HK1190734A - Anti-fgfr4 antibodies and methods of use - Google Patents

Description

anti-FGFR 4 antibodies and methods of use

Cross reference to related applications

This application claims priority to U.S. patent application No.61/473,106, filed on 7/4/2011, the contents of which are hereby incorporated by reference.

Sequence listing

This application contains a sequence listing, has been filed in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. The ASCII copy created on day 22/3/2012 was named p4524r1wo. txt and was 37,020 bytes in size.

Technical Field

The present invention relates to anti-FGFR 4 antibodies and methods of use thereof.

Background

Fibroblast Growth Factor (FGF) constitutes a family of 22 structurally related polypeptides with diverse biological activities; most of these signaling molecules function by binding to and activating their cognate receptor (FGFR; named FGFR 1-4), a family of receptor tyrosine kinases (Ewarakumar et al, 2005; Ornitz and Itoh, 2001). These receptor-ligand interactions lead to receptor dimerization and autophosphorylation, complex formation with membrane-bound and cytoplasmic accessory proteins, and initiation of multiple signaling cascades (Powers et al, 2000). The FGFR-FGF signaling system plays an important role in development and tissue repair by modulating cellular functions/processes such as growth, differentiation, migration, morphogenesis, and angiogenesis.

Alterations (i.e., over-expression, mutations, translocations, and truncations) in FGFR are associated with a variety of human cancers, including myeloma, breast, gastric, colon, bladder, pancreatic, and hepatocellular (Bangeet al, 2002; Cappellen et al, 1999; Chesi et al, 2001; Chesi et al, 1997; Gowardhan et al, 2005; Jaakkola et al, 1993; Jang et al, 2001; Jang et al, 2000; Jeffers et al, 2002; Xiao et al, 1998). Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, causing over 50 million deaths each year (Shariff et al, 2009). Although the role of FGFR4 in cancer remains to be fully elucidated, several findings suggest that this receptor may be an important role in the development and/or progression of HCC. FGFR4 is the predominant FGFR isoform present in human hepatocytes (Kan et al, 1999); we have also previously reported that liver tissues have the highest transcript levels of FGFR4 (Lin et al, 2007). In addition to FGFR4 that is overexpressed in liver cancer (and several other types of human tumors), several missense genetic alterations were observed in HCC patient samples; notably, a highly frequent G388R single nucleotide polymorphism in FGFR4 was identified (associated with reduced survival of head and neck cancer, and a more aggressive phenotype of colon, soft tissue, prostate, and breast cancer) (Ho et al, 2009). Moreover, it has been previously demonstrated that ectopic expression of FGF19 (i.e., a FGFR 4-specific ligand) in mice promotes hepatocyte proliferation, hepatocyte dysplasia, and neoplasia (nicholese et al, 2002).

It is clear that there is still a need for agents with clinical properties that are most suitable for development into therapeutic agents. The invention described herein satisfies this need and provides other benefits.

All references (including patent applications and publications) cited herein are incorporated by reference in their entirety.

Summary of The Invention

The invention provides anti-FGFR 4 antibodies and methods of use thereof.

In one aspect, the invention provides an isolated antibody that binds FGFR4, wherein the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 1 nM. In some embodiments, the anti-FGFR 4 antibody binds human, mouse, and cynomolgus FGFR4 with an affinity of ≤ 1 nM. In some embodiments, the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 0.05 nM.

In some embodiments, the anti-FGFR 4 antibody does not substantially bind to a mouse C3 protein having the amino acid sequence shown in fig. 12D.

In some embodiments, the anti-FGFR 4 antibody binds to denatured FGFR 4. In some embodiments, the anti-FGFR 4 antibody binds to reduced, denatured FGFR 4. In some embodiments, Western blotting is used to determine the binding of anti-FGFR 4 antibodies to FGFR 4.

In some embodiments, the anti-FGFR 4 antibody does not substantially bind to human FGFR4 comprising the G165A mutation. In some embodiments, the anti-FGFR 4 antibody binds to a polypeptide having at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity to a sequence comprising amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence, consisting essentially of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence, or consisting of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence. In some embodiments, the anti-FGFR 4 antibody binds to a polypeptide comprising, consisting essentially of, or consisting of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, or amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR 4.

In some embodiments, the anti-FGFR 4 antibody is an antagonist of FGFR4 activity. In some embodiments, the FGFR4 activity is FGF-induced cell proliferation, FGF binding to FGFR4, FGF 19-mediated inhibition of CYP7 α 7 expression in cells exposed to FGF19, or FGF 19-induced colony formation. In some embodiments, binding of FGF1 and/or FGF19 to FGFR4 is inhibited. In some embodiments, the IC that inhibits FGF1 binding to FGFR4₅₀IC inhibiting FGF19 binding to FGFR4 at about 0.10nM₅₀About 0.10 nM.

In some embodiments, the anti-FGFR 4 antibody is a monoclonal antibody.

In some embodiments, the anti-FGFR 4 antibody is a human, humanized, or chimeric antibody.

In some embodiments, the anti-FGFR 4 antibody is an antibody fragment that binds FGFR 4.

In some embodiments, the antibody comprises (a) HVR-H3 comprising amino acid sequence SEQ ID NO:3, (b) HVR-L3 comprising amino acid sequence SEQ ID NO:6, and (c) HVR-H2 comprising amino acid sequence SEQ ID NO: 2.

In some embodiments, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO:1, (b) HVR-H2 comprising amino acid sequence SEQ ID NO:2, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 3. In some embodiments, the antibody further comprises (a) HVR-L1, comprising the amino acid sequence of SEQ ID No. 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6.

In some embodiments, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6. In some embodiments, the antibody further comprises a light chain variable domain framework sequence of SEQ id nos 9, 10, 11, and/or 12.

In some embodiments, the antibody further comprises a heavy chain variable domain framework sequence of SEQ ID NO 13, 14, 15 and/or 16.

In some embodiments, the antibody comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ id No. 7; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO 8; or (c) the VH sequence in (a) and the VL sequence in (b). In some embodiments, the antibody comprises the VH sequence SEQ ID NO 7. In some embodiments, the antibody comprises the VL sequence SEQ ID NO 8.

In some embodiments, the antibody comprises the VH sequence SEQ ID NO 7 and the VL sequence SEQ ID NO 8.

In some embodiments, the antibody is a full length IgG1 antibody.

The invention also provides an isolated nucleic acid encoding any of the antibodies described herein.

The invention also provides a host cell comprising any of the nucleic acids described herein.

The invention also provides a method of producing an antibody comprising culturing a host cell as described herein such that the antibody is produced. In some embodiments, the method further comprises recovering the antibody from the host cell.

The invention also provides an immunoconjugate comprising any of the antibodies described herein and a cytotoxic agent.

The invention also provides pharmaceutical formulations comprising any of the antibodies described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises an additional therapeutic agent.

The invention also provides any of the antibodies described herein for use as a medicament.

In some embodiments, the antibody is used to treat cancer.

In some embodiments, the antibody is used to inhibit cell proliferation.

In some embodiments, the antibody is used in the manufacture of a medicament.

In some embodiments, the medicament is for treating cancer.

In some embodiments, the medicament is for inhibiting cell proliferation.

The invention also provides a method of treating an individual having cancer comprising administering to the individual an effective amount of any of the antibodies described herein. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent.

The invention also provides a method of inhibiting cell proliferation in an individual comprising administering to the individual an effective amount of any of the antibodies described herein to inhibit cell proliferation.

In some embodiments, the cancer is breast cancer, lung cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, pituitary cancer, fibroadenomas of the breast, head and neck cancer, soft tissue cancer, neuroblastoma, melanoma, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, and/or liver cancer.

In certain embodiments, the cancer displays FGFR4 expression (such as overexpression), amplification, or activation. In certain embodiments, the cancer displays FGFR4 amplification.

Brief Description of Drawings

FIG. 1: FGFR4 is required for hepatic tumorigenesis mediated by FGF 19. A, multiple, large, prominent tumors (arrows) protruding from the liver surface of 10-month old FGF19-TG: FGFR4-WT mice (left panel). Liver from 10-month-old FGF19-TG: FGFR4-KO mice (right panel). B, BrdU incorporation in female (left panel) and male (right panel) FGF19-TG or wild-type mice mated with FGFR4-KO or FGFR4-WT mice. C, 4-month old DEN-treated FGF19-TG multiple, large, prominent tumors on the surface of the liver of FGFR4-WT mice (arrows). D, prevalence of liver tumors in men and female FGF19-TG mice treated with DEN, determined by visual and histological examination. E, liver weight from DEN-treated FGF19-TG or wild-type female (left panel) and male (right panel) mice. Asterisks indicate that liver weight of male FGF19-TG mice treated with DEN cannot be measured from the 7 month time point, since none survived beyond 6 months of age. F, liver weight of FGF19-TG or wild-type female (left panel) and male (right panel) FGFR4-KO mice treated with DEN.

FIG. 2: LD1 binds FGFR 4. A, LD1 binds to human (h), mouse (m), and cynomolgus monkey (c) FGFR4, but not to hFGFR1, hFGFR2, or hFGFR 3. Binding of LD1 to the immobilized FGFR Fc chimeric protein was determined by a solid phase binding assay. B, the affinity of LD1 for binding mouse, cynomolgus, and human FGFR4 was determined by surface plasmon resonance. C, LD1 binding to hFGFR4 expressed on the cell surface of stably transfected HEK293 cells, determined by FACS (RFU = relative fluorescence units). D, binding of LD1 to the immobilized hFGFR4-Flag chimeric protein carrying a point mutation, measured by a solid phase binding assay. E, binding of LD1 to hFGFR4-Flag chimeric protein carrying a point mutation, assessed by Western blot. The muteins were electrophoresed and sequentially immunoblotted using LD1, anti-FGFR 4 (8G 11), and anti-Flag antibody. F, dimer model, showing the position of G165 (black) on FGFR4 (black and white) bound to FGF19 (grey).

FIG. 3: LD1 inhibits FGFR4 activity. A, LD1 inhibited FGFR4 binding to FGF1 and FGF19 as determined by a solid phase binding assay. B, LD1 inhibited the growth of BaF3 cells stably expressing FGFR4/R1 stimulated by FGF 1. C, LD1 inhibited FGFR4 signaling in L6 cells stably expressing FGFR 4. D, cell surface expression of FGFR4 protein in a subset of liver tumor cell lines, determined by FACS analysis using LD1.

FIG. 4: LD1 inhibits FGFR4 biological activity in liver cancer cell lines. A, LD1 inhibited FGFR4 signaling in HEP3B cells, assessed by Western blot. B, LD1 inhibited CYP7 α 1 expression regulated by FGFR4 in HEP3B cells. CYP7 α 1 levels were expressed as fold expression relative to levels in untreated cells. C, LD1 inhibited FGFR 4-regulated C-Fos expression in a group of liver cancer cell lines. Results are expressed as fold expression relative to c-Fos levels in untreated cells. D, inhibition of colony formation by repressing FGFR4 expression in JHH5 cells stably transfected with FGFR4shRNA doxycycline inducible vector. E, LD1 inhibits HCC cell line colony formation. F, count of liver cancer cell line colony formation inhibited by LD1. Values are expressed as a percentage of the number of colonies counted without the addition of LD1.

FIG. 5: in vivo efficacy of LD1. A, LD1 inhibited FGF 19-regulated c-Fos expression in mouse liver. Results are expressed as fold expression relative to c-Fos levels in the liver of untreated mice. B, LD1 (30 mg/kg; twice weekly) inhibited HUH7 xenograft tumor growth in vivo. C, effect of LD1 on mRNA expression of FGFR4, CYP7 α 1, C-Fos, and egr-1 in HUH7 xenograft tumors from FIG. 5B. Multiple, large, prominent tumors (arrows) protruding from the liver surface of DEN-accelerated FGF19-TG: FGFR4-WT mice treated with control antibodies (upper panel). Liver of DEN-accelerated FGF19-TG: FGFR4-WT mice treated with LD1 (lower panel). E, liver weight of DEN-accelerated FGF19-TG: FGFR4-WT mice treated with control antibody, LD1, or 1A6 (anti-FGF 19 antibody).

FIG. 6: FGFR4 expression is deregulated in cancer. Line-box plot (whisker-box plot) shows FGFR4 expression in human tumor and normal tissues, determined by mRNA analysis of the BioExpress database. The center line indicates the median; boxes represent the interquartile range between the first and third quartile. A "line" extends from between the quartile to the location of the extremum. B, FGFR4 immunostaining in samples of breast (100-fold magnification) and pancreatic (100-fold magnification) adenocarcinoma and hepatocellular carcinoma (200-fold magnification and 400-fold magnification). C, FGFR4mRNA expression in a group of human normal liver and liver tumors, as determined by qRT-PCR. The values for each sample are expressed as fold-expression relative to the level observed in sample N1.

FIG. 7: FGFR expression in liver cancer cell lines. FGFR4mRNA expression in a panel of liver tumor cell lines, determined by qRT-PCR. Values are expressed as fold expression of FGFR1 levels relative to JHH4 cell line. B, FGFR4 protein expression in the same set of cell lines as in fig. 7A, as determined by Western blot.

FIG. 8: LD1 inhibits FGFR4 biological activity in HUH7 cells. LD1 inhibits CYP7 α 1 repression regulated by FGFR4 in HUH7 cells. CYP7 α 1 levels were expressed as fold expression relative to levels in untreated cells.

FIG. 9: in vivo efficacy of LD1. LD1 (30 mg/kg) inhibited HUH7 xenograft tumor growth in vivo. The anti-tumor efficacy of LD1 was evaluated in a biweekly modality.

FIG. 10: variable domain sequences of mouse and humanized variants against FGFR 4. The amino acid sequences of mouse LD1 and humanized variants hld1.vb and hld1.v22 were aligned to (a) human kappa i (huki) and (B) human VH subgroup iii (huiii) variable domain framework used in trastuzumab. Differences are highlighted in dashed boxes and position numbered according to Kabat. The hypervariable regions grafted from mouse LD1 into the human variable kappa I and subgroup III consensus framework were selected based on the combination of sequence, structure and contact CDR definitions (MacCallumRM et al, J of mol Biol (1996);262: 732-45) and are indicated in boxes. Three fine-tuned (vernier) positions in the light chain were changed during humanization to restore affinity; these locations are not expected to be surface exposed.

FIG. 11: pharmacokinetics and profiles of anti-FGFR 4 antibody variants. (A) Binding of chLD1 and hld1.vb to FGFR4 was compared using FGFR4 ELISA. (B) Comparison of chLD1, hLD1.vB and vehicle 16 day tumor volumes in HUH7 human hepatocellular carcinoma xenograft model in CRL nu/nu mice. Antibodies were administered at 30mg/kg twice weekly (10 mice per group). Only chLD1 was effective in reducing tumor growth relative to PBS control (p-value = 0.014), whereas hld1.vb was not significantly effective (p-value = 0.486). (C) Pharmacokinetics, administration of 1 or 20mg/kg IV to chLD1 and hld1.vbncr nude mice, and analysis of samples using FGFR4 ELISA. Similar results were obtained using IgG ELISA (not shown). (D) In NCR nude mice¹²⁵I-chLD1 and¹²⁵tissue distribution of I-hLD1. vB. Administered to mice¹²⁵I-chLD1 or¹²⁵vB, and the percent injected dose per gram tissue (% ID/g) was determined 2 hours after dosing, as described in methods.

FIG. 12: identification of the interaction between hld1.vb and mouse C3 d. (A) Detection of chLD1 (hatched bars) and hld1.vb (white bars) after 48 hours incubation in PBS/BSA or NCR nude mouse, rat, human and cynomolgus plasma. Percent recovery was determined using FGFR4 ELISA. (B)¹²⁵I-chLD1 (solid line) and¹²⁵plasma binding assay of I-hLD1.vB (dotted line).Trace offset (off-set); dots indicate the position of the 150kDa peak. Antibodies were incubated in mouse plasma in 0 and 48 mice, followed by analysis using size exclusion HPLC. For the incubation in PBS/BSA, human plasma or cynomolgus plasma, see FIG. 15. All incubations produced a peak at the expected 150kDa corresponding to IgG. Only higher molecular weight peaks were observed in mouse plasma samples containing hld1. vb. The initial time point revealed additional peaks at approximately 270kDa and approximately 550kDa, whereas at 48 hours only a 270kDa peak was observed. No high molecular weight peaks were observed upon incubation of hld1. vb/mouse plasma at pH4, indicating that the presence of these high molecular weight peaks is pH dependent (fig. 15). (C) Immunoprecipitation of mouse plasma. chLD1 and hld1.vb were incubated in mouse plasma for 24 hours at 37 ℃ and analyzed by size exclusion HPLC. Protein G beads were then added to the fractions, followed by SDS-PAGE analysis. A band at approximately 37kDa was detected in the fraction corresponding to the 270kDa peak present in the hld1. vb/mouse plasma sample (lane 3), but not in samples from cynomolgus monkey or human plasma or PBS/BSA (figure 15). This band was not observed in either mouse plasma alone (lane 4) or mouse plasma incubated with chLD1 (lane 2). Lane 1 runs protein molecular weight markers. (D) MS/MS sequence coverage of mouse C3 obtained from hld1.vb immunoprecipitation. The sequence of mouse C3 (SEQ ID NO: 38) is shown, and the region encoding C3d is underlined. The peptides identified were as follows:

DVPAADLSDQVPDTDSETRIILQGSPVVQMAEDAVDGER(SEQ ID NO:17)

RQEALELIKKGYTQQLAFK(SEQ ID NO:18)

AAFNNRPPSTWLTAYVVK(SEQ ID NO:19)

AANLIAIDSHVLCGAVK(SEQ ID NO:20)

QKPDGVFQEDGPVIHQEMIGGFR(SEQ ID NO:21)

EADVSLTAFVLIALQEARDICEGQVNSLPGSINKAGEYIEASYMNLQRPYTVAIAGYALALMNK(SEQ ID NO:22)

WEEPDQQLYNVEATSY(SEQ ID NO:23)

YYGGGYGSTQATFMVFQALAQYQTDVPDHK(SEQ ID NO:24)

GTLSVVAVYHAK(SEQ ID NO:25)

DLELLASGVDR(SEQ ID NO:26)

NTLIIYLEK(SEQ ID NO:27)。

FIG. 13: the affinity matured anti-FGFR 4 variant lacks C3d binding. (A) Detection of FGFR4 binding, chLD1, hld1.vb and hld1.v22 were incubated in NCR nude, C3 wild type (wt) and C3ko mouse serum for 16 hours before assessment using FGFR4 ELISA. Samples were normalized to the same samples incubated in PBS/0.5% BSA. (B) Vl 22 deficient mice C3d immunoprecipitates. Immunoprecipitates from NCR nude mouse plasma were analyzed by SDS-PAGE using chLD1 (lane 2), hld1.vb (lane 3) and hld1.v22 (lane 4). A-37 kDa band was detected only in hLD1. vB/mouse plasma samples. Lane 1 runs protein molecular weight markers.

FIG. 14: loss of C3d binding restores pharmacokinetics and efficacy. (A) Pharmacokinetic analysis of chLD1 and hld1.vb in C3wt and C3ko mice. Administering the antibody at 20mg/kg IV; FGFR4ELISA was used to monitor their concentration in blood. Clearance of hld1.vb in C3ko mice was similar to that of chLD1 in both C3ko and C3wt mice. (B) Pharmacokinetic analysis of chld1hld1.vb and hld1.v22 in NCR nude mice. Administering the antibody at 20mg/kg IV; FGFR4ELISA was used to monitor their concentration in blood. Clearance of hld1.v22 is similar to that of chLD 1. (C) Comparison of chld1hld1.vb and hld1.v22 in HUH7 human HCC xenograft model in CRL nu/nu mice. Antibodies were administered at 30mg/kg once a week (10 mice per group) and tumor volume was monitored for 4 weeks. Day 21, chLD1 and hld1.v22 (p-value =7x10, respectively)^-7And 3x10^-5) Tumor growth was effectively reduced relative to PBS control (open squares), whereas hld1.vb was only slightly effective (p-value = 0.011).

FIG. 15: size exclusion HPLC analysis of radiolabeled chLD1 and hld1.vb in plasma from in vitro (a-B) and in vivo study (C) samples. Will be provided with¹²⁵I-hLD1.vB (A) and¹²⁵I-chLD1(B) addition to PBS/BSA or mouse, humanAnd cynomolgus monkey plasma, and analyzed by size exclusion HPLC at 0 or 48 hours. All traces revealed a peak at 150kDa expected for IgG (peak on the right). Peaks of higher molecular weight at approximately 270kDa (middle peak) and approximately 550kDa (left peak) were observed only in the initial mouse plasma sample containing hld1. vb; at 48 hours, only a further 270kDa peak was observed. These high molecular weight peaks were not observed for hld1.vb in the plasma of mice at pH4.0, indicating that the generation of high molecular weight peaks is pH dependent. No high molecular weight peak was detected in cynomolgus monkey or human plasma or in any plasma to which chLD1 was added; (C) in vivo mouse plasma samples. Mice were dosed with-0.1 mg/kg antibody, specific activity chLD1 at 12.52 μ Ci/μ g, hld1.vb at 9.99 μ Ci/ug. Serum samples were collected at 0.25, 2,5, 24, 72 and 120 hours. All traces revealed a peak at 150kDa expected for peak IgG (peak on the right). Higher molecular weight peaks at 270kDa (middle peak) and 550kDa (left peak) were observed only in hLD1.vB serum samples. No high molecular weight peak was observed for chLD 1.

FIG. 16: immunoprecipitation and SDS-PAGE analysis. Antibodies were incubated in plasma for 24 hours at 37 ℃ and then fractionated by size exclusion HPLC. Protein G beads were added to the size exclusion HPLC fractions containing high molecular weight peaks. The pull-down sample was analyzed by SDS-PAGE (pull-down sample). (A) In vitro rat plasma; (B) in vitro macaque plasma; (C) human plasma in vitro. The-37 kDa protein was detected in rat samples incubated with hld1.vb, but not in cynomolgus and human samples (lane 4 of their respective gels). No band of-37 kDa protein was observed in any of the blank plasma (lane 2 per gel) or in the chLD1 samples (lane 3 per gel). Lane 1 runs protein molecular weight markers. (D) In vivo mouse plasma. Mice were administered either hld1.vb or chLD1 as described in methods. Protein G beads were then added to serum samples collected 2 hours after the dosing time point and analyzed by SDS-PAGE. A band of 37kDa was observed in hLD1.vB (lane 3), but the chLD1 serum sample (lane 2) was not. Lane 1 runs protein molecular weight markers.

FIG. 17: ELISA assay of chLD1 and hld1.vb added to C3wt serum, C3ko mouse serum, and PBS/BSA. Antibodies were incubated overnight in serum or PBS/BSA and then analyzed by ELISA. hLD1.vB was recovered completely in C3ko mouse serum relative to PBS/BSA, but not in C3wt mouse serum. chLD1 was completely recovered from all matrices.

FIG. 18: an exemplary human FGFR4 amino acid sequence is shown for GenBank accession No. AAB 25788.

Detailed Description

I. Definition of

For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) which result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such alterations.

Anti-angiogenic agents refer to compounds that block or interfere to some extent with vascular development. The anti-angiogenic factor can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds Vascular Endothelial Growth Factor (VEGF), such as bevacizumab

The terms "anti-FGFR 4 antibody" and "antibody that binds FGFR 4" refer to an antibody that is capable of binding FGFR4 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting FGFR 4. In one embodiment, the anti-FGFR 4 antibody binds to an unrelated, non-FGFR 4 protein to less than about 10% of the binding of the antibody to FGFR4 as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds FGFR4 has a mass of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In certain embodiments, the anti-FGFR 4 antibody binds to an FGFR4 epitope that is conserved among FGFR4 from different species.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

As used herein, the term "FGFR 4" refers to any native FGFR4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed FGFR4 as well as any form of FGFR4 that results from processing in a cell. The term also encompasses naturally occurring variants of FGFR4, such as splice variants or allelic variants. The amino acid sequence of an exemplary human FGFR4 is shown in figure 18.

"FGFR 4 activation" refers to the activation or phosphorylation of FGFR4 receptors. In general, FGFR4 activation results in signal transduction (e.g., phosphorylation of a tyrosine residue in FGFR4 or substrate polypeptides, caused by the intracellular kinase domain of the FGFR4 receptor). FGFR4 activation can be mediated by FGFR4 ligand (Gas 6) binding to the FGFR4 receptor of interest. Binding of Gas6 to FGFR4 activates the kinase domain of FGFR4 and thereby results in phosphorylation of tyrosine residues in FGFR4 and/or phosphorylation of tyrosine residues in other substrate polypeptides.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatoma), leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cyclophosphamide)Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetalmamine; annonaceous acetogenins (especially bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol),) (ii) a Beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including the synthetic analogue topotecan)CPT-11 (irinotecan),) Acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, particularly calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Nicolaou et al, Angew. chem Intl. Ed. Engl.,33:183-186 (1994)); CDP323, an oral α -4 integrin inhibitor, anthracyclines (dynemicin), including dynemicin A; esperamicin), and neocarzinostain (neocarzinostatin) and related chromophorins of chromenes, aclacinomycin (acrinomycin), actinomycins (actinomycin), anthranomycin (anthramycin), azaserine (azazaine), bleomycin (bleomycin), actinomycin C (capnocomycin), carminomycin, carvachromycin (carinomycin), and carvachromycin (carcinomycin)(chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditobicin (detorubicin), 6-diaza-5-oxo-L-norleucine, doxorubicin (doxorubicin) (includingMorpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrol doxorubicin and doxorubicin hydrochloride liposome injectionLiposomal doxorubicin TLC D-99PEGylated liposomal doxorubicinAnd doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozotocin), tubercidin (tubicidin), ubenimex (enimebendamusex), purified staudin (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate, gemcitabine (gemcitabine)Tegafur (tegafur)Capecitabine (capecitabine)Epothilone (epothilone) and 5-fluorouracil (5-FU); folic acid analogues, such as diformic acid (denopt)erin), methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; an epothilone; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS natural products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (tria)ziquone); 2,2',2' ' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); vindesine (vindesine) ((vindesine))) (ii) a Dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxols (taxoids), such as paclitaxel (paclitaxel)Albumin engineered nanoparticle dosage form paclitaxel (ABRAXANE)^TM) And docetaxel (doxetaxel)Chlorambucil (chlorambucil); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs, such as cisplatin (cissplatin), oxaliplatin (oxaliplatin) (e.g., cisplatin)) And carboplatin (carboplatin); vinblastines (vincas), which prevent tubulin polymerization to form microtubules, include vinblastine (vinblastine)Vincristine (vincristine)Vindesine (vindesine)And vinorelbine (C)vinorelbine)Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovorin); oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as tretinoin acid (Retinoic acid), including bexarotene (bexarotene)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate sodium (etidronate)NE-58095, zoledronic acid/zoledronate (zoledronic acid/zoledronate)Alendronate (alendronate)Pamidronate (pamidronate)Tiludronate (tirudronate)Or risedronate (risedronate)And kojisha(ii) troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, e.g.Vaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine; topoisomerase 1 inhibitors (e.g. topoisomerase 1 inhibitors)) (ii) a rmRH (e.g. rmRH)）；BAY439006(sorafenib;Bayer)；SU-11248(sunitinib,Pfizer); perifosine (perifosine), COX-2 inhibitors (such as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (such as PS 341); bortezomibCCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersensodiumpixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (rapam)ycin)(sirolimus,) (ii) a Farnesyl transferase inhibitors such as lonafarnib (SCH6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

Chemotherapeutic agents, as defined herein, include the class of "anti-hormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth. They may themselves be hormones, including but not limited to: antiestrogens with mixed agonist/antagonist properties including tamoxifen (tamoxifen) (NOLVADEX), 4-hydroxytamoxifen, toremifene (toremifene)Idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene)Trioxifene (trioxifene), naloxifene (keoxifene), and Selective Estrogen Receptor Modulators (SERMs), such as SERM 3; pure antiestrogens without agonist properties, such as fulvestrantAnd EM800 (such agents may block Estrogen Receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane (formestane) and exemestane (exemestane)And non-steroidal aromatase inhibitors, such as anaquAzole (anastrozole)Letrozole (letrozole)And aminoglutethimide (aminoglutethimide), and other aromatase inhibitors, including vorozole (vorozole)Megestrol acetate (megestrol acetate)Fadrozole (fadrozole) and 4(5) -imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (leuprolide) ((leuprolide))And) Goserelin (goserelin), buserelin (buserelin) and triptorelin (triptorelin); sex steroids (sex steroids) including pregnanins (progestines) such as megestrol acetate and medroxyprogesterone acetate (medroxyprogesterone), estrogens such as diethylstilbestrol (diethylstilbestrol) and pramlins (premarin), and androgens/retinoids such as fluoxymesterone (fluoroxymesterone), all trans retinoic acid (transretinic acid) and fenretinide (fenretinide); onapristone (onapristone); anti-pregnenones; estrogen receptor down-regulators (ERD); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The constant domains of heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "cytostatic agent" refers to a compound or composition that blocks cell growth in vitro or in vivo. As such, the cytostatic agent may be an agent that significantly reduces the percentage of cells in S phase. Other examples of cytostatics include agents that block cell cycle progression by inducing G0/G1 arrest or M-phase arrest. Humanized anti-Her 2 antibody trastuzumabIs an example of a cytostatic agent that induces G0/G1 arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Certain agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechlorethamine (mechloroethylamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see, e.g., The eds of Mendelsohn and Israel, The Molecular Basis of Cancer, Chapter 1, entitled "cell regulation, oncogenes, and antioxidant drugs", Murakanii et al, W.B. Saunders, Philadelphia, 1995, e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from taxus baccata (c)Rhone-Poulenc Rorer) is paclitaxel ((R)Semi-synthetic analogs of Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

A cancer or biological sample "exhibiting FGFR4 expression, amplification, or activation" refers to a cancer or biological sample that expresses (including overexpresses) FGFR4, has an amplified FGFR4 gene, and/or otherwise exhibits FGFR4 activation or phosphorylation in a diagnostic test.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published healthcare service 5 th edition, National Institutes of Health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, Sequences of Proteins of immunologicalcatest, fifth edition, NIH Publication91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable regions are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at 24-34 of amino acid residues L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1, and 95-102 of H3 (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition public Health Service, National Institutes of Health, Bethesda, Md (1991)). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called a shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and Fransson, Front. biosci.13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

By "inhibiting cell growth or proliferation" is meant reducing the growth or proliferation of a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

An "isolated antibody" refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B848:79-87 (2007).

An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-FGFR 4 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in different vectors, and such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the phrase "little to no binding" with respect to an antibody of the invention means that the antibody does not exhibit a biologically significant amount of binding. As will be appreciated in the art, the amount of activity can be determined quantitatively or qualitatively, so long as a comparison between the antibodies of the invention and the reference counterpart can be made. The activity can be measured or detected according to any assay or technique known in the art, such as those described herein. For the antibodies of the invention and their reference counterparts, the amount of activity can be determined in parallel or in separate runs.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH 1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging for a therapeutic product that contains information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document, where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other components having unacceptable toxicity to a subject that will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variations thereof, such as "treating" or "treatment") refers to clinical intervention in an attempt to alter the natural course of the treated individual, which may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset/progression of disease, or to slow the progression of disease.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). (see, e.g., Kindt et al KubyImmunology, 6 th edition, W.H.Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, for example, Portolano et al, J.Immunol.150:880-887(1993); Clarkson et al, Nature352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

A "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable heavy subgroup III of Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:13) -H1-WVRQAPGKGLEWV (SEQ ID NO:14) -H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:15) -H3-WGQGTLVTVSS (SEQ ID NO: 16).

The "VL subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable light kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:28) -L1-WYQQKPGKAPKLLIY (SEQ ID NO:29) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:30) -L3-FGQGTKVEIK (SEQ ID NO: 31).

The term "wasting/wasting" disorder (e.g., wasting syndrome, cachexia, sarcopenia (sarcopenia)) refers to a disorder resulting from an undesired and/or unhealthy loss of body weight or loss of body cell mass. In the elderly and in AIDS and cancer patients, wasting diseases can lead to undesirable weight loss, including both fatty and non-fatty compartments. Wasting diseases can result from improper feeding and/or metabolic changes associated with malaise and/or the aging process. Cancer patients and AIDS patients, as well as patients who have undergone extensive surgery or have chronic infections, immunological diseases, hyperthyroidism, crohn's disease, psychological diseases, chronic heart failure, or other serious trauma, often suffer from wasting diseases, sometimes also referred to as cachexia, metabolic disorders, and eating disorders. Cachexia is also manifested by hypermetabolism and hypercatabolism. Although cachexia and wasting disease are often used interchangeably and refer to wasting disorders, at least one study has distinguished cachexia from wasting syndrome, i.e., loss of non-fatty substances, particularly somatic substances (Mayer,1999, j.nutr.129(1S supply.): 256S-259S). Sarcopenia, another such condition affecting aging individuals, is often manifested as loss of muscle mass. End-stage wasting disease as described above can occur in individuals with cachexia or sarcopenia.

Compositions and methods

In one aspect, the invention is based, in part, on the identification of a plurality of FGFR4 binding agents (such as antibodies and fragments thereof). In certain embodiments, antibodies that bind FGFR4 are provided. The antibodies of the invention are useful, for example, in the diagnosis and treatment of cancer, liver disease and wasting (wasting disease).

A. Exemplary anti-FGFR 4 antibodies

In one aspect, the invention provides isolated antibodies that bind FGFR 4.

In certain embodiments, the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 1 nM. In some embodiments, the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 0.05 nM.

In certain embodiments, the anti-FGFR 4 antibody binds mouse FGFR4 with an affinity of ≦ 1 nM.

In certain embodiments, the anti-FGFR 4 antibody binds cynomolgus FGFR4 with an affinity of ≦ 1 nM.

In certain embodiments, the anti-FGFR 4 antibody binds human, mouse, and cynomolgus FGFR4 with an affinity of ≤ 1 nM.

In certain embodiments, the anti-FGFR 4 antibody does not substantially bind to human FGFR1, human FGFR2, and/or human FGFR 3. In a certain embodiment, the anti-FGFR 4 antibody exhibits little or no binding to human FGFR1, human FGFR2, and/or human FGFR 3.

In certain embodiments, the anti-FGFR 4 antibody does not substantially bind to mouse C3 protein (in some embodiments, mouse C3 protein having the amino acid sequence shown in fig. 12D). In a certain embodiment, the anti-FGFR 4 antibody exhibits little or no binding to the mouse C3 protein (in some embodiments, the mouse C3 protein having the amino acid sequence shown in fig. 12D).

In certain embodiments, the anti-FGFR 4 antibody is a humanized anti-FGFR 4 antibody, wherein the monovalent affinity of the antibody for human FGFR4 is substantially the same as the monovalent affinity of a murine antibody comprising the light and heavy chain variable sequences set forth in SEQ ID NOs 8 and 7, respectively. In some embodiments, the anti-FGFR 4 antibody is a humanized and affinity matured antibody.

In certain embodiments, the anti-FGFR 4 antibody is an antagonist of FGFR4 activity.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGF binding to FGFR 4. In some embodiments, binding of FGF1 and/or FGF19 to FGFR4 is inhibited. In some embodiments, the IC that inhibits FGF1 binding to FGFR4₅₀About 0.10 nM. In some embodiments, the IC that inhibits FGF19 binding to FGFR4₅₀About 0.10 nM.

In certain embodiments, the anti-FGFR 4 antibody inhibits cell proliferation. In some embodiments, the proliferation is FGF-induced cell proliferation. In some embodiments, the cell proliferation is BAF3/FGFR4 transgenic cell proliferation.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGF (e.g., FGF 1) -stimulated proliferation of FGFR 4-expressing cells. In some embodiments, the cell is a HUH7 cell.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGF 19-mediated inhibition of CYP7 α 7 expression in a cell exposed to FGF 19.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGF 19-induced phosphorylation of FGFR4, MAPK, FRS2, and/or ERK2 in cells exposed to FGF 19.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGF 19-induced colony formation. In some embodiments, the colonies are formed as HCC cell line colonies. In some embodiments, the HCC cell line is JHH 5.

In certain embodiments, the anti-FGFR 4 antibody binds to denatured FGFR 4. In some embodiments, the anti-FGFR 4 antibody binds to reduced, denatured FGFR 4. In some embodiments, Western blotting is used to determine binding to reduced, denatured FGFR 4.

In certain embodiments, the anti-FGFR 4 antibody binds FGFR4 expressed on the surface of a cell. In some embodiments, the cell is a HUH7 or JHH5 cell.

In certain embodiments, the anti-FGFR 4 antibody does not substantially bind to human FGFR4 comprising the G165A mutation. In certain embodiments, the anti-FGFR 4 antibody exhibits little or no binding to human FGFR4 comprising the G165A mutation.

In certain embodiments, the anti-FGFR 4 antibody binds to a polypeptide comprising, consisting essentially of, or consisting of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, or amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR 4. In certain embodiments, the anti-FGFR 4 antibody binds to a polypeptide comprising, consisting essentially of, or consisting of amino acid numbers 145 to 180 of the amino acid sequence of mature human FGFR4, amino acid numbers 145 to 180 of the amino acid sequence of mature human FGFR4, or amino acid numbers 145 to 180 of the amino acid sequence of mature human FGFR 4.

In certain embodiments, the anti-FGFR 4 antibody binds to a polypeptide having at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity to a sequence comprising amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence, consisting essentially of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence, or consisting of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence. In certain embodiments, the anti-FGFR 4 antibody binds to a polypeptide having at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity to a sequence comprising amino acid number 145-180 of the mature human FGFR4 amino acid sequence, consisting essentially of amino acid number 145-180 of the mature human FGFR4 amino acid sequence, or consisting of amino acid numbers 145 to 180 of the mature human FGFR4 amino acid sequence.

In certain embodiments, the anti-FGFR 4 antibody inhibits FGFR4 dimerization.

In certain embodiments, the anti-FGFR 4 antibody binds at the FGFR4 dimerization interface.

In certain embodiments, the anti-FGFR 4 antibody inhibits tumor growth. In some embodiments, the tumor growth is a liver tumor growth.

In one aspect, the invention provides an anti-FGFR 4 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence NHWMN (SEQ ID NO: 1); (b) HVR-H2 comprising amino acid sequence MILPVDSETTLEQKFKD (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence GDISLFDY (SEQ ID NO: 3); (d) HVR-L1 comprising amino acid sequence RTSQDISNFLN (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence YTSRLHS (SEQ ID NO: 5); and (f) HVR-L3, comprising amino acid sequence QQGNALPYT (SEQ ID NO: 6).

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO 1; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO 2; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3. In another embodiment, the antibody comprises HVR-H3 and HVR-L3, the HVR-H3 comprises the amino acid sequence of SEQ ID NO. 3, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO. 6. In yet another embodiment, the antibody comprises HVR-H3, HVR-L3, and HVR-L2, the HVR-H3 comprises amino acid sequence SEQ ID NO:3, the HVR-L3 comprises amino acid sequence SEQ ID NO:6, and the HVR-H2 comprises amino acid sequence SEQ ID NO: 2. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO 1; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO 2; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 3.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:1, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:2, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:4, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:5, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 6.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO. 1; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO. 2; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO. 3; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 4; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO 5; and (f) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO 6.

In another aspect, the anti-FGFR 4 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% sequence identity to amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTNHWMNWVRQAPGKGLEWVGMILPVDSETTLEQKFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRGDISLFDYWGQGTLVTVSS (SEQ ID NO: 7). In certain embodiments, a VH sequence having at least 90%,91%,92%,93%,94%,95%,96%,97%,98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-FGFR 4 antibody comprising the sequence retains the ability to bind FGFR 4. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 7. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-FGFR 4 antibody comprises the VH sequence in SEQ ID No.7, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO:1, (b) HVR-H2 comprising amino acid sequence SEQ ID NO:2, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 3.

In another aspect, an anti-FGFR 4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% sequence identity to amino acid sequence DIQMTQSPSSLSASVGDRVTITCRTSQDISNFLNWYQQKPGKAFKILISYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNALPYTFGQGTKVEIKR (SEQ ID NO: 8). In certain embodiments, a VL sequence having at least 90%,91%,92%,93%,94%,95%,96%,97%,98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-FGFR 4 antibody comprising the sequence retains the ability to bind FGFR 4. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in SEQ ID NO. 8. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-FGFR 4 antibody comprises the VL sequence in SEQ ID No. 8, including post-translational modifications of this sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO 4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6.

In another aspect, there is provided an anti-FGFR 4 antibody, wherein the antibody comprises a VH in any embodiment provided above and a VL in any embodiment provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO 7 and SEQ ID NO 8, respectively, including post-translational modifications of those sequences.

In any of the above embodiments, the anti-FGFR 4 antibody is humanized. In one embodiment, the anti-FGFR 4 antibody comprises the HVRs of any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. An antibody of the invention may comprise any suitable framework variable domain sequence provided that the binding activity to FGFR4 is substantially retained, e.g., in some embodiments, an antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, the framework consensus sequence comprises a substitution at position 71, 73, and/or 78. In some embodiments of these antibodies, a is at position 71, a T is at position 73 and/or a is at position 78. In one embodiment, the antibodies comprise huMAb4D5-8 (h)Genentech, Inc., South San Francisco, Calif., USA) (see also U.S. Pat. Nos. 6,407,213 and 5,821,337 and Lee et al, J.mol.biol. (2004)340(5): 1073-. In one embodiment, the framework sequence comprises the following acceptor human framework: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:9) -L1-WYQQKPGKAFKILIS (SEQ ID NO:10) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:11) -L3-FGQGTKVEIK (SEQ ID NO: 12).

In yet another aspect, the invention provides anti-FGFR 4 antibodies that bind to the same epitope as the anti-FGFR 4 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind the same epitope as an anti-FGFR 4 antibody comprising the VH sequence SEQ ID No.7 and the VL sequence SEQ ID No. 8. In certain embodiments, antibodies are provided that bind to an epitope within a fragment of FGFR4 consisting of amino acids 145-180 of the sequence set forth in FIG. 18 (SEQ ID NO: 39).

In yet another aspect, the invention provides an anti-FGFR 4 antibody that competes for binding to human FGFR4 with an anti-FGFR 4 antibody comprising the VH sequence SEQ ID No.7 and the VL sequence SEQ ID No. 8.

In yet another aspect of the invention, the anti-FGFR 4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-FGFR 4 antibody is an antibody fragment, such as an Fv, Fab ', scFv, diabody, or F (ab') 2 fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody class or isotype, as defined herein.

In yet another aspect, an antibody according to any of the above embodiments may incorporate any of the features described in sections 1-7 below, singly or in combination:

1. affinity of antibody

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) (e.g., 10) of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M）。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with Fab versions of the antibody of interest and its antigen as described in the assays described below. By using the minimum concentration of (in the presence of unlabeled antigen in the titration series¹²⁵I) Antigen-balanced Fab is labeled and the solution binding affinity of the Fab for the antigen is measured by capturing the bound antigen with an anti-Fab antibody coating plate (see, e.g., Chen et al, J.mol.biol.293: 865)-881(1999)). To establish the assay conditions, theWells (Thermo Scientific) were coated with 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) in 50mM sodium carbonate (pH9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorbed plate (Nunc #269620), 100pM or 26pM 125I-antigen was mixed with serial dilutions of Fab of interest (e.g., consistent with the evaluation of Presta et al, Cancer Res.57:4593-4599(1997), anti-VEGF antibody, Fab-12). The Fab of interest was then incubated overnight; however, incubation may continue for longer periods of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and treated with 0.1% polysorbate 20 in PBSThe plate was washed 8 times. After drying the plates, 150. mu.l/well scintillation fluid (MICROSCINT-20) was added^TMPackard) and then in TOPCOUNT^TMPlates were counted on a gamma counter (Packard) for 10 minutes. The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays.

According to another embodiment, Kd is determined using a surface plasmon resonance assay-2000 or-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then at 5. mu.l/minThe flow rate was injected to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, polysorbate 20 (TWEEN-20) was injected at 25 ℃ at a flow rate of about 25. mu.l/min at 0.05%^TM) Two-fold serial dilutions of Fab (0.78 nM to 500 nM) in surfactant PBS (PBST). Using a simple one-to-one Langmuir (Langmuir) binding model (Evaluation Software version3.2) calculation of the Association Rate (k) by Simultaneous fitting of Association and dissociation sensorgrams_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1S^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO^TMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS ph7.2 at 25 ℃ in the presence of increasing concentrations of antigen.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')₂Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al nat. Med.9: 129-. For reviews of scFv fragments, see, for example, Pluckth ü n, The Pharmacology of monoclonal antibodies, Vol.113, Rosenburg and Moore, eds (Springer-Verlag, New York), p.269-315 (1994), see also WO93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. With respect to compositions comprising salvage receptor binding epitope residues and having extended half-life in vivoFab and F (ab')₂See U.S. Pat. No.5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP404,097, WO1993/01161, Hudson et al, nat. Med.9: 129-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1).

Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No.4,816,567, and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and Methods for their production are reviewed, for example, in Almagro and Fransson, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329(1988), Queen et al, Proc.Nat' l Acad.Sci.USA86:10029-10033(1989), U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409, Kashmiri et al, Methods36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36:61-68(2005) and Klimka et al, Br.J. cancer,83:252-260(2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies from the light or heavy chain variable regions (see, e.g., Carter et al Proc. Natl. Acad. Sci. USA,89:4285(1992); and Presta et al J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in van Dijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describesTechnology, and U.S. patent application publication No. US2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol.,133:3001(1984); Brodeur et al, Monoclonal Antibody Production Techniques and applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiaondai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlens, Histology and Histopathology,20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature348:552-554, Clackson et al, Nature352:624-628(1991), Marks et al, J.mol.biol.222:581-597(1992), Marks and Bradbury in Methods in Molecular Biology248:161-175(Lo eds., Human Press, Totowa, NJ,2003), Sidhu et al, J.mol.338 (2): 299-2004 (2004); Lee et al, J.mol.340 (5):1073 (Act.) and Fec. 124101.55): Nature: (USA) 132, and Methods in U.31: 119-132 (USA).

In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for FGFR4 and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind two different epitopes of FGFR 4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing FGFR 4. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature305:537(1983)), WO93/08829, and Traunecker et al, EMBO J.10:3655 (1991)), and "bump-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering for the generation of antibody Fc-heterodimer molecules (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al J.Immunol.147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds FGFR4 and another different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

a)Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading of "conservative substitutions". More substantial variations are provided in table 1 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 1

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196 (2008)), and/or SDRs (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom, et al, Methods in Molecular Biology178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1,2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

b)Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case of antibodies comprising an Fc region, the carbohydrate to which they are attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al TIBTECH15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108(Presta, L.); US2004/0093621(KyowaHakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108, WO2000/61739, WO2001/29246, US2003/0115614, US2002/0164328, US2004/0093621, US2004/0132140, US2004/0110704, US2004/0110282, US2004/0109865, WO2003/085119, WO2003/084570, WO2005/035586, WO2005/035778, WO2005/053742, WO2002/031140, Okazaki et al J.mol.biol.336:1239-1249(2004), Yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al Arch. biochem. Biophys.249:533-545(1986); U.S. patent application No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, inter alia, in example 11), and knock-out cell lines such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al Biotech. Bioeng.87:614(2004); Kanda, Y. et al, Biotechnol. Bioeng. 94(4):680-688(2006); and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al); U.S. Pat. No.6,602,684 (Umana et al); and US2005/0123546 (Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al); WO1998/58964(Raju, S.); and WO1999/22764(Raju, S.).

c)Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I., et al ProNat' l Acad. Sci. USA83: 7059-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al Proc. nat' l Acad. Sci. USA95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, e.g., the C1q and C3C binding ELISAs in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods202:163(1996); Cragg, M.S. et al, Blood101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those having substitutions in one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO2004/056312, and Shields et al, J.biol. chem.9(2):6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO99/51642, and Idusogene et al J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al), the neonatal Fc receptor (FcRn) and are responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

See also Duncan and Winter, Nature322:738-40(1988), U.S. Pat. No.5,648,260, U.S. Pat. No.5,624,821, and WO94/29351, which focus on other examples of Fc region variants.

d)Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

e)Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to generate antibodies, for example, as described in U.S. Pat. No.4,816,567. In one embodiment, isolated nucleic acids encoding the anti-FGFR 4 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH (e.g., a light and/or heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-FGFR 4 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture broth).

For recombinant production of anti-FGFR 4 antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199 and 5,840,523 (see also Charlton, Methods in molecular biology, Vol.248 (compiled in B.K.C.Lo., Humana Press, Totowa, NJ,2003), pp.245-254, which describes expression of antibody fragments in E.coli (E.coli)). After expression, the antibody can be isolated from the bacterial cell mass paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells as described, e.g., in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM 4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; bovine rat (buffalo rate) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells, as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells, and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc.Natl.Acad.Sci.USA77:4216(1980)), and myeloma cell lines such as Y0, NS0, and 2/0. for reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.K.248, Human C.255, Towa 268, Prewako J.2003 (Tornaya), Vol.S.S.J.268).

C. Assay method

The anti-FGFR 4 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

1. Binding assays and other assays

In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA or Western blot.

In another aspect, a competition assay can be used to identify antibodies that compete with antibody LD1 for binding to FGFR 4. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as that bound by antibody LD1. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular biology vol.66(Humana Press, Totowa, NJ).

In one exemplary competition assay, immobilized FGFR4 is incubated in a solution comprising a first labeled antibody (which binds FGFR4, e.g., antibody LD 1) and a second unlabeled antibody (which is to be tested for the ability to compete with the first antibody for binding to FGFR 4). The second antibody may be present in the hybridoma supernatant. As a control, immobilized FGFR4 was incubated in a solution comprising the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind FGFR4, excess unbound antibody is removed and the amount of label associated with immobilized FGFR4 is measured. If the amount of marker associated with immobilized FGFR4 in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to FGFR 4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity assay

In one aspect, assays for identifying anti-FGFR 4 antibodies having biological activity are provided. Biological activity may include, for example, inhibiting: FGF (e.g., FGF 1) -stimulated FGFR 4-expressing cells (e.g., HUH7 cells) proliferation, FGF 19-mediated inhibition of CYP7 α 7 expression in FGF 19-exposed cells, FGF 19-induced phosphorylation of FGFR4, MAPK, FRS2, and/or ERK2 in FGF 19-exposed cells, and FGF 19-induced colony formation (in some embodiments, HCC cell line colony formation).

Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, antibodies of the invention are tested for such biological activity. Assays for testing such biological activities are described herein.

In certain embodiments, the antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Certain assays for cell proliferation, exemplified by the "cell killing" assay described herein, measure cell viability (viability). One such assay is CellTiter-Glo^TMA luminescent cell viability assay, commercially available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells. See Crouch et al (1993) J.Immunol.meth.160:81-88; U.S. Pat. No. 6602677. The assay can be performed in 96-well or 384-well format, adapting it to automated High Throughput Screening (HTS). See Cree et al (1995) AntiCancer Drugs6: 398-404. The assay protocol involves adding a single reagent directly to the cultured cells: (Reagent). This results in cell lysis and generation of a luminescent signal generated by the luciferase reaction. The luminescent signal is directly proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data may be recorded by a photometer or a CCD camera imaging device. Luminous output is expressed as Relative Light Units (RLU).

Another assay for cell proliferation is the "MTT" assay, a method that measures the oxidation of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium nitrogen bromide to formazan by mitochondrial reductase(formalzan) in a colorimetric assay. Like CellTiter-Glo^TMAs with the assay, this assay indicates the number of metabolically active cells present in the cell culture. See, e.g., Mosmann (1983) J.Immunol.Meth.65:55-63, and Zhang et al (2005) Cancer Res.65: 3877-.

Cells for use in any of the above in vitro assays include cells or cell lines that naturally express FGFR4 or that are engineered to express FGFR 4. Such cells include tumor cells that overexpress FGFR4 relative to normal cells of the same tissue origin. Such cells also include cell lines that express FGFR4 (including tumor cell lines) and cell lines that do not normally express FGFR4 but are transfected with a nucleic acid encoding FGFR 4. Exemplary cell lines provided herein for use in any of the above in vitro assays include the HCC cell line HUH 7.

In one aspect, the anti-FGFR 4 antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-FGFR 4 antibodies of the invention are tested for their ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such tests. In an exemplary xenograft system, human tumor cells are introduced into an appropriately immunocompromised non-human animal, such as a athymic "nude" mouse. Administering an antibody of the invention to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the xenograft system described above, the human tumor cell is a tumor cell from a human patient. Such xenograft models are available from OncotestGmbH (Frieberg, Germany). In certain embodiments, the human tumor cell is a cell from a human tumor cell line, such as a HUH7HCC tumor cell. In certain embodiments, the human tumor cells are introduced into the appropriate immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site (such as a breast fat pad). In certain embodiments, the xenograft model is a transgenic mouse overexpressing FGF19, e.g., as described herein and in nucleotides et al, Am J Pathol160: 2295-.

It is to be understood that any of the above assays may be performed using the immunoconjugate of the invention instead of or in addition to the anti-FGFR 4 antibody.

It is to be understood that any of the above assays may be performed using the anti-FGFR 4 antibody and other therapeutic agents, such as chemotherapeutic agents.

D. Immunoconjugates

The invention also provides immunoconjugates comprising the anti-FGFR 4 antibodies herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In another embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP 0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342(1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin, or fragment thereof, including but not limited to diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin (ricin) a chain, abrin (abrin) a chain, modeccin (modeccin) a chain, α -sarcin (sarcin), aleurites (aleurites fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolaccai americana) protein (papapi, PAPII and PAP-S), Momordica charantia (mordica charrantia) localized inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapacicularia), leptinolide (phycin) inhibitor, gelonin (gelonin) inhibitor, gelonin (gelonin), gelonin (e) localized protein (S), gelonin (trichomycin (sancin), or a protein (sanmycin), or fragment thereof, or a toxin (sanmycin) or a) inhibitor, or a toxin (sanmycin) or a) or, Enomycin (enomycin) and trichothecenes (trichothecenes).

In one embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res52: 127-.

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-FGFR 4 antibodies provided herein can be used to detect the presence of FGFR4 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as breast/breast, pancreas, esophagus, lung, and/or brain.

In one embodiment, anti-FGFR 4 antibodies are provided for use in a diagnostic or detection method. In yet another aspect, methods of detecting the presence of FGFR4 in a biological sample are provided. In certain embodiments, the method comprises contacting the biological sample with an anti-FGFR 4 antibody under conditions permissive for binding of the anti-FGFR 4 antibody to FGFR4, as described herein, and detecting whether a complex is formed between the anti-FGFR 4 antibody and FGFR 4. Such methods may be in vitro or in vivo. In one embodiment, the anti-FGFR 4 antibody is used to select a subject suitable for treatment with an anti-FGFR 4 antibody, e.g., wherein FGFR4 is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancers (e.g., breast, lung, pancreatic, brain, kidney, stomach, leukemia, endometrial, colon, prostate, pituitary, breast fibroadenomas, head and neck, soft tissue, neuroblastoma, melanoma, endometrial, testicular, bile duct, gall bladder, and liver cancers).

In certain embodiments, labeled anti-FGFR 4 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H. And¹³¹I. fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine (rhodamine) and derivatives thereof, dansyl, umbelliferone, luciferases, e.g., firefly and bacterial luciferases (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to an enzyme employing a hydrogen peroxide oxidation dye precursor such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical formulations

Pharmaceutical formulations of anti-FGFR 4 antibodies as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)) in a lyophilized formulation or in an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenols, butanols or benzyl alcohols; p-butyl alcohol; benzylHydrocarbyl hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersant such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those compounds whose activities are complementary and do not adversely affect each other. For example, it may be desirable to further provide an EGFR antagonist (such as erlotinib), an anti-angiogenic agent (such as a VEGF antagonist, such as an anti-VEGF antibody), or a chemotherapeutic agent (such as a taxane or platinum agent). Such active components are suitably present in combination in amounts effective for the desired purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any of the anti-FGFR 4 antibodies provided herein can be used in a therapeutic method.

In one aspect, anti-FGFR 4 antibodies for use as a medicament are provided. In other aspects, anti-FGFR 4 antibodies for use in treating cancer are provided. In other aspects, anti-FGFR 4 antibodies for use in treating liver disease (e.g., cirrhosis) are provided. In other aspects, anti-FGFR 4 antibodies for use in treating wasting disorders are provided. In certain embodiments, anti-FGFR 4 antibodies for use in methods of treatment are provided. In certain embodiments, the invention provides an anti-FGFR 4 antibody for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of an anti-FGFR 4 antibody. In certain embodiments, the invention provides anti-FGFR 4 antibodies for use in a method of treating an individual having a liver disorder (such as cirrhosis), the method comprising administering to the individual an effective amount of an anti-FGFR 4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In still other embodiments, the invention provides anti-FGFR 4 antibodies for use in inhibiting cell proliferation.

In certain embodiments, the invention provides an anti-FGFR 4 antibody for use in a method of inhibiting cell proliferation in an individual, comprising administering to the individual an effective amount of an anti-FGFR 4 antibody to inhibit cell proliferation. An "individual" according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides the use of an anti-FGFR 4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating cancer. In yet another embodiment, the medicament is for use in a method of treating cancer, comprising administering to an individual having cancer an effective amount of the medicament. In one embodiment, the medicament is for the treatment of a liver disorder (such as cirrhosis, non-alcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis). In yet another embodiment, the medicament is for use in a method of treating a liver disorder (such as cirrhosis, non-alcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis), comprising administering to an individual with the liver disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In yet another embodiment, the medicament is for inhibiting cell proliferation. In yet another embodiment, the medicament is for use in a method of inhibiting cell proliferation in an individual, the method comprising administering to the individual an effective amount of the medicament to inhibit cell proliferation. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides a method of treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an anti-FGFR 4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides methods of treating liver disorders (such as cirrhosis, non-alcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis). In one embodiment, the method comprises administering to an individual having such a liver disorder an effective amount of an anti-FGFR 4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides a method for treating a wasting disorder. In one embodiment, the method comprises administering to an individual having such a wasting disorder an effective amount of an anti-FGFR 4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides a method for inhibiting cell proliferation in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-FGFR 4 antibody to inhibit cell proliferation. In one embodiment, the "individual" is a human.

In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-FGFR 4 antibodies provided herein, e.g., for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-FGFR 4 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-FGFR 4 antibodies provided herein and at least one other therapeutic agent, e.g., as described below.

The antibodies of the invention may be used alone or in combination with other agents in therapy. For example, an antibody of the invention can be co-administered with at least one other therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are contained in the same or different formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiotherapy.

The antibodies of the invention (and any other therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The antibodies of the invention should be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical status of the individual patient, the cause, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to practitioners. The antibody need not be, but may optionally be, formulated with one or more drugs currently used to prevent or treat the condition. The effective amount of such other drugs will depend on the amount of antibody present in the formulation, the type of condition being treated, and other factors discussed above. These agents are generally used at the same dosage and with the administration routes described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route, as empirically determined/clinically determined appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the prophylactic or therapeutic purpose for which the antibody is administered, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg to 10 mg/kg) of the antibody may be administered to the patient as a first candidate amount, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment should generally be continued until the desired suppression of the condition occurs. The above-described dose can be administered intermittently, such as once per week or once every three weeks (e.g., such that the patient receives from about 2 to about 20, or, for example, about 6 doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosing regimens may be used. The progress of the treatment is readily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods may be practiced using the immunoconjugates of the invention in vitro in place of the anti-FGFR 4 antibody and in anti-FGFR 4 antibody.

H. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the conditions described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention, and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates the use of the composition to treat the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is to be understood that any of the above-described preparations may include an immunoconjugate of the invention in place of or in addition to the anti-FGFR 4 antibody.

Example III

A. Treatment with anti-FGFR 4 antibody inhibits hepatocellular carcinoma

Materials and methods

And (4) carrying out computer expression analysis. For expression analysis, the expression vector was obtained from Bioexpress^TMNormalized Gene expression data extracted from databases (Gene Logic, Gaithersburg, Md.) generated a box plot (box-plot) and a line plot (whisker-plot) for FGFR 4. The distribution of FGFR4 expression in normal and cancer tissues was assessed using signals associated with probe 204579_ at.

Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue sections were treated with Trilogy (Cell Marque, Rocklin, CA) for antigen retrieval and then incubated with 10 μ G/ml anti-FGFR 4 antibody (8G 11; Genentech, South San Francisco, CA). Immunostaining was achieved using biotinylated secondary antibody ABC-HRP reagent (Vector Laboratories, Burlingame, Calif.) and metal-enhanced DAB colorimetric peroxidase substrate (Thermo Fisher Scientific, Rockford, IL).

Semi-quantitative RT-PCR. Total RNA was extracted using RNeasy kit (Qiagen, Valencia, CA). Gene expression was amplified and quantified using specific primers and fluorescent probes (31). Gene-specific signals were normalized to RPL19 housekeeping genes. All TaqMan qRT-PCR reagents were purchased from applied biosystems (Foster City, Calif.). A minimum of three sets of data were analyzed for each condition. Data are expressed as mean ± SEM.

Immunoprecipitation and immunoblotting. Lysates of cultured cells or frozen tissues were prepared with RIPA lysis buffer (Millipore, Billerica, MA) supplemented with the complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN), phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich, st. louis, MO), 2mM sodium fluoride, and 2mM sodium orthovanadate. Equal amounts of protein determined by the BCA assay (Thermo Fisher Scientific) were analyzed by immunoblot analysis using antibodies against FGFR4 (8G 11; Genentech), FGFR3 (Santa Cruz Biotechnology, Santa Cruz, CA), FGFR2 (GeneTex, Irvine, CA), and FGFR1 (Santa Cruz Biotechnology). For human liver lysates, the immunoblot analysis was preceded by FGFR4 immunoprecipitation as previously described (16).

Production of FGFR4 monoclonal antibodies. FGFR4 null mice were immunized with recombinant human and mouse FGFR4-Fc chimeric proteins (Genentech). Spleens were harvested 8 weeks later and hybridomas were generated. Culture supernatants were collected and screened for immunogens by a solid phase antibody binding assay. Positive cell lines were further screened for their efficacy in inhibiting FGF1 and FGF19 binding to human and mouse FGFR4 using a solid phase receptor binding assay. The LD 1-producing hybridoma was subcloned twice to ensure monoclonality.

Molecular cloning of LD1. Total RNA was extracted from muLD 1-producing hybridoma cells using RNeasy mini kit (Qiagen). The light and heavy chain variable domains were amplified using reverse transcription-PCR (RT-PCR). The forward primers are specific for the NH2 terminal amino acid sequences of the light and heavy chain variable regions. Light chain and heavy chain reverse primers were designed to anneal to regions of the light chain constant domain and the heavy chain first constant domain, respectively, that are highly conserved across species. The amplified light chain variable domain is cloned into a mammalian expression vector comprising a human kappa constant domain. The amplified heavy chain variable domain was inserted into a mammalian expression vector encoding the constant domain of full-length human IgG 1. Chimeric antibodies were transiently expressed as previously described (16). The experiments described in this section a of the examples (including figures 1-9) used chimeric LD1.

Solid phase antibody binding assays. Maxisorp96 well plates were coated overnight at 4 ℃ with 50. mu.l of 2. mu.g/ml anti-human immunoglobulin Fc fragment specific (Jackson ImmunoResearch Laboratories, West Grove, Pa.) or anti-FLAG antibody (Sigma-Aldrich). Non-specific binding sites were saturated with 200. mu.l PBS/3% Bovine Serum Albumin (BSA) for 1 hour, and FGFR-IgG (Genentech and R & D Systems, Minneapolis, MN) or FLAG-tagged FGFR4 (FGFR 4. DELTA. TM. -FLAG) in PBS/0.3% BSA was incubated for 1 hour. Plates were washed and incubated with anti-FGFR 4 antibody in PBS/0.3% BSA for 1 hour. Bound antibodies were detected using HRP conjugated anti-igg (jackson immunoresearch laboratories) and TMB peroxidase chromogenic substrate (KPL, Gaithersburg, MD).

Flow cytometry analysis. Cells for flow cytometry analysis were resuspended in PBS containing 5mM EDTA and washed with PBS containing 2% heat-inactivated Fetal Bovine Serum (FBS). All subsequent steps were performed on ice. Cells (1X 10)⁶Individual) were incubated with primary antibody (LD 1 or isotype control) for 30 minutes followed by Phycoerythrin (PE) conjugated anti-human IgG antibody (Jackson ImmunoResearch). Cells were analyzed by FACScan flow cytometry (BD Biosciences, San Jose, Calif.).

A DNA construct. Human FGFR4 (hFGFR 4) cDNA (16) was cloned as previously described. The extracellular domain of FGFR4 was also subcloned into the expression vector pCMV-Tag4A (Stratagene, La Jolla, Calif.) to obtain a secreted form bearing a FLAG Tag at the C-terminusFGFR4 (FGFR 4. DELTA. TM. -Flag). Single nucleotide mutations were introduced in FGFR4 Δ TM-Flag constructs using the QuikChange XL site-directed mutagenesis kit (Stratagene). We also generated a human FGFR4FGFR1 chimeric construct (hFGFR 4/R1) comprising the extracellular and transmembrane domains of human FGFR4 (amino acid residues M1-G392 of FGFR 4) and the cytoplasmic domain of human FGFR1 (amino acid residues K398-R820 of FGFR 1) fused together. Linking FGFR4 (bold) and FGFR1 (m) ((m))In general) Has an amino acid sequence of (A) · AVLLLLAGLYRGKMKSG32 (SEQ ID NO: 32). The hFGFR4cDNA or hFGFR4/R1cDNA was ligated into pQCXIP retrovirus bicistronic expression vector (Clontech Laboratories, Mountain View, Calif.).

FGFR4 Δ TM-Flag conditioned medium. HEK293 cells were transfected with wild type or mutant FGFR4 Δ TM-Flag constructs or the corresponding empty vectors and maintained in serum-free PS25 medium for 72 to 96 hours. The resulting medium was filtered, supplemented with HEPES pH7.2 (final concentration 40 mM) and protease inhibitors, and stored at 4 ℃ until use.

Cell culture and stable cell lines. HEK293, HEPG2, and HEP3B cells were obtained from the American type culture Collection (ATCC, Manassas, Va.) and maintained in F12: DMEM mixture (50: 50) supplemented with 10% FBS and 2mM L-glutamine. HUH7 and PLC/PRF/5 cells were cultured in DMEM high glucose, 10% FBS. JHH4, JHH5, and JHH7 cells were purchased from the Japanese cancer research resource Bank (Tokyo, Japan) and maintained in Williams Medium E supplemented with 10% FBS and 2mM L-glutamine. SNU449 cells were obtained from ATCC and maintained in RPMI1640 containing 10% FBS and 2 ml-glutamine. BaF3 cells were maintained in RPMI1640 (Life Technologies, Carlsbad, Calif.) supplemented with 10% FBS, 1ng/ml IL-3, and 2mM L-glutamine. L6 cells were obtained from ATCC and maintained in DMEM high glucose supplemented with 10% FBS.

Cultures of BaF3 and L6 cells were infected with empty, hFGFR4, or hFGFR4/R1 retroviral expression vectors according to the manufacturer's recommendations and selected for 10 to 12 days in medium containing 2.5 μ g/mL puromycin (life technologies). The highest expressing cells in the fifth percentile were isolated from the selected pool by Fluorescence Activated Cell Sorting (FACS) using an anti-FGFR 4 antibody (8G 11; Genentech). The resulting pool of cells expressing high levels of FGFR4, high levels of FGFR4/R1 and control cells stably transfected with empty vector were maintained in complete medium containing 2.5 μ g/mL puromycin.

Mitogenic assays. BaF 3/control, BaF3/FGFR4, and BaF3/FGFR4/R1 cells were washed twice and 96-well plates (22,500 cells/well) were seeded in RPMI1640 supplemented with 10% FBS, 2mM L-glutamine, and 2 μ g/ml heparin. FGF was added to each well and cells were incubated for 72 hours at 37 ℃. Relative cell density was measured using CellTiter Glo (Promega, Madison, WI) according to the manufacturer's recommendations.

FGF pathway activated anti-FGFR 4 antibody inhibition. Cells were serum starved for 24 hours in the absence or presence of LD1 or isotype control antibody. They were then stimulated with 5ng/ml FGF1 (FGF acidic, R & DSystems) and 10. mu.g/ml heparin for 5 minutes. Cells were lysed with RIPA lysis buffer (Millipore) supplemented with intact EDTA-free protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich), 2mM sodium fluoride, and 2mM sodium orthovanadate. Equal amounts of protein were analyzed by immunoblotting using antibodies against ERK1/2 phosphate, FRS2 phosphate, ERK1/2 (Cell Signaling Technology, Danvers, MA), and FRS2 (Millipore).

Clonogenic (clonogenic) assay. HUH7 (5,000 cells/well), PLC/PRF/5 (2,000 cells/well), JHH5 (500 cells/well), or JHH5/hFGFR4shRNA (500 cells/well) cells were seeded in triplicate in 2ml medium/well in 6-well plates. 3 hours after inoculation, HUH7 and PLC/PRF/5 cells were treated with or without anti-FGFR 4 antibody (chLD 1; Genentech). The antibodies were changed twice a week during the experiment (14 days). For JHH5 and JHH5/hFGFR4shRNA cells, treatment was initiated 3 hours post inoculation with or without 2mg/ml doxycycline and replaced three times per week during the experiment. Cells were washed with PBS and stained with 0.5% crystal violet solution. Colonies were counted using MetaMorph software (Molecular Devices, Sunnyvale, Calif.).

In vivo experiments. All animal protocols were approved by the research animal care and use committee. Female nu/nu mice 5 to 6 weeks old were obtained from Charles River Laboratories International (Wilmington, Mass.). Mice were provided with standard feed and water ad libitum until 12 hours prior to injection, at which time the feed was removed. Mice were given Intraperitoneal (IP) injections (10 mg/kg) of either control or anti-FGFR 4 (chLD 1) antibody. After 18 hours, the mice received either vehicle (PBS) or 1mg/kg FGF19 Intravenously (IV). After 30 minutes, mice from all groups were necropsied and tissue samples were collected, frozen in liquid nitrogen, and stored at-70 ℃. Total RNA from frozen tissue samples was prepared using RNeasy kit (Qiagen). Each condition was analyzed for groups of 3 to 5 animals. Data are expressed as mean ± SEM and analyzed by Student's t assay.

For xenograft experiments, 6 to 8 week old nu/nu female mice (Charles river laboratories International) were inoculated subcutaneously with 5x10⁶Individual cells (200 ul/mouse) and matrigel (bdbiosciences). After 7 days, an equivalent volume (. about.150 mm) will be carried³) Mice of tumors (n = 10) were randomized into groups and IP-treated twice weekly. Tumors were measured with an electronic caliper (Fowler Sylvac Ultra-Cal MarkIII; Fred V.Fowler Company, Newton, Mass.) and using the formula (W)²x L)/2 the mean tumor volume was calculated, where W and L are the smaller and larger diameters, respectively. Data are expressed as mean tumor volume ± SEM and analyzed by Student's t assay.

FGF19 transgenic mice were generated as previously described (32). FGFR4 deletion-free mutant (FGFR 4-KO) animals were constructed as previously reported (33) and were provided by w.l. mckeehan in accordance with the material transfer protocol (University of Texas south western Medical Center, Dallas, TX). Mice that overexpress both FGF19 and lack FGFR4 receptors (FGF 19-TG: FGFR 4-KO) were generated by crossing young adult FGF19-TG males with young adult FGFR4-KO females. The presence of two genetic engineering events was confirmed by tail DNA PCR at weaning.

Results

FGFR4 is required for liver cancer development in FGF19 transgenic mice. It was previously shown that exogenous expression of FGF19 in transgenic mice elicits HCC at 10 months of age (18). To assess whether FGFR4 was involved in this FGF 19-mediated tumorigenesis, we mated FGF19 transgenic (FGF 19-TG) mice with FGFR4 knockout (FGFR 4-KO) mice or FGFR4 wild type (FGFR 4-WT) mice. Mice were necropsied at various time points and hepatoma incidence was assessed by performing visual and histological examination and by measuring pre-neoplastic (prenoplastic) hepatocyte proliferation (i.e., BrdU incorporation). The development of HCC in FGF19-TG: FGFR4-WT mice was previously described (18). In contrast to FGF19-TG: FGFR4-WT mice, FGF19-TG: FGFR4-KO mice did not develop macroscopic or histological evidence of hepatoma formation at any time during this experiment (FIG. 1A). Also, pre-neoplasia hepatocyte proliferation was significantly elevated in FGF19-TG mice with the genotype FGFR4-WT, but not in FGF19-TG: FGFR4-KO littermates (FIG. 1B). In agreement with the previously reported higher frequency and severity of tumor development in female FGF19-TG mice (18), BrdU incorporation was increased in FGF19-TG: FGFR4-WT females compared to corresponding males (compare the left and right panels of FIG. 1B). We also evaluated the effect of the potent liver carcinogen Diethylnitrosamine (DEN) on the development of HCC in FGF19-TG mice. Administration of DEN accelerated HCC development in FGF19-TG: FGFR4-WT mice. Pre-neoplastic and neoplastic lesions-altered (basophilic) liver foci, central peripheral hepatocyte dysplasia, well differentiated hepatomas, and aggressive hepatocellular carcinomas were seen over the entire range in the liver from all the DEN-treated FGF19-TG: FGFR4-WT animals by 4 months of age compared to the 10 months of age of mice of FGF19-TG: FGFR4-WT without DEN treatment (fig. 1D). The main morphological feature of the liver from almost all FGF19-TG: FGFR4-WT mice at all time points was the presence of macroscopically evident HCC nodules on multiple leaves (fig. 1C). Tumor burden was assessed by measuring liver weight. Relative liver weights were progressively increased in FGF19-TG: FGFR4-WT mice treated with DEN at all time points (FIG. 1E). Interestingly, the liver weight increase was more pronounced in females (2.7-fold at 6 months) than in males (1.8-fold at 6 months) (compare the left and right panels of fig. 1E). It should be noted that none of the males survived more than 6 months of age (fig. 1E). The hepatoma development observed in FGF19-TG: FGFR4-WT mice treated with DEN was abolished by abolishing FGFR4 expression in FGFR4-KO mice. Thus, the relative liver weights of FGF19-TG: FGFR4-KO mice remained constant during adulthood (FIG. 1F). These results suggest that FGFR4 expression is required for liver cancer development promoted by FGF19 in mice.

Production of neutralizing monoclonal antibodies against FGFR 4. To assess whether targeting FGFR4 could have a therapeutic effect in HCC, we generated FGFR 4-specific monoclonal antibodies by immunizing FGFR4-KO mice with recombinant mouse and human FGFR 4. One of the resulting clones (designated LD 1) was selected based on the specificity of binding to mouse, cynomolgus, and human FGFR4 (fig. 2A). Such antibodies do not bind mouse or human FGFR1, FGFR2, or FGFR3 (fig. 2A). Surface plasmon resonance analysis revealed that LD1 binds mouse, cynomolgus, and human FGFR4 with comparable affinity (fig. 2B). We used flow cytometry to assess whether LD1 binds FGFR4 present on the cell surface. Specific binding of LD1 to HEK293 cells stably transfected with human FGFR4 was directly proportional to the added antibody concentration (fig. 2C). LD1 did not bind to control HEK293 cells stably transfected with the empty vector. Together, these data demonstrate that LD1 specifically binds mouse, cynomolgus, and human FGFR4 and also recognizes human receptors expressed on the cell surface.

To locate the FGFR4 epitope for LD1, we compared the amino acid sequences of mouse and human FGFR1, FGFR2, FGFR3, and FGFR 4. Eight amino acids were selected based on their similarity between the FGFR4 orthologs and their dissimilarity among the FGFR1-3 orthologs. These amino acids in FGFR4 were replaced with the amino acid present at the equivalent position in FGFR3 to generate eight different mutant constructs of human FGFR 4. These constructs were expressed and LD1 binding was assessed using a solid phase binding assay. LD1 bound equally well to wild-type FGFR4 and most mutant constructs; G165A was the only FGFR4 mutant with impaired LD1 binding (fig. 2D). LD1 did not bind to the negative control wild-type FGFR3 (fig. 2D). We also tested LD1 binding to the mutant construct using immunoblot analysis. All previously described protein constructs were reduced, denatured, electrophoresed, and electrotransferred to nitrocellulose. The nitrocellulose membranes were incubated sequentially with LD1, an anti-FGFR 4 antibody (8G 11) recognizing a different epitope, or an anti-FLAG antibody. Wild-type FGFR4 and all FGFR4 mutant constructs were detected by anti-FLAG antibody and 8G11, while LD1 detected all constructs equally well with the exception of the G165A mutant (fig. 2E). No protein bands were detected by any antibody in the control lane (fig. 2E). We generated a three-dimensional model of FGFR4 dimer binding to two molecules of FGF19 to visualize the position of G165 (fig. 2F). G165 is located in the center of the FGFR4-FGF19 complex, at the point of contact between the two FGFR4 subunits. Together, these results show that G165 is critical for the interaction of LD1 with human FGFR 4. LD1 binds to reduced and denatured FGFR4 also suggesting that the epitope is not dependent on tertiary conformation.

We next tested whether LD1 could block FGF1 and FGF19 from binding FGFR4 using a solid phase receptor binding assay. LD1 inhibited FGF binding dose-dependently and IC50 reached 0.093 ± 0.006nM for FGF1 and 0.102 ± 0.003nM for FGF19 (fig. 3A). To assess whether LD1 could inhibit the function of FGFR4 expressed on the cell surface, we first utilized a BaF3 mouse pre-B cell line (pro-bclell line) stably transfected with a chimeric construct encoding the extracellular domain of FGFR4 and the intracellular domain of FGFR1 (BaF 3/FGFR 4/R1). The wild-type BaF3 cell line is an interleukin-3 (IL-3) -dependent cell line that does not express any FGFR. BaF3 cells transfected with FGFR proliferate in the absence of IL-3 upon stimulation with FGF and heparin (21). Transfection of this construct allowed us to replace IL-3 with FGF to support the growth of BaF3 cells. LD1 inhibited BaF3/FGFR4/R1 cell proliferation in the presence of 5nM FGF1, with an IC50 of 17.4 ± 5.4nM (fig. 3B).

We also used L6 rat skeletal muscle cell line (L6/FGFR 4) stably transfected with a vector expressing FGFR4 to assess the effect of LD1 on FGF signaling. Addition of FGF1 and heparin to L6/FGFR4 cell cultures activated the FGFR pathway as demonstrated by phosphorylation of FGFR substrate 2 (FRS 2) and extracellular signal-regulated kinase 1/2 (ERK 1/2), while LD1 inhibited ligand-induced phosphorylation of these second messengers in a dose-dependent manner (fig. 3C). Interestingly, addition of LD1 also triggered an increase in total FRS2 content in these cells (fig. 3C).

Using flow cytometry, we evaluated the binding of LD1 and confirmed the expression of FGFR4 on the cell surface of a subset of HCC cell lines. LD1 bound PLC/PRF/5 to the highest extent, while HUH7 and JHH5 cells to a lesser extent (FIG. 3D). Binding of control antibodies to the surface of these cells was negligible (fig. 3D). Furthermore, binding of LD1 and control antibodies to the surface of BaF3 cells (which served as a negative control because they did not express FGFR 4) was also negligible (fig. 3D).

LD1 inhibits FGFR4 function in liver cancer cell lines. The inhibitory activity of LD1 was characterized using liver cancer cell lines with various levels of endogenous FGFR (i.e., FGFR 1-4) expression (fig. 7). In HEP3B cells, addition of FGF19 triggered phosphorylation of FRS2 and ERK1/2, while LD1 inhibited FRS2 phosphorylation stimulated by FGF19 (fig. 4A), similar to its effect on L6/FGFR4 cells. However, LD1 did not appreciably alter ERK1/2 phosphorylation (FIG. 4A).

The expression of cytochrome P4507 α 1 (CYP 7 α 1) and c-Fos in the liver cell line is regulated by FGF19 (16, 22). We tested whether LD1 could inhibit this FGF 19-mediated gene regulation. In HEB3B cells, addition of FGF19 reduced CYP7 α 1 expression by 81% (fig. 4B). Addition of LD1 restored 67% of CYP7 α 1 basal expression (fig. 4B). LD1 increased CYP7 α 1 expression 2-fold without FGF19 addition (fig. 4B). Although addition of FGF19 did not affect CYP7 α 1 expression in HUH7 cells, addition of LD1 had a similar effect as in HEP3B cells, i.e., increased expression of this gene by 2.9 and 3.5 fold in the presence or absence of FGF19, respectively (fig. 8). The addition of negative control antibody had no effect on CYP7 α 1 expression in either HEP3B or HUH7 cells (fig. 4B and 8, respectively). Interestingly, addition of LD1 resulted in up-regulation of CYP7 α 1 expression in both HEP3B and HUH7 cells without exogenous addition of FGFR4 ligand. This indicates that LD1 inhibits the basal activity of FGFR4, which is likely to be maintained by the FGFR4 ligand autocrine/paracrine loop.

To further assess the effect of LD1 on the basal activity of FGFR4, we measured c-Fos expression without exogenous addition of FGFR4 ligand. Activation of the FGFR4 pathway was previously shown to increase c-Fos expression (16). The addition of LD1 reduced basal expression of c-Fos by 50% in JHH5, JHH7, and HUH7 cell lines and by 75% in PLC/PRF/5 cell lines; addition of the control antibody had no effect on basal C-Fos expression (fig. 4C). These results demonstrate the ability of LD1 to inhibit FGFR4 basal activity.

LD1 inhibited colony formation. We first measured colony formation by JHH5 cells stably transfected with doxycycline-inducible FGFR 4-specific shRNA or control shRNA. Although JHH5 cells transfected with the control construct did not differ in their ability to form colonies in the absence or presence of doxycycline, addition of doxycycline to JHH5 cells transfected with the FGFR4shRNA construct inhibited colony formation by 76% compared to cells in the absence of doxycycline (fig. 4D). This result suggests that FGFR4 is involved in colony formation of liver cancer cell lines.

We next tested the ability of LD1 to inhibit colony formation for a panel of liver cancer cell lines. Addition of LD1 to cultures of JHH5, HUH7, and PLC/PRF/5 cells caused a dose-dependent decrease in dose formation, reaching maximum inhibition of 26%, 50%, and 82%, respectively (fig. 4F). FIG. 4E shows representative examples of PLC/PRF/5 and HUH7 cell cultures. Addition of control antibody did not affect colony formation (fig. 4E and 4F). These results indicate that LD1 inhibits colony formation mediated by FGFR4 in liver cancer cell lines.

LD1 inhibits FGFR4 activity in vivo. We evaluated the in vivo efficacy of LD1 by measuring c-Fos induction triggered by FGF19 in the liver of mice injected with LD1 or a control antibody. We chose to monitor the response of c-Fos to FGF19, since c-Fos induction in the liver was sensitive to FGF19 stimulation (16). c-Fos expression in the liver of mice treated with FGF19 was 53-fold higher compared to the liver of mice treated with Phosphate Buffered Saline (PBS) (fig. 5A). LD1 administered 18 hours prior to FGF19 injection reduced c-Fos induction by 3.5 fold (fig. 5A). LD1 also reduced the basal level of c-Fos expression in untreated mice by 6-fold (fig. 5A). Injection of control antibody did not alter basal or FGF 19-stimulated c-Fos expression compared to untreated mice (fig. 5A). These data demonstrate the in vivo efficacy of LD1 in inhibiting basal and FGF 19-stimulated FGFR4 activity.

LD1 inhibits tumor growth in vivo. To examine the in vivo efficacy of LD1 in inhibiting tumor growth, we first utilized the HUH7 liver cancer cell line xenograft model. To carry an established tumor (approximately 150 mm)³) The mice were administered 30mg/kg LD1, 30mg/kg control antibody, or PBS once a week. After 13 days, HUH7 tumors from mice treated with either PBS or control antibody grew to an average size of 720mm³(FIG. 5B). However, HUH7 tumors of LD 1-treated mice grew to an average size of 28mm³Tumor growth was inhibited by 96% compared to control antibody or PBS (fig. 5B). In repeated experiments, 30mg/kg LD1 administered twice weekly caused complete tumor growth inhibition (FIG. 9). At necropsy, tumors were excised and the effect of LD1 on FGFR4 and the expression of genes regulated by FGFR4 was evaluated. Administration of LD1 did not affect FGFR4 expression in HUH7 xenograft tumors (fig. 5C). However, LD1 increased CYP7 α 1 expression by 3-fold compared to CYP7 α 1 expression levels measured in tumors of mice treated with PBS (fig. 5C). LD1 also reduced the expression of C-Fos and egr-1 by 17 and 6 fold, respectively, compared to mice treated with PBS (FIG. 5C).

To further assess the in vivo efficacy of LD1, we used the FGF19-TG mouse model. FGF19-TG mice were treated with DEN at 15 days of age to accelerate tumorigenesis, and then randomized into 3 groups at 4 weeks of age. One group received control antibody and the other two groups received either LD1 or anti-FGF 19 antibody (1 a 6) on a weekly basis. 1A6 was previously shown to prevent tumor formation in FGF19-TG mice (23). After 6 months, mice were necropsied and livers excised for analysis. The livers of mice treated with control antibody had macrosuberculi apparent from an eye on multiple leaves (fig. 5D). However, there was no evidence of neoplasia in the livers of mice treated with LD1 (fig. 5D) or 1a 6. We also measured liver weight to assess tumor burden, as this parameter was previously shown to correlate strongly with percent tumor volume in the FGF19-TG model (18, 23). The weight of the liver from mice treated with LD1 or 1a6 (p =0.035 and p =0.052, respectively) was significantly reduced compared to the weight of the liver from mice treated with the control antibody (fig. 5E). The difference in liver weight between mice treated with LD1 and mice treated with 1a6 was not significant (p = 0.439) (fig. 5E). Together, these data clearly demonstrate the in vivo efficacy of LD1 in inhibiting hepatocellular carcinoma in preclinical models.

FGFR4 expression is altered in cancer. We assessed FGFR4 expression in a variety of human normal and cancerous tissues by analyzing the BioExpress database (GeneLogic, inc., Gaithersburg, MD, USA). FGFR4 expression is highly variable in most types of cancer. FGFR4 expression was elevated in liver, colorectal, gastric, esophageal, and testicular cancers, but decreased in kidney, lung, lymphoid, and small intestine cancers compared to normal tissues (fig. 6A). Using Immunohistochemistry (IHC), we localized FGFR4 in a panel of lung, breast, pancreatic, and ovarian adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, thyroid carcinoma, and normal lung, pancreatic, and thyroid samples. FGFR4 assays gave membrane and cytoplasmic staining in normal and neoplastic epithelial cells (representative example is shown in fig. 6B). Higher levels of staining are typically found in tumor samples compared to normal tissue. Moderate to significant markers against FGFR4 were evident in tumors from pancreas (41% of specimens), breast (46%), lung (31%), ovary (41%), colon (90%), liver (33%), and thyroid (11%) (table 2 and reference 23).

Table 2: FGFR4 expression in normal and cancer tissues. Prevalence of FGFR4 expression in normal and cancer tissues as determined by histopathological assessment of FGFR4 immunostaining.

Widespread expression of FGFR4 in human HCC was also previously confirmed by in situ hybridization (23). Since a link between FGFR4 and HCC was soon suggested, we decided to further assess FGFR4 expression in 23 primary human liver tumors and 11 normal livers using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of FGFR4 in each sample was normalized to the expression of this receptor in the first normal liver sample (N1). The mean level of FGFR4 in liver tumors (1.22 ± 0.05 fold) was moderately elevated compared to normal liver (0.90 ± 0.04 fold), but the difference did not reach statistical significance when the population was considered as a whole (p = 0.23) (fig. 6C). However, FGFR4 expression was significantly higher (more than 2-fold) in one tumor subset (7/23; 30%). These results indicate that FGFR4 expression is deregulated in several types of cancer. Elevated expression of FGFR4 in a subset of liver tumors suggests that it may represent an attractive target for the treatment of liver cancer in a selected patient population.

In this study, we provide evidence that FGFR4 is involved in hepatocellular carcinoma and that treatment with FGFR4 inactivating antibodies can provide anti-tumor effects. To assess the involvement of FGFR4 in liver tumorigenesis, we used a genetically engineered mouse model. Exogenous expression of FGF19 was shown to promote hepatocyte proliferation, hepatocyte dysplasia, and HCC development in mice. Furthermore, we and others demonstrated that Klotho β is required for liver-specific activity of FGF19 (16,19, 24). Since KLB and FGFR4 are most highly expressed in the liver, we hypothesized that deregulation of the FGFR4 pathway is responsible for hepatic tumorigenesis mediated by FGF 19. To test this hypothesis, we mated FGF19-TG mice with FGFR4-KO mice. Pre-neoplastic (prenoplastic) hepatocyte proliferation and hepatoma formation occurred only in FGF19-TG mice with FGFR4-WT background. FGFR4-KO mice abolished liver tumorigenesis. We further challenged mice by administering the potent liver carcinogen, diethylnitrosamine. Treatment with DEN accelerated HCC development in FGF19-TG mice with an FGFR4-WT background, whereas no evidence of hepatoma formation was found in FGFR4-KO mice. It is clearly concluded that FGFR4 is required for FGF 19-promoted liver tumorigenesis.

Together, these data suggest a link between FGFR4, liver tumorigenesis, and liver cancer progression. Therefore, FGFR4 is a potential therapeutic target, and its inhibition may provide therapeutic benefits to liver cancer patients. To this end, we developed an anti-FGFR 4 neutralizing antibody (LD 1). LD1 binds FGFR4 and inhibits ligand binding, pathway activation, gene expression regulation, cell proliferation, and colony formation (in vitro). By assessing the interaction of LD1 with FGFR4 constructs carrying point mutations at sites that are similar between FGFR4 orthologs but dissimilar in FGFR1-3 orthologs, the site at which LD1 binds FGFR4 was located; these amino acid residues in FGFR4 were replaced with the amino acid residues present at equivalent positions in FGFR 3. LD1 binds to the wild-type FGFR4 and all mutant FGFR4 constructs except the G165A mutant. Substitution of glycine 165 of FGFR4 with alanine almost abolished LD1 binding. The strong specificity of LD1 for FGFR4 combined with the high identity of this region between FGFRs underscores the importance of this residue for LD1 binding. Glycine 165 in FGFR4 corresponds to alanine 171 in FGFR 1. Interestingly, alanine 171 is the closest residue in the FGFR1 dimer interface (25). Traversing the dimer axis, the side chain of alanine 171 of one receptor makes a hydrophobic contact with alanine 171 of the adjacent receptor. Sequence conservation in this region of FGFR is consistent with this region forming the receptor-receptor interface (25). Thus, binding of LD1 to this equivalent region in FGFR has the potential to disrupt receptor dimerization. Receptor dimerization induced by ligands is crucial for FGFR activation (26, 27). Therefore, inhibition of FGFR4 dimerization is a potential mechanism of action for LD1. Similar mechanisms of action have been described for other therapeutic antibodies (28).

We show that LD1 acts on hepatoma xenograft tumors in vivo by inhibiting regulation of genes downstream of FGFR4 and by blocking tumor growth. Furthermore, administration of LD1 inhibited the formation and development of HCC in FGF19-TG mice.

These data demonstrate that FGFR4 is involved in promoting tumorigenesis and cancer progression. In particular, our results suggest that FGFR4 may play an important role in hepatocellular carcinoma. Several lines of evidence support this hypothesis. FGFR4 is the predominant FGFR isoform present in human hepatocytes (15). We previously reported that liver tissue had the highest FGFR4 and KLB transcript levels, both of which are critical for ligand-stimulated activity achieved by this signaling complex (16). Furthermore, ectopic expression of mice in FGF19 (i.e., a ligand specific for FGFR 4) promoted hepatocyte proliferation, hepatocyte dysplasia, and neoplasia (18), and it was reported that hepatocyte proliferation induced by FGF19 is exclusively mediated by FGFR4 (24). A recent report suggests that FGFR4 also contributes significantly to HCC progression by regulating alpha-fetoprotein secretion, proliferation, and anti-apoptosis (17). FGFR4 expression was also shown to promote resistance to chemotherapy (29). It should be noted that there was a panel reporting a protective effect of FGFR4 in mice, but not HCC promoting effects (30). It is likely that background factors (including the identity and concentration of the ligand as well as co-receptor expression and levels of FGFR) might modulate the role of FGFR4 in tumorigenesis. For example, we found that FGFR4 expression was significantly elevated in a subset of primary liver tumors, suggesting that FGFR4 might represent an attractive target for the treatment of liver cancer in a selected patient population. Given the increasing evidence that FGFR4 is involved in liver tumorigenesis and HCC progression, we believe that therapeutic intervention, including anti-FGFR 4 neutralizing antibodies, may be beneficial in liver cancer therapy.

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B. Highly specific off-target binding identified and eliminated during humanization of antibodies to FGF receptor 4

Materials and methods

FGFR4 was biotinylated using Sulfo-NHS-LC-biotin (Pierce; Cat 21335).

Generation of chimeric LD 1-Balb/c mice were immunized as described herein with the extracellular domain of human FGF receptor 4 (FGFR 4) expressed in CHO cells and purified as described 14. LD1 expressing hybridomas were identified when clones were screened for the ability to block FGF19 binding to FGFR4 in a protein-based ELISA.

Mouse LD1 variable domain was cloned from total RNA extracted from LD1 generating hybridoma cells using standard methods. The light chain variable domain (VL) and heavy chain variable domain (VH) were amplified using RT-PCR with degenerate reverse primers for the light chain constant domain (CL) and heavy chain first constant domain (CH 1) and forward primers specific for the N-terminal amino acid sequences of the VL and VH regions. These variable domains are then cloned in-frame into vectors containing the respective human light and heavy chain constant regions.

Humanization and affinity maturation of LD 1-grafting of the hypervariable regions of LD1 into the human kappa i (huki) and human VH subgroup iii (huiii) variable domain frameworks used in trastuzumab. Optimization of FGFR4 binding affinity using framework repair via addition of mouse fine-tuning (vernier) positions until the identification of the least framework-changed combination that fully restores FGFR4 binding affinity³³。

Affinity maturation was performed on hLD1.vB displayed on phage as monovalent Fab-P3 fusions using a soft randomization strategy. The sequence diversity is introduced separately into each hypervariable region so that the propensity towards murine hypervariable region sequences is maintained, using a contaminating oligonucleotide synthesis strategy³⁴. For each variegated position, contamination of codons encoding wild-type amino acids with a 70-10-10-10 nucleotide mixture resulted in an average of 50% mutation rate at each position.

Panning of hLD1.vB variegated phage library Using a soluble selection method³⁵. This approach relies on a short binding period of low concentrations of biotinylated FGFR4 in solution followed by a short 5 minute capture of phage bound FGFR4 on immobilized neutravidin. Excess unlabeled FGFR4 (over 100 nM) was added prior to the capture step to increase the stringency of dissociation selection. Bound phage were eluted by incubating the wells with 100mM HCl for 30 min, neutralized with 1M Tris pH8, and amplified using XL1-Blue cells and M13/KO7 helper phage. The phage antibodies were reformatted to full-length IgG, transiently expressed in mammalian cells, and purified by protein a chromatography.

Affinity assay-Using BIAcore^TM2000 affinity assays were performed by surface plasmon resonance. Approximately 50RUhLD1.vB IgG was immobilized on a CM5 sensor chip in 10mM sodium acetate pH4.8 and serial 2-fold dilutions (0.48-1000 nM) of FGFR4 in PBST were injected at a flow rate of 30. mu.l/min. Each sample was analyzed for 4 minutes binding and 10 minutes dissociation. After each injection, the chip was regenerated using 10mM glycine pH 1.7. Binding response was corrected by subtracting RU from flow cell immobilized irrelevant IgG at similar density. Kinetic analysis was performed using a 1:1 langmuir (Languir) model fitted with both kon and koff.

Xenograft experiments-all animal protocols were approved by the Genentech scientific research animal care and use committee. 7 weeks of age was obtained from Charles River Laboratories International (Strain code 088)Female nu/nu (nude-CRL) mice. Mice were maintained under specific pathogen free conditions. HUH7 cells (5X 10) in HBSS/Matrigel (1: 1 v/v; BD Biosciences, Cat. 354234) in a volume of 0.2mL⁶(ii) a Japan Health science Research Resources Bank, catalog JCRB 0403) was subcutaneously implanted in the mouse side. Tumors were measured twice weekly with calipers and tumor volumes were calculated using the formula: v =0.5xLxW²Wherein L and W are the length and width of the tumor, respectively. When the mean tumor volume reaches 145mm³Mice were randomized (n = 15) and treated once weekly with 0.2mL intraperitoneal injection vehicle (PBS), 30mg/kg chLD1, 30mg/kg hld1.vb, or 30mg/kg hld1.v 22. After treatment, tumor volume was measured as described above. Percent tumor growth inhibition (% TGI) was calculated using the following formula, where C = day 21 mean tumor volume for the control vehicle group and T = day 21 mean volume for each group from mice given test treatment: % TGI =100x ((C-T)/C). Data were analyzed and tumor doubling differences between groups were assessed using a time series test with JMP software version 6.0 (SAS Institute; Cary, NC). Data are presented as mean tumor volume ± SEM.

Pharmacokinetic studies in mice-NCR nude mice were supplied by Taconic (catalog NCRNU). C3 knockout mice³⁶Backcrossing with C57BL/6 mice for at least 10 passages. The progeny were intercrossed to generate C3 knockout mice and wild-type controls. In this study, they were designated as C3ko and C3wt mice, respectively.

Mice weighing 15.5-38.3g via the tail vein were administered a bolus of 1,5 or 20mg/kg body weight IV anti-FGFR 4 antibody. At selected time points, up to 28 days post-dose, blood samples were collected via retro-orbital bleeding or cardiac puncture (n =3 mice per time point) and sera were isolated. Serum samples were stored at-80 ℃ until anti-FGFR 4 antibody serum concentrations were determined using ELISA.

PK parameters were assessed using WinNonlin enterprise version 5.2.1 (Pharsight corp.) using an anti-FGFR 4 antibody serum concentration-time profile. Since one concentration-time profile was determined for each group, one estimate for each PK parameter was obtained and reported, along with the Standard Error (SE) of the fit for each PK parameter. The nominal dose administered for each group was used for modeling.

radioiodination-Using an Indirect Iodogen addition method³⁷The antibody is radioiodinated. Using NAP5 pre-equilibrated in PBS^TMThe radiolabeled protein was purified on a column (GE Healthcare Life Sciences, Cat 17-0853-01). The specific activity of the molecules used in the in vitro study was 14.38. mu. Ci/. mu.g for chLD1 and 15.05. mu. Ci/. mu.g for hld1. vb. The specific activity of the molecules used in the in vivo study was 12.52. mu. Ci/. mu.g for chLD1 and 9.99. mu. Ci/. mu.g for hld1. vb. Following radioiodination, the radioiodinated antibody was characterized by size exclusion High Performance Liquid Chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ELISA, to be intact and retain antigen binding comparable to unlabeled antibody.

In vitro incubation-for incubations in serum, antibodies were added to NCR nude mouse, C3ko, or C3wt mouse serum³⁶And PBS +0.5% BSA to a final concentration of 200. mu.g/ml. Aliquots (100. mu.l) were prepared and incubated at 37 ℃ under gentle rotation. Samples were transferred to dry ice at 0,4, 8, 24, 48, and 96 hours and stored at-70 ℃ until ELISA analysis.

For incubation in plasma, antibodies were added to cynomolgus monkey, human, rat (Bioretrieval LLC, catalogues CYNPLLIHP, HMLLIHP, and RATPLLIHP, respectively), and NCR nude mouse plasma (Taconic, catalogues NCRNU-E) and PBS +0.5% BSA at a final concentration of 200. mu.g/ml ±)¹²⁵I-antibody, final concentration 5X10⁶CPM/ml. Aliquots (100. mu.l) were prepared and incubated at 37 ℃ under gentle rotation. Samples were transferred to dry ice at 0, 24, and 48 hours and stored at-70 ℃ until analysis by size exclusion HPLC (¹²⁵I-antibody + unlabeled antibody sample) or protein G extraction followed by SDS-PAGE (unlabeled antibody sample).

Tissue distribution study-female NCR nude mice receive one IV bolus¹²⁵I-chLD1 (300. mu. Ci/kg). + -. unlabeled chLD 1(20 mg/kg) or¹²⁵I-hLD1.vB (300. mu. Ci/kg). + -. unlabeled hLD1.vB (20 mg/kg). Blood was collected at 15 minutes and 2,5, 24, 72, and 120 hours post-dose and processed for serum. Sera were frozen at-70 ℃ until analysis by size exclusion HPLC and protein G extraction, followed by SDS-PAGE separation. Wallac1480Wizard3 "(EC) was also used&G Wallac, catalog 1480-. Liver, lung, kidney, and heart were collected at 2, 72, and 120 hours post-dose and frozen at-70 ℃ until total radioactivity was analyzed. All animals were operated according to IACUC guidelines.

IgG and FGFR4 ELISA-FGFR 4 specific IgG was measured using FGFR4 ELISA. FGFR4 was immobilized on microtiter plates, LD1 standards (chLD 1, hLD1.vB, or hLD1. v22) and samples diluted in Magic buffer +0.35M NaCl (1 XPBspH7.4, 0.5% BSA, 0.05% Tween-20, 0.25% Chaps, 5mM EDTA, 0.2% BgG, 0.35M NaCl, 15ppm Proclin) were added, and samples were incubated with F (ab') conjugated to horseradish peroxidase₂Goat anti-huIgG Fc (HRP, Jackson, catalog 109-.

Determination of Total antibodies using human Fc ELISA, i.e.using F (ab')₂Rabbit anti-huIgG Fc (Jackson, cat. No. 309-₂Goat anti-huIgG Fc (Jackson, catalog # 109-036-098) was detected. Color development was performed using TMB peroxidase substrate solution (Moss, Cat. No. TMBE-1000), and 1M H was added₃PO₄To terminate the reaction. Plates were read at 450/620nm on a microplate reader (Biotek EL311 or equivalent).

Size exclusion HPLC-Using Phenomenex^TMBioSep-SEC-S3000 column (300 X7.8mm, 5 μm column; Torrance, Cat 00H-2146-KO) size exclusion HPLC was performed in PBS and samples were started at pH4.0 and pH 7.0. Diluting the pH7.0 sample in PBS; a sample of pH4.0 was generated by lowering the pH with 200mM citric acid pH 3.0. Flow rate 0.5ml/min for 30 minutes, isocratic. Simulation of ChemStationThe digital converter was set at 25,000U/mV, peak width 2 seconds, and crack 4nM (Agilent Technologies; catalog 35900E). Radioactivity was detected by raytest Ramona90 (raytest USA inc.; Wilmington, NC) inline with a standard Agilent1100HPLC module system (Santa Clara, CA).

Protein G bead extraction and SDS-PAGE separation-Triton X-100 (final concentration 1% (v/v)) was added to the fraction collected from size exclusion HPLC followed by protein G beads (GE Lifesciences Inc, product catalog 17-0885-01). Samples were incubated overnight at 4 ℃ under gentle rotation, and then beads were washed four times with PBS +1% Triton X-100. Each sample was split and half of the samples were usedReduction with sample reduction reagent (catalog NP 0004). By using4 XLDS sample buffer (pH8.4) (catalog NP 00007). + -.)Sample reduction reagent treatment beads, and 99 degrees C temperature in 5 minutes; then useApplication of 1 XMOPS SDS running buffer (catalog NP 0001)4-12% Bis-Tris gel (catalog NP0321 BOX). The gel was stained with coomassie blue R250 dye. All ofReagents were obtained from Invitrogen corporation.

Mass spectrometry and bioinformatic analysis-as previously described³⁸Samples excised from SDS-PAGE were processed. Briefly, after rapid solution microwave-assisted trypsin digestion, a reverse phase layer is passedThe peptide was separated and eluted directly into a nanospray ionization source at a spray voltage of 2kV and analyzed using an LTQ XL-Orbitrap mass spectrometer (ThermoFisher). Precursor ions were analyzed in FTMS at 60,000 resolution. MS/MS was performed in LTQ, running the instrument in a data-dependent mode, in which the first 10 ions, which are most abundant, were fragmented. The data was searched using Mascot search algorithm (Matrix Sciences) or by re-interpretation.

For searching Mascot data: search criteria included full MS tolerance of 20ppm, MS/MS tolerance of 0.5Da (GlyGly on Lys), methionine oxidation, phosphorylation of +57Da and ST on Cys, and Y as variable modifications with up to 3 mis-cleavages. Data were searched against a subset of mammals from the Swissprot database.

Results

Humanization and standardization of LD1. The human chimeric antibody LD1 (chLD 1) has been shown to bind human FGFR4, block signaling by FGF19 and other FGF ligands, and suppress tumor growth in a HUH7 human hepatocellular carcinoma (HCC) xenograft model¹⁴. As a first step in the humanization of LD1, the light and heavy chain variable domains of chLD1 were aligned with the human kappa i (huki) and human VH subgroup iii (huiii) variable domains framework used in trastuzumab (fig. 10). Grafting hypervariable regions from chLD1 into these human variable frameworks to generate direct CDR grafts (hld 1. va)^15,16. Binding to FGFR4 by surface plasmon resonance was approximately 5-fold lower in affinity for hld1.va compared to chLD1 (not shown). The substitution of mouse sequences at multiple fine tuning positions in both the light and heavy chain variable domains was explored as a means to improve binding and led to the identification of three important mouse fine tuning positions in LC: P44F, L46I and Y49S. These changes were introduced in hld1.vb with an affinity for FGFR4 comparable to chLD1 (table 3).

Table 3: binding kinetics of anti-FGFR 4 antibody variants. The binding and dissociation rates of human FGFR4 binding to immobilized antibody variants were measured using surface plasmon resonance.

Surprisingly, despite similar FGFR4 binding affinity (fig. 11A), hld1.vb reduced antitumor efficacy in the HUH7 human HCC xenograft model in nu/nu mice compared to chLD1 (fig. 11B). After 9 days, HUH7 tumors from PBS-treated mice grew to an average volume of approximately 700mm³. In the chLD 1-treated group, the mean HUH7 tumor volume was approximately 400mm³Tumor growth was inhibited by 43% compared to tumors in PBS treated animals. However, the mean tumor volume in mice treated with hld1.vb was about 600mm³Tumor growth was inhibited by 14% compared to tumors in PBS treated animals.

Pharmacokinetic assessments of chLD1 and hld1.vb in athymic NCR nude mice revealed rapid clearance of both chLD1 and hld1.vb at 1mg/kg IV (140 and 132mL/d/kg, respectively), suggesting a clearance mechanism mediated by the target. This clearance mechanism appears to saturate at higher doses of 20mg/kg with chLD 1. At this dose, the observed clearance (11.7 mL/d/kg; FIG. 11C) was within the range of target independent clearance observed in mice for a typical humanized antibody (6-12 mL/d/kg) (literature 17 and P.Theil personal communication). However, hLD1.vB still cleared rapidly (34.2 mL/d/kg; FIG. 11C). This suggests that there may be other clearance mechanisms for hld1.vb responsible for the apparent lack of efficacy in the mouse xenograft model.

Consistent with PK discovery, use¹²⁵I-chLD1 and¹²⁵biodistribution studies performed by I-hld1.vb revealed a significantly different distribution profile (fig. 11D). Due to the high expression of FGFR4 on hepatocytes,¹²⁵I-chLD1 rapidly and specifically distributed to the liver, with only a limited amount found in the liver at equivalent doses up to 2 hours¹²⁵I-hLD1.vB (. about.80 vs.35% ID/g). In contrast, the observed distribution of these antibodies in blood was reversed, suggesting that there is a competitive interaction preventing h, as opposed to the loss of stability of the antibodies in vivo, which would lead to a loss of overall radioactivityDistribution of vb to the liver.

Identification of C3 interference. In an attempt to explain the observed in vivo differences between chLD1 and hld1.vb, we assessed the stability of antibodies in plasma and potential off-target plasma or tissue interactions that may affect their function. Plasma stability was assessed by incubating chLD1 or hld1.vb in mouse, rat, monkey or human plasma for 48 hours at 37 ℃, followed by assessment of both FGFR4 binding activity and total human IgG concentration. Although the total chLD1 or hld1.vb concentration measured by IgG ELISA was unchanged (not shown), hld1.vb recovery detected by FGFR4ELISA was significantly reduced (by-30%) in mouse and rat plasma compared to control incubations in PBS/BSA (fig. 12A). In contrast, there was no loss of chLD1FGFR4 binding activity in any of the conditions tested. A significant reduction in vb recovery (particularly from rodent plasma) suggests that the loss is not due to degradation and is more likely to be the formation of interfering complexes in rodent plasma. Because the interaction of hld1.vb with mouse plasma can lead to the formation of higher molecular weight complexes, iodinated chLD1 and hld1.vb were also incubated in plasma and analyzed using size exclusion HPLC. Only at the position of¹²⁵A high molecular weight peak was detected in a mouse plasma sample of I-hLD1.vB, but¹²⁵I-chLD1 is not. In addition to the expected antibody peak at 150kDa, peaks corresponding to about 270 and about 550kDa were initially detected (fig. 12B), however, by 48 hours, only the 150 and 270kDa peaks remained; the 550kDa peak was no longer observed. These higher molecular weight peaks were not detectable in cynomolgus and human plasma or PBS/BSA containing hld1.vb or in any sample containing chLD1 (fig. 15). Interestingly, the presence of these high molecular weight peaks was directly related to the antibody recovery data obtained in the FGFR4 ELISA. Moreover, the presence of these peaks was reduced when the assay was performed at ph4.0 (fig. 15), further supporting hld1. vb-dependent interaction with mouse serum.

Immunoprecipitation of mouse plasma revealed a protein of about 37kDa that was selectively pulled down using hLD1.vB but not using chLD1 (FIG. 12C). Consistent with findings from size exclusion HPLC, this 37kDa protein band was observed in rats, but not cynomolgus and human plasma samples (fig. 16A-C). Furthermore, the protein was detected in plasma from mice administered hld1.vb (fig. 16D). MS/MS analysis of trypsin digested peptide derived from 37kDa mouse plasma protein identified this band as derived from mouse complement C3 (fig. 12D). Direct involvement of C3 was supported by complete recovery of hld1.vb incubated in plasma from C3 knock-out (ko) mice (fig. 17).

Affinity maturation and reassessment of C3 binding. Both chLD1 and hld1.vb share the same human constant regions and Complementarity Determining Regions (CDRs), and therefore differ only in their variable domain frameworks. Moreover, the light and heavy chain variable domain frameworks used for humanizing hld1.vb share a high degree of homology with several humanized antibodies, including trastuzumab which has not been reported to exhibit interactions with mouse serum proteins. Thus, off-target interaction of hld1.vb with mouse C3 most likely results from a specific combination of mouse LD1 CDRs and human variable domain framework.

We reasoned that some changes in CDR sequences of hld1.vb (resulting from affinity maturation of Fab fragments displayed on phage) could lead to improved affinity for FGFR4, with concomitant loss of mouse C3 binding. As variants of IgG expressing phage selection and screening for FGFR4 binding affinity and potential interaction with mouse C3 using an immunoprecipitation assay coupled with SDS-PAGE analysis. One variant with 3 amino acid changes in CDR-H2 (H52L, S53V and D60E, fig. 10) compared to hld1.vb, i.e. hld1.v22 shows both improved binding affinity to FGFR4 (table 3) and loss of bound complement C3B (fig. 13B).

The extent to which mouse complement C3 altered in vivo clearance of hld1.vb and hld1.v22 was evaluated in a pharmacokinetic study comparing C3ko mice to C3wt mice at 20 mg/kg. As previously observed in NCR mice, hld1.vb cleared rapidly from circulation in C3wt mice (29 mL/d/kg); however, both chLD1 and hld1.vb had similar pharmacokinetic profiles in C3ko mice (clearance 8.7 and 9.3mL/d/kg, respectively) (fig. 14A and table 4).

Table 4: pharmacokinetic parameters of the anti-FGF. R4 variant dosed at 20mg/kg IV

These data are consistent with tissue distribution data and confirm that specific interaction with mouse complement C3 in vivo results in rapid clearance of hld1. vb. Since clearance of hld1.vb was significantly improved in C3ko mice and hld1.v22 did not bind C3, we expected hld1.v22 would have a similar pharmacokinetic profile in NCR nude mice as chLD 1. As shown in FIG. 14B, clearance of chlD1 and hLD1.v22 in NCR nude mice was similar (11.8 and 11.3mL/d/kg, respectively), while hLD1.vB was rapidly cleared (46.7 mL/d/kg), consistent with our previous findings.

The ability of hld1.v22 to inhibit tumor growth in the HUH7HCC xenograft model was evaluated, compared to hld1 and hld1.v b. After 21 days, HUH7 tumors from PBS-treated mice grew to an average volume of approximately 2,100mm³(FIG. 14C). In 15 animals of the PBS-treated group, due to tumor volume limitation (approximately 2,500 mm)³)3 animals were euthanized prior to the end of the study. Average HUH7 tumor volume in hLD1.vB treated group was approximately 1,200mm³Representing 43% inhibition of tumor growth compared to tumors in PBS treated animals. However, the mean tumor volume in mice treated with hld1.v22 was about 530mm³. This result was comparable to chLD 1-treated mice, with an average HUH7 tumor volume of approximately 350mm³. For both hld1.v22 and chLD1 treated groups, this represents a 75% and 83% reduction in tumor size compared to the PBS-vehicle treated group, respectively. The tumor doubling time was significantly greater in the group treated with hld1.vb (12.2 days), hld1.v22 (15.8 days) or chLD1 (17.1 days) than in the PBS treated group (8.2 days). In additionIn addition, tumor doubling times were significantly longer in the hld1.v22 or chLD1 treated groups than in the hld1.v b treated group. Similar in vivo performance of both hld1.v22 and chLD1 compared to hld1.v b strongly suggests that specific off-target interaction with mouse complement C3 results in increased clearance, resulting in lower hld1.v b exposure and reduced efficacy.

Thus, the production of the anti-FGFR 4 antibody hld1.v22 has the following steps: 1) generating human chimeric LD1 (chLD 1) comprising LD1 murine VL and VH domains and a human IgG1 constant domain; 2) the 6 murine HVRs from chLD1 were grafted into human VL kappa I and human VH subgroup III variable domain frameworks to generate direct HCR grafts, generating the antibody hld1. va. Antibody hld1.va binds FGFR4 about 5-fold less than ChLD 1; 3) introduction of the light chain mutations P44F, L46I and Y49S produced the antibody hld1. vb. Antibody hld1.vb binding to human FGFR4 was about 2-fold weaker than antibody chLD1, but hld1.vb had reduced in vivo tumor efficacy compared to chLD1, rapid in vivo clearance, and was found to bind mouse complement protein C3; 4) affinity maturation was performed and three changes were added to HVR H2 (H52L, S53V, D60E) to improve binding affinity to FGFR4 and abolish binding to the mouse complement c3 protein. The resulting humanized and affinity matured antibody (antibody hld1.v 22) had in vivo efficacy, pK and tissue distribution comparable to chLD 1. Furthermore, the antibody hld1.v22 has been determined to have at least comparable biological activity as the parent chLD1 antibody, e.g. to inhibit cancer in human xenograft tumor studies.

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (41)

1. An isolated antibody that binds FGFR4, wherein the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 1 nM.

2. The isolated antibody of claim 1, wherein the anti-FGFR 4 antibody binds human, mouse, and cynomolgus FGFR4 with an affinity of ≤ 1 nM.

3. The isolated antibody of claim 2, wherein the anti-FGFR 4 antibody binds human FGFR4 with an affinity of ≤ 0.05 nM.

4. The isolated antibody of any one of claims 1-3, wherein the anti-FGFR 4 antibody does not substantially bind to a mouse C3 protein having the amino acid sequence set forth in figure 12D.

5. The isolated antibody of any one of claims 1-4, wherein the anti-FGFR 4 antibody does not substantially bind to human FGFR4 comprising a G165A mutation.

6. The isolated antibody of any one of claims 1-5, wherein the anti-FGFR 4 antibody binds to a polypeptide having at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity to: a sequence comprising amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, a sequence consisting essentially of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4 or a sequence consisting of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR 4.

7. The isolated antibody of claim 6, wherein the anti-FGFR 4 antibody binds to a polypeptide that: a polypeptide comprising amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, a polypeptide consisting essentially of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR4, or a polypeptide consisting of amino acid numbers 150 to 170 of the amino acid sequence of mature human FGFR 4.

8. The antibody of any one of claims 1-7, wherein the anti-FGFR 4 antibody is an antagonist of FGFR4 activity.

9. The antibody of claim 8, wherein FGFR4 activity is FGF-induced cell proliferation, FGF-binding to FGFR4, FGF 19-mediated inhibition of CYP7 α 7 expression in cells exposed to FGF19, or FGF 19-induced colony formation.

10. The antibody of claim 9, wherein the binding of FGF1 and/or FGF19 to FGFR4 is inhibited.

11. The antibody of claim 10, wherein the IC that inhibits FGF1 binding to FGFR4 is₅₀IC of about 0.10nM and inhibiting FGF19 binding FGFR4₅₀About 0.10 nM.

12. The antibody of any one of claims 1-11, which is a monoclonal antibody.

13. The antibody of any one of claims 1-11, which is a human, humanized, or chimeric antibody.

14. The antibody of any one of claims 1-11, which is an antibody fragment that binds FGFR 4.

15. The antibody of any one of claims 1-14, wherein the antibody comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID No. 3, (b) HVR-L3 comprising the amino acid sequence of SEQ ID No.6, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID No. 2.

16. The antibody of any one of claims 1-14, wherein the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. 1, (b) HVR-H2 comprising the amino acid sequence of SEQ ID No.2, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID No. 3.

17. The antibody of claim 16, further comprising (a) HVR-L1, comprising the amino acid sequence of seq id No. 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6.

18. The antibody of any one of claims 1-14, comprising (a) HVR-L1, comprising the amino acid sequence of seq id NO 4; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO 5; and (c) HVR-L3, comprising amino acid sequence SEQ ID NO: 6.

19. The antibody of any one of claims 1-18, further comprising a light chain variable domain framework sequence of SEQ ID NOs 9, 10, 11 and/or 12.

20. The antibody of any one of claims 1-19, further comprising a heavy chain variable domain framework sequence of SEQ ID NOs 13, 14, 15, and/or 16.

21. The antibody of any one of claims 1-20, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of seq id No. 7; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence SEQ ID NO. 8; or (c) the VH sequence in (a) and the VL sequence in (b).

22. The antibody of claim 21, comprising the VH sequence SEQ ID NO 7.

23. The antibody of claim 21, comprising the VL sequence of SEQ ID NO 8.

24. An antibody comprising the VH sequence SEQ ID NO 7 and the VL sequence SEQ ID NO 8.

25. The antibody of any one of claims 1-24, which is a full length IgG1 antibody.

26. An isolated nucleic acid encoding the antibody of any one of claims 1-24.

27. A host cell comprising the nucleic acid of claim 26.

28. A method of producing an antibody comprising culturing the host cell of claim 27 such that the antibody is produced.

29. The method of claim 28, further comprising recovering the antibody from the host cell.

30. An immunoconjugate comprising the antibody of any one of claims 1-24 and a cytotoxic agent.

31. A pharmaceutical composition comprising the antibody of any one of claims 1-24 and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 16, further comprising an additional therapeutic agent.

33. The antibody of any one of claims 1-24 for use as a medicament.

34. The antibody of any one of claims 1-24 for use in the treatment of cancer.

35. The antibody of any one of claims 1-24 for use in inhibiting cell proliferation.

36. The antibody of any one of claims 1-24 for use in the manufacture of a medicament.

37. The use of claim 36, wherein the medicament is for the treatment of cancer.

38. The use of claim 36, wherein the medicament is for inhibiting cell proliferation.

39. A method of treating an individual having cancer comprising administering to the individual an effective amount of the antibody of any one of claims 1-24.

40. The method of claim 39, further comprising administering to the individual an additional therapeutic agent.

41. A method of inhibiting cell proliferation in an individual comprising administering to the individual an effective amount of the antibody of any one of claims 1-24 to inhibit cell proliferation.