connection with antibody substances and polypeptides of the present disclosure refers to polypeptides chemically modified by such techniques as ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
  • Derivatives retain the binding properties of underivatized molecules of the disclosure.
  • Detectable moiety or a “label” refers to a composition detectable by
  • useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a FGFR4.
  • the detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantitate the amount of bound detectable moiety in a sample.
  • FGFR4-specific composition of the disclosure that is effective to ameliorate or lessen symptoms or signs of disease associated with FGFR4 signaling.
  • treat refers to eliminating, reducing, suppressing or ameliorating, either temporarily or permanently, either partially or completely, a clinical symptom, manifestation or progression of an event, disease or condition associated with FGFR4 signaling. Such treating need not be absolute to be useful.
  • the present disclosure provides a, FGFR4-specific antibody, which may include the sequences set out in Table 1, fragments, variants and derivatives thereof, pharmaceutical formulations including a FGFR4-specific antibody recited above, methods of preparing the pharmaceutical formulations, and methods of treating patients with the pharmaceutical formulations and compounds.
  • immunoglobulins can be assigned to different classes, IgA, IgD, IgE, IgG and IgM, which may be further divided into subclasses or isotypes, e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • IgGl immunoglobulins
  • IgG2 immunoglobulins
  • IgG3, IgG4 immunoglobulins
  • IgAl and IgA2 immunoglobulins
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, IgGl and IgG3 isotypes have ADCC activity.
  • An antibody disclosed herein, if it comprises a constant domain may be of any of these subclasses or isotypes.
  • the antibodies of the present disclosure may exhibit binding affinity to FGFR4 of a
  • Kd of less than or equal to about 10 M, less than or equal to about 10 M, or less than or
  • affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay using 1-125 labeled FGFR4; or by another method set forth in the examples below or known to the skilled artisan.
  • SPR surface plasmon resonance
  • the affinity data may be analyzed, for example, by the method of Scatchard et al., (Ann N.Y. Acad. Sci., 51 :660, 1949).
  • a KinExA kinetic exclusion assay is also useful to measure the affinity of an antibody for its antigen.
  • KinExA technology measures binding events in the solution phase, rather than binding events between a solution phase and a solid phase.
  • many methods for measuring binding events require at least one reactant be modified through immobilization or labeling, the KinExA method does not require modification of molecules under study.
  • the KinExA method is believed to allow a wider range of binding constants to be measured than other methods currently available. Additional description about KinExA devices and operation for antibody characterization is available from the manufacturer (Sapidyne Instruments, Inc., Boise, ID) and can be found in the published literature, for example U.S. Patent No. 6,664,114 and Darling et al., "Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions.” Assay and Drug Development Technologies, 2004, 2:647-657.
  • Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) polypeptides of three example antibodies of the present disclosure (XPA.48.056, XPA.48.148 and XPA.48.117) and four comparator antibodies (BM-1, BM-2, BM-3 and BM-4) are provided in Table 1. CDRs are underlined and in order from CDR1 , CDR2, and CDR3.
  • HWYQQKSGQQPKLLIYSASNTESGPSRFSGSG S GTDFTLTIDP VE ADDI AN Y YC 00 SNELP WT FGGGTKLELKR
  • FGFR4 Fibroblast growth factor 4 receptor
  • NP 002002.3; UniProt: P22455 is a tyrosine protein kinase that acts as cell-surface receptor for fibroblast growth factors and plays a role in the regulation of cell proliferation, differentiation and migration, and in regulation of lipid metabolism, bile acid biosynthesis, and glucose metabolism.
  • FGFR4 is a member of the fibroblast growth factor receptor family with highly conserved amino acid sequences between members and throughout evolution. FGFR family members differ from one another in their ligand affinities and tissue
  • a full-length representative protein would consist of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain.
  • the extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation.
  • the genomic organization of this gene compared to FGFR 1-3, encompasses 18 exons rather than 19 or 20.
  • Ligands for FGFR4 include FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF16, FGF17, FGF18, FGF19, FGF21 and FGF23.
  • FGFR4 binds to FGF19 with particularly high binding affinity.
  • FGFR4 phosphorylates FRS2 and possibly PLCG1. Ligand binding leads to the activation of several signaling cascades. Activation of PLCG1 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate. FGFR4 promotes SRC-dependent phosphorylation of the matrix protease MMP14 and its lysosomal degradation. FGFR4 signaling is down-regulated by receptor internalization and degradation; MMP14 generally promotes internalization and degradation of FGFR4. Mutations that lead to constitutive kinase activation or impair normal FGFR4 inactivation lead to aberrant signaling.
  • FGFR4 has been observed in cells/tissues including adrenal cortex, adrenal gland, bile duct, cervix, cornea, corneal endothelial cell, corneal epithelial cell, heart, hepatocyte, intestine, islets of langerhans, kidney, lamina basement, liver, lung, lymph node, mammary gland, muscle, muscularis mucosa, ovary, pituitary gland, renal tubular epithelium, retina, skin, spleen, stomach, sublingual gland, ureter, urothelium and uterus.
  • a regulator of FGFR4 signaling is klotho-beta (KLB), a 130 kDa transmembrane protein that exhibits a more restricted expression profile in adipose, liver and pancreas tissues.
  • KLB and FGFR4 are both expressed at high levels in mature hepatocytes, where KLB stabilizes FGF19-FGFR4 binding to regulate production of cholesterol 7a-hydroxylase (CYP7A1) and hepatocyte proliferation.
  • the FGFR4 associated with cancer includes a Gly388Arg polymorphism.
  • Gly388Arg polymorphism the presence of the Gly388Arg polymorphism in cancer patients in a population of 58 cases (29% heterozygous and 24% homozygous) and 88 controls (43% heterozygous and 27% homozygous) was associated with a poor prognosis in hepatocellular carcinoma patients.
  • overexpression of FGFR4 was observed in 33% of such cancer patients.
  • the FGFR4 Arg388 polymorphism was detected in 83 out of 185 (45%) melanoma patients and was significantly associated with tumor thickness and the nodular melanoma subtype.
  • the analysis of 137 melanoma tissues by immunohistochemistry showed that 45% of the specimens expressed FGFR4 at different levels and correlated with pTNM tumour stages, metastases, number of primary tumors and survival.
  • FGFR4 is overexpressed in low- and high-metastatic colorectal cancer (CRC) cell lines, with higher expression at late CRC stages.
  • CRC colorectal cancer
  • FGFR4 is a tumor-associated antigen of autoantibodies in colorectal cancer patients and may serve for the diagnosis of colorectal cancer at early stages in combination with other tumor- associated antigens. In addition, FGFR4 is overexpressed in 50-70%) of pancreatic cancer cell lines and pancreatic carcinomas. FGFR4 is significantly increased in high-grade pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma, where FGFR4 stimulation by FGF19 increased cell adhesion to extracellular matrix and decreased cell migration.
  • FGFR4 signaling is also associated with non-cancer disease, conditions or disorders including, but not limited to, irritable bowel syndrome (IBS), coronary artery disease, and gallstone disease.
  • IBS irritable bowel syndrome
  • coronary artery disease CAD
  • gallstone disease CAD
  • the present disclosure encompasses amino acid molecules encoding target
  • FGFR4 specific antibodies e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody.
  • An FGFR4 specific antibody of the disclosure may include a human kappa ( ⁇ ) or a human lambda ( ⁇ ) light chain or an amino acid sequence derived therefrom, or a human heavy chain or a sequence derived therefrom, or both heavy and light chains together in a single chain, dimeric, tetrameric or other form.
  • a heavy chain and a light chain of a target specific immunoglobulin are different amino acid molecules.
  • the same amino acid molecule contains a heavy chain variable region and a light chain variable region of a target specific antibody.
  • the amino acid sequence of the anti-FGFR4 antibody comprises one or more CDRs of the amino acid sequence of the mature (i.e., missing signal sequence) light chain variable region (VL) of antibodies XPA.48.056, XPA.48.148, and XPA.48.117 set out in Table 1 (SEQ ID NOs: 2, 4 and 6, respectively, with CDRs underlined) or variants thereof, including CDR grafted, modified, humanized, chimeric, or Human Engineered antibodies or any other variants described herein.
  • the VL comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of the light chain of any one of the foregoing antibodies.
  • the FGFR4 specific antibody comprises a light chain CDR1, CDR2 or CDR3 ((LCDRl, LCDR2, LCDR3), each of which are independently selected from the CDR1, CDR2 and CDR3 regions of an antibody having a light chain variable region comprising the amino acid sequence of the VL region set out in SEQ ID NOs: 2, 4 and 6, a nucleic acid encoding the VL region set out in SEQ ID NOs: 2, 4, and 6, or encoded by a nucleic acid molecule encoding the VL region.
  • CDR1, CDR2 or CDR3 ((LCDRl, LCDR2, LCDR3), each of which are independently selected from the CDR1, CDR2 and CDR3 regions of an antibody having a light chain variable region comprising the amino acid sequence of the VL region set out in SEQ ID NOs: 2, 4 and 6, a nucleic acid encoding the VL region set out in SEQ ID NOs: 2, 4, and 6, or encoded by a nucleic acid molecule encoding the
  • the light chain CDRs are located at approximately residues 26-33 (LI), 51-53 (L2) and 90- 100 (L3) in the light chain variable domain of an antibody light chain of approximately similar length to those disclosed herein. In another aspect, it is contemplated that the light chain CDRs are located at approximately residues 26-34 (LI), 52-58 (L2) and 97-106 (L3) in the light chain variable domain of an antibody light chain of approximately similar length to those disclosed herein.
  • a polypeptide of the target specific antibody may comprise the CDR1, CDR2 and CDR3 regions of an antibody comprising the amino acid sequence of the VL region selected from the group consisting of XPA.48.056, XPA.48.117, and XPA.48.148.
  • Antibodies of the present disclosure may have a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: l as defined by Kabat et al. ⁇ supra).
  • the present disclosure also encompasses antibodies having a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: l numbered according to ImMunoGenTics (IMGT; supra) numbering.
  • Antibodies of the present disclosure may have a light chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2 as defined by Kabat et al. ⁇ supra).
  • antibodies of the present disclosure have a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:3 as defined by Kabat et al. ⁇ supra).
  • the present disclosure also encompasses antibodies having a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:3 numbered according to ImMunoGenTics (IMGT; supra) numbering.
  • Antibodies of the present disclosure may have a light chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4 as defined by Kabat et al. ⁇ supra).
  • Antibodies of the present disclosure may have a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5 as defined by Kabat.
  • the present disclosure also encompasses antibodies having a heavy chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:5 numbered according to ImMunoGenTics (IMGT) numbering.
  • Antibodies of the present disclosure may have a light chain variable region polypeptide having a CDRl, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:6 as defined by Kabat et al. ⁇ supra).
  • the human target specific antibody comprises one or more CDRs of the amino acid sequence of the mature (i.e., missing signal sequence) heavy chain variable region (VH) of antibody XPA.48.056, XPA.48.148, and XPA.48.117 set out in Table 1 or SEQ ID NOs: 1, 3 and 5, respectively, or variants thereof.
  • the VH comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any one of the heavy chain of the foregoing antibodies.
  • the target specific antibody comprises a heavy chain CDR1, CDR2 or CDR3 (HCDRl, HCDR2, HCDR3), each of which are independently selected from the CDR1, CDR2 and CDR3 regions of an antibody having a heavy chain variable region comprising the amino acid sequence of the VH region set out in SEQ ID NOs: 1, 3 and 5, a nucleic acid encoding the VH region set out in SEQ ID NOs: 1, 3 and 5, or encoded by a nucleic acid molecule encoding the VH region.
  • HCDRl, HCDR2, HCDR3 heavy chain variable region comprising the amino acid sequence of the VH region set out in SEQ ID NOs: 1, 3 and 5, a nucleic acid encoding the VH region set out in SEQ ID NOs: 1, 3 and 5, or encoded by a nucleic acid molecule encoding the VH region.
  • a target specific antibody comprises a heavy chain CDR1, CDR2 or CDR3, each of which are independently selected from the CDR1, CDR2 and CDR3 regions of an antibody having a heavy chain variable region comprising the amino acid sequence of the VH region set out in SEQ ID NOs: 1, 3 and 5.
  • the heavy chain CDRs are located at approximately residues 26-33 (HI), 51-58 (H2) and 97-107 (H3) in the heavy chain variable domain of an antibody heavy chain of approximately similar length to those disclosed herein.
  • the heavy chain CDRs are located at approximately residues 26-33 (HI), 51-58 (H2) and 97-115 (H3) in the heavy chain variable domain of an antibody heavy chain of approximately similar length to those disclosed herein.
  • a polypeptide of the target specific antibody may comprise the CDR1, CDR2 and CDR3 regions of an antibody comprising the amino acid sequence of the VH region selected from the group consisting of XPA.48.056, XPA.48.117, and XPA.48.148.
  • the antibody comprises a mature light chain variable region as disclosed above and a mature heavy chain variable region as disclosed above, optionally paired as set forth in Table 1.
  • a monoclonal monoclonal antibody that retains any one, two, three, four, five, or six of HCDRl, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of antibodies XPA.48.056, XPA.48.117, and/or XPA.48.148, optionally including one or two mutations in any of such CDR(s), e.g., a conservative or non- conservative substitution, and optionally paired as set forth in Table 1.
  • CDRs for the heavy and light chains of XPA.48.056, XPA.48.117, and/or XPA.48.148 are underlined in Table 1 and shown in FIG. 8.
  • a monoclonal antibody that retains all of HCDR1, HCDR2, HCDR3, or the heavy chain variable region of any one of SEQ ID NOs: 1, 3, and 5, optionally including one or two mutations in any of such CDR(s), optionally further comprising any suitable heavy chain constant region, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, or IgE, a human sequence thereof, or a hybrid thereof;
  • a monoclonal antibody that retains all of LCDRl , LCDR2, LCDR3, or the light chain variable region of any one SEQ ID NOs: 2, 4 and 6, optionally including one or two mutations in any of such CDR(s), optionally further comprising any suitable light chain constant region, e.g., a kappa or lambda light chain constant region, a human sequence thereof, or a hybrid thereof.
  • the antibody comprises all three light chain CDRs, all three heavy chain CDRs, or all six CDRs of the light and heavy chain, paired as set forth in Table 1.
  • two light chain CDRs from an antibody may be combined with a third light chain CDR from a different antibody.
  • a LCDRl from one antibody can be combined with a LCDR2 from a different antibody and a LCDR3 from yet another antibody, particularly where the CDRs are highly homologous.
  • two heavy chain CDRs from an antibody may be combined with a third heavy chain CDR from a different antibody; or a HCDR1 from one antibody can be combined with a HCDR2 from a different antibody and a HCDR3 from yet another antibody, particularly where the CDRs are highly homologous.
  • an antibody comprises a polypeptide having an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more identical to the heavy chain variable region set out in SEQ ID NOs: 1, 3, and 5 and/or an amino acid sequence an amino acid sequence at least about 65%>, 70%>, 75%>, 80%>, 81%>, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more identical to the light chain variable region set out in SEQ ID NOs: 2, 4 and 6, the antibody further comprising at least one, two, three, four, five or all of HCDR1, HCDR2, HCDR
  • the amino acid sequence with percentage identity to the light chain variable region may comprise one, two or three of the light chain CDRs. In other embodiments, the amino acid sequence with percentage identity to the heavy chain variable region may comprise one, two, or three of the heavy chain CDRs.
  • an antibody in another embodiment, comprises a polypeptide having an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more identical to all three HCDRs in a heavy chain variable region set out in Table 1.
  • an antibody comprises a polypeptide having an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more identical to the all three LCDRs in the light chain variable region set out in Table 1.
  • an antibody comprises a polypeptide having an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more identical to all six CDRs in the heavy chain and light chain variable regions set out in Table 1.
  • the antibodies of the disclosure may have one, or two or more amino acid substitutions in the CDR regions of the antibody, e.g., non-conservative or conservative substitutions.
  • the residues of the framework are altered.
  • the heavy chain framework regions which can be altered lie within regions designated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chain CDR residues, and the residues of the light chain framework regions which can be altered lie within the regions designated L-FRl, L-FR2, L-FR3 and L-FR4, which surround the light chain CDR residues.
  • An amino acid within the framework region may be replaced, for example, with any suitable amino acid identified in a human framework or human consensus framework.
  • a subject anti-FGFR4 antibody binds FGFR4 with an affinity of
  • a subject anti-FGFR4 antibody binds to FGFR4 with at least 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more higher affinity (e.g., preferentially binds to FGFR4) compared to binding to any one of FGFR1 , FGFR2 and FGFR3.
  • Heavy and light chain amino acid sequences of XPA.48.056 are set out in SEQ ID NOs: 1 and 2, respectively.
  • Heavy and light chain amino acid sequences of XPA.48.148 are set out in SEQ ID NOs: 3 and 4, respectively, and heavy and light chain amino acid sequences of XPA.48.1 17 are set out in SEQ ID NOs: 5 and 6, respectively.
  • an antibody of the present disclosure specifically binds to FGFR4 and results in greater inhibition of FGF19-induced ERK 1/2 phosphorylation as compared to an antibody comprising variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO:7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO: 9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 1 1 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • the inhibition of FGF19-induced ERK 1/2 phosphorylation may be, e.g., 10% or greater, 20% or greater, 30%> or greater, 40%> or greater, 50%> or greater, 60%> or greater, 70%> or greater, 80%> or greater, 90%> or greater, 100% or greater, 150% or greater, 200%) or greater, or 500% or greater than an antibody having the variable heavy chain and variable light chain sequences of SEQ ID NOs:7 and 8, respectively, SEQ ID NOs:9 and 10, respectively, SEQ ID NOs: l 1 and 12, respectively, or SEQ ID NOs: 13 and 14, respectively.
  • the subject antibody which results in the greater inhibition of FGF19-induced ERK 1/2 phosphorylation is XPA.48.1 17, XPA.48.148, XPA.48.056, or an antibody having one, two, three, four, five or six CDRs of XPA.48.1 17, XPA.48.148, or XPA.48.056 (which CDRs are underlined in Table 1).
  • Any suitable assay may be employed to determine whether an antibody results in greater inhibition of FGF19-induced ERK 1/2 phosphorylation as compared to an antibody.
  • An example assay is set forth below in the Examples section.
  • an antibody of the present disclosure specifically binds to FGFR4 and reduces or inhibits cell proliferation (e.g., the proliferation of cancer cells, in which the cancer is associated with FGFR4 signaling, such as any of the cancers listed in Tables 3A, 3B, 3C, 3D and 4).
  • variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO:7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO:9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 11 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • the reduction or inhibition of cell proliferation may be, e.g., 10% or greater, 20% or greater, 30% or greater, 40%) or greater, 50%> or greater, 60%> or greater, 70%> or greater, 80%> or greater, 90%> or greater, 100% or greater, 150% or greater, 200% or greater, or 500%) or greater than an antibody having the variable heavy chain and variable light chain sequences of SEQ ID NOs:7 and 8, respectively, SEQ ID NOs:9 and 10, respectively, SEQ ID NOs: l 1 and 12, respectively, or SEQ ID NOs: 13 and 14, respectively.
  • the subject antibody which results in the reduction or inhibition of cell proliferation is XPA.48.117, XPA.48.148, XPA.48.056, or an antibody having one, two, three, four, five or six CDRs of XPA.48.117, XPA.48.148, or XPA.48.056 (which CDRs are underlined in Table 1).
  • an antibody of the present disclosure specifically binds to FGFR4 and reduces or inhibits tumor growth (e.g., the reduction or inhibition of growth of a tumor associated with FGFR4 signaling, such as any of the cancers listed in Tables 3A, 3B, 3C, 3D and 4).
  • variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO: 7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO:9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 11 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • the reduction or inhibition of tumor growth may be, e.g., 10%> or greater, 20%> or greater, 30%> or greater, 40%> or greater, 50%> or greater, 60%> or greater, 70%> or greater, 80%> or greater, 90%> or greater, 100% or greater, 150% or greater, 200% or greater, or 500% or greater than an antibody having the variable heavy chain and variable light chain sequences of SEQ ID NOs:7 and 8, respectively, SEQ ID NOs:9 and 10, respectively, SEQ ID NOs: l 1 and 12, respectively, or SEQ ID NOs: 13 and 14, respectively.
  • the subject antibody which results in the reduction or inhibition of tumor growth is XPA.48.117, XPA.48.148, XPA.48.056, or an antibody having one, two, three, four, five or six CDRs of XPA.48.117, XPA.48.148, or XPA.48.056 (which CDRs are underlined in Table 1).
  • Any suitable assay may be employed to determine whether an antibody is able to reduce or inhibit tumor growth, and to determine a difference in the reduction or inhibition as compared to a different antibody.
  • An example assay is set forth below in the Examples section.
  • a subject antibody specifically binds to FGFR4 and binds to an epitope of FGFR4 that is different from an epitope bound by an antibody comprising variable heavy chain and variable light chain sequences selected from: (a) a variable heavy chain sequence as shown in SEQ ID NO: 7 and a variable light chain sequence as shown in SEQ ID NO: 8; (b) a variable heavy chain sequence as shown in SEQ ID NO: 9 and a variable light chain sequence as shown in SEQ ID NO: 10; (c) a variable heavy chain sequence as shown in SEQ ID NO: 11 and a variable light chain sequence as shown in SEQ ID NO: 12; and (d) a variable heavy chain sequence as shown in SEQ ID NO: 13 and a variable light chain sequence as shown in SEQ ID NO: 14.
  • the antibody that binds to a different epitope may be XPA.48.056, or an antibody having one, two, three, four, five or six CDRs of XPA.48.056 (which CDRs are underlined in Table 1).
  • Any suitable assay may be employed to determine whether an antibody binds to a different epitope as compared to a different antibody.
  • competitive binding assays find use in making such determinations, and an example competitive binding assay is set forth below in the Examples section.
  • the present disclosure also encompasses nucleic acid molecules encoding FGFR4 specific antibodies.
  • different nucleic acid molecules encode a heavy chain variable region and a light chain variable region of a FGFR4 specific antibody.
  • the same nucleic acid molecule encodes a heavy chain and a light chain variable regions of a FGFR4 specific antibody.
  • the nucleic acid encodes a FGFR4 specific antibody of the present disclosure, as well as any of the polypeptides encoded by the nucleic acids described herein.
  • a nucleic acid molecule of the present disclosure comprises a nucleotide sequence that encodes the VL amino acid sequence of antibodies XPA.48.056, XPA.48.148, and XPA.48.117 set out in SEQ ID NOs: 2, 4 and 6 or a portion thereof.
  • the VL amino acid sequence is a consensus sequence.
  • the nucleic acid encodes the amino acid sequence of the light chain CDRs of the antibody.
  • the portion is a contiguous portion comprising CDR1-CDR3.
  • said portion comprises at least one, two or three of a light chain CDR1, CDR2, or CDR3 region, optionally with a different human or human consensus framework, and optionally with 1, or up to 2, or up to 3 mutations in the collective 3 CDRs.
  • the present disclosure provides antigen-binding compounds, including functional fragments, having a variable region amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5 and 6., e.g., SEQ ID NOS: 1 and 2; 3 and 4; or 5 and 6.
  • an aforementioned antigen binding compound is selected from the group consisting of a fully assembled tetrameric antibody, a monoclonal antibody a humanized antibody; a human antibody; a chimeric antibody; a multispecific antibody, an antibody fragment, Fab, F(ab')2; Fv; scFv or single-chain antibody fragment; a diabody; triabody, tetrabody, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), a binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or derivative of any one of these antibodies, that comprise one or more CDR sequences of the disclosure and exhibit the desired biological activity, or a mixture of two or more antibodies.
  • SMIP small modular immunopharmaceutical
  • the antigen binding compounds of the present disclosure can retain binding affinity of 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 , 10 ⁇ 10 , 10 "11 M or less for FGFR4 (e.g., as measured by surface plasmon resonance).
  • the antibodies of the present disclosure comprise a heavy chain variable region or light chain variable region as set out in amino acid sequences SEQ ID NOs: 1, 3, and 5 and SEQ ID NOs: 2, 4 and 6, respectively, as paired in Table 1. It is further contemplated that the antibodies may comprise all or part of the antibodies set out in the above amino acid sequences. In one embodiment, the antibodies comprise at least one of CDR1, CDR2, or CDR3 of the heavy chain of SEQ ID NOs: 1, 3, and 5, or at least one of CDR1, CDR2 or CDR3 of the light chain of SEQ ID NOs: 2, 4 and 6, as paired in Table 1.
  • any of the heavy chain CDR and light chain CDR sequences described above may also include amino acids added to either end of the CDRs.
  • Preparation of variants and derivatives of antibodies and antigen-binding compounds of the present invention, including affinity maturation or preparation of variants or derivatives containing amino acid analogs, is described in further detail herein.
  • Examples of variants include those containing a conservative or non-conservative substitution of a corresponding amino acid within the amino acid sequence, or a replacement of an amino acid with a corresponding amino acid of a different human antibody sequence.
  • the present disclosure contemplates a purified polypeptide comprising at least one HCDR or LCDR (e.g., at least on CDR (underlined) present in the heavy and/or light chain variable regions set out in Table 1), wherein the framework regions of the heavy chain variable region and the framework regions of the light chain variable region comprise framework regions from a human antibody.
  • the framework regions of the heavy chain variable region and the framework regions of the light chain variable region are chemically altered by amino acid substitution to be more homologous to a different human antibody sequence.
  • each heavy chain framework region (H-FRl-4) it is contemplated that at least one, at least two, at least three, at least four, at least five, or at least six native framework region residues of the heavy chain variable region have been altered by amino acid substitution, and wherein within each light chain framework region (L-FR1-4), at least one, at least two, at least three, at least four, at least five or at least six native framework residues of the light chain variable region have been altered by amino acid substitution.
  • the light chain comprising a light chain CDR3 sequence described above may also comprise both (a) a light chain CDRl sequence described above and (b) a light chain CDR2 sequence described above.
  • Antibodies comprising any one of the light chain variable regions described above may further comprise a heavy chain variable region, optionally paired as described in Table 1, e.g., a heavy chain variable region that binds to target antigen, e.g., a heavy chain variable region comprising heavy chain CDR sequences described above.
  • the antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1, 3, and 5 and a light chain variable region selected from the group consisting of SEQ ID NOs: 2, 4 and 6.
  • the nucleic acid molecule comprises a nucleotide sequence that encodes the light chain variable amino acid sequence of one of SEQ ID NOs: 2, 4 and 6 or a portion thereof, and/or a nucleotide sequence that encodes the heavy chain variable amino acid sequence of one of SEQ ID NOs: 1, 3 and 5 or a portion thereof.
  • the nucleic acid molecule encodes a VL amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 97, 98 or 99% identical to a VL amino acid sequence set out in SEQ ID NOs: 2, 4 and 6.
  • Nucleic acid molecules of the disclosure include nucleic acids that hybridize under highly stringent conditions, such as those described herein, to a nucleic acid sequence encoding the light chain variable region amino acid sequence of SEQ ID NOs: 2, 4 and 6.
  • the nucleic acid molecule encodes a VH amino acid sequence of any one of antibodies XPA.48.056, XPA.48.117, and XPA.48.148, or a portion thereof.
  • the nucleic acid encodes the amino acid sequence of the heavy chain CDRs of the antibody.
  • the portion is a contiguous portion comprising heavy chain CDR1-CDR3.
  • said portion comprises at least one, two or three of a heavy chain CDR1, CDR2, or CDR3 region, optionally with a different human or human consensus framework, and optionally with 1, or up to 2, or up to 3 mutations in the collective 3 CDRs.
  • the nucleic acid molecule comprises a nucleotide sequence that encodes the heavy chain amino acid sequence of one of heavy chain of SEQ ID NOs: 1, 3, and 5 or a portion thereof.
  • the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VH amino acid sequence set out in SEQ ID NOs: 1, 3, and 5.
  • the VH amino acid sequence is a consensus sequence.
  • Nucleic acid molecules of the disclosure further include nucleic acids that hybridize under highly stringent conditions, such as those described herein, to a nucleic acid sequence encoding the heavy chain variable region amino acid sequence of SEQ ID NOs: 1, 3, and 5.
  • nucleic acids of the disclosure may encode a full- length light chain or heavy chain having one or more CDRs present in an antibody selected from XPA.48.056, XPA.48.117, and XPA.48.148, where a full-length light chain or full- length heavy chain comprises a light chain constant region or a heavy chain constant region, respectively, light chain constant regions optionally include unmodified or modified kappa or lambda regions, and heavy constant regions include unmodified or modified constant regions of any of the classes, such as IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, or IgE.
  • an anti-FGFR4 antibody of the present disclosure includes a light chain variable region and/or a heavy chain variable region, wherein (a) the light chain variable region comprises at least a CDR1 shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto, a CDR2 shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto, and/or a CDR3 shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto; and/or wherein (b) the heavy chain variable region comprises at least a CDRl shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto, a CDR2 shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto, and/or a CDR3 shown in Table 1 or FIG. 8 or sequences at least 80% identical thereto.
  • an anti-FGFR4 antibody of the present disclosure includes a light chain variable region and/or a heavy chain variable region, wherein (a) the light chain variable region comprises at least a CDRl shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto, a CDR2 shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto, and/or a CDR3 shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto; and/or wherein (b) the heavy chain variable region comprises at least a CDRl shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto, a CDR2 shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto, and/or a CDR3 shown in Table 1 or FIG. 8 or sequences at least 90% identical thereto.
  • An antibody of the disclosure can comprises a human kappa ( ⁇ ) or a human lambda ( ⁇ ) light chain or an amino acid sequence derived therefrom, or a human heavy chain or a sequence derived therefrom, or both heavy and light chains together in a single chain, dimeric, tetrameric or other form.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are generally highly specific, and may be directed against a single antigenic site, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against the same or different determinants (epitopes). In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.
  • Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. (Nature, 256:495-7, 1975) (Harlow & Lane; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1988); Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al, (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581-597, 1991). Additional methods for prodicing monoclonal antibodies are well-known to a person of ordinary skill in the art.
  • Monoclonal antibodies such as those produced by the above methods, are suitably separated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.
  • antibodies of the present disclosure may be used as smaller antigen binding fragments of the antibody that are well-known in the art and described herein.
  • Antibody fragments comprise a portion of an intact full length antibody and can include an antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecfic, trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies); minibody; chelating recombinant antibody; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins;
  • SMIP small modular immunopharmaceuticals
  • camelized antibodies camelized antibodies; VHH containing antibodies; and other polypeptides formed from antibody fragments. See, e.g., Holliger & Hudson (Nat. Biotech. 23: 1126-36 (2005)).
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, monovalent fragments consisting of the VL, VH, CL and CH domains each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, that has two "Single-chain Fv” or "scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which a VL and VH region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al, Science 242:423-426, 1988, and Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
  • scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).
  • An Fd fragment consists of the VH and CHI domains.
  • Additional antibody fragments include a domain antibody (dAb) fragment (Ward et al., Nature 341 :544-546, 1989) which consists of a VH domain.
  • Diabodies are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93/11161; Holliger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak et al, Structure 2: 1121-1123, 1994). Diabodies can be bispecific or monospecific.
  • these antibodies form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only having the structure H2L2 (referred to as “heavy-chain antibodies” or “HCAbs”).
  • HCAbs heavy-chain antibodies
  • Camelid VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CHI domain (Hamers-Casterman et al., supra).
  • llama IgGl is a conventional (H2L2) antibody isotype in which VH recombines with a constant region that contains hinge, CHI, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lack CHI domains and that contain no light chains.
  • Camelid VHH domains have been found to bind to antigen with high affinity (Desmyter et al, J. Biol. Chem. 276:26285-90, 2001) and possess high stability in solution (Ewert et al, Biochemistry 41 :3628-36, 2002).
  • Classical VH-only fragments are difficult to produce in soluble form, but improvements in solubility and specific binding can be obtained when framework residues are altered to be more VHH-like. (See, e.g., Reichman, et al., J Immunol Methods 1999, 231 :25-38.) Methods for generating antibodies having camelid heavy chains are described in, for example, in U.S. Patent Publication Nos. 20050136049 and
  • variable domain of an antibody heavy-chain is the smallest fully functional antigen-binding fragment with a molecular mass of only 15 kDa, this entity is referred to as a nanobody (Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004).
  • a nanobody library may be generated from an immunized dromedary as described in Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods as described in Revets et al, Expert Opin. Biol. Ther. 5(1): 111-24 (2005).
  • tribody a scFv molecule is fused to one or both of the VL-CL (L) and VH-CHl (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab.
  • a "minibody” consisting of scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al, Protein Eng Des Sel. 17(4):315-23, 2004.
  • Intrabodies are single chain antibodies which demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al, Proc Natl Acad Sci U S A. 101 : 17616-21 , 2004). Intrabodies, which comprise cell signal sequences which retain the antibody construct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J 14: 1542-51, 1995) and Wheeler et al. (FASEB J. 17: 1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domain (PTD) is fused with single chain variable fragment (scFv) antibodies Heng et al, (Med Hypotheses. 64: 1105-8, 2005).
  • PTD protein transduction domain
  • scFv single chain variable fragment
  • immunoglobulin fusion proteins specific for target protein are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions. See e.g., WO03/041600, U.S. Patent publication 20030133939 and US Patent Publication 20030118592.
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin.
  • An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
  • the CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.
  • compositions comprising one, two, and/or three CDRs (e.g., a single CDR alone or in tandem, 2, 3, or other multiple repeats of the CDRs,; or combinations of 2 or 3 CDRs alone or in tandem repeats; optionally, with a spacer amino acid sequence between the CDRs or repeats) of a heavy chain variable region or a light chain variable region of an antibody may be generated by techniques known in the art.
  • bispecific antibodies may bind to two different epitopes of the FGFR4.
  • an FGFR4-specific antibody arm may be combined with an arm which binds to a cell surface molecule so as to focus cellular defense mechanisms to the target (FGFR4).
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express or take up the target.
  • bispecific antibodies possess a target-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten).
  • cytotoxic agent e.g., saporin, anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the interface can comprise at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See, e.g., WO 1996/027011.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al. (Science 229:81-83, 1985) describe a procedure wherein intact antibodies are proteo lyrically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies.
  • bispecific antibodies have been produced using leucine zippers. (Kostelny et al, J. Immunol. 148: 1547-1553, 1992).
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re -oxidized to form the antibody
  • the fragments comprise a heavy chain variable region (VH) connected to a light- chain variable region (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • scFv single-chain Fv
  • the bispecific antibody may be a "linear antibody” produced as described in Zapata et al. Protein Eng. 8: 1057-62 (1995).
  • Linear antibodies comprise a pair of tandem Fd segments (VH -CH1-VH -CHI) which form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • the bispecific antibody may be a chelating recombinant antibody (CRAb).
  • CRAb chelating recombinant antibody
  • a chelating recombinant antibody recognizes adjacent and non- overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously (Neri et al, J Mol Biol. 246:367-73, 1995).
  • Antibodies with more than two valencies are also contemplated.
  • trispecific antibodies can be prepared. See, e.g., Tutt et al, J. Immunol. 147:60, 1991.
  • chimeric or humanized antibodies are less immunogenic in humans than the parental non-human (e.g., mouse) monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis.
  • Chimeric monoclonal antibodies in which the variable Ig domains of a non-human (e.g., mouse)monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison et al, Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984); and, Boulianne et al, Nature 312, 643-646, (1984)).
  • Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as humanizing through “CDPv grafting”), (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”), or, alternatively, (3) substituting human amino acids at positions determined to be unlikely to adversely effect either antigen binding or protein folding, but likely to reduce immunogenicity in a human environment (e.g., HUMAN),
  • humanized antibodies will include both “humanized,” “veneered” and “HUMAN ENGINEEREDTM” antibodies. These methods are disclosed in, e.g., Jones et al, Nature 321 :522 525 (1986); Morrison et al, Proc. Natl. Acad. Sci., U.S.A., 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92 (1988);
  • CDR grafting involves introducing one or more of the six CDRs from the mouse heavy and light chain variable Ig domains into the appropriate four framework regions of human variable Ig domains. This technique (Riechmann, et al., Nature 332:323-27 (1988)), utilizes the conserved framework regions (FR1-FR4) as a scaffold to support the CDR loops which are the primary contacts with antigen.
  • FR1-FR4 conserved framework regions
  • a disadvantage of CDR grafting is that it can result in a humanized antibody that has a substantially lower binding affinity than the original mouse antibody, because amino acids of the framework regions can contribute to antigen binding, and because amino acids of the CDR loops can influence the association of the two variable Ig domains.
  • the CDR grafting technique can be improved by choosing human framework regions that most closely resemble the framework regions of the original mouse antibody, and by site- directed mutagenesis of single amino acids within the framework or CDRs aided by computer modeling of the antigen binding site (e.g., Co et al, J. Immunol. 152, 2968-2976 (1994)).
  • Human antibodies to target protein can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci.
  • WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci.
  • WO 91/00906 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated.
  • 6,091,001 disclose the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule.
  • WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci.
  • U.S. Patent No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. See also, U.S. Patent Nos. 6,114,598 6,657,103 and 6,833,268.
  • an immune response can be produced to a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
  • Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735.
  • This publication discloses monoclonal antibodies against a variety of antigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin, gp39, and tetanus toxin. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.
  • WO 96/33735 discloses that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice immunized with IL-8, blocked IL-8 induced functions of neutrophils.
  • Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096 and U.S. patent application no. 20030194404; and U.S. patent application no. 20030031667.
  • Additional transgenic animals useful to make monoclonal antibodies include the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429 and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), which contains gene sequences from unrearranged human antibody genes that code for the heavy and light chains of human antibodies. Immunization of a HuMAb-MOUSE® enables the production of fully human monoclonal antibodies to the target protein.
  • TCMOUSETM TransChromo Mouse which comprises megabase-sized segments of human DNA and which incorporates the entire human immunoglobulin (hlg) loci.
  • TCMOUSETM has a fully diverse repertoire of hlgs, including all the subclasses of IgGs (IgGl-G4). Immunization of the TCMOUSETM with various human antigens produces antibody responses comprising human antibodies. [0191] See also Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al, Year in Immunol, 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Patent No. 5,589,369, U.S. Patent No.
  • U.S. Patent Publication No. 20030092125 describes methods for biasing the immune response of an animal to the desired epitope. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • the antibodies produced by phage technology are produced as antigen binding fragments-usually Fv (e.g., scFv) or Fab fragments-in bacteria and thus lack effector functions.
  • Effector functions can be introduced by one of two strategies: the fragments can be engineered, for example, into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
  • the present disclosure provides methods for producing target- specific antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with target protein or a portion thereof, isolating phage that bind target, and obtaining the antibody from the phage.
  • one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with target antigen or an antigenic portion thereof to create an immune response, extracting antibody producing cells from the immunized animal; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using a primer, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage.
  • Recombinant target-specific antibodies of the disclosure may be obtained in this way.
  • antibody producing cells can be extracted from non-immunized animals, RNA isolated from the extracted cells and reverse transcribed to produce cDNA, which is amplified using a primer, and inserted into a phage display vector such that antibodies are expressed on the phage.
  • Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • WO 99/10494 describes the isolation of high affinity and functional agonistic antibodies for MPL and msk receptors using such an approach.
  • Antibodies of the disclosure can be isolated by screening of a recombinant combinatorial antibody library, such as a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. See e.g., U.S. Patent No. 5,969,108. There are commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage display kit, catalog no. 240612).
  • a human VH and VL library are screened to select for antibody fragments having the desired specificity.
  • the antibody libraries used in this method can be scFv libraries prepared and screened as described herein and in the art (McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al, (Nature 348:552-554 (1990)); and Griffiths et al, (EMBO J 12:725-734 (1993)).
  • the scFv antibody libraries can be screened using target protein as the antigen.
  • the Fd fragment (VH-CH1) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen.
  • the Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them.
  • selection of Fab by antigen binding co-selects for the Fab encoding sequences, which can be amplified subsequently.
  • panning Fab specific for the antigen are enriched and finally isolated.
  • Huminization of of antibodies can be accomplished using "guided selection", which utilizes a phage display technique for the humanization of a mouse monoclonal antibody (See Jespers, L. S., et al, Bio/Technology 12, 899-903 (1994)).
  • the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen.
  • the mouse Fd fragment thereby provides a template to guide the selection.
  • the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.
  • Fv fragments are displayed on the surface of phage, by the association of one chain expressed as a phage protein fusion (e.g., with Ml 3 gene III) with the complementary chain expressed as a soluble fragment.
  • the phage may be a filamentous phage such as one of the class I phages: fd, M13, fl, Ifl, Ike, ZJ/Z, Ff and one of the class II phages Xf, Pfl and Pf3.
  • the phage may be Ml 3, or fd or a derivative thereof.
  • VL and VH segments of the VL/VH pair(s) of interest can be randomly mutated, e.g., within the any of the CDR1, CDR2 or CDR3 region of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response.
  • This in vitro affinity maturation can be accomplished by amplifying VL and VH regions using PCR primers complimentary to the VH CDR1, CDR2, and CDR3, or VL CDR1, CDR2, and CDR3, respectively, which primers have been "spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VL and VH segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VL and VH segments can be rescreened for binding to target antigen.
  • nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the disclosure, as described below.
  • the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described herein.
  • the phage display method may be carried out in a mutator strain of bactaria or host cell.
  • a mutator strain is a host cell which has a genetic defect which causes DNA replicated within it to be mutated with respect to its parent DNA.
  • Example mutator strains are NR9046mutD5 and NR9046 mut Tl .
  • the phage display method may be carried out using a helper phage.
  • This is a phage which is used to infect cells containing a defective phage genome and which functions to complement the defect.
  • the defective phage genome can be a phagemid or a phage with some function encoding gene sequences removed.
  • helper phages are M13K07, M13K07 gene III no. 3; and phage displaying or encoding a binding molecule fused to a capsid protein.
  • Antibodies are also generated via phage display screening methods using the hierarchical dual combinatorial approach as disclosed in WO 92/01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described therein. This technique is also disclosed in Marks et al, (Bio/Technology, 10:779-783 (1992)).
  • antibodies may be isolated using in vitro display methods and microbial cell display, including ribosome display and mRNA display (Amstutz et al, Curr. Op. Biotech. 12: 400-05 (2001)). Selection of polypeptide using ribosome display is described in Hanes et al, (Proc. Natl Acad Sci USA, 94:4937-4942 (1997)) and U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is also useful for rapid large scale mutational analysis of antibodies. The selective mutagenesis approach also provides a method of producing antibodies with improved activities that can be selected using ribosomal display techniques.
  • methods for isolating antibodies specific to FGFR4 is carried out using a phage display method identical or similar to the methods described in the Examples section hereinbelow.
  • modified polypeptide compositions comprising one, two, three, four, five, and/or six CDRs of an antibody are generated, wherein a CDR is altered to provide increased specificity or affinity to the target molecule.
  • Sites within antibody CDRs are typically modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid substituted for a non-identical hydrophobic amino acid) and then with more dissimilar choices (e.g., hydrophobic amino acid substituted for a charged amino acid), and then deletions or insertions may be made at the target site.
  • PCR primers complementary to these consensus sequences are generated to amplify the antigen-specific CDR sequence located between the primer regions.
  • Techniques for cloning and expressing nucleotide and polypeptide sequences are well-established in the art [see e.g. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York (1989)].
  • the amplified CDR sequences are ligated into an appropriate plasmid.
  • the plasmid comprising one, two, three, four, five and/or six cloned CDRs optionally contains additional polypeptide encoding regions linked to the CDR.
  • Antibody substances comprising the modified CDRs are screened for binding affinity for the original antigen. Additionally, the antibody or polypeptide is further tested for its ability to neutralize the activity of the target antigens. For example, antibodies of the disclosure may be analyzed as set out in the Examples to determine their ability to interfere with the biological activity of target antigen.
  • Modifications may be made by conservative or non-conservative amino acid substitutions described in greater detail below.
  • “Insertions” or “deletions” can in the range of about 1 to 20 amino acids, and can be about 1 to 10 amino acids. The variation may be introduced by systematically making substitutions of amino acids in an antibody polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity. Nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody (including antibody fragment) fused to an epitope tag or a salvage receptor epitope.
  • Other insertional variants of the antibody molecule include the fusion to a polypeptide which increases the serum half-life of the antibody, e.g. at the N-terminus or C-terminus.
  • epitope tag polypeptide has enough residues to provide an epitope against which an antibody there against can be made, yet is short enough such that it does not interfere with activity of the antibody.
  • the epitope tag can be selected to be sufficiently unique so that the antibody there against does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (e.g., between about 9-30 residues). Examples include the flu hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (Field et al, Mol. Cell. Biol.
  • tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
  • tags such as the FLAG® tag (Eastman Kodak, Rochester, NY), well known and routinely used in the art, are embraced by the disclosure.
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • variants Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Substitutional mutagenesis within any of the hypervariable or CDR regions or framework regions is contemplated. Conservative substitutions involve replacing an amino acid with another member of its class. Non-conservative substitutions involve replacing a member of one of these classes with a member of another class.
  • nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (He, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M);
  • polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gin, Q); positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (Hi
  • cysteine residues not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • Affinity maturation generally involves preparing and screening antibody variants that have substitutions within the CDRs of a parent antibody and selecting variants that have one or more improved biological properties such as binding affinity relative to the parent antibody.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) may be mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity). See e.g., WO 92/01047, WO 93/112366, WO 95/15388 and WO 93/19172.
  • Affinity maturation via panning methods Affinity maturation of recombinant antibodies is commonly performed through several rounds of panning of candidate antibodies in the presence of decreasing amounts of antigen. Decreasing the amount of antigen per round selects the antibodies with the highest affinity to the antigen thereby yielding antibodies of high affinity from a large pool of starting material.
  • Affinity maturation via panning is well known in the art and is described, for example, in Huls et al. (Cancer Immunol Immunother. 50: 163-71 (2001)). Methods of affinity maturation using phage display technologies are described elsewhere herein and known in the art (see e.g., Daugherty et al, Proc Natl Acad Sci U S A. 97:2029-34 (2000)).
  • LTM Look-through mutagenesis
  • Mutated CDRs are combined to generate combinatorial single-chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all variants.
  • scFv single-chain variable fragment
  • Error-prone PCR involves the randomization of nucleic acids between different selection rounds. The randomization occurs at a low rate by the intrinsic error rate of the polymerase used but can be enhanced by error-prone PCR (Zaccolo et al.,. J. Mol. Biol. 285:775-783 (1999)) using a polymerase having a high intrinsic error rate during transcription (Hawkins et al., J Mol Biol. 226:889-96 (1992)). After the mutation cycles, clones with improved affinity for the antigen are selected using routine mehods in the art.
  • DNA Shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce variant polynucleotides. DNA shuffling has been described in US Patent No. 6,605,449, US Patent 6,489,145, WO 02/092780 and Stemmer, Proc. Natl. Acad. Sci. USA, 91 : 10747-51 (1994).
  • DNA shuffling is comprised of 3 steps: fragmentation of the genes to be shuffled with DNase I, random hybridization of fragments and reassembly or filling in of the fragmented gene by PCR in the presence of DNA polymerase (sexual PCR), and
  • DNA shuffling differs from error-prone PCR in that it is an inverse chain reaction. In error-prone PCR, the number of polymerase start sites and the number of molecules grows exponentially. In contrast, in nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time. [0225] In the case of an antibody, DNA shuffling allows the free combinatorial association of all of the CDRls with all of the CDR2s with all of the CDR3s, for example. It is contemplated that multiple families of sequences can be shuffled in the same reaction.
  • shuffling generally conserves the relative order, such that, for example, CDR1 will not be found in the position of CDR2.
  • Rare shufflants will contain a large number of the best (e.g. highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity.
  • the template polynucleotide which may be used in DNA shuffling may be DNA or RNA. It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.
  • the template polynucleotide can be from 50 bp to 50 kb. The template polynucleotide often are double-stranded.
  • polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, during the initial step of gene selection. It is also contemplated that two different but related polynucleotide templates can be mixed during the initial step.
  • Alanine scanning - Alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Cunningham and Wells, (Science 244: 1081-1085 (1989)). A residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen.
  • affinity maturation techniques known in the art may be used, including for example techniques described in published patent applications WO2009/088933; WO2009/088928; WO2009/088924; as well as Clackson et al, Nature 352:624-628, 1991; Marks et al, Biotechnology 10:779-783, 1992; Virnekas et al, Nucleic Acids Res. 22:5600-5607, 1994; Glaser et al, J. Immunol. 149:3903-3913, 1992; Jackson et al, J. Immunol. 154:3310-3319, 1995; Schier et al, J. Mol. Biol. 255:28-43, 1996; and Yang et al., J. Mol. Biol. 254:392-403, 1995, incorporated by reference herein in their entirety.
  • Antibody variants can also be produced that have a modified glycosylation pattern relative to the parent antibody, for example, deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine -X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the
  • glycosylation sites may be added to an antibody by altering the amino acid sequence such that it contains one or more of these tripeptide sequences.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may also be used.
  • O-linked glycosylation sites may be added to an antibody by inserting or substituting one or more serine or threonine residues to the sequence of the original antibody.
  • Fc glycans influence the binding of IgG to Fc receptors and Clq, and are therefore important for IgG effector functions.
  • Antibody variants with modified Fc glycans and altered effector function may be produced.
  • antibodies with modified terminal sugars such as sialic acids, core fucose, bisecting N-acetylglucosamine, and mannose residues may have altered binding to the FcyRIIIa receptor and altered ADCC activity.
  • antibodies with modified terminal galactose residues may have altered binding to Clq and altered CDC activity (Raju, Curr. Opin. Immunol. 20: 471-78 (2008).
  • ADCC effector activity is mediated by binding of the antibody molecule to the FcyRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Asn-297 of the CH2 domain.
  • Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcyRIII-mediated effector functions more efficiently than native, fucosylated antibodies.
  • homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., (Cancer Research 53: 2560-2565 (1993)).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, (Anti-Cancer Drug Design 3: 219-230 (1989)).
  • sequences within the CDR can cause an antibody to bind to MHC Class II and trigger an unwanted helper T-cell response. A conservative substitution can allow the antibody to retain binding activity yet lose its ability to trigger an unwanted T- cell response.
  • Steplewski et al (Proc Natl Acad Sci U S A. 85:4852-56 (1998)), which described chimeric antibodies wherein a murine variable region was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions.
  • an antibody fragment rather than an intact antibody, to increase tumor penetration, for example.
  • This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478).
  • a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478).
  • the salvage receptor binding epitope can constitute a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. In one example, three or more residues from one or two loops of the Fc domain are transferred.
  • the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CHI, CH3, or VH region, or more than one such region, of the antibody.
  • the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment. See also International applications WO 97/34631 and WO
  • antibodies of the present disclosure may comprise a human Fc portion, a human consensus Fc portion, or a variant thereof that retains the ability to interact with the Fc salvage receptor, including variants in which cysteines involved in disulfide bonding are modified or removed, and/or in which the a met is added at the N-terminus and/or one or more of the N-terminal 20 amino acids are removed, and/or regions that interact with complement, such as the Clq binding site, are removed, and/or the ADCC site is removed [see, e.g.,Sarmay et al, Molec. Immunol. 29:633-9 (1992)].
  • Mutation of residues within Fc receptor binding sites can result in altered effector function, such as altered ADCC or CDC activity, or altered half-life.
  • potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position).
  • IgGl residues that affected binding to Fc receptor II are as follows: (largest effect) Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298, and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339, Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reduced binding.
  • IgGl residues that reduced binding to Fc receptor IIIA by 40% or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376.
  • Variants that improved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A, and A339T.
  • Lys414 showed a 40% reduction in binding for FcRIIA and FcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30% reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23% improvement to FcRIIIA. See also Presta et al., (Biochem. Soc. Trans. 30:487-490, 2001), incorporated herein by reference in its entirety, which described several positions in the Fc region of IgGl were found which improved binding only to specific Fc gamma receptors (R) or simultaneously improved binding to one type of Fc gamma R and reduced binding to another type.
  • R Fc gamma receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • U.S. Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced Clq binding or CDC activity.
  • U.S. Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced Clq binding or CDC activity.
  • U.S. Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced Clq binding or CDC activity.
  • a mutation at amino acid position 238, 265, 269, 270, 327 or 329 are stated to reduce binding to FcRI
  • a mutation at amino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 are stated to reduce binding to FcRII
  • a mutation at amino acid position 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.
  • 20040132101 describes variants with mutations at amino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering), of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding to an Fc gamma receptor.
  • FcgammaRIIa at least 10-fold less efficiently than wildtype IgGl but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Mutations were made in the region of amino acids 233-236 and/or at amino acid positions 327, 330 and 331. See also WO 99/58572, incorporated by reference herein in its entirety.
  • Covalent modifications of the antibody are also included within the scope of this disclosure. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, a-bromo-P-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5- 7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction can be performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino -terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing . alpha. -amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione,l,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R-N.dbd.C.dbd.N-R'), where R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N- acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Hakimuddin, et al, (Arch. Biochem. Biophys. 259: 52 (1987)) and by Edge et al., (Anal. Biochem. 118: 131 (1981)).
  • Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo- glycosidases as described by Thotakura et al, (Meth. Enzymol. 138: 350 (1987)).
  • Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorb
  • derivative refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine.
  • derivatives of the antibody substance of the invention such as a bispecific antibody, are also useful as therapeutic agents and may be produced by the methods herein.
  • the conjugated moiety can be incorporated in or attached to an antibody substance either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
  • Polyethylene glycol may be attached to the antibody substances to provide a longer half-life in vivo.
  • the PEG group may be of any convenient molecular weight and may be linear or branched.
  • the average molecular weight of the PEG can range from about 2 kiloDalton ("kD") to about 100 kDa, from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10 kDa.
  • the PEG groups will generally be attached to the antibody substances of the disclosure via acylation or reductive alkylation through a natural or engineered reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the antibody substance (e.g., an aldehyde, amino, or ester group).
  • a natural or engineered reactive group on the PEG moiety e.g., an aldehyde, amino, thiol, or ester group
  • Addition of PEG moieties to antibody substances can be carried out using techniques well-known in the art. See, e.g., International Publication No. WO 96/11953 and U.S. Patent No. 4,179,337.
  • Ligation of the antibody substance with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC.
  • the PEGylated substances are purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
  • an antibody may be administered in its "naked” or unconjugated form, or may be conjugated directly to other therapeutic or diagnostic agents, or may be conjugated indirectly to carrier polymers comprising such other therapeutic or diagnostic agents.
  • the antibody is conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Suitable chemotherapeutic agents include: daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).
  • Suitable toxins include: bacterial toxins such as diphtheria toxin; plant toxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al J. Natl. Cancer Inst. 92(19): 1573-81 (2000); Mandler et al, Bioorg. Med. Chem. Letters 10: 1025-1028 (2000); Mandler et al, Bioconjugate Chem.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent or luminescent or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art; for example, see (Sternberger, L.A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E.A. et al, Meth. Enzym. 62:308 (1979); Engval, E. et al, Immunol. 109: 129 (1972); Goding, J.W. J. Immunol. Meth. 13:215 (1976)).
  • affinity labels such as biotin, avidin, etc.
  • enzymatic labels such as horseradish
  • This general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
  • the carrier polymer may be, for example, an aminodextran or polypeptide of at least 50 amino acid residues.
  • Various techniques for conjugating a drug or other agent to the carrier polymer are known in the art.
  • a polypeptide carrier can be used instead of
  • the polypeptide carrier should have at least 50 amino acid residues in the chain, and can be about 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues.
  • the pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent.
  • suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and conjugate.
  • agents to which the antibody can be conjugated include any of the cytotoxic or
  • conjugated antibodies can be prepared by directly conjugating an antibody component with a therapeutic agent.
  • the general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
  • a therapeutic agent is directly attached to an oxidized antibody component.
  • a carbohydrate moiety of an antibody can be attached to polyethyleneglycol to extend half-life.
  • a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation, or using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al, Int. J. Cancer56:244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal Antibodies:
  • bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyldithiol) propionate
  • I
  • Antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al, Ann. Oncol. 6:945 (1995), Nicolet et al, Cancer Gene Ther. 2: 161 (1995), Becker et al, Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al, Clin. Cancer Res. 2: 1951 (1996), and Hu et al, Cancer Res. 56:4998 (1996). In addition, Yang et al, (Hum.
  • antibody-toxin fusion proteins in which a recombinant molecule comprises one or more antibody components and a toxin or chemotherapeutic agent also are known to those of skill in the art.
  • antibody-Pseudomonas exotoxin A fusion proteins have been described by Chaudhary et al, Nature 339:394 (1989), Brinkmann et al, Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al, Proc. Nat'l Acad. Sci. USA 89:5867 (1992), Friedman et al, J. Immunol. 150:3054 (1993), Wels et al, Int. J. Can. 60: 137
  • toxins which are suitably employed in the preparation of such fusion proteins are ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al, Cell 47:641 (1986), and Goldenberg, CA--A Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins are known to those of skill in the art.
  • Antibodies of the present disclosure may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to an active anti-cancer drug.
  • a prodrug e.g., a peptidyl chemotherapeutic agent, See WO81/01145
  • an active anti-cancer drug See, for example, WO88/07378 and U.S. Patent No. 4,975,278.
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Enzymes that are useful in the present disclosure include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as a-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs;
  • antibodies with enzymatic activity can be used to convert the prodrugs of the disclosure into free active drugs (See, e.g., Massey, Nature 328: 457-458 (1987).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
  • fusion proteins comprising at least the antigen binding region of an antibody of the disclosure linked to at least a functionally active portion of an enzyme of the disclosure can be constructed using recombinant DNA techniques well known in the art (See, e.g., Neuberger et al, Nature 312:604-608 (1984))
  • DNA encoding an antibody of the present disclosure may be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies). Usually this requires cloning the DNA or mRNA (e.g., as cDNA) encoding the antibodies. Cloning and sequencing is carried out using standard techniques, such as for example polymerase chain reaction (PCR) (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press; Ausubel, et al. (Eds.), and Protocols in Molecular Biology, John Wiley & Sons (1994)), which are incorporated herein by reference.
  • PCR polymerase chain reaction
  • Nucleotide probe reactions and other nucleotide hybridization reactions are carried out at conditions enabling the identification of polynucleotides which hybridize to each other under specified conditions.
  • Oe example of a set of conditions is as follows: stringent hybridization at 42°C in 50% formamide, 5X SSC, 20 mM Na » P04, pH 6.8; and washing in IX SSC at 55°C for 30 minutes.
  • Formula for calculating equivalent hybridization conditions and/or selecting other conditions to achieve a desired level of stringency are well known. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.
  • hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51
  • an "isolated" nucleic acid molecule or “isolated” nucleic acid sequence is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined, is considered isolated.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • RNA used for cloning and sequencing is a hybridoma produced by obtaining a B cell from the transgenic mouse and fusing the B cell to an immortal cell.
  • An advantage of using hybridomas is that they can be easily screened, and a hybridoma that produces a human monoclonal antibody of interest selected.
  • RNA can be isolated from B cells (or whole spleen) of the immunized animal.
  • sources other than hybridomas it may be desirable to screen for sequences encoding immunoglobulins or immunoglobulin polypeptides with specific binding characteristics.
  • Phage display is described further herein and is also well-known in the art.
  • cDNA from an immunized transgenic mouse e.g., total spleen cDNA
  • the polymerase chain reaction is used to amplify a cDNA sequences that encode a portion of an immunoglobulin polypeptide, e.g., CDR regions, and the amplified sequences are inserted into a phage vector.
  • cDNAs encoding peptides of interest e.g., variable region peptides with desired binding characteristics, are identified by standard techniques such as panning.
  • the sequence encoding an entire variable region of the immunoglobulin polypeptide is determined, however, it will sometimes by adequate to sequence only a portion of a variable region, for example, the CDR-encoding portion.
  • the portion sequenced will be at least 30 bases in length, more often based coding for at least about one- third or at least about one -half of the length of the variable region will be sequenced.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, human embryonic kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art.
  • host cells such as E. coli cells, simian COS cells, human embryonic kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. Recombinant production of antibodies is well known in the art.
  • Expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Cell, cell line, and cell culture are often used interchangeably and all such designations herein include progeny.
  • Transformants and transformed cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • amino acid sequence of an immunoglobulin of interest may be determined by direct protein sequencing. Suitable encoding nucleotide sequences can be designed according to a universal codon table.
  • Amino acid sequence variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the molecule, such as changing the number or position of glycosylation sites.
  • Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide -mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.
  • the present disclosure also provides isolated nucleic acid encoding antibodies of the disclosure, optionally operably linked to control sequences recognized by a host cell, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies, which may comprise culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture or culture medium.
  • recombinant techniques for the production of the antibodies which may comprise culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture or culture medium.
  • Various systems and methods for antibody production are reviewed by Birch & Racher (Adv. Drug Deliv. Rev. 671-685 (2006)).
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selective marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Antibodies of the present disclosure may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence selected can be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence may be substituted by a signal sequence selected, for example, from the group of the pectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the native signal sequence may be substituted by, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces a-factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO90/ 13646.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Expression and cloning vectors may contain a selective gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, tetracycline, G418, geneticin, histidinol, or mycophenolic acid (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs methotrexate, neomycin, histidinol, puromycin, mycophenolic acid and hygromycin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody-encoding nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, such as primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
  • host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with DNA sequences encoding antibody of the disclosure, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.
  • APH aminoglycoside 3 '-phosphotransferase
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature, 282: 39 (1979)).
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, (Genetics 85: 12 (1977)).
  • the presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
  • Ura3 -deficient yeast strains are complemented by plasmids bearing the ura3 gene.
  • vectors derived from the 1.6 ⁇ circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts.
  • an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis Van den Berg,
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody-encoding nucleic acid.
  • Promoters suitable for use with prokaryotic hosts include the arabinose (e.g., araB) promoter phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • arabinose e.g., araB
  • trp tryptophan
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody of the disclosure.
  • Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3 ' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruv
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as Abelson leukemia virus, polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an
  • immunoglobulin promoter from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978.
  • Enhancer sequences are known from mammalian genes (globin, elastase, albumin, alpha- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5 ' or 3 ' to the antibody-encoding sequence, and can be located at a site 5 ' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/11026 and the expression vector disclosed therein. Another is the mouse immunoglobulin light chain transcription terminator.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g.,
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K.
  • thermotolerans and K. marxianus
  • yarrowia EP 402,226
  • Pichia pastors EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms.
  • examples of invertebrate cells include plant and insect cells.
  • baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as hosts.
  • Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including CHOKl cells (ATCC CCL61), DXB-11 , DG-44, and Chinese hamster ovary cells/- DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, (Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
  • buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed or transfected with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • vectors and transfected cell lines with multiple copies of transcription units separated by a selective marker can be useful for the expression of antibodies that bind target.
  • the host cells used to produce the antibody of this disclosure may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium, including from microbial cultures. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or
  • the antibody composition prepared from microbial or mammalian cells can be purified using, for example, hydroxylapatite chromatography cation or avian exchange chromatography, and affinity chromatography, with affinity chromatography.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ , j2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al, EMBO J. 5: 15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH 3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • Antibodies may be screened for binding affinity by methods known in the art. For example, gel-shift assays, Western blots, radiolabeled competition assay, co- fractionation by chromatography, co-precipitation, cross linking, ELISA, and the like may be used, which are described in, for example, Current Protocols in Molecular Biology (1999) John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
  • methods of screening for antibodies which modulate the activity of a target antigen comprise contacting test antibodies with a target polypeptide and assaying for the presence of a complex between the antibody and the target ligand.
  • the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular antibody to bind to the target ligand.
  • high throughput screening for antibody fragments or CDRs having suitable binding affinity to a target polypeptide is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test antibodies are contacted with a target polypeptide and washed. Bound polypeptides are then detected by methods well known in the art. Purified antibodies of the disclosure can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the target and immobilize it on the solid support.
  • Methods for assessing neutralizing, reducing, or inhibiting biological activity of FGFR4 are known in the art.
  • the methods may include assaying the phosphorylation and/or activity of intracellular components (e.g., ER phosphorylation, GSK3P phosphorylation, and the like) associated with FGFR4 activity.
  • intracellular components e.g., ER phosphorylation, GSK3P phosphorylation, and the like
  • neutralization of FGFR4 activity by an antibody of the present disclosure is at least 2-50 fold, 10-100 fold, 2- fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more potent than neutralization by a comparator antibody (e.g., a comparator antibody selected from BM-1, BM-2, BM-3 and/or BM-4 as described elsewhere herein).
  • a comparator antibody e.g., a comparator antibody selected from BM-1, BM-2, BM-3 and/or BM-4 as described elsewhere herein.
  • an antibody of the present disclosure is administered with a second agent useful to treat a disease or disorder as described herein. If more than one antibody effective at binding the target (FGFR4) is identified, it is contemplated that two or more antibodies to different epitopes of the target and/or which bind preferentially to different isoforms of FGFR4 may be mixed such that the combination of antibodies together to provide still improved efficacy against a condition or disorder associated with the target polypeptide.
  • an anti-FGFR4 antibody and an antibody that binds to an FGFR4 co-receptor e.g., klotho-beta (KLB)
  • KLB klotho-beta
  • Compositions comprising one or more antibody of the invention may be administered to persons or mammals suffering from, or predisposed to suffer from, a condition or disorder to be treated associated with the FGFR4
  • the present disclosure provides methods of treating cancer (e.g., hepatocellular carcinoma, etc.) where the method includes administering an anti-FGFR4 antibody provided by the present disclosure in combination with radiotherapy, which may have a synergistic effect in treating a subject (e.g., a human subject) having cancer.
  • cancer e.g., hepatocellular carcinoma, etc.
  • radiotherapy which may have a synergistic effect in treating a subject (e.g., a human subject) having cancer.
  • Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.
  • a second agent may be other therapeutic agents, such as cytokines, growth factors, antibodies to other target antigens, anti-inflammatory agents, anti-coagulant agents, agent that inhibit extracellular matrix production, agents that will lower or reduce blood pressure, agents that will reduce cholesterol, triglycerides, LDL, VLDL, or lipoprotein(a) or increase HDL, agents that will increase or decrease levels of cholesterol-regulating proteins, anti-neoplastic drugs or molecules.
  • cytokines such as cytokines, growth factors, antibodies to other target antigens, anti-inflammatory agents, anti-coagulant agents, agent that inhibit extracellular matrix production, agents that will lower or reduce blood pressure, agents that will reduce cholesterol, triglycerides, LDL, VLDL, or lipoprotein(a) or increase HDL, agents that will increase or decrease levels of cholesterol-regulating proteins, anti-neoplastic drugs or molecules.
  • second therapeutic modalities such as radiotherapy, chemotherapy, photodynamic therapy, or surgery is also contemplated.
  • the antibody of the present disclosure and the second agent may be given simultaneously, in the same formulation. It is further contemplated that the agents are administered in a separate formulation and administered concurrently, with concurrently referring to agents given within 30 minutes of each other.
  • the second agent is administered prior to administration of the antibody composition.
  • Prior administration refers to administration of the second agent within the range of one week prior to treatment with the antibody, up to 30 minutes before administration of the antibody. It is further contemplated that the second agent is
  • Subsequent administration is meant to describe administration from 30 minutes after antibody treatment up to one week after antibody administration.
  • adjunct therapies may be administered, where appropriate.
  • the patient may also be administered an extracellular matrix degrading protein, surgical therapy, chemotherapy, a cytotoxic agent, or radiation therapy where appropriate.
  • the administration when the antibody is administered in combination with a second agent, such as for example, wherein the second agent is a cytokine or growth factor, or a chemotherapeutic agent, the administration also includes use of a radiotherapeutic agent or radiation therapy.
  • the radiation therapy administered in combination with an antibody composition is administered as determined by the treating physician, and at doses typically given to patients being treated for cancer.
  • a cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • a non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and/or does not cause destruction of cells.
  • a non-cytotoxic agent may include an agent that can be activated to be cytotoxic.
  • a non-cytotoxic agent may include a bead, liposome, matrix or particle (see, e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein).
  • Such agents may be conjugated, coupled, linked or associated with an anti-FGFR4 antibody according to the disclosure.
  • Chemotherapeutic agents contemplated for use with the antibodies of the present disclosure include, but are not limited to, those listed in Table 2:
  • prednisone and equiv-alents interferon ( ⁇ , ⁇ , ⁇ )
  • the present disclosure provides a method for inhibiting FGFR4 activity by administering an FGFR4-specific antibody to a patient in need thereof.
  • the target specific antibody is a human, chimeric or humanized antibody.
  • the target is human (i.e., human FGFR4) and the patient is a human patient.
  • the patient may be a mammal that expresses a target protein with which the target-specific antibody cross-reacts.
  • the antibody may be administered to a non-human mammal expressing a target protein with which the antibody cross-reacts (i.e. a primate) for veterinary purposes or as an animal model of human disease.
  • Such animal models may be useful for evaluating the therapeutic efficacy of target specific antibodies of the disclosure.
  • the disclosure provides a method for treating a condition or disorder associated with FGFR4 signaling comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or a pharmaceutical composition as described herein.
  • Examples of diseases, conditions or disorders associated with FGFR4 signaling that can be treated with an antibody substance that binds FGFR4 include cancers, such as hepatocellular (liver) cancer, breast cancer, prostate cancer, ovarian cancer, thyroid cancer, gastric cancer, pituitary cancer, pancreatic cancer, colorectal cancer, stomach cancer, adrenocortical (adrenal gland) cancer, glioma, lung cancer, bladder cancer, head and neck cancer, oral cancer, uterine cancer, cervical cancer, brain cancer, neuroma (nerve tissue) cancer, kidney cancer, bladder cancer, skin cancer, endocrine cancer, neuroendocrine cancer, testis cancer, soft tissue sarcoma, bone sarcoma, Hodgkin's lymphoma, pituitary adenomas, breast fibroa
  • cancers such as hepatocellular (liver) cancer, breast cancer, prostate cancer, ovarian cancer, thyroid cancer, gastric cancer, pituitary cancer, pancreatic cancer
  • the antibodies of the present invention find use in treating diseases, conditions or disorders associated with FGFR4 signaling including, but not limited to, those listed in Tables 3-5.
  • Tables 3A-3D provides a list of solid tumors associated with FGFR4 signaling/dysregulation.
  • Table 3A lists carcinomas originating from epithelial tissues.
  • Table 3B lists sarcomas (cancerous tumors originating from connective tissue like bone, cartilage, fat, muscle, or blood vessels).
  • Table 3C lists lymphomas (bone marrow-derived cells that mature in the lymphatic system).
  • Table 3D lists adenomas (non-cancerous tumors).
  • Table 4 lists blood cancers associated with FGFR4 signaling.
  • Table 5 lists other indications associated with FGFR4 signaling.
  • treatment of these disorders or conditions in an animal in need of said treatment comprises administering to the animal an effective amount of an antibody (e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody) or a composition comprising an antibody described herein.
  • an antibody e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • a composition comprising an antibody described herein.
  • the treatment includes administering an anti-FGFR4 antibody that reduces or inhibits the binding of an FGFR4 ligand (e.g., an FGF, such as FGF19) to FGFR4.
  • an FGFR4 ligand e.g., an FGF, such as FGF19
  • Such treatment is effective in treating diseases, conditions or disorders associated with the binding of an FGFR4 ligand (e.g., an FGF, such as FGF 19) to FGFR4, which may include, but are not limited to, any of the diseases, conditions or disorders described herein (e.g., including those listed in Tables 3A-3D, 4 and 5).
  • the conditions treatable by methods of the present disclosure can be in a mammalian subject.
  • Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle.
  • the antibodies of the present disclosure may be used as affinity purification agents for target or in diagnostic assays for target protein, e.g., detecting its expression in specific cells, tissues, or serum.
  • the antibodies may also be used for in vivo diagnostic assays.
  • the antibody is labeled with a radionuclide (such as 1U In, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S) so that the antibody can be localized using a radionuclide (such as 1U In, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S) so that the antibody can be localized using a radionuclide (such as 1U In, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S) so that the antibody can be localized using a radionuclide (such as 1U In, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S) so that the antibody can be local
  • the antibodies of the present disclosure may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, such as ELISAs, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987). The antibodies may also be used for immunohistochemistry, to label tissue or cell samples using methods known in the art.
  • the FGFR4-specific antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation, which are all techniques well-known in the art.
  • the antibodies of the disclosure can be used to detect FGFR4 (or any protein associated therewith) in humans and other mammals.
  • the present disclosure provides methods for detecting FGFR4 in a biological sample comprising contacting a biological sample with an FGFR4 specific antibody of the disclosure and detecting the bound antibody.
  • the FGFR4 specific antibody is directly labeled with a detectable label.
  • the FGFR4 specific antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the FGFR4 specific antibody is labeled.
  • a second antibody is chosen that is able to specifically bind the particular species and class of the first antibody.
  • the FGFR4 specific antibody is a human IgG
  • the secondary antibody could be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co.
  • the immunoassays disclosed above are used for a number of purposes.
  • the FGFR4 specific antibodies can be used to detect FGFR4 in cells or on the surface of cells in cell culture, or secreted into the tissue culture medium.
  • the FGFR4 specific antibodies can be used to determine the amount of FGFR4 on the surface of cells or secreted into the tissue culture medium that have been treated with various compounds.
  • This method can be used to identify compounds that are useful to inhibit or activate FGFR4 expression or secretion. According to this method, one sample of cells is treated with a test compound for a period of time while another sample is left untreated.
  • the total FGFR4 level may be measured using one of the immunoassays described above. The total level of FGFR4 in the treated versus the untreated cells is compared to determine the effect of the test compound. Labels
  • a subject anti-FGFR4 antibody e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • a "label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • labels suitable for use in the present disclosure include, radioactive labels (e.g., 32P), fluorophores (e.g., fluorescein), electron dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens as well as proteins which can be made detectable, e.g., by incorporating a radiolabel into the hapten or peptide, or used to detect antibodies specifically reactive with the hapten or peptide.
  • radioactive labels e.g., 32P
  • fluorophores e.g., fluorescein
  • electron dense reagents e.g., enzymes (e.g., as commonly used in an ELISA)
  • enzymes e.g., as commonly used in an ELISA
  • biotin e.g., as commonly used in an ELISA
  • haptens as well as proteins which can be made detectable, e.g., by incorporating a radiol
  • labels suitable for use in the present invention include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the
  • radiolabels e.g., H, I, S, C, or P
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • the label in one embodiment is covalently bound to the biopolymer using an isocyanate reagent for conjugation of an active agent according to the disclosure.
  • the bifunctional isocyanate reagents of the disclosure can be used to conjugate a label to a biopolymer to form a label biopolymer conjugate without an active agent attached thereto.
  • the label biopolymer conjugate may be used as an intermediate for the synthesis of a labeled conjugate according to the disclosure or may be used to detect the biopolymer conjugate.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • the compounds of the present disclosure can also be conjugated directly to signal- generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes suitable for use as labels include, but are not limited to, hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
  • Fluorescent compounds, i.e., fluorophores, suitable for use as labels include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • fluorophores include, but are not limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine yellow, lissamine rhodamine, B sulfonyl chloride erythroscein, ruthenium (tris, bipyridinium), Texas Red, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, etc.
  • Chemiluminescent compounds suitable for use as labels include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means for detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film, as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the f uorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels may be detected simply by observing the color associated with the label.
  • Other labeling and detection systems suitable for use in the methods of the present disclosure will be readily apparent to those of skill in the art.
  • Such labeled modulators and ligands can be used in the diagnosis of a disease or health condition.
  • anti-FGFR4 antibody substances of the present disclosure e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • the antibody substances can be formulated in a composition comprising one or more
  • “pharmacologically acceptable” refer to molecular entities and compositions that do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below.
  • “Pharmaceutically acceptable carriers” include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. [0361] In addition, compounds may form solvates with water or common organic solvents. Such solvates are contemplated as well.
  • the antibody e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • parenteral administration e.g., injection, infusion
  • parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration.
  • the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody.
  • the dosing can be given by injections, such as intravenous or subcutaneous injections.
  • the route of administration can be selected according to various factors, such as whether the administration is brief or chronic.
  • Other administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral or local administration e.g. through a catheter placed close to the desired site. Injection, especially intravenous, is of interest.
  • compositions of the present disclosure containing an antibody substance of the disclosure as an active ingredient may contain pharmaceutically acceptable carriers or additives depending on the route of administration.
  • carriers or additives include water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a
  • HSA human serum albumin
  • additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present disclosure.
  • Formulation of the pharmaceutical composition will vary according to the route of administration selected (e.g., solution, emulsion).
  • An appropriate composition comprising the antibody to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers.
  • aqueous carriers e.g., sterile phosphate buffered saline solutions, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like.
  • Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid and methionine
  • preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzy
  • polypeptides such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g., Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, and which may be selected to
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, micro emulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, micro emulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, micro emulsions, nano-particles and nanocapsules
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Aqueous suspensions may contain the active compound (e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody) in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • the active compound e.g., an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl- eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • the aqueous suspensions may also contain one or more preservatives, for example eth
  • the antibodies of the present disclosure can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, ka
  • the concentration of antibody e.g., an XPA.48.056, XPA.48.117, and/or
  • XPA.48.148 antibody in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for parenteral injection could be made up to contain 1 ml sterile buffered water, and 50 mg of antibody.
  • a typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of antibody.
  • parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
  • An effective dosage of antibody is within the range of 0.01 mg to 1000 mg per kg of body weight per administration.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous, oleaginous suspension, dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, vegetable oils, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the form In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. It must be stable under the conditions of
  • compositions useful for administration may be formulated with uptake or absorption enhancers to increase their efficacy.
  • Such enhancers include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85: 1282-1285 (1996)) and Oliyai and Stella (Ann. Rev.
  • Antibody compositions contemplated for use to inhibit FGFR4 activity including disrupting binding of the FGFR4 to its cognate ligand (e.g., FGF19), disrupting FGFR4- mediated signaling, and the like.
  • the compositions exhibit inhibitory properties at concentrations that are substantially free of side effects, and are therefore useful for extended treatment protocols.
  • co-administration of an antibody composition with another, more toxic, cytotoxic agent can achieve beneficial inhibition of a condition or disorder being treated, while effectively reducing the toxic side effects in the patient.
  • compositions contemplated for use in the present disclosure are well balanced, thereby enhancing their utility for both in vitro and especially in vivo uses, while other compositions lacking such balance are of substantially less utility.
  • compositions contemplated for use in the disclosure have an appropriate degree of solubility in aqueous media which permits absorption and bioavailability in the body, while also having a degree of solubility in lipids which permits the compounds to traverse the cell membrane to a putative site of action.
  • antibody compositions contemplated e.g., those including an XPA.48.056,
  • XPA.48.117, and/or XPA.48.148 antibody are effective (e.g., maximally effective) when they are delivered to the site of FGFR4 activity.
  • methods of the present disclosure include a step of administering a pharmaceutical composition (e.g., a pharmaceutical composition that includes an
  • the pharmaceutical composition is a sterile composition.
  • Methods of the present disclosure are performed using any medically-accepted means for introducing a therapeutic directly or indirectly into a mammalian subject, including but not limited to injections, oral ingestion, intranasal, topical, transdermal, parenteral, inhalation spray, vaginal, or rectal administration.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, and intracisternal injections, as well as catheter or infusion techniques. Administration by, intradermal, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well.
  • administration e.g., of an XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody
  • administration is performed at the site of a cancer, bowel, coronary artery, gallstone or other affected tissue needing treatment by direct injection into the site or via a sustained delivery or sustained release mechanism, which can deliver the formulation internally.
  • a sustained delivery or sustained release mechanism which can deliver the formulation internally.
  • compositions capable of sustained delivery of a composition (e.g., a soluble polypeptide, antibody, or small molecule) can be included in the formulations of the disclosure implanted near or at site of the cancer, fibrosis or affected tissue or organ.
  • a composition e.g., a soluble polypeptide, antibody, or small molecule
  • Therapeutic compositions may also be delivered to the patient at multiple sites.
  • the multiple administrations may be rendered simultaneously or may be administered over a period of time. In certain cases it is beneficial to provide a continuous flow of the therapeutic composition.
  • Additional therapy may be administered on a period basis, for example, hourly, daily, weekly, every 2 weeks, every 3 weeks, monthly, or at a longer interval.
  • Also contemplated in the present disclosure is the administration of multiple agents, such as an antibody composition in conjunction with a second agent as described herein, including but not limited to a chemotherapeutic agent or an agent useful to treat, e.g., cancer, irritable bowel syndrome, coronary artery disease, gallstone disease, and/or the like.
  • agents such as an antibody composition in conjunction with a second agent as described herein, including but not limited to a chemotherapeutic agent or an agent useful to treat, e.g., cancer, irritable bowel syndrome, coronary artery disease, gallstone disease, and/or the like.
  • antibody composition e.g., a composition that includes an
  • XPA.48.056, XPA.48.117, and/or XPA.48.148 antibody) in a given dosage may vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated.
  • the antibody may administered in a doage regeimn of about 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000 mg/day.
  • concentrations may be administered as a single dosage form or as multiple doses. Standard dose-response studies, first in animal models and then in clinical testing, reveal optimal dosages for particular disease states and patient populations.
  • kits may be modified if traditional therapeutics are administered in combination with therapeutics of the disclosure.
  • kits which comprise one or more compounds or compositions packaged in a manner which facilitates their use to practice methods of the disclosure.
  • a kit includes a compound or composition described herein (e.g., a composition comprising a target-specific antibody alone or in combination with a second agent), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method.
  • the compound or composition can be packaged in a unit dosage form.
  • the kit may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay.
  • the kit contains a label that describes use of the antibody composition.
  • lambda phage display library and 10 cfu of phage particles from a scFv kappa phage display library were blocked for 1 hour at room temperature in 1 mL of 5% milk/PBS (Sigma-Aldrich, St. Louis, MO) containing 50-100 ⁇ g/mL human IgG-Fc fragment (Bethyl Labs, Montgomery, TX) with gentle rotation.
  • Nunc MaxiSorpTM Immunoplates (Nalge Nunc International, Rochester, NY) were coated with 50 ⁇ g rhFGFR4-Fc or rhFGFR4-ECD in 5-7 wells overnight. The next day the plate was blocked with 5% milk/PBS and incubated at room temperature for 1 hour. For the second round of panning, 20-25 ⁇ g/mL rhFGFR4-Fc or rhFGFR4-ECD was used to coat 3 wells of a MaxiSorpTM Immunoplate overnight.
  • rhFGFR4-Fc or rhFGFR4-ECD was used to coat 1-2 wells of a MaxiSorpTM Immunoplate overnight.
  • the deselected phage were added to the immunoplate immobilized with either rhFGFR4-Fc or rhFGFR4-ECD and incubated for 2 hours at room temperature or 37 °C.
  • the Immunoplate containing rhFGFR4-Fc or rhFGFR4-ECD was then washed five times with PBS-0.1% TWEEN followed by five washes with PBS.
  • the Immunoplate was washed ten times with PBS-0.1% TWEEN followed by ten quick washes with PBS.
  • the Immuno Plate was washed ten to twenty times with PBS-0.1% TWEEN followed by ten to twenty washes with PBS.
  • Phage were eluted with a volume of 100 mM triethylamine (TEA) equivalent to the coating volume for 20 minutes at room temperature with gentle rotation. The eluate was pooled and neutralized with one half the coating volume of 1M Tris-HCl (pH 8.0). The eluted phage was used to infect TGI bacterial cells (Stratagene, La Jolla, CA) at an OD 600 of 0.5. Infection occurred for 30 minutes at 37 °C without shaking and for 30 minutes at 37 °C with shaking at 90 rpm. Cells were pelleted and resuspended in 2YT media supplemented with 100 ⁇ g/ml carbenicillin and 2% glucose. The resuspended TGI cells were plated on 2YT agar plates with 100 ⁇ g/ml carbenicillin and 2% glucose and incubated overnight at 30 °C.
  • TGI bacterial cells (Stratagene, La Jolla, CA) at an OD 600 of 0.5
  • helper phage M13K07 New England Biolabs, MA
  • MOI multiplicity of infection
  • Helper phage infection of TGI cells occurred at an OD 6 oo of -0.5 followed by an incubation at 37 °C for 30 minutes without shaking and an incubation at 37 °C with shaking at 100 rpm.
  • TGI cell pellets were resuspended in 2YT media supplemented with 100 ⁇ carbenicillin and 50 ⁇ kanamycin and allowed to grow overnight at 25 °C or 30 °C and 250 rpm.
  • the next day, phage were recovered using vigorous centrifugation to remove cell debris. The phage were used directly in the next round of panning. In order to monitor the enrichment resulting from the phage selections, the amount of input and output phage was titered for all three rounds of panning.
  • Solution panning was performed using a biotinylated rhFGFR4-Fc fusion protein or biotinylated FGFR4 extracellular domain alone (rhFGFR4-ECD).
  • rhFGFR4-ECD biotinylated FGFR4 extracellular domain alone
  • Each protein was biotinylated to an average of 1 biotin per molecule using an excess of NHS-PEG 4 -Biotin (Pierce, Rockford, IL).
  • SPR surface plasmon resonance
  • lambda phage display library and 10 cfu of phage particles from a scFv kappa phage display library were blocked for 1 hour at room temperature in 1 mL of 5% milk/PBS containing 50 ⁇ g/mL human IgG-Fc fragment with gentle rotation.
  • Blocked phage were deselected three times for 30 minutes against streptavidin-coated magnetic Dynabeads® M-280 (Invitrogen Dynal AS, Oslo, Norway).
  • Phage were mixed with 200 pmol biotinylated rhFGFR4-Fc or biotinylated rhFGFR4-ECD for 90 minutes at room temperature with gentle rotation.
  • 50 pmoles of biotin-rfiFGFR4-Fc or biotin-rfiFGFR4-ECD was used.
  • 10 pmoles of biotin-rfiFGFR4-Fc or biotin-rfiFGFR4-ECD was used.
  • Streptavidin-coated M-280 Dynabeads® were added to the phage and mixed for 1 hour at room temperature to capture rhFGFR4-Fc or rhFGFR4-ECD bound phage. The beads were then washed.
  • the beads were washed quickly (i.e. beads are pulled out of solution using a magnet and resuspended in 1 ml wash buffer) three times with PBS-0.1% TWEEN, followed by three washes with PBS.
  • the beads were washed three to six times with PBS-0.1% TWEEN for 0 - 2 minutes a wash followed by three 0 - 2 minute washes with PBS.
  • beads were washed three times with PBS-0.1% TWEEN for 2-10 minutes and three times with PBS for 2-10 minutes.
  • FGFR4-Streptavidin bead-bound phage were eluted with 0.5 mL 100 mM triethylamine (TEA) for 30 minutes at room temperature with gentle rotation. The beads were separated from the eluate. The eluate was removed and neutralized with 0.25 to 0.5 mL 1M Tris-HCl (pH 8.0). The eluted phage was used to infect TGI bacterial cells at an OD 600 of -0.5. Infection occurred for 30 minutes at 37 °C without shaking and for 30 minutes at 37 °C with shaking at 90 rpm.
  • TAA triethylamine
  • helper phage M13K07 at a multiplicity of infection (MOI)of 10.
  • Helper phage infection of TGI cells occurred at an OD 6 oo of -0.5 followed by an incubation at 37 °C for 30 minutes without shaking and an incubation at 37 °C with shaking at 100 rpm.
  • TGI cell pellets were resuspended in 2YT media supplemented with 100 ⁇ g/ml carbenicillin and 50 ⁇ g/ml kanamycin and allowed to grow overnight at 25 °C or 30 °C and 250 rpm. The next day, phage were recovered using vigorous centrifugation to remove cell debris. The phage supernatant was used directly in the next round of panning. In order to monitor the enrichment resulting from the phage selections, the amount of input and output phage was titered for all three rounds of panning.
  • the output from the first round of immobilized or solution scFv panning was used.
  • phage were recovered using vigorous centrifugation and blocked for 1 hour at room temperature with 5% fetal bovine serum (Thermo Scientific, Rockford, IL) in PBS.
  • Blocked phage were deselected with 5xl0 7 parental CHO-K1 cells that were washed 3 times with 5% fetal bovine serum/PBS. Phage and CHO-Klwere incubated for 45 minutes with 5% fetal bovine serum/PBS at room temperature with gentle rotation.
  • the output from the first round of immobilized or solution Fab panning was used. After rescue of 50 to 100 times the phage output, phage were recovered using vigorous centrifugation and blocked for 1 hour at room temperature with 5% fetal bovine serum/PBS. Blocked phage were deselected with 5xl0 7 parental CHO-K1 cells that were washed 3 times with 5% fetal bovine serum/PBS. Phage and CHO-K1 were incubated for 45 minutes with 5% fetal bovine serum/PBS at room temperature with gentle rotation.
  • Bound phage were eluted with 0.5 ml 100 mM glycine, pH 2.2, for 10 minutes at room temperature with gentle rotation. After centrifugation to remove the cells, the eluate was neutralized with 0.5 mL 1M Tris-HCl (pH 8.0) and used to infect TGI bacterial cells at an OD 6 oo of -0.5. Infection occured for 30 minutes at 37 °C without shaking and for 30 minutes at 37 °C with shaking at 90 rpm. Cells were pelleted and resuspended in 2YT media supplemented with 100 ⁇ g/ml carbenicillin and 2% glucose. The resuspended TGI cells were plated on 2YT agar plates with 100 ⁇ g/ml carbenicillin and 2% glucose and incubated overnight at 30 °C.
  • helper phage M13K07 at a multiplicity of infection (MOI) of 10.
  • Helper phage infection of TGI cells occurred at an 0 D6 oo of -0.5 followed by an incubation at 37 °C for 30 minutes without shaking and an incubation at 37 °C with shaking at 100 rpm.
  • TGI cell pellets were resuspended in 2YT media supplemented with 100 ⁇ g/ml carbenicillin and 50 ⁇ g/ml kanamycin and allowed to grow overnight at 25 °C or 30 °C and 250 rpm. The next day, phage were recovered using vigorous centrifugation to remove cell debris. The phage supernatant was used directly in the next round of panning. In order to monitor the enrichment resulting from the phage selections, the amount of input and output phage was titered for all three rounds of panning.
  • Bacterial periplasmic extracts were added to the coated ELISA plate (50 ⁇ /well) and allowed to bind to either FGFR4 on the ELISA plate for 2 hour at room temperature. Bound scFv or Fab fragments were detected with murine anti-c-myc mAb (Roche, Indianapolis IN) for 1 hour at room temperature followed by goat anti-mouse HRP-conjugated antisera (Thermo Scientific, Rockford, IL). Three washes with PBS-0.1% Tween 20 (Teknova, Hollister, CA; Sigma-Aldrich, St. Louis, MO) were performed following every stage of the ELISA screens.
  • Anti-FGFR4 antibodies from phage display libraries were screened in flow cytometric (FACS) based assays to measure antibody binding to cells.
  • the cells were cultured overnight in this media and as such are designated as "serum-starved.” These cells were washed once and resuspended at 2xl0 6 cells/ml in PBS containing 0.5% BSA and 0.01% sodium azide (FACS buffer).
  • the cells were then washed once with FACS buffer and the binding of the anti-c-myc revealed by the addition of an Alexa Fluor647-conjugated anti-mouse IgG. After a further 15 minutes incubation on ice the cells were washed and the pellets resuspended in FACS buffer. The cells were analyzed on a FACSCANTM (Becton- Dickinson, Milipitas, CA) and the data analyzed in both FLOWJOTM (Treestar, Ashland, OR) and Microsoft EXCELTM (Microsoft, Redmond, WA).
  • FACSCANTM Becton- Dickinson, Milipitas, CA
  • FLOWJOTM Testar, Ashland, OR
  • Microsoft EXCELTM Microsoft, Redmond, WA.
  • VH variable heavy
  • VL variable heavy
  • VL variable heavy
  • VL variable heavy
  • VL variable heavy
  • VH variable heavy
  • VL variable light
  • Binding of the reformatted antibodies to hFGFR4 was assessed by FACS as described above using CHO-hFGFR4, HepG2, or HuH7 cells. The data was fit to a sigmoidal dose-response curve to determine EC50 values (Table 6). Assay results (FIG. 1) show that anti-FGFR4 antibodies XPA.48.056, XPA.48.117 and XPA.48.148 bind to the CHO-hFGFR4, HepG2, and HuH7 with low nanomolar affinity.
  • Anti-FGFR4 antibody kinetics were screened in two different ways using surface plasmon resonance (SPR) technology.
  • Antibody fragments were first prepared as bacterial periplasmic extracts (PPE) and screened by their dissociation rate constants against rhFGFR4. A select number of clones was then reformatted as full IgG antibodies to characterize their binding affinity and kinetics in a second method.
  • PPE bacterial periplasmic extracts
  • Recombinant human rhFGFR4-Fc a recombinant Fc fusion protein consisting of the extracellular domain (ECD; amino acids 22-369) of human FGFR4 (R&D Systems,
  • rhFGFR4-ECD hFGFR4 extracellular domain alone
  • rhFGFR4-ECD hFGFR4 extracellular domain alone
  • Either goat anti-human IgG (Fab specific) antibody or anti- histidine tag IgG (anti-his) was covalently coupled on spot 5 of all four flow cells, depending on which phage library was used.
  • rhFGFR4-Fc or rhFGFR4-ECD was immobilized to a density of between 1000 to 3000 Response Units (RU). Spot 3 was reserved as a reference spot for spots 1 and 2 while spot 4 was reserved as a reference for spot 5.
  • the sensor chip was activated with a 1 : 1 mixture of 0.1M N-hydroxysuccinimide (NHS) and 0.4M l-ethyl-3- (3 -dimethyl aminopropyl) carbodiimide (EDC).
  • rhFGFR4-Fc was diluted to 5 ⁇ g/mL in 0.01 M sodium acetate pH 4.5 and injected over spot 1 at 10 ⁇ /minute for 5 minutes followed by blocking with 1M ethanolamine. Subsequently, rhFGFR4-ECD was coupled to spot 2 in a similar fashion.
  • PPEs containing antibody fragments were injected over the sensor, resulting in binding of the Fab or single chain Fv to the immobilized rhFGFR4 on spots 1 and 2 and capture of Fab or single chain on spot 5.
  • the running buffer for screening was a HEPES Buffered Saline (HBS-EP+) with lOmM Hepes, 150mM Sodium Chloride, 3mM EDTA, 0.05% Polysorbate 20 (Teknova, Hollister, CA) and 1 mg/m bovine serum albumin (BSA) (Sigma Aldrich, St. Louis MO).
  • the instrument settings were as follows: the flow rate was set to 30 micro liters/minute, and sample injection time was five minutes with a dissociation time of 10 minutes.
  • the instrument temperature was set to 25°C.
  • Nonspecific binding of the antibody fragment to the sensor surface was corrected by subtracting the interaction of the antibody fragment with a reference spot (having no immobilized ligand) from the interaction of the antibody fragment with the appropriate lig
  • Binding kinetics were determined for a select number of anti-FGFR4 antibodies by SPR using the ProteOnTM XPR36 interaction array system (Bio-Rad, Hercules, CA). Briefly, a GLM sensor chip (GLM Sensor Chip #176-5012) was preconditioned with successive injections of 10 mM SDS, 50mM NaOH, 100 mM Tris pH 9.5, and running buffer at 100 ⁇ / ⁇ flow rate. Running buffer for immobilization was a HEPES Buffered Saline (HBS-EP+) with lOmM Hepes, 150mM Sodium Chloride, 3mM EDTA, and 0.05%
  • rhFGFR4-ECD For kinetic analysis, antibodies were immobilized onto the vertical flow channel by anti-human Fc capture with targeted coating concentration of 200-300 RU. After switching to a horizontal orientation rhFGFR4-ECD samples were injected at a flow rate of 30 ⁇ / ⁇ for four minutes with a 600 second dissociation time. rhFGFR4-ECD was injected at concentrations of 100, 33.3, 11.11, 3.7, and 1.23 nM with 1-2 regeneration injections between cycles to remove antibody/rfiFGFR4 complexes. Regeneration injections were each 30 seconds of 100 mM HC1.
  • Double-referenced data curves were fit using ProteOnTM software with a simple 1 : 1 Langmuir binding model to yield kinetic parameters for on-rate (k a ) and off-rate (k d ).
  • Equilibrium binding constant (K D ) values were calculated from a ratio of the kinetic parameters (kd/k a ).
  • Anti-FGFR4 antibodies were characterized using a DELFIA® (Dissociation- Enhanced Lanthanide Fluorescent Immunoassay) assay that measured their ability to inhibit FGF19 binding to rhFGFR4 receptor.
  • DELFIA® Dissociation- Enhanced Lanthanide Fluorescent Immunoassay
  • High-binding 384-well plates (Corning, Tewksbury, MA) were coated with 100 of 2 ⁇ g/mL anti-human immunoglobulin Fc fragment specific (Jackson Immunoresearch, West Grove, PA) in carbonate coating buffer (15 mM Na 2 C0 3 and 34 mM NaHC0 , pH 9.0) (Sigma Aldrich, St. Louis, MO) for 16 hours at 4°C.
  • carbonate coating buffer 15 mM Na 2 C0 3 and 34 mM NaHC0 , pH 9.0
  • the plates were blocked with 200 of PBS-0.1% Tween 20 containing 10% of BSA (Sigma Aldrich, St. Louis MO) and 1 g/ml FGFR4-Fc. Following a 1 hour incubation, the plates were washed 3 times using PBS-0.1%> Tween 20.
  • FGF19 binding affinity Assay for FGF19 binding affinity, increasing concentrations of FGF19 were added to the plate in the presence or absence of 100 ⁇ g/ml heparin (Sigma Aldrich, St. Louis MO). The plates were then washed three times with PBS-0.1% Tween 20, and incubated for 1 hour at room temperature with biotinylated FGF19-specific polyclonal antibody (R&D Systems, Minneapolis, MN) at 1 ⁇ g/ml and Europium (EU)-labeled Streptavidin (Perkin Elmer, Santa Clara, CA) diluted 1 : 1000 in DELFIA® assay buffer (Perkin Elmer, Santa Clara, CA).
  • biotinylated FGF19-specific polyclonal antibody R&D Systems, Minneapolis, MN
  • EU Europium-labeled Streptavidin
  • the plates were then washed 3 times with DELFIA® wash solution (Perkin Elmer, Santa Clara, CA) and incubated with 100 ⁇ DELFIA® enhancement solution (Perkin Elmer, Santa Clara, CA) for 15 minutes at room temperature with agitation.
  • the plates were read on a FlexStation 3 (Molecular Devices, Sunnyvale, CA) plate Reader in TRF (time -resolved fluorescence) mode.
  • the data were plotted and the IC 5 o values were calculated using Prism software (GraphPad Software, La Jolla, CA).
  • periplasmic extracts (PPE) containing antibody fragments
  • 30-50 ⁇ of each PPE was mixed with the same volume of FGF19/heparin solution prepared with an FGF19 concentration corresponding to the EC80 (40nM) obtained from the titration curve described above.
  • the resulting mixture was added to 384 plates coated with rhFGFR4-Fc that was captured using an anti-human Fc antibody and incubated for 1 hour at RT.
  • the plates were washed 3 times using PBS-0.1% Tween 20 buffer.
  • the ligand was detected using biotinylated FGF19-specific polyclonal antibody and Eu-labeled Streptavidin procedure described previously.
  • Anti-FGFR4 antibodies were further characterized using an SPR assay for their ability to inhibit FGF19 binding to the FGFR4 receptor. Briefly, samples of rhFGFR4-Fc receptor and FGF19 ligand are drawn in sequence from separate vials and then injected consecutively over the chip surface containing immobilized anti-FGFR4. rhFGFR4-Fc was injected in presence and absence of 100 ⁇ g/ml heparin. The binding of rhFGFR4-Fc and FGF19 was monitored as an increase in SPR response over time for each antibody.
  • FIG. 2 shows sensorgrams comparing SPR signal (RU in Y-axis) caused by rhFGFR4 and FGF19 binding to immobilized anti-FGFR4 antibodies over time (X-axis).
  • the first injection shows capture of rhFGFR4-Fc by anti-FGFR4 antibodies in the presence and absence of heparin.
  • Binding of FGF19 in the second injection is indicative of an antibody that does not block ligand binding to the captured FGFR4-Fc, while inhibition of FGF19 binding is indicative of an antibody that blocks ligand binding.
  • antibody XPA.48.056 exhibits greater ligand blocking activity compared to an anti- FGFR4 antibody 4FR6D3 (BioLegend, San Diego, CA) as suggested by the lower binding of FGF19 to rhFGFR4 shown by SPR.
  • Antibodies were immobilized in spots 2 and 4 using amine coupling on an A4000 Biacore instrument. Amine coupling was performed by activating the chip with EDC/NHS (GE Healthcare, Piscataway, NJ) for five minutes and injecting anti-FGFR4 antibodies at 5 ⁇ g/mL in pH 5.0 Acetate (GE Healthcare, Piscataway, NJ) for 5 minutes. After blocking the surface using ethanolamine, 100 nM of rhFGFR4-ECD was injected over all spots including reference subtraction spot 1 and 5 of each flow-cell. Binding to the immobilized antibody was detected, followed by injection of the second antibody to test for pairing with the immobilized antibody. Pairing results were evaluated using the Biacore A4000 evaluation software.
  • Table 8 illustrates the results of a binning assay conducted with 3 antibodies examined in a pair-wise SPR binding assay. Binding is expressed in response units (RU). In this assay antibodies XPA.48.056, XPA.48.117, and comparator antibodies BM-2 and BM-3 were immobilized on the chip. Anti-FGFR4 antibodies were grouped in different bins according to their ability to pair with each other using SPR. "NB" means no binding.
  • Antibodies identified in Bin 1 appeared to be partial inhibitors of FGF19 mediated pERK signaling ( ⁇ 50 %).
  • the FGFR4 receptor is a type I transmembrane receptor tyrosine kinase that undergoes dimerization and autophosphorylation upon binding of the ligand FGF-19. Ligand binding subsequently catalyzes the phosphorylation and recruitment of molecules such as FRS2, Grb, and Sos. Each of these proteins serves as a docking site for the recruitment of downstream signaling molecules resulting in the activation of various signaling pathways such as the Ras/RAF/MAPK pathways. These pathways ultimately regulate cell growth and differentiation, survival, and migration.
  • test antibody The effects of a test antibody on signaling can be measured by assessing the ability of the antibody to neutralize FGF-19 induced phosphorylation of specific intracellular proteins, such as MAPK (ERKl/2), which are specific to the FGFR4 signaling pathway.
  • MAPK ERKl/2
  • the phosphorylation of these proteins can be measured and quantified by
  • HepG2 hepatocellular carcinoma cells were cultured in RPMI 1640 Medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS for normal maintenance. Cells were seeded at a density of 4 x 10 5 cells/well in a 96-well plate and allowed to re-attach in a 37°C, 5% C02 incubator. After 48 hours, the cells were washed one time with pre -warmed PBS and incubated with "starvation media" consisting of RPMI 1640 Medium supplemented with 0.5% BSA for an additional 24 hours. To screen anti-FGFR4 antibodies, both test antibody and ligand were formulated in starvation media.
  • the media was removed from the cells and test antibody was added at the desired concentration in ⁇ / ⁇ 1 for approximately 15 minutes prior to the addition of rfiFGF-19 (R&D Systems, Minneapolis, MN). After incubation for an additional 10 minutes in the presence of 100 ng/ml rfiFGF-19 in a 37 °C, 5% C0 2 incubator, the treated cells were lysed in a buffer containing 20 mM Tris- HC1 (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 2mM Phenylmethanesulfonylfluoride (Sigma-Aldrich, St. Louis, MO) in DMSO,
  • Phosphatase Inhibitor Cocktails 1 and 2 Sigma-Aldrich, St. Louis, MO
  • Complete Mini Protease Inhibitor Roche Diagnostics Corporation, Indianapolis, IN
  • the lysates were clarified by centrifuging for 3 minutes. The amount of
  • phosphorylated ER l/2 or GSK-3P present was quantified using electrochemiluminescence with the MesoScale Discovery Multi-spot Assay System (Meso Scale Discovery,
  • Antibodies were initially tested at a single 50 ⁇ g/mL dilution point to identify both agonist and antagonist antibodies. Select antibodies were then confirmed in a 6 point dilution series examining ERK 1/2 (FIG. 4, Panel A) or GSK-3p neutralization (FIG. 4, Panel B). These assays confirmed that antibodies XPA.48.117, XPA.48.148, and XPA.48.056 were all able to neutralize both ERK 1/2 and GSK-3P phosphorylation in a dose dependent manner. Antibody XPA.48.117, however, was by far more potent than any other benchmark comparator tested in the ERK 1/2 neutralization assay.
  • Antibodies were tested for their ability to mediate antibody-dependent cellular cytotoxicity in human hepatocellular carcinoma cell lines endogenously expressing FGFR4, such as HepG2 or HuH-7, or in HCTl 16 colorectal adenocarcinoma cells that were stably transfected with FGFR4 (HCTl 16-FGFR4) in order to provide a cell line over-expressing the target of interest.
  • FGFR4 endogenously expressing FGFR4
  • HCTl 16 colorectal adenocarcinoma cells that were stably transfected with FGFR4 (HCTl 16-FGFR4) in order to provide a cell line over-expressing the target of interest.
  • HepG2 hepatocellular carcinoma cells were cultured in RPMI 1640 Medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS for normal maintenance.
  • HuH-7 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) containing glucose at 4.5 g/L supplemented with 10% FBS and 2 mM glutamine (Invitrogen, Carlsbad, CA) for normal maintenance.
  • DMEM Dulbecco's Modified Eagles Medium
  • HCTl 16-FGFR4 colorectal adenocarcinoma cells stably expressing FGFR4 were cultured in McCoy's 5 A Medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS for normal maintenance.
  • McCoy's 5 A Medium Invitrogen, Carlsbad, CA
  • each cell line was run in RPMI 1640 Medium supplemented with 10% serum.
  • PBMCs Peripheral blood mononuclear cells isolated from buffy coats (American Red Cross, Oakland, CA) using standard methods were activated overnight in RPMI 1640 Medium (Invitrogen, Carlsbad, CA) supplemented with 10%> FBS plus 20 IU/ml rhIL-2 (Roche, Indianapolis, IN). Test antibody was placed in a 96-well plate at a concentration of lor 10 ⁇ g/mL followed by addition of Versene-detached cells (Invitrogen, Carlsbad, CA) at a density of 2 x 10 4 cells/well. Antibody and cells were allowed to complex at room
  • Cytotoxicity ((experimental - (T+E)) / (target max - target spontaneous)) * 100.
  • Adherent AML12 mouse hepatocytes were maintained in a 1 : 1 mixture of
  • Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) and Ham's F12 medium (Life Technologies, Grand Island, NY) supplemented with 0.005 mg/mL insulin, 0.005 mg/mL transferrin 5 ng/mL selenium, and 40 ng/mL dexamethasone, 10% fetal bovine serum (Hyclone, Logan, UT). Prior to use in assays the cells were washed in PBS containing 0.5%) BSA and 0.01% sodium azide (FACS buffer), counted, and the concentration adjusted to 2 x 10 6 cells/mL in FACs Buffer.
  • FACS buffer sodium azide
  • HEK293E cells were transfected with mouse FGFR4 cDNA (Origene Technologies Inc., Rockville, MD) according to standard protocols. Briefly, 20mL of HEK293E cells at 3 x 10 5 cells/mL were transfected with lOug DNA using lug/ml lipofectamine 2000 reagent. Forty-eight hours after transfection, the cells were washed in FACS buffer (PBS with 0.5% BSA and 0.01% sodium azide), counted, and the concentration adjusted to 2 x 10 6 cells/mL in FACS buffer.
  • FACS buffer PBS with 0.5% BSA and 0.01% sodium azide
  • the cells were analyzed on a FACSCantoTM (Becton-Dickinson, Milpitas, CA) and the data analyzed in FLOWJOTM (Treestar, Ashland, OR) and normalized to the degree of binding observed in untransfected cells. Data was graphed using GraphPad Prism® (GraphPad Software Inc., La Jolla, CA). Representative data at a concentration of 50nM antibody is shown in FIG. 6, Panel B.
  • Example 10 Tumor inhibition by FGFR4 antibodies in a xenograft mouse model
  • Antibodies XPA.48.117 and XPA.48.148 were evaluated for their ability to inhibit tumor growth in the HuH7 xenograft model (Bumbaca, D. et al. MAbs. 3, 1-11; 2011). Eight to nine week old BALB/c Nu/Nu mice (Charles River Laboratories, Wilmington, MA) were implanted subcutaneously with 5x 10 6 HuH7 cells in BD MATRIGELTM (1 : 1 ,200 uL) per animal. When tumors reached an average size of approximately 150 mm animals were randomized into test groups of fifteen mice each.
  • Each group was administered anti-KLH human IgGi isotype control, XPA.48.117, or XPA.48.148 intraperitoneally at 30/mg/kg/wk for a total of 4 doses. Tumor volume were measured twice weekly. Animals were sacrificed one day after the last dose (day 35). For all measurements, statistical significance was determined by one -tailed Student's t-test for change in tumor size (V/V 0 ).
  • tumors from mice treated with XPA.48.117 were significantly smaller than tumors from mice treated with anti-KLH human IgGl control XPA.48.148.
  • Tumors treated with XPA.48.117 inhibited growth by 73.8% as compared to control IgG (p ⁇ 0.01) at day 35.

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Abstract

La présente invention concerne des méthodes et des compositions destinées au traitement d'états pathologiques, de maladies ou de troubles associés à la signalisation/au dérèglement du FGFR4. La présente invention concerne également des anticorps se liant au FGFR4. De manière inattendue, il s'est avéré que les anticorps spécifiques contre le FGFR4 modulent la signalisation du FGFR4 et sont capables de neutraliser la phosphorylation des protéines intracellulaires induite par le FGF-19, ainsi que d'inhiber la croissance tumorale in vivo. Lesdits anticorps selon l'invention sont également proposés pour le traitement de tumeurs associées à la signalisation du FGFR4, ainsi que d'autres états pathologiques, maladies ou troubles associés à la signalisation du FGFR4.
PCT/US2013/077552 2012-12-28 2013-12-23 Anticorps spécifiques contre le fgfr4 et méthodes d'utilisation Ceased WO2014105849A1 (fr)

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WO2017100642A1 (fr) 2015-12-11 2017-06-15 Regeneron Pharmaceuticals, Inc. Méthodes pour ralentir ou empêcher la croissance de tumeurs résistantes au blocage de l'egfr et/ou d'erbb3
US9708318B2 (en) 2015-02-20 2017-07-18 Incyte Corporation Bicyclic heterocycles as FGFR4 inhibitors
US9890156B2 (en) 2015-02-20 2018-02-13 Incyte Corporation Bicyclic heterocycles as FGFR4 inhibitors
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US10851105B2 (en) 2014-10-22 2020-12-01 Incyte Corporation Bicyclic heterocycles as FGFR4 inhibitors
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WO2024114546A1 (fr) * 2022-11-29 2024-06-06 中国科学院深圳先进技术研究院 Anticorps monoclonal anti-fgfr4, lymphocytes t porteurs de récepteur antigénique chimérique ciblant fgfr4 et leur utilisation
WO2024114509A1 (fr) * 2022-11-28 2024-06-06 中国科学院深圳先进技术研究院 Anticorps monoclonal anti-fgfr4, lymphocyte t porteur d'un récepteur antigénique chimérique prenant fgfr4 en tant que cible et utilisation associée
US12012409B2 (en) 2020-01-15 2024-06-18 Incyte Corporation Bicyclic heterocycles as FGFR inhibitors
US12065494B2 (en) 2021-04-12 2024-08-20 Incyte Corporation Combination therapy comprising an FGFR inhibitor and a Nectin-4 targeting agent
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EP4509142A1 (fr) 2023-08-16 2025-02-19 Ona Therapeutics S.L. Fgfr4 comme cible dans le traitement du cancer
US12428420B2 (en) 2021-06-09 2025-09-30 Incyte Corporation Tricyclic heterocycles as FGFR inhibitors

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090214428A1 (en) * 2005-03-14 2009-08-27 Dimitrov Dimiter S Human Monoclonal Antibodies Against Hendra and Nipah Viruses
US20100169992A1 (en) * 2006-11-03 2010-07-01 Johannes Bange Fgfr4 antibodies
US20110076284A1 (en) * 2009-09-25 2011-03-31 Xoma Technology Ltd. Novel Modulators
US20110150903A1 (en) * 2008-07-08 2011-06-23 Sanofi-Aventis Fgf-r4 receptor-specific antagonists

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090214428A1 (en) * 2005-03-14 2009-08-27 Dimitrov Dimiter S Human Monoclonal Antibodies Against Hendra and Nipah Viruses
US20100169992A1 (en) * 2006-11-03 2010-07-01 Johannes Bange Fgfr4 antibodies
US20110150903A1 (en) * 2008-07-08 2011-06-23 Sanofi-Aventis Fgf-r4 receptor-specific antagonists
US20110076284A1 (en) * 2009-09-25 2011-03-31 Xoma Technology Ltd. Novel Modulators

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BUMBACA ET AL.: "Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4", MABS, vol. 3, no. 4, 1 July 2011 (2011-07-01), pages 376 - 386 *

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