BR112015032789B1 - COMPOSITIONS, METHOD FOR PRODUCING ANTIBODIES, PHARMACEUTICAL COMPOSITIONS AND USE OF A PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

COMPOSITIONS, HOST CELL, METHOD FOR PRODUCING IR ANTIBODIES, PHARMACEUTICAL COMPOSITIONS AND USE OF A PHARMACEUTICAL COMPOSITION. The present invention provides antibodies that bind to FGFR2IIIb, wherein the antibodies are non-fucosylated. The present invention provides compositions comprising antibodies that bind to FGFR2IIIb, wherein at least 95% of the antibodies in the composition are non-fucosylated. In some embodiments, methods are provided for treating cancer comprising administering non-fucosylated anti-FGFR2IIIb antibodies.

Description

FIELD OF THE INVENTION

[001] Immunoconjugates comprising anti-FGFR2IIIb antibodies are provided.

BACKGROUND OF THE INVENTION

[002] Members of the fibroblast growth factor (FGF) family bind to four known receptor tyrosine kinases, fibroblast growth factor receptor 1-4 (FGFR1-4) and its isoforms, with different FGFs binding to different FGFRs to different degrees (Zhang et al., J. Biol. Chem. 281:15694, 2006). A human FGFR2 protein sequence is provided, for example, at GenBank Locus AF487553. Each FGFR consists of an extracellular domain (ECD) comprising three immunoglobulin-like (Ig) domains (D1, D2, and D3), a unique transmembrane helix, and an intracellular catalytic kinase domain ( Mohammadi et al., Cytokine Growth Factor Revs, 16:107, 2005 ). There is a contiguous stretch of acidic amino acids in the linker between D1 and D2, called the “acid box” (AB). The region containing D1 and AB is believed to be involved in receptor autoinhibition, which is alleviated by ligand binding. FGFs bind to receptors primarily through the D2 and D3 regions of the receptors. FGFRs are characterized by various alternative splicing of their mRNAs, leading to a variety of isoforms (Ornitz et al., J. Biol. Chem. 271:15292, 1996; see also Swiss-Prot P21802 and P21802 isoforms-1 to -20 for FGFR2 sequences and their isoforms). Notably, there are forms containing all three Ig domains (α isoform) or only the two Ig D2 and D3 domains and no D1 domain (β isoform). In FGFR1-FGFR3, all forms contain the first half of D3 denoted IIIa, but two alternative exons can be used for the second half of D3, leading to forms IIIb and IIIc. For FGFR2, these are respectively denoted FGFR2IIIb and FGFR2IIIc (or just FGFR2b and FGFR2c); the corresponding beta forms are denoted FGFR2(beta)IIIb and FGFR2(beta)IIIc. The FGFR2IIIb form of FGFR2 (also denoted K-sam-II) is a high affinity receptor for both FGF1 and KGF family members (FGF7, FGF10 and FGF22), whereas FGFR2IIIc (also denoted K-sam-I) binds well to both FGF1 and FGF2, but does not bind to KGF family members (Miki et al., Proc. Natl. Acad. Sci. USA 89:246, 1992). In fact, FGFR2IIIb is the only receptor for members of the KGF family (Ornitz et al., 1996, op. cit.) and is therefore also called KGFR.

[003] FGFRs and their isoforms are differentially expressed in different tissues. FGFR2IIIb (and the IIIb forms of FGFR1 and FGFR3) is expressed in epithelial tissues, whereas FGFRIIIc is expressed in mesenchymal tissue ( Duan et al., J. Biol. Chem. 267:16076, 1992; Ornitz et al., 1996, op. cit.). Certain FGF ligands of these receptors have an inverse pattern of expression. Thus, members of the KGF subfamily, including FGF7 (KGF), FGF10 and FGF22, bind only to FGFRIIIb (Zhang et al., op. cit.) and are expressed in mesenchymal tissues, so that they may be paracrine effectors of epithelial cells (Ornitz et al., 1996, op. cit.). In contrast, FGF4-6 members of the FGF4 subfamily bind FGFR2IIIc and are expressed in both epithelial and mesenchymal lineages, so they have both autocrine and paracrine functions. Due to the fact of the expression patterns of the FGFR2 isoforms and their ligands, FGFR2 plays an important role in epithelial-mesenchymal interactions (Finch et al., Dev. Dyn. 203:223, 1995), so it is not surprising that knock-out of FGFR2IIIb in mice leads to severe embryonic defects and lethality (De Moerlooze et al., Development 127:48 3, 2000).

[004] KGF (FGF7) and KGFR (FGFR2IIIb) are overexpressed in many pancreatic cancers (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and their coexpression correlates with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Somatic mutations of the FGFR2 gene have been found in 12% of a large panel of endometrial (uterine) carcinomas, and several cases tested were necessary for tumor cell survival ( DUTT et al., Proc. Natl. Acad. Sci. USA 105:8713, 2008 ). In two tumors, the FGFR2 mutation was found to be the same S252W substitution associated with Apert syndrome. FGFR2 amplification and overexpression is associated with a diffuse, undifferentiated type of gastric cancer that has a particularly poor prognosis, and inhibition of FGFR2 activity by small molecule compounds potently inhibited the proliferation of these cancer cells ( Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006 ).

SHORT DESCRIPTION OF THE INVENTION

[005] In some embodiments, an anti-FGFR2IIIb antibody is provided, wherein the heavy chain variable region comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: (6); (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein the antibody is non-fucosylated. In some embodiments, the antibody is fucose-depleted at Asn297. In some embodiments, compositions are provided comprising a plurality of anti-FGFR2IIIb antibodies, wherein the heavy chain variable region of each anti-FGFR2IIIb antibody in the composition comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: (6); (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region of each anti-FGFR2IIIb antibody in the composition comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein at least 95% of the antibodies in the composition are non-fucosylated. In some embodiments, the composition can be a supernatant from an antibody producing cell line. In some embodiments, the composition may be a buffered composition. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. amino acid sequence of SEQ ID NO: 3. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 2 and the light chain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, there is provided a composition comprising a plurality of non-fucosylated anti-FGFR2IIIb antibodies, wherein the anti-FGFR2IIIb antibodies compete for binding to FGFR2IIIb with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5.

[006] In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are chimeric antibodies. In some embodiments, the antibodies are humanized antibodies. In any of the embodiments described in the present application, the antibodies can comprise a K light chain constant region. In any of the embodiments described in the present application, the antibodies can comprise an IgG1 heavy chain constant region.

[007] In some embodiments, the antibodies have enhanced ADCC activity in vitro compared to fucosylated anti-FGFR2IIIb antibodies that have the same amino acid sequence. In some embodiments, the non-fucosylated anti-FGFR2IIIb antibodies cause specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with fucosylated anti-FGFR2IIIb antibodies cosylated. In some embodiments, ADCC activity is determined using Ba/F3 cells that express FGFR2III as target cells and isolated human PBMCs as effector cells.

[008] In some embodiments, the antibodies have improved affinity for Fc gamma RIIIA compared to fucosylated anti-FGFR2IIIb antibodies that have the same amino acid sequence. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies bind Fc gamma RIIIA with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17-fold, or at least 20-fold more affinity than fucosylated anti-FGFR2IIIb antibodies. In some embodiments, affinity for Fc gamma RIIIA is determined using surface plasmon resonance. In some embodiments, Fc gamma RIIIA is selected from Fc gamma RIIIA(V158) and Fc gamma RIIIA(F158). In some embodiments, Fc gamma RIIIA is Fc gamma RIIIA(V158).

[009] In any of the embodiments described in the present application, the antibodies can bind to FGFR2IIIb but not to FGFR2IIIc.

[010] In any of the embodiments described in the present application, a composition comprising a plurality of non-fucosylated anti-FGFR2IIIb antibodies comprises at least 95% non-fucosylated antibodies. In any of the embodiments described in the present application, a composition comprising a plurality of non-fucosylated anti-FGFR2IIIb antibodies can have undetectable fucosylation. In some embodiments, the presence of fucose can be determined by a method comprising high performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.

[011] In some embodiments, host cells are provided that comprise nucleic acid encoding an anti-FGFR2IIIb antibody described in the present application, wherein the functional host cell lacks an alpha-1,6-fucosyltransferase (FUT8) gene. In some embodiments, the host cell is a CHO cell.

[012] In some embodiments, methods for producing non-fucosylated anti-FGFR2IIIb antibodies are provided. In some embodiments, a method comprises culturing a host cell under conditions suitable for expressing nucleic acid encoding the anti-FGFR2IIIb antibody, wherein the host cell lacks an alpha-1,6-fucosyltransferase (FUT8) gene. In some embodiments, a method of producing non-fucosylated anti-FGFR2IIIb antibodies comprises culturing the host cell under conditions suitable for producing non-fucosylated anti-FGFR2III antibody. In some embodiments, the method further comprises recovering anti-FGFR2IIIb antibody produced by the host cell. In some embodiments, less than 5% of the anti-FGFR2IIIb antibodies produced by the host cell comprise fucose. In some embodiments, at least 95% of the anti-FGFR2IIIb antibodies produced by the host cell are fucose-depleted (i.e., are non-fucosylated). In some embodiments, fucose is undetectable in anti-FGFR2IIIb antibodies produced by the host cell. In some embodiments, the presence of fucose is detected by a method comprising HPLC, capillary electrophoresis, or MALDI-TOF mass spectrometry.

[013] In some embodiments, pharmaceutical compositions are provided wherein the pharmaceutical composition comprises non-fucosylated anti-FGFR2IIIb antibodies described in the present application and a pharmaceutically acceptable carrier.

[014] In some embodiments, methods for treating cancer are provided. In some embodiments, a method comprises administering an effective amount of a pharmaceutical composition comprising non-fucosylated anti-FGFR2IIIb antibodies described in the present application and a pharmaceutically acceptable carrier. In some embodiments, the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer comprises an amplification of the FGFR2 gene. In some embodiments, the FGFR2 amplification comprises a ratio of FGFR2:CEN10 (chromosome 10 centromere) >3. In some embodiments, the cancer overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but does not comprise amplification of the FGFR2 gene. In some embodiments, FGFR2IIIb expression or overexpression is determined by IHC. In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, the IHC stain is scored as described in Example 6.

[015] In some embodiments, a method of treating cancer further comprises administering at least one additional therapeutic agent selected from a platinum agent, paclitaxel, ABRAXANE®, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C and doxorubicin hydrochloride. In some embodiments, the platinum agent is selected from cisplatin, oxaliplatin and carboplatin. In some embodiments, a method of treating cancer further comprises administering paclitaxel. In some embodiments, a method of treating cancer further comprises administering cisplatin and/or 5-FU.

[016] In some embodiments, uses of a pharmaceutical composition comprising non-fucosylated anti-FGFR2IIIb antibodies described in the present application and a pharmaceutically acceptable carrier are provided. In some embodiments, such use is to treat cancer in a cancer subject. In some embodiments, the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer comprises an amplification of the FGFR2 gene. In some embodiments, the FGFR2 amplification comprises a ratio of FGFR2:CEN10 (chromosome 10 centromere) >3. In some embodiments, the cancer overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but does not comprise amplification of the FGFR2 gene. In some embodiments, FGFR2IIIb expression or overexpression is determined by IHC. In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, the IHC stain is scored as described in Example 6.

[017] In some embodiments, pharmaceutical compositions for treating cancer are provided, wherein the pharmaceutical composition comprises non-fucosylated anti-FGFR2IIIb antibodies described in the present application and a pharmaceutically acceptable carrier. In some embodiments, the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer comprises an amplification of the FGFR2 gene. In some embodiments, the FGFR2 amplification comprises a ratio of FGFR2:CEN10 (chromosome 10 centromere) >3. In some embodiments, the cancer overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but does not comprise amplification of the FGFR2 gene. In some embodiments, FGFR2IIIb expression or overexpression is determined by IHC. In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, the IHC stain is scored as described in Example 6.

BRIEF DESCRIPTION OF THE FIGURES

[018] Figures 1 to 1C show ADCC activity of αffr2ba non -fuckelated and αffr2bf fuckelated against B/F3 cells expressing FGFR2IIIB, as discussed in example 3. In subtitles, “αffr2bf/fgfr2b” indicates that the antibody αfgfr2bf fucket /F3 expressing FGFR2IIIB.

[019] Figures 2A to 2D show the efficacy of non-fucosylated αFGFR2bA and fucosylated αFGFR2bF in an OCUM-2M gastric cancer xenograft model, at (A and B) 10 mg/kg and (C and D) 3 mg/kg, as discussed in Example 4.

[020] Figures 3A and 3B show the dose-dependent efficacy of non-fucosylated αFGFR2bA in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.

[021] Figures 4A and 4B show the efficacy of combination therapy with non-fucosylated αFGFR2bA and paclitaxel in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.

[022] Figures 5A and 5B show the efficacy of combination therapy with non-fucosylated αFGFR2bA and 5-FU/cisplatin in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.

[023] Figures 6A and 6B show the efficacy of non-fucosylated αFGFR2bA in an MFM-223 gastric cancer xenograft model, as discussed in Example 4.

[024] Figure 7 shows the glycan profile of αFGFR2b antibody produced in (A) Potelligent® CHOK1SV cells and (B) CHOK1SV cells, as described in Example 1.

[025] Figure 8 shows schematic diagrams of N-linked glycans found in antibodies.

[026] Figure 9 shows ADCC of Ba/F3 FGF2b cells with increasing concentrations of αFGFR2bA or αFGFR2bF. Assays were performed with normal human PBMC at an E:T ratio of 25:1 as described in Example 5. Data are plotted as LDH release.

[027] Figure 10 shows ADCC of OCUM-2M cells with increasing concentrations of αFGFR2bA or αFGFR2bF. Assays were performed with normal human PBMC at an E:T ratio of 25:1 as described in Example 5. Data are plotted as percent specific lysis.

[028] Figures 11A to 11F show the detection of FGFR2IIIb in tumor tissue samples using immunohistochemistry, as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

[029] Non-fucosylated antibodies that bind to FGFR2IIIb are provided. In some embodiments, non-fucosylated antibody heavy chains and light chains that are capable of forming antibodies that bind to FGFR2IIIb are also provided. In some embodiments, heavy chains and light chains of non-fucosylated antibodies comprising one or more specific hypervariable regions (HVRs) are provided. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity relative to fucosylated anti-FGFR2IIIb antibodies. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA over fucosylated anti-FGFR2IIIb antibodies. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA(V158) relative to fucosylated anti-FGFR2IIIb antibodies. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA(F158) relative to fucosylated anti-FGFR2IIIb antibodies. In certain embodiments, non-fucosylated anti-FGFR2IIIb antibodies do not bind to FGFR2IIIc.

[030] Polynucleotides encoding antibodies that bind to FGFR2IIIb are provided. Polynucleotides encoding antibody heavy chains or light chains are also provided. Host cells expressing non-fucosylated anti-FGFR2IIIb antibodies are provided. Methods of treatment using non-fucosylated antibodies to FGFR2IIIb are provided. These methods include, but are not limited to, methods of treating cancer such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.

[031] The section headings used herein are for organizational purposes and should not be construed as limiting the subject described.

[032] All references cited in this application, including patent applications, patent publications, and GenBank accession number are incorporated into this application by reference, as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[033] The techniques and procedures described or referred to in this application are generally well known and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel, et al. eds., (2003)); the Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993); and updated versions thereof.

I. DEFINITIONS

[034] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art. In addition, unless otherwise required by context or expressly indicated, singular terms must include pluralities and plural terms must include the singular.

[035] It is understood that aspects and embodiments of the invention described in the present application include "consists of" and/or "consists essentially of" aspects and embodiments. As used in this application, the singular form "a", "an", and "the", "the" includes plural references unless otherwise indicated.

[036] In this order, the use of “or” means “and/or”, unless expressly stated or understood by experts in the field. In the context of a multiple dependent claim, the use of “or” again refers to more than one prior independent or dependent claim.

[037] As understood by one skilled in the art, reference to "about" a value or parameter in this application includes (and describes) embodiments that are directed to that value or parameter itself. For example, the description referring to "about X" includes the description of "X".

[038] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" can be used interchangeably, and refer to a polymer of nucleotides. Such nucleotide polymers can contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear nucleotide sequence comprising the nucleic acid molecule or polynucleotide.

[039] The terms "polypeptide" and protein are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum size. Such amino acid residue polymers can contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both whole proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein retains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or they can be accidental, such as through mutations in the hosts that produce the proteins or errors due to PCR amplification.

[040] “FGFR2IIIb” or “FGFR2b” are used interchangeably to refer to the splice form of fibroblast growth factor receptor 2 IIIc. An exemplary human FGFR2IIIb is shown in GenBank accession number NP_075259.4, dated July 7, 2013. An exemplary non-limiting mature human FGFR2IIIb amino acid sequence is shown in SEQ ID NO: 1.

[041] “FGFR2IIIc” or “FGFR2c” are used interchangeably to refer to the splice form of fibroblast growth factor receptor 2 IIIc. An exemplary human FGFR2IIIc is shown in GenBank accession number NP_000132.3, dated July 7, 2013. An exemplary non-limiting mature FGFR2IIIc amino acid sequence is shown in SEQ ID NO: 12.

[042] The term "epitope" refers to a site on a target molecule (for example, an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (for example, an antibody, antibody fragment, or antibody-containing framework protein that binds to binding regions. specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from either contiguous or non-contiguous juxtaposed residues (eg, amino acids, nucleotides, sugars, lipid component) of the target molecule. Epitopes formed from contiguous residues (eg, amino acids, nucleotides, sugars, lipid component) typically are held on exposure to denaturing solvents whereas epitopes formed by enovel Tertiary growths are typically lost on treatment with denaturing solvents. An epitope can include, but is not limited to, at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides). In some examples an epitope is less than 20 residues (eg, amino acids or nucleotides) in length, less than 15 residues, or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding for the antigen.

[043] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and/or sugars within the antigenic protein to which a specific antibody binds the epitope.

[044] A "linear epitope" comprises contiguous polypeptides, amino acids and/or sugars within the antigenic protein to which a specific antibody binds the epitope.

[045] The term "antibody" in this application is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided they exhibit the desired antigen-binding activity.

[046] The term antibody includes, but is not limited to, fragments that are capable of binding to antigen, such as Fv, single-chain Fv (scFv), Fab, Fab' and (Fab')2. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a unique antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, cynomolgus monkey, etc.

[047] The term "heavy chain variable region" refers to a region comprising heavy chain structure (FR) HVR1 2, HVR2, FR3 and HVR3. In some embodiments, the heavy chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4.

[048] The term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, CH2 and CH3. Exemplary non-limiting heavy chain constant regions include Y, δ and α. Exemplary non-limiting heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a y constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. Furthermore, an antibody comprising a constant ε region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further divided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (which comprise a y1 constant region), IgG2 (which comprise a y2 constant region), IgG3 (which comprise a y3 constant region), and IgG4 (which comprise a y4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[049] The term "heavy chain" refers to a polypeptide comprising at least one heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[050] The term "light chain variable region" refers to a region comprising light chain structure (FR) HVR1 2, HVR2, FR3 and HVR3. In some embodiments, a light chain variable region also comprises an FR1 and/or an FR4.

[051] The term "light chain constant region" refers to a region comprising a light chain constant domain, CL. Exemplary non-limiting light chain constant regions include A and K.

[052] The term "light chain" refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[053] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and/or forms structurally defined as loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of higher sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 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) occur at amino acid residues 24 to 34 of L1, 50 to 56 of L2, 89 to 97 of L3, 31 to 35B of H1, 50 to 65 of H2, and 95 to 102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The terms hypervariable regions (HVRs) and complementarity determining regions (CDRs) are used interchangeably in the present application in reference to portions of the variable region that form the antigen-binding regions.

[054] A "acceptor human framework" for purposes in the present application is 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. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence therewith or may contain changes in amino acid sequence. In some embodiments, the number of amino acid exchanges 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 human VL acceptor framework is identical in sequence to the human VL immunoglobulin framework sequence or human consensus framework sequence.

[055] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site on a molecule (eg, an antibody) and its binding partner (eg, an antigen). In some embodiments, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described in the present application.

[056] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) and/or complementarity determining regions (CDRs), compared to a parent antibody that lacks these alterations, such as alterations that result in an enhancement in the affinity of the antibody for the antigen.

[057] A "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is derived from a specific source or species, while the remainder of the heavy chain and/or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are of a first species and all of the constant regions of the chimeric antibody are of a second species.

[058] A "humanized antibody" refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced by the corresponding amino acid of a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is a Fab, an scFv, a (Fab')2, etc.

[059] An "HVR grafted antibody" refers to a humanized antibody in which one or more hypervariable regions (HVRs) from a first (non-human) species has been grafted onto framework regions (FRs) from a second (human) species.

[060] A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; C1q binding, CDC; ADCC; phagocytosis, downregulation of cell surface receptors (eg, B cell receptor; BCR), etc. These effector functions generally require the Fc region to be combined with a binding domain (eg, an antibody variable domain) and can be assessed using various assays.

[061] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgG1 Fc region (A and non-A allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[062] A "variant Fc region" comprises an amino acid sequence that differs from that native sequence Fc region by virtue of at least one amino acid modification.

[063] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FCYR is a native human FcR. In some embodiments, an FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FCYRI, FCYRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FCYRII receptors include FCYRIIA (an "activation receptor") and FCYRIIB (an "inhibition receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activation receptor FcyRIIA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. Receptor inhibition FcyRIIB contains a tyrosine-based immunoreceptor inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and from Haas et al., J. Lab. clinic Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" in this application.

[064] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO document 2004/92219 (Hinton et al.).

[065] "Effector functions" refer to the biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of effector functions of antibodies include: Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[066] “Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FCYRIII and perform ADCC effector function(s). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), Natural Killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a native source, for example, blood.

[067] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., (NK) cells, neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to an antigen-producing target cell and subsequently eliminate the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FCYRIII, whereas monocytes express FCYRI, FCYRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, as described in US patents 5,500,362, 5,821,337 or 6,737,056 (Presta). Useful effector cells for these assays include PBMC and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in an animal model as disclosed in Clynes et al. Proc. Natl. academic Sci. (USA) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in US Patent 7,923,538 and US Patent 7,994,290.

[068] An antibody that has an "enhanced ADCC activity" refers to an antibody that is more effective in mediating ADCC in vitro or in vivo compared to the parent antibody, where the antibody and the parent antibody differ in at least one structural respect, and when the amounts of that antibody and parent antibody used in the assay are essentially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is non-fucosylated whereas the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using the in vitro ADCC assay as disclosed in the present application, but other assays or methods for determining ADCC activity, e.g., in an animal model, etc., are contemplated. In some embodiments, an antibody with enhanced ADCC activity has improved affinity for Fc gamma RIIIA. In some embodiments, an antibody with enhanced ADCC activity has improved affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with enhanced ADCC activity has improved affinity for Fc gamma RIIIA (F158).

[069] An antibody with "altered" FcR binding affinity or ADCC activity is one that has either improved or decreased FcR binding activity and/or ADCC activity compared to a parent antibody, wherein the antibody and the parent antibody differ in at least one structural respect. An antibody that "exhibits increased binding" to an FcR binds at least one FcR with better affinity than the parent antibody. An antibody that "displays decreased binding" to an FcR binds at least one FcR with lower affinity than a parent antibody. Those antibodies that exhibit decreased binding to an FcR may have little or no notable binding to an FcR, for example, 0 to 20% FcR binding compared to a native sequence IgG Fc region.

[070] "Improved affinity for Fc gamma RIIIA" refers to an antibody that has greater affinity for Fc gamma RIIIA (also referred to, in some cases, as CD16a) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural respect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is non-fucosylated whereas the parent antibody is fucosylated. Any suitable method for determining Fc gamma RIIIA affinity can be used. In some embodiments, affinity for Fc gamma RIIIA is determined by a method described in the present application. In some embodiments, an antibody with improved affinity for Fc gamma RIIIA has improved ADCC activity. In some embodiments, an antibody with improved affinity for Fc gamma RIIIA has improved affinity for Fc gamma RIIIA(V158). In some embodiments, an antibody with improved affinity for Fc gamma RIIIA has improved affinity for Fc gamma RIIIA(F158).

[071] "Non-fucosylated" antibody or a "fucose-depleted" antibody refers to an IgG1 or IgG3 isotype antibody that is devoid of fucose in its glycosylation constant region. Glycosylation of human IgG1 or IgG3 occurs at Asn297, as glycosylation of a central fucosylated biantennary complex oligosaccharide terminated with up to two Gal residues. In some embodiments, a non-fucosylated antibody is devoid of fucose in Asn297. These structures are designated as G0, G1 (α1,6 or α1,3) or G2 glycan residues, depending on the amount of terminal Gal residues. See, for example, Raju, T.S., BioProcess Int. 1: 44-53 (2003)) CHO-like glycosylation of Fc antibody is described, for example, in Routier, F.H., Glycoconjugate J. 14: 201-207 (1997). In some embodiments, at least 85% of a batch of antibodies expressed recombinantly in non-glycomodified CHO host cells are fucosylated to Asn297. When referring to a composition comprising a plurality of antibodies, antibodies are considered to be non-fucosylated if < 5% of the antibodies in the composition comprise fucose in Asn297. Methods for measuring fucose include any methods known in the art, including the methods described in the present application. In some embodiments, fucose is detected by the method described in Example 1. In some embodiments, fucose is undetectable in a composition comprising a plurality of non-fucosylated antibodies. In some embodiments, a non-fucosylated antibody has enhanced ADCC activity. In some embodiments, a non-fucosylated antibody has improved affinity for Fc gamma RIIIA. In some embodiments, a non-fucosylated antibody has improved affinity for Fc gamma RIIIA (V158). In some embodiments, a non-fucosylated antibody has improved affinity for Fc gamma RIIIA (F158).

[072] “Complement-dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the traditional complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass), which then bind to their cognate antigens. To assess complement activation, a CDC assay, for example, as described in Gazzano-Santoro et al, J. Immunol. Methods 202:163 (1996), can be done. Antibodies with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described, for example, in US patent 6,194,551 B1, US patent 7,923,538, US patent 7,994,290 and WO 1999/51642. See also, for example, Idusogie et al., J. Immunol. 164: 4178-4184 (2000))

[073] The term "substantially similar" or "substantially the same", as used in the present application, denotes a sufficiently high degree of similarity between two or more numerical values such that a person skilled in the art would consider the difference between the two or more values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said value. In some embodiments, the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25% or 50%.

[074] The expression "substantially reduced" or "substantially different", as used in the present application, denotes a sufficiently high degree of similarity between two numerical values so that a person skilled in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the two substantially different numerical values differ by more than about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

[075] The terms "leader sequence" and "signal sequence" are used interchangeably to refer to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence can be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences can be natural or synthetic, and they can be heterologous or homologous to the protein to which they are attached.

[076] A "native sequence" polypeptide comprises a polypeptide that has the same amino acid sequence as a polypeptide found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence polypeptide" specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms), and allelic variants of the naturally occurring polypeptide.

[077] A "variant" polypeptide means a biologically active polypeptide that has at least about 80% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant will have at least about 80% amino acid sequence identity. In some embodiments, a variant will have at least about 90% amino acid sequence identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.

[078] As used in the present application, "Percentage (%) of amino acid sequence identity" and "homology" in relation to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment, for the purposes of determining percent amino acid sequence identity, can be achieved in a number of ways which are within the art, for example, with the use of publicly available computer programs such as the BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) computer programs. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the entire sequences being compared.

[079] An amino acid replacement can include, but is not limited to, replacing one amino acid in a polypeptide with another amino acid. Conservative substitutions are shown in Table 1 under the heading "preferred substitutions". More substantial changes are provided in Table 1, under the heading of "exemplary substitutions", and as further described below in reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products selected for a desired activity, eg, retained/enhanced antigen binding, decreased immunogenicity, and improved ADCC or CDC. TABLE 1

[080] Amino acids can be grouped according to the common properties of the side chain: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acid: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[081] Non-conservative substitutions will imply the exchange of a member of one of these classes for another class.

[082] The term "vector" is used to describe a polynucleotide that can be genetically engineered to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell.

[083] A "host cell" refers to a cell that can be or has been a recipient of an isolated vector or polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as cells from primate or non-primate animals; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.

[084] The term "isolated" refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. When a polypeptide is secreted by a cell after expression, it is considered "isolating" the polypeptide, physically separating the supernatant containing the polypeptide from the cell that produced it. Similarly, a polynucleotide is referred to as "isolated" when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector within a host cell can be referred to as "isolated".

[085] The terms “individual” or “subject” are used interchangeably in this application to refer to an animal; for example, a mammal. In some embodiments, methods are provided for treating mammals, including, but not limited to, humans, rodents, apes, felines, canines, equines, cattle, swine, sheep, goats, laboratory mammalian animals, farm mammalian animals, sport mammalian animals, and pet mammalian animals. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder.

[086] An “illness” or “disorder” refers to a condition where treatment is required.

[087] The term "cancer" refers to a malignant proliferative dysfunction associated with uncontrolled cell proliferation, unrestricted cell growth, and impaired cell death through apoptosis. Exemplary non-limiting cancers include gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and esophageal cancer. In some embodiments, a cancer comprises an amplification of the FGFR2 gene. In some embodiments, the FGFR2 amplification comprises a ratio of FGFR2:CEN10 (chromosome 10 centromere) >3. In some embodiments, a cancer comprising an amplification of the FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, a cancer overexpresses FGFR2IIIb, but does not comprise amplification of the FGFR2 gene. In some embodiments, a gastric cancer comprises an amplification of the FGFR2 gene. In some embodiments, a gastric cancer comprising an amplification of the FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, a gastric cancer comprising amplification of FGFR2 overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, a gastric cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, a gastric cancer overexpresses FGFR2IIIb, but does not comprise amplification of the FGFR2 gene. In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression.

[088] The term "tumor" is used in the present application to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. A tumor can be benign, premalignant, or malignant; malignant tumor cells are cancerous. The tumor cells can be solid tumor cells or leukemic tumor cells. The term "tumor growth" is used in the present application to refer to proliferation or growth by a cell or cells comprising a tumor that leads to a corresponding increase in tumor size.

[089] As used in this application "treatment" is an approach to achieving beneficial or desired clinical outcomes. "Treatment" as used in the present application encompasses any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of: alleviation of one or more symptoms, lessening the extent of disease, preventing or slowing the spread (e.g., metastasis, e.g., metastasis to the lung or lymph nodes) of the disease, preventing or delaying recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing down the disease or its progression, stopping its development and remission (whether partial or total). Also encompassed by "treatment" is a reduction in the pathological consequence of a proliferative disease. Methods of the invention contemplate any one or more of these aspects of treatment.

[090] In the context of cancer, the term "treat" includes any or all of: inhibiting growth of tumor cells or cancer cells, inhibiting replication of tumor cells or cancer cells, decreasing the total tumor mass, and ameliorating one or more symptoms associated with the disease.

[091] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree or probability of that characteristic. “Reduce” or “inhibit” is to decrease, reduce or stop an activity, function and/or amount compared to a reference. In certain embodiments, it is meant by "reducing" or "inhibiting" the ability to cause an overall decrease of 20% or more. In another embodiment, it is meant by "reducing" or "inhibiting" the ability to cause an overall decrease of 50% or more. In yet another embodiment, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more.

[092] A “reference”, as used in this application, refers to any sample, standard or level that is used for comparison purposes. A reference can be obtained from a healthy and/or non-diseased sample. In some instances, a reference may be obtained from an untreated sample. In some instances, a reference is obtained from an undiseased or untreated sample from an individual subject. In some examples, a referral is obtained from one or more healthy individuals who are not the subject or patient.

[093] As used in the present application, "delaying the development of a disease" means postponing, preventing, delaying, delaying, stabilizing, suppressing and/or postponing the development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or the individual being treated. As is evident to one skilled in the art, sufficient or significant delay can actually encompass prevention, where the individual does not develop the disease. For example, a late-stage cancer, such as metastatic development, may be delayed.

[094] “Prevention”, as used in the present application, includes providing prophylaxis in relation to the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease.

[095] As used in this application, “suppressing” a function or activity is to reduce the function or activity when compared otherwise to the same conditions except for one condition or parameter of interest, or alternatively, compared to another condition. For example, an antibody that suppresses tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[096] An "effective amount" of an agent refers to an amount effective, in dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[097] A "therapeutically effective amount" of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the subject. A therapeutically effective amount is also one at which any toxic or harmful effects of the substance/molecule, agonist or antagonist, are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be distributed in one or more administrations.

[098] A "prophylactically effective amount" refers to an amount effective, in dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, as long as a prophylactic dose is used in subjects, either before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[099] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in such a form to allow the biological activity of the active ingredient(s) to be effective, and that does not contain additional components that are unacceptably toxic to a subject to which the formulation would be administered. These formulations may be sterile.

[0100] A "pharmaceutically acceptable carrier" refers to a non-toxic solid material, semi-solid, diluent, or liquid filler, auxiliary formulation, or vehicle conventional in the art for use with a therapeutic agent which together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients in the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0101] A “sterile” formulation is aseptic or essentially free of living microorganisms and their spores.

[0102] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration, in any order.

[0103] The term "simultaneously" is used in the present application to refer to the administration of two or more therapeutic agents, when at least part of the administration overlaps in time or when the administration of the therapeutic agent falls within a short period of time in relation to the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered no more than about 60 minutes apart, such as no more than about any one of 30, 15, 10, 5, or 1 minute apart.

[0104] The term "sequentially" is used in the present application to refer to the administration of two or more therapeutic agents, when the administration of one or more agents continues after the administration of one or more other agents is discontinued. For example, administration of two or more therapeutic agents are administered more than about fifteen minutes apart, such as about any one of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month or longer.

[0105] As used in the present application, "in conjunction with" refers to the administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to the administration of one treatment modality before, during, or after administration of the other treatment modality to the subject.

[0106] The term “package insert” is used to refer to the instructions included as usual in commercial packages of therapeutic products, which contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of these therapeutic products.

[0107] An "article of manufacture" is any fabrication (for example, a package or container) or kit that comprises at least one reagent, for example, a drug for treating a disease or disorder (for example, cancer), or a probe for specifically detecting a biomarker described in this application. In certain embodiments, the fabrication or kit is promoted, distributed or sold as a unit to perform the methods described in this application.

II. ANTI-FGFR2IIIB ANTIBODIES

[0108] In some aspects, the invention provides a non-fucosylated antibody directed against FGFR2IIIb. Non-fucosylated anti-FGFR2IIIb antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, and antibodies comprising heavy chain and/or light chain HVRs (e.g., CDRs) discussed in the present application. In one aspect, the invention provides isolated non-fucosylated antibodies that bind to FGFR2IIIb. In certain embodiments, a non-fucosylated anti-FGFR2IIIb antibody modulates FGFR2IIIb activity. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody has enhanced ADCC activity. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody has improved affinity for Fc gamma RIIIA. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody has improved affinity for Fc gamma RIIIA(V158). In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody has improved affinity for Fc gamma RIIIA(F158).

[0109] The anti-FGFR2IIIb antibody designated "αFGFR2b" described in the present application in the Examples and the sequence list is intended to have the same amino acid sequence as the HuGAL-FR21 antibody in US patent 8,101,723 B2, issued on January 24, 2012. 13 and 14 of US Patent 8,101,723 B2, which set forth the amino acid sequences of the variable regions and the entire mature antibody chains of HuGAL-FR21, are incorporated by the present application by reference for all purposes. Furthermore, the HVR sequences of the HuGAL-FR21 antibody, which are underlined in Figure 13 of US patent 8,101,723 B2, are specifically incorporated into the present application by reference for all purposes.

[0110] In one aspect, the invention provides a non-fucosylated anti-FGFR2IIIb antibody comprising at least one, two, three, four, five or six HVRs (e.g., CDRs) selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0111] In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0112] In one aspect, the invention provides a non-fucosylated anti-FGFR2IIIb antibody comprising six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the non-fucosylated anti-FGFR2IIIb antibody comprises six HVRs, as described above, and binds to FGFR2IIIb. In some embodiments, the non-fucosylated anti-FGFR2IIIb antibody comprises six HVRs as described above, and binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the non-fucosylated anti-FGFRIIIb antibody does not bind FGFR2IIIc.

[0113] In one aspect, the invention provides a non-fucosylated anti-FGFR2IIIb antibody that competes with an anti-FGFR2IIIb antibody comprising six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0114] In one aspect, the invention provides a non-fucosylated antibody comprising at least one, at least two, or all three of the VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0115] In another aspect, the invention provides a non-fucosylated antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0116] In another aspect, a non-fucosylated antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three of the VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 8; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0117] In another aspect, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, a V sequence L that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 4. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody comprises the VH sequence in SEQ ID NO: 5, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0118] In another aspect, there is provided a non-fucosylated anti-FGFR2IIIb antibody, wherein the antibody comprises a light chain variable domain (VL) that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments , a VL sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 5. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody comprises the VL sequence in SEQ ID NO: 4, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0119] In another aspect, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain (V L) that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, a VH sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, and a VL sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2III antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 4. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 5. In certain embodiments, substitutions, insertions or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody comprises the VH sequence in SEQ ID NO: 4 and the VL sequence in SEQ ID NO: 5, including post-translational modifications of one or both sequences. In a specific embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0120] In another aspect, there is provided a non-fucosylated anti-FGFR2IIIb antibody, wherein the antibody comprises a VH as in any one of the embodiments provided above, and a VL as in any one of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, including post-translational modifications of those sequences.

[0121] In another aspect, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2. In certain embodiments, a heavy chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but a non-fucosylated anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 2. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody heavy chain comprises the VH sequence in SEQ ID NO: 2, including post-translational modifications of that sequence. In a specific embodiment, the heavy chain comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0122] In another aspect, there is provided a non-fucosylated anti-FGFR2IIIb antibody, wherein the antibody comprises a light chain that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3. In certain embodiments, a light chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 3. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody light chain comprises the VL sequence in SEQ ID NO: 3, including post-translational modifications of that sequence. In a specific embodiment, the light chain comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0123] In another aspect, a non-fucosylated anti-FGFR2IIIb antibody comprises a heavy chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 and a light chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a light chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind FGFR2IIIb. In certain embodiments, such an anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3. In certain embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the non-fucosylated anti-FGFR2IIIb antibody heavy chain comprises the VH sequence in SEQ ID NO: 2, including post-translational modifications of that sequence, and the non-fucosylated anti-FGFR2IIIb antibody light chain comprises the VL sequence in SEQ ID NO: 3, including post-translational modifications of that sequence. In a specific embodiment, the heavy chain comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

EXEMPLARY CHIMERIC ANTIBODIES

[0124] In certain embodiments, an antibody, such as a non-fucosylated antibody, provided in the present application is a chimeric antibody. Certain chimeric antibodies are described, for example, in US patent 4,816,567; and Morrison et al., (1984) Proc. Natl. academic 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 a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

Exemplary non-limiting non-fucosylated chimeric antibodies include chimeric antibodies comprising sequences from HVR1, HVR2 and HVR3 heavy chain and/or HVR1, HVR2 and HVR3 light chain, as described in the present application. In some embodiments, the non-fucosylated chimeric anti-FGFR2IIIb antibody comprises the variable regions described above and binds to FGFR2IIIb. In some embodiments, the non-fucosylated chimeric anti-FGFR2IIIb antibody comprises the variable regions as described above, and binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the non-fucosylated chimeric anti-FGFRIIIb antibody does not bind FGFR2IIIc.

[0126] In some embodiments, a non-fucosylated chimeric anti-FGFRIIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4, wherein the antibody binds to FGFR2IIIb. In some embodiments, a non-fucosylated chimeric anti-FGFRIIIb antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, wherein the antibody binds FG FR2IIIb. In some embodiments, a non-fucosylated chimeric anti-FGFRIIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 5; wherein the antibody binds to FGFR2IIIb.

Exemplary non-fucosylated chimeric anti-FGFR2IIIb antibodies also include chimeric antibodies that compete for binding to FGFR2IIIb with an antibody or fragment thereof described in the present application. Thus, in some embodiments, a chimeric anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody competes for binding to FGFR2IIIb, but does not bind FGFR2 IIIc.

[0128] In some embodiments, a chimeric antibody described in the present application comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and A. In some embodiments, a chimeric antibody described in the present application comprises a human IgG constant region. In some embodiments, a chimeric antibody described in the present application comprises a human IgG4 heavy chain constant region. In some embodiments, a chimeric antibody described in the present application comprises a human IgG4 constant region and a human K light chain.

[0129] As noted above, whether or not effector function is desirable may depend on the specific method for treatment aimed at an antibody. Thus, in some embodiments, when effector function is desirable, a chimeric anti-FGFR2IIIb antibody is selected that comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when effector function is desirable, a chimeric anti-FGFR2IIIb antibody is selected that comprises a constant region from the heavy chain of human IgG4 or IgG2.

EXEMPLARY HUMANIZED ANTIBODIES

[0130] In some embodiments, non-fucosylated humanized antibodies that bind to FGFR2IIIb are provided. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as human anti-mouse antibody (HAMA) response), which can result in an immune response to a therapeutic antibody and decreased effectiveness of therapy.

[0131] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity for humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, where, for example, HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

[0132] Humanized antibodies and methods for producing them are reviewed, for example, in Almagro and Fransson, (2008), Front. Biosci. 13: 1619-1633, and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; US Patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 (describing SDR engraftment (a-CDR)); Padlan, (1991) Mol. Immunol. 28:489-498 (which describe "resurfacing"); Dall'Acqua et al., (2005), Methods 36:43-60 (describing "FR scrambling"); and Osbourn et al., (2005), Methods 36:61-68 and Klimka et al., 2000, Br. J. Cancer, 83:252-260 (describing the "guided selection" approach to FR scrambling).

[0133] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, for example, Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a specific subgroup of light or heavy chain variable regions (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature framework regions (somatically mutated) or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633 ); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618).

Non-limiting exemplary humanized antibodies include αFGFR2b, described in the present application. Non-limiting exemplary non-fucosylated humanized antibodies include αFGFR2bA, described in the present application, which has the same amino acid sequences as αFGFR2b comprising fucose (also referred to as αFGFR2bF). Non-limiting exemplary non-fucosylated humanized antibodies also include antibodies comprising an αFGFR2b heavy chain variable region and/or an αFGFR2b light chain variable region. Non-limiting exemplary non-fucosylated humanized antibodies include antibodies comprising a heavy chain variable region of SEQ ID NO:4 and/or a light chain variable region of SEQ ID NO:5. In some embodiments, the humanized anti-FGFR2IIIb antibody comprises the HVRs described above (i.e., (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11) and binds to FGFR2IIIb. In some embodiments, the humanized anti-FGFR2IIIb antibody comprises the HVRs described above, and binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the non-fucosylated humanized anti-FGFRIIIb antibody does not bind FGFR2IIIc.

[0135] In some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody comprises HVR1, HVR2 and HVR3 heavy chain and/or an HVR1, HVR2 and HVR3 light chain of αFGFR2b. Non-limiting exemplary non-fucosylated humanized anti-FGFR2IIIb antibodies include antibodies comprising HVR1, HVR2 and HVR3 heavy chain sets shown in SEQ ID NOs: 6, 7 and 8. 1.

[0136] In some embodiments, a non-fucosylated humanized anti-FGFRIIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, and wherein the antibody binds to FGFR2IIIb. In some embodiments, a non-fucosylated humanized anti-FGFRIIIb antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, wherein the antibody binds FGFR 2IIIb. In some embodiments, a non-fucosylated humanized anti-FGFRIIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 5; wherein the antibody binds to FGFR2IIIb.

[0137] In some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody comprises at least one of the HVRs discussed in the present application. That is, in some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody comprises at least one HVR selected from a heavy chain HVR1 discussed in the present application, a heavy chain HVR2 discussed in the present application, a heavy chain HVR3 discussed in the present application, a light chain HVR1 discussed in the present application, a light chain HVR2 discussed in the present application, and a light chain HVR3 discussed in the present application. Furthermore, in some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody comprises at least one HVR mutated based on an HVR discussed in the present application, wherein the mutated HVR comprises 1, 2, 3 or 4 amino acid substitutions relative to the HVR discussed in the present application. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. Those skilled in the art can select one or more conservative amino acid substitutions suitable for a specific HVR sequence, where suitable conservative amino acid substitutions are not expected to significantly alter the binding properties of the antibody comprising the mutated HVR.

Exemplary non-fucosylated humanized anti-FGFR2IIIb antibodies also include antibodies that compete for binding to FGFR2IIIb with an antibody or fragment thereof described in the present application. Thus, in some embodiments, a humanized anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with αFGFR2b. In some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with αFGFR2b and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the non-fucosylated humanized anti-FGFRIIIb antibody does not bind FGFR2IIIc.

[0139] In some embodiments, a non-fucosylated humanized anti-FGFR2IIIb antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and A.

[0140] In some embodiments, a non-fucosylated humanized antibody described in the present application comprises a human IgG constant region. In some embodiments, when effector function is desirable, a non-fucosylated humanized anti-FGFR2IIIb antibody is selected that comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, a non-fucosylated humanized antibody described in the present application comprises a human IgG1 constant region. In some embodiments, a humanized non-fucosylated antibody described in the present application comprises a human IgG1 constant region, wherein N297 is non-fucosylated. In some embodiments, a non-fucosylated human antibody described in the present application comprises a human IgG1 constant region and a human k light chain.

[0141] In some embodiments, a non-fucosylated humanized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:3. national.

CONSTANT REGIONS OF EXEMPLARY ANTIBODIES

[0142] In some embodiments, a non-fucosylated antibody described in the present application comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and A.

[0143] In some embodiments, a non-fucosylated antibody described in the present application comprises a human IgG constant region. In some embodiments, when effector function is desirable, a non-fucosylated anti-FGFR2IIIb antibody is selected that comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, a non-fucosylated antibody described in the present application comprises a human IgG1 constant region. In some embodiments, a non-fucosylated antibody described in the present application comprises a human IgG1 constant region, wherein N297 is non-fucosylated. In some embodiments, a non-fucosylated antibody described in the present application comprises a human IgG1 constant region and a human k light chain.

[0144] Throughout the present specification and claims, unless explicitly stated or known by those skilled in the art, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated into this application by reference. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0145] In certain embodiments, an antibody of the invention comprises a variant Fc region that has at least one amino acid substitution compared to the Fc region of a wild-type IgG or a wild-type antibody. In certain embodiments, the variant Fc region has two or more amino acid substitutions in the Fc region of the wild-type antibody. In certain embodiments, the variant Fc region has three or more amino acid substitutions in the Fc region of the wild-type antibody. In certain embodiments, the variant Fc region has at least one, two, three or more amino acid substitutions in the Fc region described in the present application. In certain embodiments, the variant Fc region in the present application will have at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent antibody. In certain embodiments, the variant Fc region in the present application will have at least about 90% homology with a native sequence Fc region and/or with an Fc region of a parent antibody. In certain embodiments, the variant Fc region in the present application will have at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent antibody.

[0146] In certain embodiments, an antibody provided in the present application is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0147] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a biantennary branched oligosaccharide that is generally N-linked to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as a fucose attached to a GlcNAc on the “trunk” of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications to an antibody of the invention can be made in order to create antibodies with certain enhanced properties.

[0148] In one embodiment, antibodies are provided that have a fucose-depleted carbohydrate structure attached (directly or indirectly) to an Fc region (i.e., non-fucosylated antibodies). For example, the amount of fucose in a composition comprising a plurality of these antibodies can be from 0% to about 5%. In some embodiments, a composition comprising a plurality of such antibodies comprises at least 95% non-fucosylated antibodies. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297, relative to the sum of all glycostructures linked to Asn297 (eg, complex, hybrid, and high-mannose structures). Exemplary non-limiting methods for detecting fucose in an antibody include MALDI-TOF mass spectrometry (see, e.g., WO 2008/077546), HPLC measurement of fluorescence-labeled oligosaccharides (see, e.g., Schneider et al., "N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection", Agilent Technologies, Inc. (2012); Lines, J. P. harm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J. Chrom., 720: 217-225 (1996), capillary electrophoresis measurement of released fluorescently labeled oligosaccharides (see, e.g., Ma et al., Anal. Chem., 71: 5185-5192 (1999)), and ampere detection HPLC pulsed metry to measure monosaccharide composition (see, e.g., Hardy, et al., Analytical Biochem., 170: 54-62 (1988)) Asn297 refers to the asparagine residue located near 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 antibodies. In αFGFR2b antibody described in the present application, Asn297 is found in the sequence QYNST, and is in bold and underlined in the Sequence Table shown below, SEQ ID NO: 2. Fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “non-fucosylated” or “fucose-deficient” antibodies include: US patent 2003/0157108; WO 2000/61739; WO 2001/29246; US patent 2003/0115614; US patent 2002/0164328; US patent 2004/0093621; US patent 2004/0132140; US patent 2004/0110704; US patent 2004/0110282; US patent 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; 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 non-fucosylated antibodies include CHO Lec13 cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US patent application 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially in Example 11), and knockout cell lines, such as cell lines lacking the functional alpha-1,6-fucosyltransferase gene, FUT8, e.g., CHO knockout 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).

[0149] Antibodies are further provided with bifurcated oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bifurcated by GlcNAc. These antibodies may have reduced fucosylation and/or enhanced ADCC function. Examples of such antibodies are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US patent 6,602,684 (Umana et al.); and US patent 2005/0123546 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. These antibodies may have enhanced CDC function. Such antibodies are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

[0150] Antibodies are also provided with amino-terminal leader extensions. For example, one or more amino-terminal leader sequence amino acid residues are present at the amino terminus of any one or more heavy or light chains of an antibody. An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present in one or both light chains of an antibody.

[0151] Binding polypeptides with high affinity to human FcRn in vivo or the stocking can be analyzed, for example, in transgenic mice, in humans or non-human primates to which polypeptides with a variant Fc region are administered. See also, for example, Petkova et al. International Immunology 18(12):1759-1769 (2006).

[0152] In some embodiments of the invention, a non-fucosylated antibody mediates ADCC in the presence of human effector cells more effectively than a parent antibody comprising fucose. Generally, ADCC activity will be determined using the in vitro ADCC assay as disclosed in the present application, but other assays or methods for determining ADCC activity, e.g., in an animal model, etc., are contemplated.

[0153] In certain embodiments, the "KD", "Kd", "Kd" or "Kd value" of the antibody is measured by using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIACORE, Inc., Piscataway, N.J.) at 25°C with CM5 chips immobilized at approximately 10 response units (RU). Short-term carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 µg/mL (approximately 0.2 µM) prior to injection at a flow rate of 5 µL/minute to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, serial dilutions of polypeptide, eg, whole antibody, are injected into PBS with 0.05% TWEEN-20TM surfactant (PBST) at 25°C at a flow rate of approximately 25 µL/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir linkage model (BIACORE® Evaluation Computing Program version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1s-1 by the above surface plasmon resonance assay, then the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm passband) at 25°C of a 20nM anti-antigen antibody in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow spectrometer (Aviv Instruments) or an SLM-AMINCOTM 8000 Series spectrophotometer (ThermoSpectronic) with a mixing crucible.

[0154] An "on-rate", "association rate", "association rate" or "kon" of the antibody can also be determined as described above using a BIACORE®-2000 system or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.).

[0155] In certain embodiments, the difference between said two values (e.g., Kd values) is substantially the same, e.g., less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%, as a function of the reference/comparison value).

[0156] In certain embodiments, the difference between said two values (e.g., Kd values) is substantially different, e.g., greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%, as a function of the value for the reference/comparison molecule.

EXAMPLE LEADING SEQUENCES

[0157] In order for some secreted proteins to express and secrete in large amounts, a leader sequence from a heterologous protein may be desirable. In some embodiments, the use of heterologous leader sequences can be advantageous in that a resulting mature polypeptide can remain unchanged as the leader sequence is removed in the ER during the secretion process. Addition of a heterologous leader sequence may be required to express and secrete some proteins.

[0158] Certain exemplary leader sequence sequences are described, for example, in the Leader Sequence Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and PCT publication WO 2006/081430.

III. PROPERTIES OF NON-FUCOSYLATED ANTI-FGFR2IIIB ANTIBODIES

[0159] In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity in vitro and/or in vivo. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity in vitro. In some embodiments, in vitro ADCC activity is determined by a method described in the present application, for example, in Example 3. Briefly, cells expressing FGFR2IIIb are contacted with freshly isolated human PBMCs at a ratio of 25:1 effector to target cells (PBMCs) in the presence of fucosylated antibody or non-fucosylated antibody. In some embodiments, Ba/F3 cells expressing FGFR2IIIb are used as target cells. In some embodiments, cytotoxicity is determined by quantification of LDH release using the CytoTox Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). In some embodiments, maximal lysis is determined using 5% Triton X-100 and spontaneous release is determined in the absence of antibody. In some embodiments, the percentage of specific lysis can be determined using the formula: (experimental - spontaneous release) / (maximum - spontaneous release) x 100 = % specific lysis. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody that has enhanced ADCC activity results in specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with the same amount of a non-fucosylated antibody, at least one concentration of antibody tested. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody that has enhanced ADCC activity results in specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with a fucosylated antibody, where each antibody is at a concentration between 0.01 and 1 µg/mL and the target cells are Ba/F3 cells expressing FGFR2IIIb. In some embodiments, the antibodies are tested at a concentration of 0.01 µg/mL, 0.1 µg/mL or 1 µg/mL.

[0160] In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA(V158). In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies have improved affinity for Fc gamma RIIIA(F158). In some embodiments, the affinity of the antibody for Fc gamma RIIIA is determined using surface plasmon resonance, for example, as described in the present application in Example 2, and/or as follows, which is described with reference to Fc gamma RIIIA(V158), but which is also suitable for affinity determination for Fc gamma RIIIA(F158). Briefly, in some embodiments, the fucosylated or non-fucosylated anti-FGFR2IIIb antibody is a protein A-coated dextran chip. Fc gamma RIIIA (V158) (available from, for example, R&D Systems) is injected at various concentrations. The association constant, dissociation constant and affinity of Fc gamma RIIIA (V158) for fucosylated and non-fucosylated anti-FGFR2IIIb antibody can be determined e.g. using computer program provided with surface plasmon resonance system (e.g. 1:1 binding model by Biacore T200 Evaluation Computer Program). In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody with enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) or Fc gamma RIIIA(F158)) binds to Fc gamma RIIIA at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17 times or at least 20 times more affinity than a fucosylated anti-FGFR2IIIb antibody. For example, if an anti-FGFR2IIIb antibody binds Fc gamma RIIIA (V158) with an affinity (KD) of 9.2 nM and a fucosylated anti-FGFR2IIIb antibody binds Fc gamma RIIIA (V158) with an affinity (KD) of 207 nM, then the anti-FGFR2IIIb antibody binds Fc gamma RIIIA (V158) with 207/9, 2=22.5 times more affinity than fucosylated anti-FGFR2IIIb antibody.

IV. ANTI-FGFR2IIIB ANTIBODY EXPRESSION AND PRODUCTION NUCLEIC ACID MOLECULES THAT ENCODE ANTI-FGFR2IIIB ANTIBODIES

[0161] Nucleic acid molecules comprising polynucleotides encoding one or more anti-FGFR2IIIb antibody chains are provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an anti-FGFR2IIIb antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an anti-FGFR2IIIb antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0162] In some of these embodiments, the heavy chain and light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.

[0163] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti-FGFR2IIIb antibody comprises a nucleotide sequence encoding a leader sequence, which, when translated, is located at the N-terminus of the heavy chain or light chain. As discussed above, the leader sequence can be the native heavy or light chain leader sequence, or it can be another heterologous leader sequence.

[0164] The nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

VECTORS

[0165] Vectors comprising polynucleotides encoding anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains are provided. Also provided are vectors comprising polynucleotides encoding anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, as, for example, when the antibody is a scFv.

[0166] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain and a second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole or mass ratio between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1:1 and 1:5 for the heavy chain encoding vector and the light chain encoding vector is used. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0167] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Examples of such vectors are described, for example, in Running Deer et al., Biotechnol. Program 20:880-889 (2004).

HOST CELLS

[0168] In various embodiments, anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells; including CHO-S, DG44. CHO LEC13 cells, and CHO FUT8 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains can be expressed in yeast. See, for example, US Patent Publication 2006/0270045 A1. In some embodiments, a specific eukaryotic host cell is selected based on its ability to produce post-translational modifications to anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialation than the same polypeptide produced in 293 cells.

[0169] The introduction of one or more nucleic acids into a desired host cell can be performed by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Exemplary non-limiting methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be temporarily or stably transfected into desired host cells according to any suitable method.

[0170] In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies are produced in cells capable of producing non-fucosylated antibodies, such as protein fucosylation-deficient CHO Lec13 cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application 2003/0157108 A1, Presta, L; and WO 2004 /056312 A1, Adams et al., especially in Example 11), and knockout cell lines, such as cell lines lacking the functional alpha-1,6-fucosyltransferase gene, FUT8, e.g., CHO knockout 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 ). In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies are produced in CHO cells lacking a functional FUT8 gene. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies are produced in CHOK1SV Potelligent® cells (BioWa/Lonza, Allendale, NJ).

PURIFICATION OF ANTI-FGFR2IIIB ANTIBODIES

[0171] Anti-FGFR2IIIb antibodies can be purified by any suitable method. These methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the FGFR2IIIb ECD and linkers that bind to antibody constant regions. For example, a Protein A, Protein G, Protein A/G or antibody affinity column can be used to bind the constant region and purify an anti-FGFR2IIIb antibody. Hydrophobic interactive chromatography, for example a butyl or phenyl column, may also be suitable for purifying some polypeptides. Many methods for purifying polypeptides are known in the art.

PRODUCTION OF CELL-FREE ANTI-FGFR2IIIB ANTIBODIES

[0172] In some embodiments, an anti-FGFR2IIIb antibody is produced in a cell-free system. Exemplary non-limiting cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003))

V. COMPOSITIONS AND THERAPEUTIC METHODS METHODS FOR TREATMENT OF DISEASES USING ANTI-FGFR2IIIB ANTIBODIES

[0173] Antibodies of the invention and compositions comprising antibodies of the invention are provided for use in methods of treatment for humans or animals. Also provided are methods of treating diseases comprising administering non-fucosylated anti-FGFR2IIIb antibodies. Exemplary non-limiting diseases that can be treated with non-fucosylated anti-FGFR2IIIb antibodies include, but are not limited to, cancer. In some embodiments, methods are provided for treating cancer comprising administering a non-fucosylated anti-FGFR2IIIb antibody. In some embodiments, the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer. In some embodiments, methods are provided for treating gastric cancer which comprise administering a non-fucosylated anti-FGFR2IIIb antibody.

[0174] In some embodiments, a cancer comprises an amplification of the FGFR2 gene. In some embodiments, a cancer comprising an amplification of the FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, a cancer overexpresses FGFR2IIIb, but does not comprise amplification of the FGFR2 gene. In some embodiments, a gastric cancer comprises an amplification of the FGFR2 gene. In some embodiments, a gastric cancer comprising an amplification of the FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, a gastric cancer comprising amplification of FGFR2 overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, a gastric cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, expression levels are normalized to GUSB. In some embodiments, a gastric cancer overexpresses FGFR2IIIb, but does not comprise amplification of the FGFR2 gene. In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression.

[0175] Amplification of the FGFR2IIIb gene can be determined by any method suitable in the art including, but not limited to, in situ hybridization (ISH). In some embodiments, the FGFR2 amplification comprises a ratio of FGFR2:CEN10 (chromosome 10 centromere) >3.

[0176] FGFR2IIIb gene mRNA overexpression can be determined by any method suitable in the art including, but not limited to, methods comprising quantitative PCR (qPCR). The term "FGFR2IIIb mRNA overexpression" means elevated levels of FGFR2IIIb mRNA, regardless of the cause of these elevated levels (i.e., whether the elevated levels are a result of increased transcription and/or decreased mRNA degradation, another mechanism, or a combination of mechanisms).

[0177] Overexpression of FGFR2IIIb protein can be determined by any method suitable in the art including, but not limited to, antibody-based methods such as immunohistochemistry (IHC). In some embodiments, the IHC stain is scored according to methods in the art. The term "FGFR2IIIb protein overexpression" means elevated levels of FGFR2IIIb protein, regardless of the cause of such elevated levels (i.e., whether the elevated levels are a result of increased translation and/or decreased protein degradation, another mechanism, or a combination of mechanisms). In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, the IHC stain is scored as described in Example 6.

PHARMACEUTICAL COMPOSITIONS

[0178] In various embodiments, compositions comprising non-fucosylated anti-FGFR2IIIb antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Li ppencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants and diluents, are available. Furthermore, various pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like are also available. Exemplary non-limiting vehicles include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0179] In various embodiments, compositions comprising non-fucosylated anti-FGFR2IIIb antibodies can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oils or other oils, synthetic aliphatic acid glycerides, higher aliphatic acid esters, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In various embodiments, the compositions can be formulated for inhalation, for example, using acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen and the like. The compositions can also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. An exemplary non-limiting biodegradable formulation includes polylactic acid-glycolic acid polymer. Exemplary non-limiting non-biodegradable formulation includes a polyglycerine fatty acid ester. Certain production methods of these formulations are described, for example, in patent EP 1125584 A1.

ADMINISTRATION ROUTES

[0180] In various embodiments, non-fucosylated anti-FGFR2IIIb antibodies can be administered in vivo by various routes, including, but not limited to, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implant or inhalation. The object compositions can be formulated into preparations in solid, semi-solid, liquid or gaseous form; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration can be selected according to the intended application.

[0181] Pharmaceutical compositions are administered in an effective amount for treatment or prophylaxis of cancer, such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer. The therapeutically effective amount is typically dependent on the weight of the subject being treated, their physical or health condition, the extent of the condition being treated, or the age of the subject being treated. In general, non-fucosylated anti-FGFR2IIIb antibodies can be administered in an amount ranging from about 10 µg/kg of body weight to about 100 mg/kg of body weight per dose. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies can be administered in an amount ranging from about 50 µg/kg of body weight to about 5 mg/kg of body weight per dose. In some embodiments, anti-FGFR2IIIb antibodies can be administered in an amount ranging from about 100 µg/kg of body weight to about 10 mg/kg of body weight per dose. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies can be administered in an amount ranging from about 100 µg/kg of body weight to about 20 mg/kg of body weight per dose. In some embodiments, non-fucosylated anti-FGFR2IIIb antibodies can be administered in an amount ranging from about 0.5 mg/kg of body weight to about 20 mg/kg of body weight per dose.

[0182] Non-fucosylated anti-FGFR2IIIb antibody compositions can be administered as needed for subjects. Determining the frequency of administration may be made by those skilled in the art, as a physician's diagnosis based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated, and the like. In some embodiments, an effective dose of an anti-FGFR2IIIb antibody is administered to a subject one or more times. In various embodiments, an effective dose of a non-fucosylated anti-FGFR2IIIb antibody is administered to the subject once a month, more than once a month, such as every two months or every three months. In other embodiments, an effective dose of a non-fucosylated anti-FGFR2IIIb antibody is administered less than once a month, such as every other week or every week. An effective dose of a non-fucosylated anti-FGFR2 antibody is administered to the subject at least once. In some embodiments, the effective dose of a non-fucosylated anti-FGFR2IIIb antibody can be administered multiple times, including for periods of at least one month, at least six months, or at least one year.

COMBINATION THERAPY

[0183] Non-fucosylated anti-FGFR2IIIb antibodies can be administered alone or with other modes of treatment. They can be given before, at substantially the same time as, or after other modes of treatment, for example surgery, chemotherapy or radiotherapy. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody is administered in conjunction with another anti-cancer agent. Exemplary non-limiting anticancer agents that may be administered with an anti-FGFR2IIIb antibody include platinum agents (such as cisplatin, oxaliplatin, and carboplatin), paclitaxel (TAXOL®), albumin-genetically engineered nanoparticle paclitaxel formulation engineered with albumin (ABRAXANE®), docetaxel (TAXOTERE®), gemcitabine (GEMZAR®), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), epirubicin (ELLENCE®, PHARMORUBICIN®), FOLFOX (oxaliplatin combined with 5-FU and leucovorin), FOLFIRI (combination of leucovorin, 5-FU and irinotecan), leucovorin, fluorouracil (5-FU, EFUDEX®), mitomycin C (MITOZYTREX™, MUTAMYCIN®) and doxorubic hydrochloride in (Adriamycin PFS, Adriamycin RDF RUBEX®). In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody is administered in conjunction with paclitaxel. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody is administered in conjunction with cisplatin and/or 5-FU. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody is administered in conjunction with cisplatin and 5-FU. In some embodiments, a non-fucosylated anti-FGFR2IIIb antibody is administered in conjunction with FOLFOX (oxaliplatin, 5-FU, and leucovorin).

KITS/MANUFACTURING ARTICLES

[0184] The invention also provides kits, medicaments, compositions and unit dosage forms for use in any of the methods described in the present application.

[0185] Kits of the invention include one or more containers comprising a non-fucosylated anti-FGFR2IIIb antibody (or unit dosage forms and/or articles of manufacture). In some embodiments, a dosage unit is provided, wherein the dosage unit contains a predetermined amount of a composition comprising a non-fucosylated anti-FGFR2IIIb antibody, with or without one or more additional agents. In some embodiments, a unit dosage is provided in a single-use, pre-filled syringe for injection. In various embodiments, the composition contained in the unit dosage can comprise saline, sucrose or the like; a buffer such as phosphate or the like; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be supplied as a lyophilized powder which can be reconstituted by adding a suitable liquid, for example, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and/or a proteoglycan.

[0186] In some embodiments, kits of the invention further comprise instructions for use in treating cancer, for example (such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer) according to any of the methods described in this application. The kit may further comprise a description of selecting a suitable individual or treatment. Instructions provided in kits of the invention are typically instructions written on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions loaded onto a magnetic or optical storage disk) are also acceptable. In some embodiments, the kit further comprises another therapeutic agent.

[0187] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, jars, bottles, jars, flexible packaging (eg, sealed Mylar bags or plastic bags), and the like. Kits can optionally provide additional components such as buffers and interpretive information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

EXAMPLES

[0188] The examples discussed below are intended to be purely exemplary of the invention and are not to be considered as limiting the invention in any way. The examples are not intended to represent that the experiments are all following or the only experiment performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.), but some experimental errors and biases must be accounted for. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or close to atmospheric pressure.

EXAMPLE 1 NON-FUCOSYLATED ANTI-FGFR2IIIB ANTIBODY PRODUCTION

[0189] Construction of expression vectors. Nucleotide sequences encoding the heavy chain (HC) and light chain (LC) of the monoclonal antibody αFGFR2b were cloned into the GS Gene Expression System (Lonza, Basel, Switzerland) according to the manufacturer's instructions. The system generates a double gene vector (DGV) containing the expression cassettes for both light and heavy chains.

[0190] Choice of host cell line To generate the non-fucosylated monoclonal antibody αFGFR2bA (the designation “A” after αFGFR2b refers to “non-fucosylated”), CHOK1SV Potelligent® cells (BioWa/Lonza, Allendale, NJ) were chosen as the host cell line. CHOK1SV Potelligent® cells lack the FUT8 gene (α1,6-fucosyltransferase) and therefore produce fucose-free antibodies (non-fucosylated antibodies).

[0191] Construction of stable cell line for non-fucosylated αFGR2bA antibody production: The expression vector comprising the nucleotide sequences encoding the heavy chain and light chain of the αFGFR2b antibody described above was transfected into CHOK1SV Potelligent® cells by electroporation according to the manufacturer's instructions. Electroporated cells were seeded in a 96-well plate at approximately 10,000 cells/50μL/well in CHO CD medium without L-glutamine. Selective CD CHO medium containing 67μM L-methionine sulfoximine (MSX, SIGMA cat #M5379, St. Louis, MO) was added the next day at 150μL/well. Cell growth and clone formation were monitored with an IN Cell Analyzer 2000 (GE Healthcare, Piscataway, NJ).

After 4 to 6 weeks, surviving colonies were screened for αFGFR2bA antibody expression using a Homogeneous Time-Resolved Fluorescence Assay (HTRF) against standard curves generated with purified αFGFR2b antibody using XL665 protein A-conjugated and cryptate-conjugated polyclonal rabbit IgG (Cisbio, Bedford, MA). The highest expressing clones were expanded in a series of scale-up production processes including 24-well plates, centrifuge tubes, shake flasks, bench scale bioreactors and finally a platform production process. At each step, only a subset comprising the highest expressing clones was taken into the next process. The final production clone was selected based on evaluation of protein product titers, cell growth characteristics, product quality, stability as well as scalability in bioreactors. The final production line had an expression level of about 3.5g/L for αFGFR2bA. The absence of fucosylation in αFGFR2bA produced in the final production cell line was confirmed using normal phase HLPC chromatography (N-HPLC).

[0193] The αFGFR2bA antibody was purified by column chromatography and ultrafiltration to concentrate the purified material, then diafiltration to exchange in formulation buffer (20 mM histidine, 150 mM L-arginine, 0.01% polysorbate 20, pH 6.0). The antibody was stored below -70°C.

[0194] αFGFR2bA glycan analysis is performed by releasing the glycans from the protein, labeling the glycans with anthranilic acid (2-AA), and then purifying the labeled glycans. Normal phase HPLC with fluorescent detection is used to separate the glycans and measure the relative amount of each glycan on the antibody. Figure 7 shows that αFGFR2b derived from the CHOK1SV Potelligent® cell line and the αFGFR2b produced from CHOK1SV had two different glycan distributions. (A) Glycan distribution of αFGFR2b derived from the CHOK1SV Potelligent® cell line shows that the antibody is devoid of fucose (“G0”). (B) Glycan distribution of αFGFR2b derived from the CHOK1SV cell line shows that the antibody contains fucose (“G0F”).

[0195] The glycan peaks from the normal phase HPLC separation were identified using two orthogonal methods. First, the αFGFR2b produced by CHOK1SV Potelligent® was labeled and separated using the normal phase HPLC method. After fluorescent detection, the glycans were passed through a QTrap mass spectrometer. The mass of each peak was determined and used to positively identify each glycan, and is shown in Table 2. TABLE 2 MASS OF GLYCANS PEAKS

[0196] The mass of each fucosylated form of glycans (G0F, G1F and G2F) was also investigated in the αFGFR2b produced by CHOK1SV Potelligent® and was not observed. Figure 8 shows schematic diagrams of the structures of G0, G1, G2, G0F, G1F, G2F and mannose-5 (or Man-5) glycans typically seen in antibodies.

[0197] The peak identities of the HPLC assay were also confirmed using standard glycans purchased from Prozyme and matching the retention time between the standards and the αFGFR2 profiles of both, CHOK1SV Potelligent® cell line and CHOK1SV cell line. These patterns were able to identify G0, G0F, Man5, G1, G1F and G2F.

[0198] The results of the HPLC assay as well as the characterization data confirmed the absence of fucosylation in αFGFR2 derived from the CHOK1SV Potelligent® cell line.

EXAMPLE 2 NON-FUCOSYLATED ANTI-FGFR2N ANTIBODY BINDING AFFINITY

[0199] The binding affinities of αFGFR2bA and αFGFR2bF (the “F” designation after αFGFR2b refers to “fucosylated”) for Fc gamma RIIIA(V158) was determined by surface plasmon resonance. Briefly, Protein A was covalently attached to a dextran chip using the Amine Coupling Kit (GE Healthcare Life Sciences, Piscataway, NJ) and 100 mM ethylenediamine (Sigma) in sodium brate buffer, pH 8.0 (Sigma, St. Louis, MO) as the blocking reagent. Approximately 600-800RU of αFGFR2bA and αFGFR2bF were captured onto separate flow cells and a Protein A derivatized flow cell (Pierce) served as a reference control. Fc gamma RIIIA (V158) (R&D Systems, Minneapolis, MN) was diluted in HBS-P + running buffer (Biacore, GE Healthcare, Piscataway, NJ) and injected at 5 concentrations (0 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, and 1000 nM) in duplicate. The association constant, dissociation constant and affinity for αFGFR2bA were calculated using a 1:1 binding model of the Biacore T200 Evaluation Computing Program. The affinity constant for αFGFR2bF binding was determined using the Biacore T200 Evaluation Software steady-state affinity model. The results are shown in Table 3. TABLE 3

[0200] As shown in Table 3, Fc gamma RIIIA(V158) bound to αFGFR2bA with more than 20-fold greater affinity than it bound to αFGFR2bF.

EXAMPLE 3 ADCC ACTIVITY OF NON-FUCOSYLATED ANTI-FGFR2B ANTIBODY

[0201] In vitro assays to determine the ADCC activity of αFGFR2bA antibody versus αFGFR2bF antibody were performed. Target cells expressing Ba/F3 FGFR2IIIb were produced as follows. Ba/F3 cells purchased from the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, cat n° ACC300, Braunschweig, Germany) were maintained in RPMI (Mediatech, cat n° 10-041-CV, Manassas, VA), supplemented with 10% fetal bovine serum (Mediatech, cat n° 35- 010-CV) 1 ng/ml murine IL-3 (Peprotech, cat # 213-13, Rocky Hill, NJ), 1X BME (Invitrogen, cat # 1047574, Grand Island, NY) and 1X Penicillin-Streptomycin (Mediatech cat # 30-002-CI). Ba/F3 cells were transfected with an expression vector that expresses FGFR2IIIb, pBNew-hFGFR2b, using the Nucleofector® Cell Line V Kit (Lonza, cat n° VCA-1003), following the manufacturer's protocol. The pBNew vector contains a CAG promoter with a chicken β-actin intron and ampicillin and puromycin growth selection genes. Transfected cells were incubated in complete growth medium for 3 days, then treated with 2 µg/ml puromycin (InVivoGen, cat # ant-pr-1, San Diego, CA). The selection of puromocoma was maintained during cultivation. To generate individual stable clones, cells were plated at the density of 1 cell per 3 wells. Fluorescent activated cell sorting (FACS) with an anti-FGFR2IIIb antibody was used to select clones with the highest level of FGFR2IIIb expression.

[0202] MFM-223 cells were obtained from the Health Protection Agency, United Kingdom; OCUM-2M cells were obtained from Osaka City University, Osaka, Japan; and KATO III cells were obtained from the ATCC, Rockville, MD. All cells were cultured using standard methods. PMBC freshly isolated from healthy donors were obtained from AllCells, Emeryville, California.

[0203] ADCC assays were conducted using effector cells from 3 independent donors on 3 different days. The ADCC assay was performed using freshly isolated human PBMCs as effector cells at an effector cell to target (E/T) ratio of 25:1. Cells were incubated for 16 hours in the presence of effectors and increasing concentrations of antibody. The ADCC assay was validated using 2 positive control antibodies, HERCEPTIN® on SKOV-3 target cells and RITUXIN® on Raji target cells. A whole human lgG1 negative control antibody (Eureka Therapeutics, Catalog ET901, Emeryville, CA) was used for non-specific cellular cytotoxicity. Cytotoxicity was determined by quantification of LDH release according to the manufacturer's instructions (CytoTox Non Radioactive Cytotoxicity Assay, Promega, Madison, WI).

[0204] Maximum lysis was determined in the presence of 5% Triton X-100, and spontaneous release was determined in the absence of the antibody. Percent specific lysis was calculated as follows, as a percentage of maximum lysis minus spontaneous release: (experimental release - spontaneous) / (maximum release - spontaneous) x 100 = % specific lysis.

[0205] The results for Ba/F3 cells are shown in Figure 1. Non-fucosylated αFGFR2bA antibody induced greater specific lysis than non-fucosylated αFGFR2bF antibody in Ba/F3 cells expressing FGFR2IIIb. In OCUM-2M, MFM 223 and KATO III cells, αFGFR2bA showed greater ADCC activity at lower concentrations than αFGFR2bF, although the maximum specific lysis of the antibodies was comparable in OCUM-2M and MFM 223 cells (data not shown). Furthermore, αFGFR2b showed little or no ADCC activity in cells expressing FGFR2IIIc. See Figure 1A.

EXAMPLE 4 NON-FUCOSYLATED ANTI-FGFR2B ANTIBODY ACTIVITY IN GASTRIC AND BREAST CANCER XENOGRAFT MODELS

[0206] Six week old female CB17 SCID mice were purchased from Charles River Laboratories (Wilmington, MA) and were acclimated for 1 week prior to the start of the study. The human gastric carcinoma cell line OCUM-2M or the breast cell carcinoma cell line MFM-223 were used as tumor models. OCUM-2M was purchased from Public University Corporation Osaka City University (OCU, Osaka, Japan) and MFM-223 was purchased from Culture Collections, Public Health England (98050130, HPA Culture Collections, Salisbury, UK). Cells were grown for up to three passages in complete growth medium until expansion for implantation. OCUM-2M and MFM-223 cells were grown in Dulbecco's Minimal Essential Medium (DMEM) and Minimal Essential Medium (MEM), respectively. All media were supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine and Penicillin-Streptomycin solution. Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 .

[0207] When the cultured cells reached 85 to 90% confluency, the cells were harvested and resuspended in phosphate buffered saline (PBS) free of Ca2+ and Mg2+, containing 50% Matrigel (BD BioSciences, San Jose, CA). OCUM-2M cells were implanted subcutaneously into the right flank of mice at 5 x 105 cells/100 µL/mouse. MFM-223 cells were implanted subcutaneously into the right flank of mice at 5 x 10 5 cells/100 μL/mouse, and 0.72 mg pellets with 17-β estradiol released within 90 days (Innovative Research of America, Sarasota, FL) were inoculated subcutaneously into the right flank. Mice were monitored twice a week after cell implantation for tumor growth. Tumor size was measured according to the formula: Tumor size (mm3) = (width (mm) x length (mm)2)/2. When the MFM-223 tumors reached 80 mm3, the mice were sorted and randomized (n = 10) and treatment started. For OCUM-2M tumors, anti-FGFR2b monotherapy was started once tumors reached a mean size of 100 mm3, and combination therapy was started once tumors reached a mean size of 250 mm3.

[0208] Humanized Anti-FGFR2IIIb antibody (non-fucosylated αFGFR2bA; fucosylated αFGFR2bF) or albumin as a negative control were administered in doses ranging from 1 to 10 mg/kg by intraperitoneal injection twice a week, as specified in the legends to the Figures. Chemotherapeutic agents were administered by intraperitoneal injection twice a week at 12 mg/kg for paclitaxel, 2.3 mg/kg for fluorouracil (5-FU) and 33 mg/kg for cisplatin. At the start of therapy, tumor sizes were measured in each mouse twice a week. The length and width of each tumor was measured using calipers and the tumor size was calculated according to the above formula. Mice were euthanized when subcutaneous tumor volumes exceeded 2000 mm3 or when tumors became excessively necrotic.

[0209] Tumor volume comparisons as a consequence were determined to be statistically significant if P < 0.05. P-values were calculated using unpaired two-tailed t-test analysis of tumor volumes calculated on the final day that tumors were measured.

[0210] Figure 2 shows the efficacy of αFGFR2bA and αFGFR2bF at (A and B) 10 mg/kg and (C and D) 3 mg/kg in a xenographic model of human gastric cancer OCUM-2M with FGFR2 amplification. (A) At 10 mg/kg, both fucosylated and non-fucosylated antibody induced immediate tumor regression. However, non-fucosylated αFGFR2bA induced a more durable response than fucosylated αFGFR2bF. (B) Non-fucosylated αFGFR2bA significantly reduced final OCUM-2M tumor size compared to fucosylated αFGFR2bF (p = 0.0153). Statistical significance was determined using the two-tailed unpaired t-test. (C) When dosed at 3 mg/kg, non-fucosylated αFGFR2bA reduced tumor growth compared to fucosylated αFGFR2bF. Indeed, non-fucosylated αFGFR2bA induced greater tumor regression and durable response compared to fucosylated αFGFR2bF (note: one animal was removed from the αFGFR2bF group at about day 42, resulting in a shift in the curve). (D) Non-fucosylated αFGFR2bA at 3 mg/kg compared to fucosylated αFGFR2bF remarkably reduced the final OCUM-2M tumor size (p = 0.0767). Statistical significance was determined using the two-tailed unpaired t-test.

[0211] Figure 3 shows dose-dependent tumor inhibition by αFGFR2bA. (A) SCID mice bearing subcutaneous OCUM-2M xenografts were treated with 1, 1.5, 2, 3, or 5 mg/kg of non-fucosylated αFGFR2bA when the mean tumor size reached approximately 100 mm3. Although all doses of non-fucosylated αFGFR2bA inhibited tumor growth, the greatest suppression and durable response was seen with 3 and 5 mg/kg, with reduced suppression of growth with 2, 1.5, and 1 mg/kg of αFGFR2bA. (B) Non-fucosylated αFGFR2bA reduced OCUM-2M tumor size with higher doses, showing more potent growth suppression. Tumor growth suppression was statistically significant for all doses (5 mg/kg, p < 0.0001; 3 mg/kg, p < 0.0001; 2 mg/kg, p < 0.0001; 1.5 mg/kg, p = 0.0003; 1 mg/kg, p = 0.0013). Statistical significance was determined using the two-tailed unpaired t-test.

[0212] Figure 4 shows the improvement of antitumor activity of paclitaxel in an OCUM-2M gastric cancer xenograft model by administration with non-fucosylated αFGFR2bA. (A) SCID mice bearing subcutaneous OCUM-2M xenografts were treated with non-fucosylated αFGFR2bA (5 mg/kg), paclitaxel (12 mg/kg), or a combination of the two when the mean tumor size reached approximately 285 mm3. Combining non-fucosylated αFGFR2bA with paclitaxel reduced tumor size compared to either αFGFR2bA or paclitaxel alone. (B) αFGFR2bA/paclitaxel combination therapy significantly reduced final OCUM-2M tumor size compared to both paclitaxel (p=0.0005) and αFGFR2bA (p=0.0009) alone. Statistical significance was determined using the two-tailed unpaired t-test.

[0213] Figure 5 shows the improvement of antitumor activity of cisplatin/5-FU in an OCUM-2M gastric cancer xenograft model by administration with non-fucosylated αFGFR2bA. (A) SCID mice bearing subcutaneous OCUM-2M xenografts were treated with non-fucosylated αFGFR2bA (5 mg/kg), fluorouracil (5-FU) plus cisplatin (2.3 and 33 mg/kg, respectively), or with a combination of the three when the mean tumor size reached approximately 260 mm3. The combination of non-fucosylated αFGFR2bA with 5-FU/cisplatin reduced tumor size compared to either αFGFR2bA or 5-FU/cisplatin alone. (B) Combination therapy of αFGFR2bA/5-FU/cisplatin significantly reduced OCUM-2M tumor size compared to 5-FU/cisplatin (p = 0.0003). The increased reduction in tumor size by αFGFR2bA/5-FU/cisplatin combination therapy compared to αFGFR2bA alone was notable, but not statistically significant. Statistical significance was determined using the two-tailed unpaired t-test.

[0214] Figure 6 shows the efficacy of αFGFR2bA in an MFM-223 human breast cancer xenograft model. (A) MFM-223, a human breast carcinoma cell line with FGFR2 amplification, was implanted subcutaneously in SCID mice and therapy with non-fucosylated αFGFR2bA was initiated when the mean tumor size reached approximately 80 mm3. Non-fucosylated αFGFR2bA (5 mg/kg) dramatically reduced the growth of MFM-223 tumors compared to albumin control (5 mg/kg). (B) Non-fucosylated αFGFR2bA significantly reduced final MFM-223 tumor size compared to albumin control (p = 0.0008). Statistical significance was determined using the two-tailed unpaired t-test.

EXAMPLE 5 NON-FUCOSYLATED ANTI-FGFR2B ANTIBODY MEASURES HIGHER ADCC THAN FUCOSYLATED ANTI-FGFR2G ANTIBODY

[0215] In vitro assays to determine the ADCC activity of αFGFR2bA antibody versus αFGFR2bF antibody were performed. Target cells expressing Ba/F3 FGFR2IIIb were generated as described above. OCUM-2M cells were obtained from Osaka City University, Osaka, Japan. All cells were cultured using standard methods. PMBC freshly isolated from healthy donors were obtained from AllCells, Emeryville, California.

[0216] ADCC assays were conducted using freshly isolated human PBMCs as effector cells at an effector cell to target (E:T) ratio of 25:1. Cells were incubated for 16 hours in the presence of effectors and increasing concentrations of antibody. All conditions were tested in triplicate. Cytotoxicity was determined by quantification of LDH release according to the manufacturer's recommendations (Promega CytoTox Non-Radioactive Cytotoxicity Assay). For OCUM-2M cells, maximal lysis was determined in the presence of 5% Triton X-100, and spontaneous release was determined in the absence of the antibody. The percentage of specific lysis was calculated as a percentage of maximum lysis minus spontaneous release, as follows: (experimental release - spontaneous) / (maximum release - spontaneous) x 100 = % specific lysis. GraphPad computer program curve-fit analysis was used to obtain EC50 values.

[0217] The results for Ba/F3 cells are shown in Figure 9. The non-fucosylated αFGFR2bA antibody showed higher potency (greater ADCC activity at lower concentrations) than the fucosylated αFGFR2bF antibody on Ba/F3 cells expressing FGFR2IIIb. Similar results are shown for OCUM-2M cells in Figure 10. Table 4 shows the fold increase in potency of non-fucosylated anti-FGFRb antibody compared to fucosylated anti-FGFR2b antibody in the two different cell lines shown in Figures 9 and 10. TABLE 4

EXAMPLE 6 IMMUNOHISTOCHEMICAL DETECTION OF FGFR2IIIB PROTEIN IN TUMOR TISSUE

[0218] The murine αFGFR2b antibody comprising the murine variable regions of GAL-FR21 (see US patent 8,101,723 B2) and a murine IgG2a constant region were used to detect the FGFR2IIIb protein in gastric tumor tissue embedded in formalin-fixed paraffin (FFPE). The FFPE tumor tissue was deparaffinized with successive xylene washes and reduced concentrations of ethanol and rehydrated with water for five minutes. Tissue sections mounted on slides were immersed in 0.1 mM citrate buffer (pH 6.0), heated at 95°C to 99°C for 15 minutes. Tissue sections were cooled and placed in 3% H2O2 for 5 minutes at room temperature, then washed twice in TBST (0.05M Tris, 0.15M NaCl, 0.05% Tween 20) and blocked in blocking buffer (2.5% normal horse serum in TBST) for 30 minutes at room temperature. Tissue sections were then incubated with 5 μg/mL of αFGFR2b antibody in blocking buffer for 30 minutes at room temperature. Incubation times between 30 minutes and 2 hours produced similar staining intensities. After three washes with TBST buffer, tissue sections were incubated with ready-to-use biotinylated secondary horse anti-mouse antibody (RTU) (10 µg/mL diluted in blocking buffer (Vector Laboratories, Burlingame, CA; Cat. No. PK-7200) for 30 minutes at room temperature. Tissue sections were washed twice with TSBT buffer and incubated with horseradish peroxidase-streptavidin (H RP; Vector Laboratories, Cat. No. PK-7200) for 30 minutes at room temperature. Detection was performed using 3',3'-diaminobenzidine (DAB) substrate (Vector Laboratories, Cat. No. PK-4105) for ten minutes at room temperature as per the manufacturer's instructions. Tissue sections were counterstained with Hematoxylin (Dako North America, Carpinteria, CA, Cat. No. K-8008) as instructed The tissue sections were dehydrated using successive washes with increasing concentrations of ethanol and xylenes and then covered with a coverslip.

[0219] Staining concentrations of 0.1 to 5 mg/ml of αFGFR2b antibody were found to be particularly effective for protein detection.

[0220] Staining was scored as follows: 0 No staining observed with αFGFR2b antibody 1+ Weak staining is observed in samples stained with αFGFR2b antibody. Staining is absent in the corresponding sections stained with the negative control antibody. 2+ Cytoplasmic membrane staining specific for the αFGFR2b antibody is more prevalent in sections. 3+ Strong specific staining with αFGFR2b antibody with distinct membrane localization in at least a subset of stained tumor cells.

[0221] Figure 11 shows exemplary staining 0 (A), 1+ (B, C), 2+ (D), and 3+ (E, F) of tumor tissues. In Figure 11A, ductal cells and scattered tumor cells showed an absence of staining with the use of the αFGFR2b antibody. In Figure 11B, which shows an interstitial-type gastric tumor sample, the tumor cells (arrows) showed some staining with the use of the αFGFR2b antibody, whereas the neighboring ductal cells did not show staining. In Figure 11C, which shows a diffuse-type gastric tumor sample (arrows), the tumor cells show some staining with the use of the αFGFR2b antibody, whereas the neighboring stromal cells (arrowheads) show no staining. In Figure 11D, tumor cells (arrows) show intermediate staining with the use of the αFGFR2b antibody, whereas neighboring stromal cells (arrowheads) show no staining. In Figure 11E, tumor cells (arrows) show high localized staining on the membrane with the use of the αFGFR2b antibody, whereas neighboring stromal cells (arrowheads) show no staining. In Figure 11F, tumor cells (arrows) show high membrane/cytoplasm staining with the use of the αFGFR2b antibody, whereas neighboring stromal cells (arrowheads) show no staining. SEQUENCE TABLE

Claims (39)

1. COMPOSITION, comprising anti-FGFR2IIIb antibodies, wherein each anti-FGFR2IIIb antibody comprises heavy chain and light chain variable regions, wherein the heavy chain variable region comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein at least 95% of the anti-FGFR2IIIb antibodies in the composition are non-fucosylated. 2. COMPOSITION according to claim 1, characterized in that the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4. 3. COMPOSITION according to claim 1 or 2, characterized in that the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. 4. COMPOSITION according to claim 1, characterized in that the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. 5. COMPOSITION according to any one of claims 1 to 4, characterized in that the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. 6. COMPOSITION according to any one of claims 1 to 5, characterized in that the light chain comprises the amino acid sequence of SEQ ID NO: 3. 7. COMPOSITION according to claim 1, characterized in that the heavy chain comprises the amino acid sequence of SEQ ID NO: 2 and the light chain comprises the amino acid sequence of SEQ ID NO: 3. 8. COMPOSITION, characterized in that it comprises non-fucosylated anti-FGFR2IIIb antibodies, wherein the anti-FGFR2IIIb antibodies compete for binding to FGFR2IIIb with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. 9. COMPOSITION according to any one of claims 1 to 8, characterized in that the antibodies are monoclonal antibodies. 10. COMPOSITION according to any one of claims 1 to 9, characterized in that the antibodies are chimeric or humanized antibodies. 11. COMPOSITION, according to any one of claims 1 to 10, characterized by the fact that the antibodies are devoid of fucose in Asn297. 12. COMPOSITION according to any one of claims 1 to 11, characterized in that the antibodies comprise a K light chain constant region, an IgG1 heavy chain constant region, or both. 13. COMPOSITION according to any one of claims 1 to 12, characterized in that the non-fucosylated antibodies have improved in vitro ADCC activity compared to a fucosylated anti-FGFR2IIIb antibody that has the same amino acid sequence. 14. COMPOSITION according to any one of claims 1 to 13, characterized by the fact that the non-fucosylated antibodies have improved Fc gamma RIIIA affinity compared to a fucosylated anti-FGFR2IIIb antibody that has the same amino acid sequence. 15. COMPOSITION according to any one of claims 1 to 14, characterized in that the non-fucosylated anti-FGFR2IIIb antibodies bind to FGFR2IIIb, but do not bind to FGFR2IIIc. 16. METHOD FOR PRODUCING NON-FUCOSYLATED ANTI-FGFR2IIIB ANTIBODIES IN VITRO, characterized in that it comprises cultivating a host cell comprising a nucleic acid encoding anti-FGFR2IIIb antibodies, as defined in any one of claims 1 to 15, and lacking a functional alpha-1,6-fucosyltransferase (FUT8) gene, under appropriate conditions to express non-fucos anti-FGFR2IIIb antibodies ilados. 17. METHOD, according to claim 16, characterized in that it further comprises recovering the anti-FGFR2IIIb antibodies produced by the host cell. 18. METHOD, according to claim 16 or 17, characterized in that at least 95% of the anti-FGFR2IIIb antibodies produced by the host cell are non-fucosylated. 19. PHARMACEUTICAL COMPOSITION, characterized in that it comprises the composition, as defined in any one of claims 1 to 15, and a pharmaceutically acceptable vehicle. 20. USE OF A PHARMACEUTICAL COMPOSITION, as defined in claim 19, characterized in that it is for the manufacture of a drug to treat cancer in an individual in need thereof. 21. USE, according to claim 20, characterized by the fact that the cancer is a solid tumor. 22. USE, according to claim 20, characterized in that the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer or esophageal cancer. 23. USE, according to claim 22, characterized by the fact that the cancer is gastric cancer. 24. USE according to any one of claims 20 to 23, characterized by the fact that the cancer overexpresses FGFR2IIIb. 25. USE, according to claim 24, characterized by the fact that the expression of FGFR2IIIb is determined by immunohistochemistry (IHC). 26. USE according to any one of claims 20 to 25, characterized by the fact that the cancer does not include amplification of the FGFR2 gene. 27. USE according to any one of claims 20 to 25, characterized in that the cancer comprises an amplification of the FGFR2 gene. 28. USE, according to any one of claims 20 to 27, characterized in that the drug further comprises at least one additional therapeutic agent selected from a platinum agent, paclitaxel, ABRAXANE®, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C and doxorubicin hydrochloride, for the treatment of cancer in an individual in need thereof. 29. USE, according to claim 28, characterized in that the at least one additional therapeutic agent is FOLFOX. 30. PHARMACEUTICAL COMPOSITION, for the treatment of cancer in an individual, characterized in that it comprises an effective amount of an anti-FGFR2IIIb antibody, as defined in any one of claims 1 to 15, and a pharmaceutically acceptable vehicle. 31. PHARMACEUTICAL COMPOSITION, according to claim 30, characterized by the fact that the cancer is a solid tumor. 32. PHARMACEUTICAL COMPOSITION, according to claim 30, characterized in that the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and esophageal cancer. 33. PHARMACEUTICAL COMPOSITION, according to claim 32, characterized in that the cancer is gastric cancer. 34. PHARMACEUTICAL COMPOSITION, according to any one of claims 30 to 33, characterized by the fact that the cancer overexpresses FGFR2IIIb. 35. PHARMACEUTICAL COMPOSITION, according to claim 34, characterized by the fact that the expression of FGFR2IIIb is determined by immunohistochemistry (IHC). 36. PHARMACEUTICAL COMPOSITION, according to claim 34 or 35, characterized by the fact that the cancer does not include amplification of the FGFR2 gene. 37. PHARMACEUTICAL COMPOSITION according to any one of claims 30 to 34, characterized in that the cancer comprises an amplification of the FGFR2 gene. 38. PHARMACEUTICAL COMPOSITION, according to any one of claims 30 to 37, characterized in that it further comprises at least one additional therapeutic agent selected from a platinum agent, paclitaxel, ABRAXANE®, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C and doxo hydrochloride rrubicin. 39. PHARMACEUTICAL COMPOSITION, according to claim 38, characterized in that the at least one additional therapeutic agent is FOLFOX.