Afucosylated Anti-FGFR2iiib antibodies

Abstract

To provide compositons comprising antibodies that bind FGFR2IIIb.SOLUTION: A composition comprising a plurality of anti-FGFR2IIIb antibodies, wherein each anti-FGFR2IIIb antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising (i) HVR-H1, (ii) HVR-H2, and (iii) HVR-H3, each comprising a specific amino acid sequence, and the light chain variable region comprising (iv) HVR-L1, (v) HVR-L2, and (vi) HVR-L3, each comprising a specific amino acid sequence, wherein at least 95% of the anti-FGFR2IIIb antibodies in the composition are afucosylated.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Afucosylated anti-FGFR2IIIb antibodies are provided. [Background technology]

[0002] Fibroblast growth factor (EGF) family members bind to different FGFRs to varying degrees. Four tyrosine kinase receptors for fibroblast growth factor receptors bind different FGFs It binds to FGFR1-4 and their isoforms (Zhang et al. l., J. Biol. Chem. 281:15694, 2006). The protein sequence of human FGFR2 is, for example, The EGFR is available at GenBank locus AF487553. The extracellular domain (ECD) contains immunoglobulin (Ig)-like domains (D1, D2, and D3). ), a single transmembrane helix, and an intracellular catalytic kinase domain (Mohammadi et al. l., Cytokine Growth Factor Revs, 16:107, 2005). The linker between D1 and D2 contains The adjacent amino acids are called the "acid box" (AB). The region containing D1 and AB is the region of the receptor that is relaxed upon binding to a ligand. FGFs are thought to be involved in autoregulation. They act mainly on the D2 and D3 domains of the receptor. FGFR binds to its receptor through its mRNA domain, which gives rise to various isoforms. It is characterized by multiple alternative splicing of NA (Ornitz et al., J. Biol. Ch em. 271:15292, 1996; Swiss-Pro See also P21802 and isoforms P21802-1 to P21802-20 In particular, forms containing all three Ig domains (α isoforms), or A form containing only two Ig domains, D2 and D3, without D1 (β-Ig) In FGFR1 to FGFR3, all forms are IIIa and IIIb. Contains the first half of D3 shown, but two alternative exons are used for the second half of D3 This results in the IIIb and IIIc forms. These are FGFR2IIIb and FGFR2IIIc (or simply FGF the corresponding beta forms are designated FGFR2 (beta FGFR2b and FGFR2c); )IIIb and FGFR2(beta)IIIc. sam-I) binds well to both FGF1 and FGF2 but not to KGF. It does not bind to other family members, but binds to FGFR2 (also designated K-sam-II). The FGFR2IIIb form inhibits FGF1 and KGF family members (FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19 ... It is a high-affinity receptor for both FGF10 and FGF22 (Miki et al., Proc. Na Acad. Sci. USA 89:246, 1992). Indeed, FGFR2IIIb is a member of the KGF family. It is the only receptor for KG members (Ornitz et al., 1996, op. cit.), and therefore It is marked FR.

[0003] FGFRs and their isoforms are differentially expressed in various tissues. R2IIIb (and the IIIb forms of FGFR1 and FGFR3) are expressed in epithelial tissues. FGFRIIIc is expressed in mesenchymal tissues, whereas FGFRIIIc is expressed in mitochondrial tissues (Duan et al., J. Biol. Chem. 267 :16076, 1992; Ornitz et al., 1996, op. cit.). Specific FGF ligands for these receptors Thus, FGF7 (KGF), FGF10, and FGF11 have opposite expression patterns. KGF subfamily members, including GF22, bind only to FGFRIIIb (Zhang et al., 2014). t al., op. cit.) and expressed in mesenchymal tissues, thus exerting paracrine effects on epithelial cells. On the other hand, FGF4 subfamily members FGF4 to 6 are FGFRs. It binds to 2IIIc and is expressed in both epithelial and mesenchymal tissues, thus acting as an autoclave. It may have either a synaptic or paracrine function. Due to the expression pattern of FGFR2 and its ligands, FGFR2 plays a role in epithelial-mesenchymal interactions. (Finch et al., Dev. Dyn. 203:223, 1995), and therefore, FG in mice Knockout of FR2IIIb causes embryonic defects and lethality (De Moerlooze et al. l., Development 127:483, 2000).

[0004] KGF (FGF7) and KGFR (FGFR2IIIb) are involved in many pancreatic cancers. , and are overexpressed (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and their co-expression FG is correlated with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Somatic mutations in the FR2 gene were found in 12% of a large panel of endometrial (uterine) cancers. In various test cases, it was found to be essential for tumor cell survival. The FGFR2 mutation was found to be the same S252W substitution associated with Apert syndrome. Amplification and overexpression of FGFR2 are associated with undifferentiated diffuse gastric cancer, which has a significantly poor prognosis. In response, inhibition of FGFR2 activity by small molecule compounds strongly inhibits the proliferation of such cancer cells. (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Zhang et al., J. Biol. Chem. 281:15694, 2006 [Non-patent document 2] Mohammadi et al., Cytokine Growth Factor Revs, 16:107, 2005 [Non-patent document 3] Ornitz et al., J. Biol. Chem. 271:15292, 1996 [Non-patent document 4] Miki et al., Proc. Natl. Acad. Sci. USA 89:246, 1992 [Non-Patent Document 5] Ornitz et al., 1996, op. cit. [Non-patent document 6] Duan et al., J. Biol. Chem. 267:16076, 1992 [Non-Patent Document 7] Zhang et al., op. cit. [Non-patent document 8] Finch et al., Dev. Dyn. 203:223, 1995 [Non-Patent Document 9] De Moerlooze et al., Development 127:483, 2000 [Non-Patent Document 10] Ishiwata et al., Am. J. Pathol. 153: 213, 1998 [Non-Patent Document 11] Cho et al., Am. J. Pathol. 170:1964, 2007 [Non-Patent Document 12] Kunii et al., Cancer Res. 68:2340, 2008 [Non-Patent Document 13] Nakamura et al., Gastroenterol. 131:1530, 2006 Summary of the Invention

[0006] In some embodiments, an anti-FGFR2IIIb antibody is provided, wherein the heavy chain variable region is (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 (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (iv) 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. The antibody contains an HVR-L3 containing the amino acid sequence; the antibody is afucosylated. In some embodiments, the antibody lacks fucose at Asn297. In the present invention, a composition comprising a plurality of anti-FGFR2IIIb antibodies is provided, wherein each anti-FGFR2IIIb antibody in the composition The heavy chain variable region of the FR2IIIb antibody comprises: (i) an HVR- comprising the amino acid sequence of SEQ ID NO: 6; H1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) SEQ ID NO: and each anti-FGFR2IIIb antibody in the composition comprises an HVR-H3 comprising an amino acid sequence of 8. The light chain variable region of the antibody is: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (vi) 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. at least 95% of the antibodies in the composition are afucosylated; In some embodiments, the composition may be the supernatant of an antibody-producing cell line. In some embodiments, the composition is a buffered composition. In some embodiments, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain variable region domain comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 4, and In some embodiments, 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. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 3. In the present invention, a composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies is provided, wherein the antibodies a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 5; It competes for binding to FGFR2IIIb with an antibody comprising a light chain variable domain containing

[0007] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In any of the embodiments described herein, the antibody may comprise a kappa light chain constant region. In any of the embodiments described herein, the antibody comprises an IgG1 heavy chain constant region. Good too.

[0008] In some embodiments, the antibody is a fucosylated anti-FGF antibody having the same amino acid sequence as It has enhanced ADCC activity in vitro compared to the R2IIIb antibody. In some embodiments, the afucosylated anti-FGFR2IIIb antibody is a fucosylated anti-FGF at least 10, at least 15, or at least 10 times higher than the specific lysis by the R2IIIb antibody At least 20, at least 25, at least 30, at least 35, at least 40, at least at least 45, at least 50, at least 60, at least 65, at least 70, or less In some embodiments, the specific lysis is at least 75 percentage points higher. The ADCC activity was observed in Ba / F3 cells expressing FGFR2IIIb as target cells, and The efficacy of immunization with IgG4-dependent IL-16 is determined using isolated human PBMCs as effector cells.

[0009] In some embodiments, the antibody is a fucosylated anti-FGF antibody having the same amino acid sequence as Has enhanced affinity for Fc gamma RIIIA compared to R2IIIb antibodies In some embodiments, the afucosylated anti-FGFR2IIIb antibody is a fucosylated at least 2-fold, at least 3-fold, at least 4-fold higher than anti-FGFR2IIIb antibody; At least 5 times, at least 7 times, at least 10 times, at least 12 times, at least 1 Binds to Fc gamma RIIIA with 5-fold, at least 17-fold, or at least 20-fold higher affinity In some embodiments, Fc gamma RIIIA is a surface plasmon resonance (SPR) antibody. In some embodiments, Fc gamma RIIIA is determined using Fc gamma RIIIA. Fc gamma RIIIA (V158) and Fc gamma RIIIA (F158). In some embodiments, Fc gamma RIIIA is Fc gamma RIIIA(V158) is.

[0010] In any of the embodiments described herein, the antibody may bind to FGFR2IIIb. It is acceptable for the antibody to bind to FGFR2IIIc, but it does not have to bind to FGFR2IIIc.

[0011] In any of the embodiments described herein, multiple afucosylated anti-FGFR2IIIb The composition comprising the antibody comprises at least 95% afucosylated antibody. In any embodiment, a composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies comprises: Fucosylation may be undetectable. In some embodiments, the presence of fucose is High-performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-T It may be determined by methods including OF mass spectrometry.

[0012] In some embodiments, a nucleic acid encoding an anti-FGFR2IIIb antibody described herein is provided. A host cell is provided that contains a nucleic acid encoding a functional alpha-1,6-fucosyltransferase. In another embodiment, the host cell is a CHO It is a cell.

[0013] In some embodiments, an afucosylated anti-FGFR2IIIb antibody is generated. In some embodiments, the method comprises injecting a host cell with an anti-FG and culturing the nucleic acid encoding the FR2IIIb antibody under conditions suitable for expression. and the host cell contains a functional alpha-1,6-fucosyltransferase (FUT8) gene. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is produced. The method for producing the afucosylated anti-FGFR2IIIb antibody comprises converting a host cell into a In some embodiments, the method comprises culturing the host cell under conditions suitable for the host cell to grow. and recovering the anti-FGFR2IIIb antibody produced by the cells. In an embodiment, at least one of the anti-FGFR2IIIb antibodies produced by the host cell 5% contains fucose. In some embodiments, the anti-F produced by the host cell At least 95% of the GFR2IIIb antibody lacks fucose (e.g., is afucosylated). In some embodiments, the anti-FGFR2 II antibody produced by the host cell In Ib antibodies, no fucose is detected. In some embodiments, the presence of fucose Currently, methods include HPLC, capillary electrophoresis, or MALDI-TOF mass spectrometry. It is more detectable.

[0014] In some embodiments, the afucosylated anti-FGFR2IIIb antibody described herein A pharmaceutical composition is provided comprising the compound and a pharmaceutically acceptable carrier.

[0015] In some embodiments, methods of treating cancer are provided. The method comprises administering to a subject an afucosylated anti-FGFR2IIIb antibody as described herein and a pharmaceutical In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a therapeutically acceptable carrier. In embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and ectopic pregnancy cancer. In some embodiments, the cancer is selected from gastric cancer. In some embodiments, the cancer comprises FGFR2 gene amplification. FGFR2 amplification is characterized by an FGFR2:CEN10 (chromosome 10 centromere) ratio of >3 In some embodiments, the cancer overexpresses FGFR2IIIb. In embodiments, the cancer containing FGFR2 amplification has a higher FGFR2 amplification rate than FGFR2IIIc. In some embodiments, the cancer comprises FGFR2 amplification. is 2-fold, 3-fold, 5-fold, or 10-fold higher than the normalized FGFR2IIIc expression level In some embodiments, the FGFR2IIIb is expressed at a high, normalized level. In some embodiments, the expression level is normalized to GUSB. overexpress FGFR2IIIb but do not contain FGFR2 gene amplification. In one embodiment, the expression or overexpression of FGFR2IIIb is determined by IHC. In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC is The color indicates overexpression of FGFR2IIIb. 2+ or 3+ staining of tumor cells indicates overexpression of FGFR2IIIb. In this embodiment, IHC staining is scored as described in Example 6.

[0016] In some embodiments, the method of treating cancer includes administering to a subject a platinum agent, paclitaxel, abdominopropyl methacrylate, or the like. ABRAXANE®, docetaxel, gemcitabine, capecitabine Irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluconazole At least one selected from the group consisting of orouracil, mitomycin C, and doxorubicin hydrochloride In some embodiments, the platinum agent further comprises administering one additional therapeutic agent. Selected from cisplatin, oxaliplatin, and carboplatin. In some embodiments, the treatment of cancer further comprises administering paclitaxel. In some embodiments, the treatment of cancer further comprises administering cisplatin and / or 5-FU. Included.

[0017] In some embodiments, the afucosylated anti-FGFR2IIIb antibody described herein The use of a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable carrier is provided. In an embodiment, such use is for treating cancer in an individual with cancer. In some embodiments, the cancer is gastric cancer, breast cancer, ovarian cancer, intrauterine cancer, In some embodiments, the cancer is selected from mesenchymal cancer, pancreatic cancer, and esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer comprises FGFR2 gene amplification. In some embodiments, FGFR2 amplification is FGFR2:CEN10 (chromosome 10 census). In some embodiments, the cancer has an FGFR2IIIb (FGFR2IIIb) ratio of >3. In some embodiments, the cancer with FGFR2 amplification is overexpressed. In some embodiments, the FGFR2IIIb is overexpressed relative to 2IIIc. , cancers with FGFR2 amplification have higher FGFR2IIIc expression levels than normalized FGFR2IIIc expression levels. Express FGFR2IIIb at normalized levels that are 2-fold, 3-fold, 5-fold, or 10-fold higher In some embodiments, the expression levels are normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but not the FGFR2 gene. In some embodiments, the expression or overexpression of FGFR2IIIb does not include gene amplification. Expression is determined by IHC. In some embodiments, tumor 1+, 2+ or 3+ staining of cells indicates overexpression of FGFR2IIIb. In an embodiment, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2III In some embodiments, IHC staining is performed as described in Example 6. The score is then calculated accordingly.

[0018] In some embodiments, the afucosylated anti-FGFR2IIIb antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, the cancer is gastric cancer, breast cancer, ovarian cancer, endometrial cancer, In some embodiments, the cancer is selected from pancreatic cancer and esophageal cancer. In some embodiments, the cancer comprises FGFR2 gene amplification. In embodiments, FGFR2 amplification is FGFR2:CEN10 (chromosome 10 centromere ) ratio >3. In some embodiments, the cancer overexpresses FGFR2IIIb In some embodiments, the cancer with FGFR2 amplification is FGFR2III In some embodiments, the FGF receptor 2IIIb is overexpressed relative to FGFR2IIIc. Cancers with R2 amplification had 2-fold, 3-fold, and 4-fold higher FGFR2IIIc expression levels than normalized FGFR2IIIc expression levels. The cells express FGFR2IIIb at normalized levels that are 2-fold, 5-fold, or 10-fold higher. In some embodiments, expression levels are normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but not FGFR2 gene amplification. In some embodiments, the expression or overexpression of FGFR2IIIb does not include In some embodiments, the presence of a tumor cell is determined by IHC. +, 2+, or 3+ staining indicates overexpression of FGFR2IIIb. In this study, 2+ or 3+ staining of tumor cells by IHC indicated excess FGFR2IIIb. In some embodiments, IHC staining is performed as described in Example 6. , is scored. [Brief explanation of the drawings]

[0019] [Figure 1] 1A to 1C show the ADCC activity of afucosylated αFGFR2bA and fucosylated αFGFR2bF against FGFR2IIIb-expressing Ba / F3 cells, as described in Example 3. In the legend, "αFGFR2bF / FGFR2b" indicates that the fucosylated αFGFR2bF antibody was tested against FGFR2IIIb-expressing Ba / F3 target cells. [Figure 2] Figures 2A to 2D show the efficacy of afucosylated αFGFR2bA and fucosylated αFGFR2bF in the OCUM-2M gastric cancer xenograft model at 10 mg / kg (A and B) and 3 mg / kg (C and D), as described in Example 4. [Figure 3] 3A and 3B show the dose-dependent efficacy of afucosylated αFGFR2bA in the OCUM-2M gastric cancer xenograft model, as described in Example 4. [Figure 4]4A and 4B show the efficacy of afucosylated αFGFR2bA and paclitaxel combination therapy in an OCUM-2M gastric cancer xenograft model, as described in Example 4. [Figure 5] 5A and 5B show the efficacy of afucosylated αFGFR2bA and 5-FU / cisplatin combination therapy in an OCUM-2M gastric cancer xenograft model, as described in Example 4. [Figure 6] 6A and 6B show the efficacy of afucosylated αFGFR2bA in the MFM-223 breast cancer xenograft model, as described in Example 4. [Figure 7] FIG. 7 shows the glycan profile of αFGFR2b antibody produced in (A) Potelligent® CHOK1SV cells and (B) CHOK1SV cells, as described in Example 1. [Figure 8] FIG. 8 shows a schematic representation of N-linked glycans typically found on antibodies. [Figure 9] 9 shows ADCC of Ba / F3 FGF2b cells with increasing concentrations of αFGFR2bA or αFGFR2bF. Assays were performed using normal human PBMCs at an E:T ratio of 25:1 as described in Example 5. Data are plotted as LDH release. [Figure 10] Figure 10 shows ADCC of OCUM-2M cells with increasing concentrations of αFGFR2bA or αFGFR2bF. Assays were performed with normal human PBMCs at an E:T ratio of 25:1, as described in Example 5. Data are plotted as percent specific lysis. [Figure 11] 11A to 11F show the detection of FGFR2IIIb in tumor tissue samples using immunohistochemistry, as described in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0020] Afucosylated antibodies that bind to FGFR2IIIb are provided. In addition, an afucosylated FGFR2IIIb antibody can be generated. Antibody heavy and light chains are provided. In some embodiments, one or more particular hypervariable Afucosylated antibodies, heavy and light chains, containing heterologous regions (HVRs) are provided. In some embodiments, the afucosylated anti-FGFR2IIIb antibody is a fucosylated anti-FGFR2IIIb antibody. In some embodiments, the antibody has enhanced ADCC activity compared to the IIb antibody. The afucosylated anti-FGFR2IIIb antibody showed a higher activity than the fucosylated anti-FGFR2IIIb antibody. In some embodiments, the antibody has enhanced affinity for Fc gamma RIIIA compared to Fc gamma RIIIA. In the present invention, the afucosylated anti-FGFR2IIIb antibody is a fucosylated anti-FGFR2IIIb Has enhanced affinity for Fc gamma RIIIA (V158) compared to antibodies In some embodiments, the afucosylated anti-FGFR2IIIb antibody is a fucosylated Enhancement to Fc gamma RIIIA (F158) compared to anti-FGFR2IIIb antibody In some embodiments, the afucosylated anti-FGFR2 III antibody has a specific affinity. b The antibody does not bind to FGFR2IIIc.

[0021] Polynucleotides encoding antibodies that bind to FGFR2IIIb are provided. Polynucleotides encoding the heavy or light chains are also provided. Host cells expressing FGFR2IIIb antibodies are provided. Methods of treatment using cosylated antibodies are provided. Such methods include those for treating gastric cancer, breast cancer, This includes methods for treating cancers such as ovarian, endometrial, pancreatic, and esophageal cancer. Not limited to these.

[0022] The section headings used herein are for organizational purposes only. and are not to be construed as limiting the subject matter described.

[0023] Patent applications, patent publications, and Genbank accessions cited herein All references, including their section numbers, are hereby incorporated by reference in their entirety as if each reference were specifically and individually incorporated by reference into the present invention. No. 6,117,793, filed on Oct. 1, 2004, and which are incorporated herein by reference as if fully set forth.

[0024] The techniques and procedures described or referenced herein are generally well understood and generally within the skill of those in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition dition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURR ENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al.eds., (2003)); es METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney , ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Mol ecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); In troduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Ple num Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffi ths, 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 Ma mmalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Ch ain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E . Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and S ons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. F inch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1 989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds. , Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harl ow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Z anetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: P Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott C Company, 1993); and their revised versions, using widely available conventional methods. I can.

[0025] I. Definition Unless otherwise defined herein, the scientific and technical Technical terms shall have the meaning commonly understood by one of ordinary skill in the art. Unless otherwise required or expressly stated in the context, singular terms shall include the plural; and , plural terms shall include the singular.

[0026] Aspects and embodiments of the present invention described herein may be used "consisting of" and / or "essentially As used herein, the singular forms "a," " "An" and "the" include plural references unless otherwise indicated.

[0027] Unless otherwise defined or understood by one of ordinary skill in the art, In this context, the use of "or" means "and / or." Use implies reference to one or more of the preceding independent or dependent claims.

[0028] As will be understood by those skilled in the art, "approximately" values or Reference to a parameter includes (describes) an embodiment for that value or the parameter itself. For example, the statement "about X" includes the statement "X."

[0029] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably. and may refer to a polymer of nucleic acids. Such a polymer of nucleic acids may be naturally occurring and / or may include non-naturally occurring nucleic acids and may include DNA, RNA, and PNA, A "nucleic acid sequence" is a linear sequence of nucleic acids, including a nucleic acid molecule or polynucleotide. Points to an array.

[0030] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. Such polymers of amino acid residues are used without any limitation on their minimum length. They may contain natural or non-natural amino acid residues, and may be peptides, oligopeptides, or peptides of amino acid residues. These may include, but are not limited to, dimers, trimers, and multimers. Both full-length proteins and fragments thereof are included by definition. Post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for purposes of this invention, a "polypeptide" refers to a protein that has a desired activity. Deletions, additions, substitutions (usually conservative in nature) to the native sequence, etc., while maintaining the identity These modifications are intentionally made by site-directed mutagenesis. or mutations in the host producing the protein or during PCR amplification. It may be accidental due to an error.

[0031] "FGFR2IIIb" or "FGFR2b" are used interchangeably and are used to describe fibroblast proliferation refers to the factor receptor 2IIIb splice form. Exemplary human FGFR2IIIb GenBank accession number NP_075259.4 dated July 7, 2013. A non-limiting exemplary mature human FGFR2IIIb amino acid sequence is shown in SEQ ID NO:1. is shown.

[0032] "FGFR2IIIc" or "FGFR2c" are used interchangeably and are used to describe a fibroblast proliferation refers to the factor receptor 2IIIc splice form. Exemplary human FGFR2IIIc GenBank accession number NP_000132.3 dated July 7, 2013 A non-limiting exemplary mature human FGFR2IIIc amino acid sequence is shown in SEQ ID NO: 12. It is shown as follows.

[0033] The term "epitope" refers to a region of an antigen-binding molecule (e.g., an antibody, an antibody fragment, including the binding region of an antibody). a target molecule (e.g., a protein, a nucleus) to which the fragment or scaffold protein binds An epitope is often a site on an amino acid or an antigen (such as a carbohydrate, a lipid, or an amino acid). It consists of chemically active surface molecular groups such as amino acids, polypeptides, or sugar side chains, and Epitopes have specific three-dimensional structural characteristics, as well as specific charge characteristics. Adjacent residues or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) Epitopes formed by tertiary structural folding can be formed from both Typically, they are lost upon treatment with reducing solvents, but adjacent residues (e.g., amino acids, nucleotides) are lost. Epitopes formed from amino acid residues (such as nucleotides, sugars, or lipid moieties) typically undergo a reduction upon exposure to reducing solvents. The epitope may be at least 3, at least 5, or 8 to 10 residues (e.g., Examples of suitable amino acids include, but are not limited to, amino acids, amino acids, or nucleotides. In the present invention, an epitope is less than 20 residues (e.g., amino acids or nucleotides) in length, The length of the antibody is less than 12 residues, or less than 12 residues. In this case, they may bind to the same epitope within an antigen.

[0034] A "non-linear epitope" or "conformational epitope" is an epitope that is specific to that epitope. Non-contiguous polypeptides, amino acids, and / or sugars within an antigenic protein to which the body binds Includes:

[0035] A "linear epitope" is an antigenic protein to which an antibody specific for that epitope binds. It includes adjacent polypeptides, amino acids, and / or sugars within a protein.

[0036] The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures. Clonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and , including, but not limited to, antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0037] The term antibody refers to a fragment capable of binding to an antigen, such as an Fv, a single chain Fv (s These include, but are not limited to, (cFv), Fab, Fab', and (Fab')2. Papain digestion of antibodies produces two identical antibody-binding fragments called "Fab" fragments, Each has a single antigen-binding site, and the remainder is designated "Fc", reflecting its ability to be readily crystallized. Papain treatment results in a fragment that has two antigen-binding sites and is still able to cross-link antigens. The term antibody also includes chimeric antibodies, humanized antibodies, and the like. Antibodies, including but not limited to antibodies of various species, such as murine, human, and cynomolgus monkey. I can't.

[0038] The term "heavy chain variable region" refers to a heavy chain HVR1, framework (FR) 2, HVR2, FR In some embodiments, the heavy chain variable region is a region comprising HVR3, HVR4, and HVR5. It also includes at least a part of FR1 and / or at least a part of FR4.

[0039] The term "heavy chain constant region" refers to a region of at least three C H 1. C H 2, and C H 3 heavy chain constant domain Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Typical exemplary heavy chain constant regions include ε and μ. Each heavy chain constant region has a specific function for each antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, and an antibody containing a delta constant region is an I antibody. Antibodies containing the α constant region are IgA antibodies. An antibody comprising the ε constant region is an IgM antibody, and an antibody comprising the ε constant region is an IgE antibody. Certain isotypes can be further divided into subclasses. For example, IgG antibodies are classified into Ig IgG1 (containing the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region) IgG4 (containing a γ4 constant region) and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include IgA1 (containing the α1 constant region) and IgA2 (containing the α2 constant region) antibodies. and IgM antibodies include, but are not limited to, IgM1 and IgM2. Not limited to.

[0040] The term "heavy chain" includes at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least one heavy chain constant region. The term "full-length heavy chain" refers to a heavy chain variable region, with or without a leader sequence. and a heavy chain constant region.

[0041] The term "light chain variable region" refers to a light chain HVR1, framework (FR) 2, HVR2, FR In some embodiments, the light chain variable region refers to a region comprising HVR3, HVR4, and HVR5. It also contains at least FR1 and / or FR4.

[0042] The term "light chain constant region" refers to the light chain constant domain C L Non-limiting exemplary light Chain constant regions include λ and κ.

[0043] The term "light chain" includes at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a light chain constant region. The term "full-length light chain" refers to a light chain variable region, with or without a leader sequence. It refers to a polypeptide comprising a mitochondrial, mitochondrial, and light chain constant region.

[0044] The term "hypervariable region" or "HVR" refers to a region of a gene that is hypervariable in sequence and / or structurally defined. The term "hypervariable loop" refers to the regions of an antibody variable domain that form defined loops ("hypervariable loops"). In general, natural four-chain antibodies have V H Three (H1, H2, H3) and V L Three (L1, L The HVRs generally consist of six HVRs derived from hypervariable loops and / or "phase" regions. The amino acid residues are derived from the complementarity-determining regions (CDRs), the latter of which exhibit the highest sequence variability. and / or involved in antigen recognition. A typical hypervariable loop is amino acid residues 26-32. (L1), 50~52 (L2), 91~96 (L3), 26~32 (H1), 53~55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:9 01-917 (1987). Typical CDRs (CDR-L1, CDR-L2, CDR-L3, CDR CDR-H1, CDR-H2, and CDR-H3) are amino acid residues 24 to 34 of L1, 50-56, 89-97 in L3, 31-35B in H1, 50-65 in H2, and 9 in H3 Occurs from 5 to 102 (Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (19 91)). The terms hypervariable region (HVR) and complementarity determining region (CDR) are used herein. The terms "variable domain" and "antigen binding domain" are used interchangeably to refer to the portion of the variable domain that forms the antigen-binding region.

[0045] For purposes herein, an "acceptor human framework" is defined below. As shown, a human immunoglobulin framework or a human consensus framework The light chain variable domain (V L ) framework or heavy chain variable domain (V H )Fre The framework contains the amino acid sequence of the human immunoglobulin framework. Acceptor human frameworks derived from the framework or human consensus framework are , may contain the same amino acid sequence, or it may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less. , 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. V L The acceptor human framework is V L Human immunoglobulin framework sequences Or the sequence is identical to a human consensus framework sequence.

[0046] "Affinity" refers to the affinity of a single binding site of a molecule (e.g., an antibody) to its binding partner (e.g., In some embodiments, the strength of the total non-covalent interactions between the antibody and the antigen. "Binding affinity" refers to the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). refers to the intrinsic binding affinity that reflects the affinity of molecule X for its partner Y. Affinity can generally be expressed as a dissociation constant (Kd). It can be measured by common methods known in the art.

[0047] An "affinity matured" antibody is one that exhibits increased affinity to an antigen compared to a parent antibody that does not possess that modification. one or more alterations in one or more hypervariable regions (HVRs) thereof that result in an improvement in affinity for the It refers to an antibody having a complementarity-determining region (CDR) and / or a complementarity-determining region (CDR).

[0048] "Chimeric" antibodies are antibodies in which some of the heavy and / or light chains are derived from a particular source or species, while the rest are derived from a particular species. In some embodiments, the heavy and / or light chains of an antibody are derived from different sources or species. In the present invention, chimeric antibodies are derived from a first species (e.g., mouse, rat, cynomolgus monkey, etc.). and at least one variable region derived from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody refers to an antibody comprising at least one constant region. contains at least one murine variable region and at least one human constant region. In one embodiment, the chimeric antibody comprises at least one cynomolgus monkey variable region and at least one In some embodiments, all variable regions of the chimeric antibody comprise at least one human constant region. The constant regions of the chimeric antibody are derived from a first species and all of the constant regions of the chimeric antibody are derived from a second species.

[0049] A "human antibody" is an antibody in which at least one amino acid in the framework region of a non-human variable region is replaced by a It refers to antibodies in which some amino acids have been replaced with the corresponding amino acids from the human variable region. In an embodiment, a humanized antibody comprises at least one human constant region, or a fragment thereof. In some embodiments, the humanized antibody is a Fab, scFv, (Fab')2, etc. do.

[0050] "HVR-grafted antibody" refers to the first One or more hypervariable regions (HVRs) of one species (non-human) are interdigitated with framework regions of a second species (human). It refers to a humanized antibody grafted into the FR region.

[0051] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; C1q binding; CDC; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors; BCR), etc. Such effector functions are typically associated with a binding domain (e.g., an antibody variable domain). The Fc region for antibody binding is required and can be assessed using a variety of assays.

[0052] A "native sequence Fc region" is an Fc region having an amino acid sequence identical to that of an Fc region found in nature. The native sequence human Fc region contains the amino acid sequence of a native sequence human IgG1 Fc region. (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human I and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Includes variants.

[0053] A "variant Fc region" is an Fc region that is modified by at least one amino acid mutation in a native sequence Fc region. It contains an amino acid sequence that is different from the amino acid sequence.

[0054] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In one embodiment, the FcγR is a native human FcR. FcγRI, FcγRII, and FcγRI bind to the gamma receptors (gamma receptors). II subclass receptors, including allelic variants and alternatively spliced versions of these receptors. FcγRII receptors include FcγRIIA ("activating receptor"). FcγRIIB ("inhibitory receptor") and FcγRIIB ("inhibitory receptor"), which are primarily involved in the cytoplasmic delivery of The activating receptor FcγRIIA has a similar amino acid sequence with a different main domain. The inhibitory receptor F contains an ITAM in its cytoplasmic domain. cγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (e.g., Daeron , Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are described, for example, in Ravetch and K inet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1 994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are referred to herein by the term "FcRs." FcR" are included in the

[0055] The term "Fc receptor" or "FcR" also refers to the receptors involved in the transport of maternal IgG to the fetus. including the neonatal receptor, FcRn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunogl. Methods for measuring binding to FcRn are known (e.g., 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); See Publication No. 2004 / 92219 (Hinton et al.).

[0056] "Effector functions" are those attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity ( ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell Activation.

[0057] "Human effector cells" are cells that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII. and carry out ADCC effector functions. Examples of human leukocytes that mediate ADCC are peripheral leukocytes (PEs). Blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic Effector cells include T cells and neutrophils. Effector cells can be derived from natural sources, such as blood. It may be isolated.

[0058] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the activation of specific cytotoxic cells (e.g., They bind to Fc receptors (FcRs) present on cells (e.g., NK cells, neutrophils, and macrophages). The combined secreted Ig allows cytotoxic effector cells to specifically target antigen-bearing target cells. Cytotoxicity, which allows binding and subsequent killing of target cells by cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII However, monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in the IL-16 / IL-2 cells was determined by the method described in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). The ADCC activity of a molecule of interest is summarized in Table 3 on page 64. No. 5,500,362 or No. 5,821,337 or U.S. Patent No. 6,737,056 (Presta) In vitro ADCC assays such as those described in may be performed. For this purpose, effective effector cells include PBMCs and NK cells. Additionally, ADCC activity of a molecule of interest can be assayed using methods such as those described in, e.g., Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998), in vivo Additional antibodies with altered Fc region amino acids and enhanced or reduced Fc region amino acids can be evaluated. The ADCC activity is described, for example, in U.S. Pat. No. 7,923,538 and U.S. Pat. No. 7,994,290. It is written.

[0059] If the amounts of such antibody and parent antibody used in the assay are identical, then "enhanced AD" An antibody with "CC activity" is one that mediates ADCC in vitro or in vivo compared to the parent antibody. The term "antibody" refers to an antibody that is more effective in treating a cancer, wherein the antibody and the parent antibody share at least one structural aspect. In some embodiments, the antibody and the parent antibody differ in amino acid sequence. The antibody has afucosylated sequences, but is afucosylated, whereas the parent antibody is fucosylated. In some embodiments, ADCC activity is measured by the in vitro ADCs described herein. C assay, but may be used in other assays, such as animal models, or ADC Methods for determining A C activity are contemplated. In some embodiments, enhanced A Antibodies with DCC activity have enhanced affinity for Fc gamma RIIIA. In some embodiments, the antibody with enhanced ADCC activity is an Fc gamma R In some embodiments, the antibody has an enhanced affinity for IIIA(V158). Therefore, antibodies with enhanced ADCC activity are directed against Fc gamma RIIIA (F158). It has an enhanced affinity for the

[0060] An antibody with "altered" FcR binding affinity or ADCC activity has an altered FcR binding affinity or ADCC activity compared to the parent antibody. , having enhanced or reduced FcR binding activity and / or ADCC activity, wherein The antibody and parent antibody differ in at least one structural aspect. An antibody that "exhibits" binding to at least one FcR binds with higher affinity than the parent antibody. An antibody that "exhibits reduced binding" to an FcR has lower binding to at least one FcR than the parent antibody. Antibodies that exhibit reduced binding to FcRs bind with almost undetectable affinity to FcRs. No or no detectable binding, e.g., 0.05 relative to the native sequence IgG Fc region. It may have ~20% binding.

[0061] "Enhanced affinity for Fc gamma RIIIA" refers to an increased affinity for Fc gamma RIIIA. It refers to an antibody that has a higher affinity than the parent antibody (in some cases, it also refers to Cd16a). where the antibody and the parent antibody differ in at least one structural aspect. In this embodiment, the antibody and the parent antibody have identical amino acid sequences, but the parent antibody is a fucoprotein. Fc gamma RIIIA is afucosylated whereas antibodies are afucosylated. Any suitable method for determining affinity may be used. In embodiments, affinity for Fc gamma RIIIA is determined by the methods described herein. In some embodiments, the enhanced affinity for Fc gamma RIIIA is In some embodiments, antibodies having F Antibodies with enhanced affinity for Fc gamma RIIIA ( In some embodiments, the Fc group has enhanced affinity for the Fc antigen. Antibodies with enhanced affinity for Fc gamma RIIIA (F1 58) has enhanced affinity for

[0062] Afucosylated or "fucose-deficient" antibodies have a low glycosylation in the constant region. Refers to IgG1 or IgG3 isotype antibodies that lack fucose and are up to 2Ga Like the predominant fucosylated biantennary complex oligosaccharide glycosylation terminated by l residues, I Glycosylation of IgG1 or IgG3 occurs at Asn297. Therefore, the antibody lacks the fucose at Asn297. Glycan residues are defined as G0, G1 (α1,6 or α1,3) or G2 depending on the amount of See, e.g., Raju, TS, BioProcess Int. 1: 44-53 (2003). HO-type glycosylation is described, for example, in Routier, FH, Glycoconjugate J. 14: 201-207 (199 In some embodiments, in a non-glycoengineered CHO host cell, At least 85% of recombinantly expressed antibody batches have a nucleotide sequence at Asn297. When referring to a composition comprising multiple antibodies, the fucose at Asn297 is An antibody is afucosylated if <5% of the antibody in the composition is afucosylated. Methods for measuring fucose include those described herein. In some embodiments, fucose is prepared by any method known in the art. In some embodiments, fucose is detected by the method described in 1. In some embodiments, the fucosylated antibody is not detected in a composition comprising the fucosylated antibody. Cosylated antibodies have enhanced ADCC activity. Cosylated antibodies have enhanced affinity for Fc gamma RIIIA. In an embodiment, the afucosylated antibody is an antibody against Fc gamma RIIIA (V158). In some embodiments, the afucosylated antibody has an Fc affinity. It has enhanced affinity for mAbRIIIA (F158).

[0063] "Complement dependent cytotoxicity" or "CDC" refers to the lysing of a target cell in the presence of complement. The canonical complement activation pathway involves the binding of the first component of the complement system (C1q) to its cognate antigen. It is initiated by binding to antibodies (appropriate subclass). To assess complement activation, e.g. For example, the CD400 described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) A C assay may be performed. Antibodies having q binding ability are disclosed in U.S. Pat. Nos. 6,194,551, 7,923,538, It is described in U.S. Pat. No. 7,994,290 and WO 1999 / 51642. See also, e.g., Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0064] As used herein, the terms "substantially similar" or "substantially the same" mean that a person of ordinary skill in the art would recognize that two or more The differences between the above values are mostly in the context of the biological properties measured by said values. 2 or more, so that one would consider there to be little or no biological and / or statistical significance In some embodiments, two or more substantial A substantially similar value is less than about 5%, 10%, 15%, 20%, 25%, or 50%. differs.

[0065] As used herein, the terms "substantially reduced" or "substantially different" refer to the degree to which a person skilled in the art can , the difference between two values is in the context of the biological property measured by said values, It means a difference between two values that is high enough to be statistically significant. In some embodiments, the two substantially different values are about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% The difference is higher than the other.

[0066] The terms "leader sequence" and "signal sequence" are used interchangeably and are used interchangeably to refer to sequences derived from mammalian cells. The sequence of amino acid residues located at the N-terminus of a polypeptide that promotes secretion of the polypeptide is The leader sequence is the sequence that is present in a polypeptide derived from a mammalian cell when forming a mature protein. The leader sequence may be natural or synthetic and may be cleaved upon translocation of the attached The protein may be heterologous or homologous to the protein with which it is coupled.

[0067] A "native sequence" polypeptide is one that contains the same amino acids as a polypeptide found in nature. Therefore, a polypeptide of a native sequence can be expressed in any animal. Such naturally occurring polypeptides may have the amino acid sequence of a naturally occurring polypeptide derived from The polypeptide of the sequence can be isolated from nature or obtained by recombinant or synthetic means. The term "native sequence" polypeptides refers, inter alia, to naturally occurring, short-chain polypeptides. secreted or secreted polypeptides (e.g., extracellular domain sequences), naturally occurring variants forms (e.g., alternatively spliced forms), and naturally occurring polynucleotides. Allelic variants of the peptides are included.

[0068] "Variants" of polypeptides may be prepared by aligning sequences and inserting gaps, if necessary. After achieving the maximum percent sequence identity, and after any conservation as part of the sequence identity is achieved, The amino acid sequence of the polypeptide is at least about 80% identical to that of the native sequence, without considering complementary substitutions. The term "variant" refers to a biologically active polypeptide having amino acid sequence identity with the polypeptide. For example, one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, the variants include polypeptides that are at least 80% In some embodiments, the variants have at least 90% amino acid sequence identity. In some embodiments, the variant has at least It has 95% amino acid sequence identity.

[0069] As used herein, the term "amino acid sequence identity" refers to a peptide, polypeptide, or antibody sequence. "Percentage of identity" and "homology" are calculated by aligning the sequences, if necessary, and After inserting gaps to achieve the maximum percent sequence identity and in a particular peptide or polypeptide without considering any conservative substitutions as part of is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the candidate sequence. Alignment for purposes of determining percentage amino acid sequence identity is , various methods within the skill of the art, such as BLAST, BLAST-2, ALIG N, or publicly available computer programs such as MEGALIGN™ (DNASTAR) software. This can be achieved by using computer software. Any algorithm required to achieve maximal alignment over the entire length of the sequences Suitable parameters for measuring alignment can be determined, including:

[0070] Amino acid substitutions include the replacement of one amino acid with another amino acid in a polypeptide. Conservative substitutions include, but are not limited to, those listed in Table 1 under the heading "preferred substitutions." More substantial changes are given in Table 1 under the heading "Exemplary Substitutions" and are Amino acid substitutions are further described below with reference to classes of amino acid side chains. and the product has the desired activity, e.g., retained / improved antigen binding, immunogenicity, etc. The patients were screened for a decrease in ADCC or improvement in CDC. [Table 1]

[0071] Amino acids can be divided into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0072] Non-conservative substitutions involve exchanging a member of one of these classes for another. This will be the case.

[0073] The term "vector" refers to a cloned polynucleotide or polynucleotide that can be propagated in a host cell. To describe a polynucleotide that may be engineered to include a nucleotide(s), A vector may contain one or more of the following elements: an origin of replication, a target polypeptide, and a vector encoding the target polypeptide. one or more regulatory sequences (e.g., promoters and / or enhancers) that regulate expression of the peptide sir), and / or one or more selectable marker genes (e.g., antibiotic resistance genes, and genes that may be used in colorimetric assays, such as β-galactosidase The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell. Refers to the vector used for

[0074] A "host cell" can be a recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; Non-limiting exemplary mammalian cells include NSO cells, fungal cells, plant cells, and insect cells. Cells, PER.C6® cells (Crucell), and 293 and CHO cells, and These modified cells include, for example, cells such as 293-6E and DG44, respectively. Not limited to these.

[0075] The term "isolated" refers to at least one substance that is typically found or produced together in nature. refers to a molecule that has been separated from some of its constituent parts, e.g., from at least one of the cells from which it was produced. A polypeptide is said to be "isolated" if it is separated from other components. In the case of a polypeptide secreted by a cell, the polypeptide is released from the cell that produces the polypeptide. Physically separating the supernatant containing the polypeptide is considered to "isolate" the polypeptide. In general, polynucleotides are not limited to larger polynucleotides typically found in nature ( For example, in the case of a DNA polynucleotide, a portion of the DNA may be a genomic DNA or a mitochondrial DNA. If the polynucleotide is not a fragment thereof, or is, for example, an RNA polynucleotide, It is said to be "isolated" when it is separated from at least some components of the cells that produce it. Therefore, a DNA polynucleotide contained in a vector within a host cell is said to be "isolated." That's fine.

[0076] The terms "individual" and "subject" are used interchangeably herein and refer to, for example, a mammal. In some embodiments, methods of treating mammals are provided, including administering to mammals The subjects include humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, and experimental mammals. Animals, including but not limited to domestic mammals, sport mammals, and pet mammals. In some embodiments, an "individual" or "subject" refers to a person in need of treatment for a disease or disorder. It refers to an individual or object.

[0077] A "disease" or "disorder" refers to a condition for which treatment is indicated.

[0078] The term "cancer" refers to the development of cancer caused by uncontrolled cell proliferation, unrestrained cell growth, and apoptosis. refers to malignant proliferative diseases associated with decreased cell death. Some non-limiting exemplary cancers include gastric cancer, Cancer is selected from breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer. In some embodiments, the cancer comprises FGFR2 gene amplification. FGFR2 amplification is associated with an FGFR2:CEN10 (chromosome 10 centromere) ratio of >3. In some embodiments, the cancer comprising FGFR2 gene amplification is FGFR2 In some embodiments, the cancer with FGFR2 amplification is In some embodiments, FGFR2IIIb is overexpressed relative to FGFR2IIIc. In these cases, cancers containing FGFR2 amplification were characterized by normalized FGFR2IIIc expression levels. at normalized levels 2-fold, 3-fold, 5-fold, or 10-fold higher than FGFR2III In some embodiments, the expression level is normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb, but In some embodiments, the gastric cancer does not comprise an FGFR2 gene amplification. In some embodiments, the gastric cancer comprising FGFR2 gene amplification is In some embodiments, the FGFR2 gene is a gene that overexpresses FGFR2IIIb. Gastric cancers overexpress FGFR2IIIb more than FGFR2IIIc. In some embodiments, the gastric cancer containing FGFR2 amplification is treated with a standardized FGFR2III cFG at normalized levels 2-fold, 3-fold, 5-fold, or 10-fold higher than the expression level In some embodiments, the expression level is relative to GUSB. In some embodiments, the gastric cancer is characterized by FGFR2IIIb overexpression. In some embodiments, the overexpression is FGFR2 gene amplification. In some embodiments, the overexpression is mRNA overexpression. It is overexpression.

[0079] The term "tumor" is used herein to describe a group of cells that exhibits abnormally high levels of proliferation and growth. Tumors may be benign, pre-malignant, or malignant; malignant tumor cells are cancerous. Tumor cells can be from solid tumors or lymphoid tumors. or) cells. The term "tumor growth" is used herein to refer to the tumor growth associated therewith. It is used to refer to the proliferation or growth of one or more cells comprising a tumor, resulting in an increase in size .

[0080] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. As used herein, "treatment" refers to a therapeutic approach for a disease in a mammal, including a human. For purposes of this invention, any beneficial or desired clinical result is encompassed by any administration or application. is: alleviation of one or more symptoms, reduction in the extent of disease, prevention or slowing of disease progression (e.g., lung or metastasis, such as metastasis to lymph nodes), prevention or delay of disease recurrence, delay or reduction of disease progression accelerating, improving the condition, preventing the disease or its progression, preventing or slowing the disease or its progression, cessation of progression, and remission (whether partial or complete), but "Treatment" also includes reducing the pathological consequences of proliferation-related diseases. The methods of the present invention contemplate any one or more modes of treatment.

[0081] In relation to cancer, the term "treat" includes inhibiting the growth of tumor or cancer cells; inhibiting tumor or cancer cell replication, reducing the overall tumor burden, and and ameliorating one or more symptoms associated with the disease.

[0082] The term "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic characteristic or the reduction or cessation of that characteristic. "Reducing" or "inhibiting" refers to the reduction or cessation of the incidence, severity, or likelihood of something occurring. refers to the reduction, decrease, or cessation of activity, function, and / or amount compared to a reference. In certain embodiments, by "reduce" or "inhibit" is meant an overall decrease of 20% or more. In another embodiment, "reducing" or "inhibiting" refers to the ability to cause a significant decrease in By "" is meant the ability to cause an overall reduction of 50% or more. In this case, "reduce" or "inhibit" means 75%, 85%, 90%, 95% or more. This means the ability to cause an overall decrease of more than

[0083] As used herein, a "reference" refers to any sample, standard, or level used for comparison. References may be obtained from healthy and / or non-diseased samples. In some embodiments, the reference may be obtained from an untreated sample. The reference may be obtained from an untreated or non-diseased sample of the subject individual. In the present invention, the reference is obtained from one or more healthy individuals who are not the subject or patient.

[0084] As used herein, "delaying disease progression" refers to preventing the progression of the disease (such as cancer). , slow down, delay, restrain, and / or postpone. The delay can be of varying lengths of time depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay effectively prevents an individual from progressing to disease. For example, late stage cancer, such as advanced or metastatic cancer, may be delayed.

[0085] As used herein, "preventing" refers to preventing a person from becoming susceptible to a disease but who has not been diagnosed with the disease. This includes providing prevention with respect to the occurrence or recurrence of a disease in a subject who has not previously experienced it.

[0086] As used herein, "inhibiting" a function or activity refers to suppressing a condition or parameter of interest. When compared with other conditions that are the same, or when compared with other conditions, the function or activity For example, an antibody that reduces tumor growth is one that reduces tumor growth in the absence of the antibody. slows the growth rate of tumors compared to the growth rate of tumors in

[0087] An "effective amount" of a drug is the dosage and concentration required to achieve the desired therapeutic or prophylactic result. It refers to an effective amount over a period of time.

[0088] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present invention is an amount that is effective in an individual. and the disease state, age, sex and weight of the individual, as well as the method of the present invention for eliciting the desired response in the individual. It may vary depending on factors such as the potency of the substance / molecule, agonist, or antagonist. A therapeutically effective amount is an amount that does not exceed the toxic or adverse effects of a substance / molecule, agonist, or antagonist. A therapeutically effective amount is an amount that has a therapeutically beneficial effect greater than the amount delivered by one or more administrations. This may be done.

[0089] A "prophylactically effective amount" refers to a compound administered to a patient, at a dosage and for a period of time necessary to achieve the desired prophylactic result. A prophylactic dose usually, but not necessarily, refers to an amount effective to treat a disease before or at an early stage. Because the compounds are used in a prophylactically effective amount, a prophylactically effective amount is less than a therapeutically effective amount.

[0090] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a pharmaceutical composition that allows the biological activity of an effective active ingredient. and other ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such preparations may be sterile.

[0091] A "pharmaceutically acceptable carrier" is a therapeutic agent that is used in conjunction with a pharmaceutical composition for administration to a subject. non-toxic solid, semi-solid, or Refers to a liquid excipient, diluent, encapsulating material, formulation aid, or carrier. Pharmaceutically acceptable Suitable carriers are nontoxic to recipients at the dosages and concentrations employed and and be compatible with the other ingredients of the formulation. It is suitable for this purpose.

[0092] A "sterile" preparation is aseptic or essentially free from live microorganisms and their spores. stomach.

[0093] Administration "in combination with" one or more further therapeutic agents may be simultaneous (concurrent), in any order, and includes sequential or sequential administration.

[0094] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents. wherein at least some of the administration overlaps in time or administration of one therapeutic agent is concurrent with administration of the other. For example, two or more therapeutic agents may be administered in a short time period, e.g., about 3 The administration is performed with a separation time of less than about 60 minutes, which is less than 0, 15, 10, 5, or 1 minute. do.

[0095] The term "sequential" is used herein to refer to the administration of two or more therapeutic agents. In some cases, administration of one or more agents continues after discontinuation of administration of one or more other agents. For example, two or more The treatment is administered for about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks The administrations are separated by a time period greater than about 15 minutes, such as one or more days, or one or more months.

[0096] As used herein, "in combination" refers to the administration of one treatment in addition to another treatment. As such, "in combination with" refers to administering to a subject before, during, or after the administration of another treatment. It refers to the administration of a treatment after administration.

[0097] The term "package insert" is used to refer to instructions customarily included in commercial packaging for a therapeutic product; Any instructions, directions for use, dosage, administration, combination therapy, contraindications and / or or includes information about precautions.

[0098] "Product" refers to any product (e.g., packaging or container) or disease or disorder (e.g., at least one drug, such as a medicament for treating a disease or condition, or a biomarker as described herein; In certain embodiments, the kit comprises a probe for specifically detecting the The product or kit is promoted and distributed as a unit for performing the methods described herein. or sold.

[0099] II. Anti-FGFR2IIIb antibody In some embodiments, the present invention is directed against FGFR2IIIb. The afucosylated anti-FGFR2IIIb antibody is an afucosylated antibody as defined herein. Humanized antibodies, chimeric antibodies, murine antibodies, and heavy and / or light chain HVRs (e.g., In one embodiment, the present invention provides an antibody comprising a CDR. provides isolated afucosylated antibodies that bind to FGFR2IIIb. In embodiments, the afucosylated anti-FGFR2IIIb antibody modulates FGFR2IIIb activity. In some embodiments, the afucosylated anti-FGFR2IIIb antibody is In some embodiments, the afucosylated anti-FGFR2 antibody has improved ADCC activity. IIIb antibodies have enhanced affinity for Fc gamma RIIIA. In an embodiment, the afucosylated anti-FGFR2IIIb antibody is Fc gamma RIIIA ( In some embodiments, afucosin has enhanced affinity for α- and β-actin. The monoclonal anti-FGFR2IIIb antibody has enhanced activity against Fc gamma RIIIA (F158). It has a strong affinity.

[0100] The anti-FGFR2IIIb antibody refers to "αFGFR2b" described in the Examples of this specification. The sequence listing is based on the HuG It has the same amino acid sequence as the AL-FR21 antibody. , which are incorporated herein by reference for any purpose and are incorporated herein by reference in their entirety. No. 8,101,723, showing the amino acid sequences of the variable regions and full-length mature antibody chains. 13 and 14 are specifically incorporated herein by reference for any purpose. , the HuGAL-FR21 antibody shown underlined in FIG. 13 of U.S. Pat. No. 8,101,723. The HVR sequences of these genes are specifically incorporated herein for any purpose.

[0101] In one embodiment, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (a) an HVR-HVR 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-H2 comprising the amino acid sequence of SEQ ID NO: 9; ) 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. At least 1, 2, 3, 4, 5, or 6 HVRs selected from HVR-L3 comprising the sequence The present invention provides an afucosylated anti-FGFR2IIIb comprising an R (e.g., a CDR).

[0102] In some embodiments, the afucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable region In some embodiments, the afucosylated anti-FGFR2I comprises a light chain variable region and a light chain variable region. The IIb antibody comprises at least one heavy chain containing a heavy chain variable region and at least one heavy chain constant region. and at least one light chain containing a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the afucosylated anti-FGFR2IIIb antibody comprises each heavy chain Two heavy chains, each containing a heavy chain variable region and at least a portion of a heavy chain constant region, and each light chain The antibody contains two light chains, each containing a light chain variable region and at least a portion of a light chain constant region. In embodiments, the afucosylated anti-FGFR2IIIb antibody has 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 afucosylated anti-FGFR2IIIb antibody has the amino acid sequence of SEQ ID NO:2. and a light chain comprising the amino acid sequence of SEQ ID NO:3.

[0103] In one embodiment, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (a) an HVR-HVR 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-H2 comprising the amino acid sequence of SEQ ID NO: 9; ) 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. Afucosylated anti-FGFR2IIIb containing six HVRs, including HVR-L3 containing the sequence In some embodiments, an afucosylated anti-FGFR2IIIb antibody is provided. In some embodiments, the FGFR2IIIb-binding domain comprises the six HVRs described above and binds to FGFR2IIIb. The afucosylated anti-FGFR2IIIb antibody contains the six HVRs and binds to FGFR2. IIIb and enhanced ADCC activity, and Fc gamma RIIIA (e.g. Fc gamma RIIIA (V158) and / or Fc gamma RIIIA (F158) and / or enhanced affinity for the antibody. In the present study, afucosylated anti-FGFR2IIIb antibodies do not bind to FGFR2IIIc. .

[0104] In one embodiment, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (a) an HVR-HVR 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-H2 comprising the amino acid sequence of SEQ ID NO: 9; ) 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. Afucosylated anti-FGFR2IIIb antibody containing six HVRs, which competes with the anti-FGFR2IIIb antibody FGFR2IIIb antibodies are provided.

[0105] In one embodiment, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (a) an HVR-HVR 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. HVR-H3 containing at least one, at least two, or three amino acid sequences selected from All V H Afucosylated antibodies comprising HVR sequences are provided.

[0106] In another embodiment, the present invention provides a method for the preparation of a human ovarian tumor suppressor comprising: (a) an HVR-HVR comprising the amino acid sequence of SEQ ID NO: 9; L1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) SEQ ID NO: 11 at least one, at least two, or All three Vs L Afucosylated antibodies comprising HVR sequences are provided.

[0107] In another embodiment, the afucosylated antibody of the invention comprises (a)(i) the antibody of SEQ ID NO: 6 (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) 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. Either one, at least two, or all three Vs H V containing HVR sequences H domain; and (b (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10 (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. At least one, at least two, or all three V selected from L V containing HVR sequences L domain, including

[0108] In another embodiment, the afucosylated anti-FGFR2IIIb antibody has the amino acid sequence of SEQ ID NO: 4. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence 97%, 98%, 99%, or 100% sequence identity between heavy chain variable domains (V H In certain embodiments, at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, or 99% identity with V H The array is Contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but includes that sequence The anti-FGFR2IIIb antibody retains the ability to bind to FGFR2IIIb. In embodiments, such anti-FGFR2IIIb antibodies do not bind to FGFR2IIIc. In certain embodiments, the antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIb. In SEQ ID NO: 4, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion, or deletion occurs outside the HVR (i.e., within the FR). Optionally, the afucosylated anti-FGFR2IIIb antibody occurs in the translation region of the sequence. V of SEQ ID NO:5, including post-modification H In certain embodiments, V H Is it the following? (A) 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: 7. HVR-H3 containing the amino acid sequence of no. 8.

[0109] In another embodiment, the amino acid sequence of SEQ ID NO: 5 is at least 90%, 91%, or %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The light chain variable domain (V L ) containing afucosylated anti-FGFR2II In certain embodiments, Ib antibodies are provided. V with 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity L Distribution The columns contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the sequence The anti-FGFR2IIIb antibody comprising the above retains the ability to bind to FGFR2IIIb. In certain embodiments, such anti-FGFR2IIIb antibodies are directed against FGFR2IIIc. In certain embodiments, the antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIb. In SEQ ID NO: 5, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion, or deletion occurs outside the HVR (i.e., F Optionally, the afucosylated anti-FGFR2IIIb antibody has the sequence V of SEQ ID NO: 4, including the post-translational modification L In certain embodiments, V L teeth, (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10 (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (d) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. It contains one, two, or three HVR sequences of choice.

[0110] In another embodiment, the afucosylated anti-FGFR2IIIb antibody has the amino acid sequence of SEQ ID NO: 4. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence 97%, 98%, 99%, or 100% sequence identity between heavy chain variable domains (V H ) sequence, and at least 90%, 91%, 92% relative to the amino acid sequence of SEQ ID NO: 5 , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity The light chain variable domain (V L In certain embodiments, at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or V with 100% identity H A sequence may contain substitutions (e.g., conservative substitutions) relative to a reference sequence. At least 90%, 91%, 92%, 93%, 94%, 95%, including insertions or deletions V with 96%, 97%, 98%, 99%, or 100% identity L The sequence is the reference sequence Anti-FG containing the sequence, but containing substitutions (e.g., conservative substitutions), insertions, or deletions. The FR2IIIb antibody retains the ability to bind to FGFR2IIIb. In the present invention, such an anti-FGFR2IIIb antibody does not bind to FGFR2IIIc, In certain embodiments, the sequence In number 4, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 5 are substituted, inserted, or In certain embodiments, the substitutions, insertions, or deletions are outside the HVRs. Optionally, an afucosylated anti-FGFR2III region of (i.e., within the FR) b The antibody comprises V of SEQ ID NO: 4, including post-translational modifications of one or both sequences. H Sequence and SEQ ID NO: 5V L In certain embodiments, V H is selected from the following: It comprises three HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) the sequence (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8. HVR-H3; and V L (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. and HVR-L3 comprising the sequence:

[0111] In another embodiment, an afucosylated anti-FGFR2IIIb antibody is provided, is V in any of the embodiments given above H and any of the embodiments given above. V in Reka L In another embodiment, the antibodies include post-translational modifications of their sequences. V, which are SEQ ID NO: 4 and SEQ ID NO: 5, respectively H and V L Includes:

[0112] In another embodiment, the afucosylated anti-FGFR2IIIb antibody has the amino acid sequence of SEQ ID NO:2. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the β-glucan concentration is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 1 Heavy chain sequences with 6%, 97%, 98%, or 99% identity to the reference sequence an afucosylated anti-FGF comprising the sequence, but including substitutions (e.g., conservative substitutions), insertions, or deletions; The R2IIIb antibody retains the ability to bind to FGFR2IIIb. In this case, such an anti-FGFR2IIIb antibody does not bind to FGFR2IIIc and selectively binds to FGFR2IIIc. In certain embodiments, the polypeptide of SEQ ID NO: 1 retains the ability to selectively bind to FGFR2IIIb. In No. 2, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In this embodiment, the substitutions, insertions, or deletions are in regions outside the HVRs (i.e., within the FRs). Optionally, the afucosylated anti-FGFR2IIIb antibody heavy chain is produced by translation of its sequence. V in SEQ ID NO: 2, including post-modification H In certain embodiments, the heavy chain comprises the sequence and one, two, or three HVRs selected from: (a) 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.

[0113] In another embodiment, the amino acid sequence of SEQ ID NO: 3 is at least 90%, 91%, or %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and afucosylated anti-FGFR2IIIb antibodies comprising a light chain having sequence identity of In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% identity to the reference sequence. Anti-FGFR2II antibodies containing the sequence, but including substitutions (e.g., conservative substitutions), insertions, or deletions. Ib antibodies retain the ability to bind to FGFR2IIIb. Such anti-FGFR2IIIb antibodies selectively bind to FGFR2IIIc without binding to FGFR2IIIc. In certain embodiments, the amino acid sequence of SEQ ID NO: 3 retains the ability to bind to FR2IIIb. In particular embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In the present invention, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., within the FR). Optionally, the light chain of the afucosylated anti-FGFR2IIIb antibody comprises a post-translational modification of the sequence , V of SEQ ID NO: 3 L In certain embodiments, the light chain comprises the sequence: (a) SEQ ID NO: 9 (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; 2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. , or three HVR sequences.

[0114] In another embodiment, the afucosylated anti-FGFR2IIIb antibody has the amino acid sequence of SEQ ID NO:2. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence %, 97%, 98%, 99%, or 100% sequence identity, and At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, Light chain sequences with 5%, 96%, 97%, 98%, 99%, or 100% sequence identity In certain embodiments, at least 90%, 91%, 92%, 93%, 94%, Heavy chain sequences with 95%, 96%, 97%, 98%, or 99% identity to the reference sequence Anti-FGFR antibodies containing the sequence, but containing substitutions (e.g., conservative substitutions), insertions, or deletions. The 2IIIb antibody retains the ability to bind to FGFR2IIIb. In this case, such an anti-FGFR2IIIb antibody does not bind to FGFR2IIIc and selectively In certain embodiments, the antibody retains the ability to specifically bind to FGFR2IIIb. Also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 9 Light chain sequences with 9% identity may contain substitutions (e.g., conservative substitutions), insertions, or fragments with respect to the reference sequence. or deletions, but containing the sequence, anti-FGFR2IIIb antibodies In certain embodiments, such anti-FGFR2IIIb antibodies retain the ability to bind to FGFR2IIIb. The body has the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 2 are , substitutions, insertions and / or deletions. A total of 1 to 10 amino acids are substituted, inserted and / or deleted. In this case, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Alternatively, the afucosylated anti-FGFR2IIIb antibody heavy chain comprises a post-translational modification of its sequence: V in SEQ ID NO:2 H and an afucosylated anti-FGFR2IIIb antibody light chain comprising the sequence , V in SEQ ID NO: 3, including post-translational modifications of that sequence L In certain embodiments, wherein the heavy chain comprises: (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: 6; (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8. R-H3; and the light chain comprises one, two, or three HVRs selected from (a) SEQ ID NO: (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; It contains two or three HVR sequences.

[0115] Exemplary Chimeric Antibodies In certain embodiments, antibodies, such as afucosylated antibodies provided herein, are chimeric. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al. et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In examples, chimeric antibodies contain non-human variable regions (e.g., mouse, rat, hamster, A further example includes a human constant region (variable region derived from a non-human primate, such as a monkey, or a heron). In this context, chimeric antibodies are antibodies whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0116] Non-limiting exemplary afucosylated chimeric antibodies include heavy chain HVR1, HVR2, and HVR3 sequences described herein. and HVR3 and / or light chain HVR1, HVR2, and HVR3 sequences. In some embodiments, the afucosylated chimeric anti-FGFR2IIIb antibody comprises In some embodiments, the variable region comprises: The afucosylated chimeric anti-FGFR2IIIb antibody comprises the above variable region and is Ib and enhanced ADCC activity, and Fc gamma RIIIA (e.g., Fc gamma RIIIA (V158) and / or Fc gamma RIIIA (F158) In some embodiments, the antibody has at least one activity selected from the group consisting of: enhanced affinity, In contrast, the afucosylated chimeric anti-FGFR2IIIb antibody does not bind to FGFR2IIIc. stomach.

[0117] In some embodiments, the afucosylated chimeric anti-FGFR2IIIb antibody is FR2IIIb and at least 90%, at least 91%, or at least 92% of SEQ ID NO:4. at least 92%, at least 93%, at least 94%, at least 95%, at least 9 variable regions that are 6%, at least 97%, at least 98%, or at least 99% identical In some embodiments, the afucosylated chimeric anti-FGFR2I comprises a heavy chain comprising the sequence The IIb antibody binds to FGFR2IIIb and binds to SEQ ID NO:5 by at least 90% and at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, or at least 99% In some embodiments, the afucosylated light chain comprises a light chain containing the same variable region sequence. The chimeric anti-FGFR2IIIb antibody binds to FGFR2IIIb and has a low affinity to SEQ ID NO:4. 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 a heavy chain comprising a variable region sequence that is at least 99% identical to SEQ ID NO: 5, and 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 and light chains that contain variable region sequences that are at least 99% identical.

[0118] Exemplary afucosylated chimeric anti-FGFR2IIIb antibodies also include the antibodies described herein. or a chimeric antibody that competes with a fragment thereof for binding to FGFR2IIIb. Thus, in some embodiments, the chimeric anti-FGFR2IIIb antibody has the sequence of SEQ ID NO:4. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. The antibody is provided to compete for binding to FGFR2IIIb with an antibody containing the antibody. In some embodiments, the antibody competes for binding to FGFR2IIIb but not to F It does not bind to GFR2III.

[0119] In some embodiments, the chimeric antibodies described herein contain one or more human constant regions. In some embodiments, the human heavy chain constant region comprises one of the following: IgA, IgG, and Ig In some embodiments, the human light chain is of an isotype selected from the group consisting of: The constant region is of an isotype selected from kappa and lambda. In some embodiments, the chimeric antibodies described herein comprise a human IgG constant region. In some embodiments, the chimeric antibodies described herein comprise a human IgG4 heavy chain constant region. In one embodiment, the chimeric antibodies described herein comprise a human IgG4 constant region and a human κB constant region. Contains light chains.

[0120] As noted above, specific antibodies may be targeted, regardless of whether effector functions are desired. Therefore, in some embodiments, the effect may depend on the particular treatment method used. If target function is desired, the human IgG1 heavy chain constant region or the human IgG3 heavy chain constant region Thus, in some embodiments, a chimeric anti-FGFR2IIIb antibody comprising If effector function is not desired, the antibody may contain a human IgG4 or IgG2 heavy chain constant region. A chimeric anti-FGFR2IIIb antibody containing the antibody is selected.

[0121] Exemplary Humanized Antibodies In some embodiments, an afucosylated humanized antibody that binds to FGFR2IIIb The humanized antibody provides an immune response to the therapeutic antibody and is effective in treating the disease. reducing the human immune response to non-human antibodies (e.g., human anti-mouse antibodies (HAMA)) Humanized antibodies are effective as therapeutic molecules because they reduce or eliminate the immune response.

[0122] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody Retains the specificity and affinity of the parent non-human antibody to reduce immunogenicity in humans Typically, a humanized antibody is humanized while retaining its HVR (or a portion thereof), such as a CDR. ) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. A humanized antibody may also optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be, for example, For example, modifications of non-human antibodies (e.g., HVR residues) can be made to restore or improve antibody specificity or affinity. The nucleotides are substituted by the corresponding residues in the antibody from which the group is derived.

[0123] Humanized antibodies and methods for producing humanized antibodies are described, for example, in Almagro and Fransson, (200 8) Front. Biosci. 13: 1619-1633, and also see, for example, Riechmann et al., (1988 ) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-1 0033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 708 No. 7409; Kashmiri et al., (2005) Methods 36:25-34 (SDR(a-CDR) graph Padlan, (1991) Mol. Immunol. 28:489-498 (describes "surface reconstruction"); Dall'Acqua et al., (2005) Methods 36:43-60 (referring to "FR shuffling") ; and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Ca Cancer, 83:252-260 (FR shuffling vs. "guided selection" This is further described in "Methods for the Detection of Ignited Polypeptides" ("Methods for the Detection of Ignited Polypeptides").

[0124] Human framework regions that may be used for humanization are designated "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296 (2296)). framework regions that are used for specific subgroups of human antibodies in the light or heavy chain variable regions; Framework regions derived from consensus sequences (e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623 human mature (somatically mutated) framework regions or human germline framework regions ark region (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633) and framework regions derived from screening of FR libraries (e.g., Baca e t al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618).

[0125] Non-limiting exemplary humanized antibodies include αFGFR2b, as described herein. The target afucosylated humanized antibody has an amino acid sequence identical to αFGFR2b, including fucose. Non-limiting exemplary afucosylated humanized antibodies include αFGFR2bA, as described herein. The subject may also comprise a heavy chain variable region of αFGFR2b and / or a light chain variable region of αFGFR2b. A non-limiting exemplary afucosylated humanized antibody includes an antibody comprising the heavy chain variable region of SEQ ID NO:4. Exemplary humanized antibodies also include antibodies comprising the VL1 domain and / or the light chain variable region of SEQ ID NO: 5. , the heavy chain HVR1, HVR2, and HVR3 of αFGFR2b, and / or the light chain HVR1, Examples of humanized antibodies include, but are not limited to, humanized antibodies comprising HVR2 and HVR3. In such a case, the humanized anti-FGFR2IIIb antibody comprises the above-mentioned HVR (i.e., (a) (b) 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) SEQ ID NO: 9 (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; L2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11), FGFR2III In some embodiments, the humanized anti-FGFR2IIIb antibody binds to the above-described A method for identifying a human Fc antigen containing an HVR, binding to FGFR2IIIb, and having enhanced ADCC activity. gamma RIIIA (e.g., Fc gamma RIIIA(V158) and / or Fc gamma RI and having at least one activity selected from the group consisting of: enhanced affinity for IgG1A (IIA(F158)); In some embodiments, the afucosylated humanized anti-FGFR2IIIb antibody is Does not bind to FGFR2IIIc.

[0126] In some embodiments, the afucosylated humanized antibody also contains the heavy chain of αFGFR2b. light chains HVR1, HVR2, and HVR3, and / or light chains HVR1, HVR2, and HVR 3. Non-limiting exemplary afucosylated humanized anti-FGFR2IIIb antibodies include those represented by SEQ ID NO: 6. , 7, and 8. Non-limiting exemplary afucosylated humanized anti-FGFR2IIIb antibodies include those set forth in SEQ ID NOs: 9, 10, and 11.

[0127] In some embodiments, the afucosylated humanized anti-FGFR2IIIb antibody is FR2IIIb and at least 90%, at least 91%, or at least 92% of SEQ ID NO:4. at least 92%, at least 93%, at least 94%, at least 95%, at least 9 variable regions that are 6%, at least 97%, at least 98%, or at least 99% identical In some embodiments, the afucosylated humanized anti-FGFR2 comprises a heavy chain comprising the sequence The IIIb antibody binds to FGFR2IIIb and binds to SEQ ID NO: 5 by at least 90% and at least 90% of the FGFR2IIIb antibody. at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 9 In some embodiments, the afucosin antibody comprises a light chain comprising a variable region sequence that is 9% identical to the afucosin antibody. The modified humanized anti-FGFR2IIIb antibody binds to FGFR2IIIb and is directed against SEQ ID NO: 4. 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, and 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 light chains comprising variable region sequences that are at least 99% identical.

[0128] In some embodiments, the afucosylated humanized anti-FGFR2IIIb antibody is In some embodiments, the antibody comprises at least one HVR as described herein. The afucosylated humanized anti-FGFR2IIIb antibody may comprise a heavy chain HVR1 as described herein, heavy chain HVR2 as described herein, heavy chain HVR3 as described herein, light chain HVR as described herein R1, HVR2 as described herein, and HVR3 as described herein. In embodiments, the afucosylated humanized anti-FGFR2IIIb antibody is and at least one mutated HVR based on an HVR, wherein the mutated HVR is an HVR described herein. In some embodiments, one or more The above amino acid substitutions are conservative amino acid substitutions. The above suitable conservative amino acids can be selected, where suitable conservative amino acid substitutions are are not expected to specifically alter the binding properties of antibodies containing mutated HVRs.

[0129] Exemplary afucosylated humanized anti-FGFR2IIIb antibodies also include the antibodies described herein. or a fragment thereof, and an antibody that competes with the fragment for binding to FGFR2IIIb. In some embodiments, the humanized anti-FGFR2IIIb antibody is In some embodiments, the αFGFR2b is provided to compete with αFGFR2b for binding to the αFGFR2b. In this study, the afucosylated humanized anti-FGFR2IIIb antibody was shown to have the activity against FGFR2IIIb. and αFGFR2b are provided to compete with αFGFR2b for binding to the FGFR2b and have enhanced ADCC activity. and Fc gamma RIIIA (e.g., Fc gamma RIIIA(V158) and / or F c) at least one selected from the group consisting of a hydroxylase and a hydroxylase having enhanced affinity for gamma RIIIA (F158) In some embodiments, the afucosylated humanized anti-FGFR2 II has an activity of 1. The Ib antibody does not bind to FGFR2IIIc.

[0130] In some embodiments, the afucosylated humanized anti-FGFR2IIIb antibody is In some embodiments, the human antibody comprises at least one human constant region, as described herein. The heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is an isotype selected from κ and λ. It belongs to Ipu.

[0131] In some embodiments, the afucosylated humanized antibodies described herein are human Ig G constant region. Thus, in some embodiments, effector function is desired. When the antibody is an afucosylated human antibody containing a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region, In some embodiments, a simulated anti-FGFR2IIIb antibody is selected. The afucosylated humanized antibodies described comprise a human IgG1 constant region. In the afucosylated humanized antibody described herein, N297 is not fucosylated. In some embodiments, the antibody comprises a human IgG1 constant region. The cosylated human antibody comprises a human IgG1 constant region and a human kappa light chain.

[0132] In some embodiments, the afucosylated humanized antibody has the amino acid sequence of SEQ ID NO:2. and a light chain comprising the amino acid sequence of SEQ ID NO: 3. The afucosylated humanized antibody is composed of the amino acid sequence of SEQ ID NO: 2 and any post-translational modifications. and a light chain consisting of the amino acid sequence of SEQ ID NO: 3 and any post-translational modifications. Cosylated antibodies are included.

[0133] Exemplary Antibody Constant Regions In some embodiments, the afucosylated antibodies described herein are selected from the group consisting of one or more human endogenous antibodies. In some embodiments, the human heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgA, IgG, and and IgD. In some embodiments, the human The light chain constant region is of an isotype chosen from kappa and lambda.

[0134] In some embodiments, the afucosylated antibodies described herein are human IgG constant In some embodiments, when effector function is desired, human I Afucosylated anti-FGFR2II containing the gG1 heavy chain constant region or the human IgG3 heavy chain constant region In some embodiments, the afucosylated Ib antibody described herein is selected. In some embodiments, the antibody comprises a human IgG1 constant region. The fucosylated antibody comprises a human IgG1 constant region in which N297 is afucosylated. In some embodiments, the afucosylated antibodies described herein are human IgG1 constant region and a human kappa light chain.

[0135] In the present specification and claims, unless expressly stated or known to those skilled in the art, immunoglobulins The numbering of residues in the purine heavy chain is based on that of Kabat et al., which is expressly incorporated herein by reference. l., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Servic e, National Institutes of Health, Bethesda, MD, 1991. The "Kabat EU index" is the numbering of residues of a human IgG1 EU antibody. Point to the number.

[0136] In certain embodiments, the antibodies of the invention have at least one IgG or IgG subunit that is at least 100% IgG subunits relative to wild-type IgG or wild-type antibodies. In certain embodiments, the mutant Fc region comprises a mutant Fc region having at least one amino acid substitution. The Fc region has two or more amino acid substitutions in the Fc region of a wild-type antibody. In embodiments, the variant Fc region comprises three or more amino acid substitutions in the Fc region of a wild-type antibody. In certain embodiments, the variant Fc region has at least one of the following: In certain embodiments, the antibodies described herein have one, two, or three Fc region amino acid substitutions. The variant Fc region may have at least one Fc region that is identical to a native sequence Fc region and / or to the Fc region of the parent antibody. In certain embodiments, the sequences described herein will have at least about 80% homology. The variant Fc region may have at least one amino acid sequence similar to that of a native sequence Fc region and / or that of a parent antibody. In certain embodiments, the mutations described herein may also have about 90% homology. The Fc region may have at least about the same sequence as the native sequence Fc region and / or as the parent antibody Fc region. It will have 95% homology.

[0137] In certain embodiments, the antibodies provided herein are glycosylated to a degree that the antibody is glycosylated. The antibody may be modified to increase or decrease its glycosylation site. Removal is achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed. This may be done simply and conveniently.

[0138] If the antibody contains an Fc region, the carbohydrate attached to it may be altered. Natural antibodies produced by human cells typically contain As in the CH2 domain of the Fc region. They contain branched, biantennary oligosaccharides commonly attached by an N-linkage to n297. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides are a variety of carbohydrates, e.g. For example, mannose, N-acetylglucosamine, and glycans linked to GlcNAc at the "stem" of the biantennary glycan structure It may contain glucosamine (GlcNAc), galactose, sialic acid, and fucose. In some embodiments, oligosaccharide modifications in the antibodies of the invention provide certain improved characteristics. This may be done to generate antibody variants with specificity.

[0139] In certain embodiments, the antibody comprises a fucose attached (directly or indirectly) to the Fc region. For example, antibodies may be provided that have carbohydrate structures that lack The amount of fucose in a composition comprising a plurality of such antibodies is from 0% to about 5%. In some embodiments, the plurality of such antibodies may be at least 95% identical. Contains fucosylated antibodies. The amount of fucose is determined based on the total amount of all glycostructures attached to Asn297. The average amount of fucose in the glycan at Asn297 (e.g., complex, hybrid, and The fucose in the antibody is determined by calculating the A non-limiting exemplary method is MALDI-TOF mass spectrometry (see, e.g., WO 2008 / 02396). 077546), fluorescently labeled free oligosaccharide HPLC methods (e.g., Schneider et al., “N-Glycan analysis of monoclonal antibodies and other glycoproteins usi ng UHPLC with fluorescence detection,” Agilent Technologies, Inc. (2012); Lines , J. Pharm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J. Chrom., 720: 217- 225 (1996)), capillary electrophoresis of fluorescently labeled free oligosaccharides (e.g., M a et al., Anal. Chem., 71: 5185-5192 (1999)), and for monosaccharide composition measurement, HPLC with pulsed amperometric detection (e.g., Hardy, et al., Analytical Bioc hem., 170: 54-62 (1988)). Asn297 is part of the Fc region (Fc region residue E Asparagine residue located at approximately position 297 in the ribosomal RNA (U numbering), Asn297 refers to It may be located at an amino acid within ±3 residues upstream or downstream of position 297, i.e., a minor amino acid sequence of the antibody. Due to a sequence mutation, it may be located between positions 294 and 300. In FGFR2b, Asn297 is the sequence QY N ST and having the following sequence: In the table, SEQ ID NO: 2 is shown in bold and underlined. Fucosylation variants have improved ADCC function. For example, see U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L. ); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.) See for examples of publications related to "afucosylated" or "fucose-deficient" antibody variants. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 and International Publication No. 2000 / 617 39, International Publication No. 2001 / 29246, U.S. Patent Application Publication No. 2003 / 01156 14, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. US Patent Application Publication No. 2004 / 0110704, US Patent Application Publication No. 2004 / 0110282, US Patent Application Publication No. 2004 / 0109865, International Publication No. 003 / 085119, International Publication No. 2003 / 084570, International Publication No. 2005 / 035586, International Publication No. 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnu ki et al. Biotech. Bioeng. 87: 614 (2004). An example of a competent cell line is Lec13 C, which is deficient in protein fucosylation. HO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US patent application Publication No. 2003 / 0157108 A1, Presta, L; and WO 2004 / 056 312 A1, Adams et al., especially Example 11), and knockout CHO cells, etc. knockouts lacking a functional alpha-1,6-fucosyltransferase gene, FUT8 Mouse cell lines (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 0 (See No. 85107).

[0140] For example, the antibody may have a biantennary oligosaccharide attached to the Fc region of the antibody, which is bisected by GlcNAc. Such antibodies are further provided with biantennary oligosaccharides that have reduced fucosylation. Examples of such antibodies include, for example, International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 660,684 (U.S. Pat. No. 660,684); Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). It has been described that the Fc region contains at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies may have improved CDC function. Antibodies can be prepared using techniques described, for example, in WO 1997 / 30087 (Patel et al.); WO 1997 / 30087 (Patel et al.); 98 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). It is written.

[0141] Antibodies may also be provided with amino-terminal leader extensions. For example, amino-terminal leader One or more amino acid residues of the sequence are present at any one or more ends of the heavy or light chain of the antibody. An exemplary amino-terminal leader extension is a three amino acid sequence present in one or both light chains of an antibody. The amino acid residue, VHS, is included or consists of the amino acid residue, VHS.

[0142] The in vivo or serum half-life of a human FcRn high affinity binding polypeptide can be determined by, for example, the mutant FcRn In a transgenic mouse, a polypeptide having a c region is administered. In a human, or non-human primates. See, e.g., Petkova et al. International Immun See Journal of Neurology 18(12):1759-1769 (2006).

[0143] In some embodiments, the afucosylated antibody has a greater potency than the parent antibody containing fucose. Typically, the antibody mediates ADCC in the presence of human effector cells, and ADCC activity is typically measured using the antibodies described herein. The ADCC activity may be determined using in vitro ADCC activity as described in the document, for example, Other assays or methods for determining ADCC activity, such as animal models, are contemplated.

[0144] In certain embodiments, the "KD "," "K d ", "Kd" or "Kd value" Approximately 10 response units (RU) of antigen-immobilized CM5 chips were used for the BIACORE™ assay at 25°C. BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscatawa y, NJ) using a surface plasmon resonance assay. A carboxymethylated dextran biosensor chip (CM5, BIACORE Inc.) was prepared from the supplier N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide salt according to the instructions The antigen is activated with EDTA (Ethylenediaminetetraacetic acid) and N-hydroxysuccinimide (NHS). Dilute to 5 μg / ml (0.2 μM) with M sodium acetate (pH 4.8) and The antigen is injected at a flow rate of 5 μL / min so that the protein response unit (RU) is approximately 10. After the injection of 1 M ethanolamine was injected to block unreacted groups. For analytical measurements, serial dilutions of a polypeptide such as a full-length antibody are incubated at 25°C for approximately 2 at a flow rate of 5 μL / min, containing 0.05% TWEEN-20™ surfactant (PBST) The association and dissociation sensorgrams were simultaneously fitted. A simple one-to-one Langmuir binding model (BIACORE® Evaluation Software) was used are version 3.2) to calculate the association rate (k on ) and dissociation rate (k off ) to calculate the equilibrium solution. Dissociation constant (Kd) is k off / k on The ratio is calculated as follows. For example, see Chen et al., J. Mol. Biol. 1999, 293:865-881. The association rate was 1.5 s by surface plasmon resonance assay. 0 6 M -1 s -1 If the association rate exceeds 100 kJ / s, the association rate can be determined by a spectrometer, e.g., a spectrophotometer with a flow stop. meter (Aviv Instruments) or 8000 Series SLM-AMINC equipped with a stirring cuvette 25 in the presence of increasing concentrations of antigen as measured by O™ spectrophotometer (ThermoSpectronic). °C, fluorescence emission intensity (excitation = 295 nm) of 20 nM anti-antigen antibody in PBS (pH 7.2) Fluorescence quenching technique was used to measure the increase or decrease in absorption (absorption = 340 nm, 16 nm bandpass). It can be measured by

[0145] The above "on-rate," "rate of association," "association rate," or is "k on ")" also refers to the BIACORE®-2000 or BIACOR Determined using an E®-3000 system (BlAcore, Inc., Piscataway, NJ) It can be done.

[0146] In certain embodiments, the difference between the two values (e.g., Kd values) is substantially the same. For example, the function of the reference / comparison value is less than about 50%, less than about 40%, less than about 30%. , less than about 20%, and / or less than about 10%.

[0147] In certain embodiments, the difference between the two values (e.g., Kd values) is substantially For example, the function of the reference / comparator molecule may be about 10% higher, about 20% higher, about 30% higher, or %, greater than about 40%, and / or greater than about 50%.

[0148] Exemplary Leader Sequences Some secretory proteins are expressed in large amounts and secreted, so the amount of heterologous protein derived from them is low. In some embodiments, a leader sequence is preferred. When removed in the ER, the resulting mature polypeptide may remain unmodified. A heterologous leader sequence may be advantageous in that: It may be required to express and secrete some proteins.

[0149] The sequences of certain exemplary leader sequences are available from, e.g., the Department of Biochemistry, National The online leader sequence database maintained by the University of Singapore Choo et al., BMC Bioinformatics, 6: 249 (2005); and International Publication No. See No. 6 / 081430.

[0150] III. Characteristics of afucosylated anti-FGFR2IIIb antibodies In some embodiments, the afucosylated anti-FGFR2IIIb antibody is and / or have enhanced ADCC activity in vivo. Afucosylated anti-FGFR2IIIb antibodies exhibit enhanced ADCC activity in vitro In some embodiments, in vitro ADCC activity is measured using the methods described herein, e.g. For example, it can be determined by the method described in Example 3. Briefly, FGFR2IIIb-expressing cells The cells were incubated with fucosylated antibodies or afucosylated antibodies at a ratio of 25:1 effector (PBMC) to target cells. The antibody is contacted with freshly isolated human PBMCs in the presence of the cosylated antibody. In some embodiments, FGFR2IIIb-expressing Ba / F3 cells are used as target cells. In some embodiments, the cytotoxicity is determined by CytoTox non-radioactive cytotoxicity. The activity was determined by quantifying LDH release using a cytotoxicity assay (Promega, Madison, WI). In some embodiments, maximum lysis is determined using 5% Triton X-100. In some embodiments, specific antibody release is determined in the absence of antibody. The percentage of spontaneous dissolution may be determined by the following formula: (experimental value - spontaneous release) / (Maximum value - spontaneous release) x 100 = specific lysis (%). In some embodiments, the enhancement Afucosylated anti-FGFR2IIIb antibodies with improved ADCC were tested at least At a concentration of 1, specific lysis was at least 10 times greater than that induced by the same amount of fucosylated antibody. 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, Produces at least 70, or at least 75 percentage points greater specific lysis. In some embodiments, the afucosylated anti-FGFR2 II antibody has enhanced ADCC. The Ib antibody, at least at one concentration tested, exhibited a specific binding affinity to the same amount of fucosylated antibody. 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 The antibody concentration ranged from 0.01 to 1 μg / ml, resulting in highly specific lysis. and the target cell is a FGFR2IIIb-expressing Ba / F3 cell. The antibody concentrations were 0.01 μg / ml, 0.1 μg / ml, or 1 μg / ml. be.

[0151] In some embodiments, the afucosylated anti-FGFR2IIIb antibody is an Fc gamma In some embodiments, the afucosin has an enhanced affinity for RIIIA. The monoclonal anti-FGFR2IIIb antibody has enhanced activity against Fc gamma RIIIA (V158). In some embodiments, the afucosylated anti-FGFR2IIIb antibody The body has an enhanced affinity for Fc gamma RIIIA (F158). In this embodiment, the antibody affinity for Fc gamma RIIIA is determined, for example, by the methods described herein. As determined using the surface plasmon resonance assay described in Example 2 and / or below. The following description is for Fc gamma RIIIA (V158), but Fc gamma RII It is also suitable for determining the affinity for IA(F158). In one embodiment, the fucosylated or afucosylated anti-FGFR2IIIb antibody is a protein Captured on a coated dextran chip. Fc gamma RIIIA (V 158) (available, for example, from R&D Systems) is injected at various concentrations. and Fc gamma RIIIA (V158) against afucosylated anti-FGFR2IIIb. The binding constant, dissociation constant, and affinity can be determined, for example, using a surface plasmon resonance system (e.g., Bi acore T200 Evaluation Software (1:1 binding model) In some embodiments, Fc gamma RIIIA (e.g., Fc gamma RIIIA) may be determined. Enhanced response to Fc gamma RIIIA (V158) or Fc gamma RIIIA (F158) The afucosylated anti-FGFR2IIIb antibody with excellent affinity was compared with the fucosylated anti-FGFR2I At least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold higher than antibody IIb , at least 7 times, at least 10 times, at least 12 times, at least 15 times, at least binds to Fc gamma RIIIA with 17-fold, or at least 20-fold, higher affinity. The afucosylated anti-FGFR2IIIb antibody had an affinity (K D ) and Fc gamma The fucosylated anti-FGFR2IIIb antibody that binds to RIIIA(V158) was 7 nM affinity (K D ) binds to Fc gamma RIIIA (V158) and The fucosylated anti-FGFR2IIIb antibody was significantly more effective than the fucosylated anti-FGFR2IIIb antibody. / 9.2 = 22.5-fold higher affinity to Fc gamma RIIIA (V158).

[0152] IV. Anti-FGFR2IIIb Antibody Expression and Production Nucleic acid molecules encoding anti-FGFR2IIIb antibodies Nucleic acid molecules comprising polynucleotides encoding one or more chains of an anti-FGFR2IIIb antibody In some embodiments, the nucleic acid molecule is selected from the group consisting of an anti-FGFR2IIIb antibody, In some embodiments, the nucleic acid comprises a polynucleotide encoding a heavy chain or a light chain. The molecule contains a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain of an anti-FGFR2IIIb antibody. In some embodiments, the first nucleic acid molecule comprises both a polynucleotide that encodes the first nucleic acid molecule and a polynucleotide that encodes the second nucleic acid molecule. a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule encoding a light chain; 2 polynucleotides.

[0153] In some such embodiments, the heavy and light chains are two separate polypeptides. The vector may be expressed as a single nucleic acid molecule or as two separate nucleic acid molecules. In this embodiment, where the antibody is an scFv, a single polynucleotide The polypeptide encodes a single polypeptide comprising a heavy chain and a light chain that binds to the

[0154] In some embodiments, a nucleic acid encoding the heavy or light chain of an anti-FGFR2IIIb antibody is The polynucleotide may contain a leader sequence located at the N-terminus of the heavy or light chain when translated. As noted above, the leader sequence may be a nucleotide sequence encoding a naturally occurring heavy chain or It may be the light chain leader sequence or another heterologous leader sequence.

[0155] The nucleic acid molecules may be constructed using recombinant DNA techniques that are conventional in the art. In some embodiments, the nucleic acid molecule is engineered for expression in a selected host cell. It is a suitable expression vector for this purpose.

[0156] vector Polynucleotides encoding anti-FGFR2IIIb heavy chains and / or anti-FGFR2IIIb light chains Also provided are vectors containing the peptides. A vector comprising a polynucleotide encoding the GFR2IIIb light chain is provided. Such vectors include DNA vectors, phage vectors, virus vectors, retrovirus vectors, and In some embodiments, the vector The vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains comprise two separate polypeptide sequences. In some embodiments, the heavy and light chains are expressed as peptides from a vector. , expressed as part of a single polypeptide, such as when the antibody is an scFv. .

[0157] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain. and the second vector comprises a polynucleotide encoding the light chain. In the method, the first vector and the second vector are introduced into the host in equal amounts (approximate molar amounts or approximate mass amounts). In some embodiments, the cells are transfected at a molar ratio of 5:1 to 1:5. Alternatively, a mass ratio of the first vector and the second vector is transfected into the host cell. In some embodiments, the vector encoding the heavy chain and the vector encoding the light chain are In some embodiments, a mass ratio of 1:1 to 1:5 is used. A 1:2 mass ratio of vector encoding the light chain to vector encoding the light chain is used.

[0158] In some embodiments, the vector is derived from a CHO, or CHO-derived cell, or an NSO The gene is selected to be optimized for expression of the polypeptide in the cell. Such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-88 9 (2004).

[0159] host cell In various embodiments, the anti-FGFR2IIIb heavy chain and / or the anti-FGFR2IIIb light chain is expressed in a bacterial cell, etc. prokaryotic cells, eukaryotic cells such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells Such expression can be achieved, for example, by procedures well known in the art. Exemplary eukaryotic cells that may be used to express the polypeptide are: COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO-S, DG 44. CHO cells, including Lec13 CHO cells and FUT8 CHO cells; PER.C 6® cells (Crucell); and NSO cells. In some embodiments, the anti-FGFR2IIIb heavy chain and / or the anti-FGFR2IIIb light chain The chains may be expressed in yeast. See, e.g., U.S. Patent Application Publication No. 2006 / 0270 See, e.g., 04 A1. In some embodiments, certain eukaryotic host cells are to the anti-FGFR2IIIb heavy chain and / or the anti-FGFR2IIIb light chain. For example, in some embodiments, CHO cells are selected based on their ability to , which has a higher level of sialic acid than the same polypeptide produced in 293 cells. The polypeptide produced by the method is

[0160] Introduction of one or more nucleic acids into a desired host cell can be accomplished by calcium phosphate transfection. , DEAE-dextran mediated transfection method, anionic lipid mediated transfection filtration, electroportation, transduction, infection, etc. The method may be performed in any manner, including, but not limited to, The method is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be prepared by any suitable method. The vector may be transiently or stably transfected into the desired host cell according to various methods.

[0161] In some embodiments, the afucosylated anti-FGFR2IIIb antibody is, for example, Protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biology) ophis. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 A1, Pre sta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11 ), and knockout CHO cells, etc. A knockout cell line lacking the enzyme gene, FUT8 (e.g., Yamane-Ohnuki et al. Biol. tech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-6 88 (2006); and WO 2003 / 085107). In some embodiments, the afucosin is produced in a cell capable of viably activating the afucosin. Monoclonal anti-FGFR2IIIb antibody produced in CHO cells lacking a functional FUT8 gene In some embodiments, the afucosylated anti-FGFR2IIIb antibody is telligent® CHOK1SV cells (BioWa / Lonza, Allendale, NJ) It is produced in

[0162] Purification of anti-FGFR2IIIb antibody The anti-FGFR2IIIb antibody may be purified by any suitable method. Methods include the use of affinity matrix or hydrophobic interaction chromatography, which Suitable affinity ligands include, but are not limited to, FGFR2IIIbECD, and antibody constants. Ligands that bind to the region, e.g., Protein A, Protein G, Protein A / G, or antibody affinity column to bind the constant region, and anti-FGFR2II Hydrophobic interaction chromatography, e.g., may be used to purify Ib antibodies. For example, butyl or phenyl columns are also suitable for purifying some polypeptides. Numerous methods for purifying polypeptides are well known in the art.

[0163] Cell-free production of anti-FGFR2IIIb antibody In some embodiments, the anti-FGFR2IIIb antibody is produced in a cell-free system. Non-limiting exemplary cell-free systems include, for example, 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).

[0164] V. Therapeutic Compositions and Methods Disease treatment method using anti-FGFR2IIIb antibody The antibodies of the present invention and compositions comprising the antibodies of the present invention are useful in methods of treatment for humans or animals. The present invention provides a method for administering an afucosylated anti-FGFR2IIIb antibody to a patient in need thereof, the method comprising administering the afucosylated anti-FGFR2IIIb antibody to a patient in need thereof; Also provided is a method for treating a disease, comprising administering an afucosylated anti-FGFR2 antibody to a mammal. Non-limiting exemplary diseases that can be treated with IIIb antibodies include, but are not limited to, cancer. In some embodiments, the afucosylated anti-FGFR2IIIb antibody In some embodiments, a method of treating cancer is provided, comprising administering: The cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is administered.

[0013] Methods for treating gastric cancer are provided, comprising administering

[0165] In some embodiments, the cancer comprises FGFR2 gene amplification. In humans, cancers containing FGFR2 gene amplification overexpress FGFR2IIIb. In some embodiments, the cancer comprising FGFR2 amplification is more potent than FGFR2IIIc. In some embodiments, FGFR2 amplification is highly overexpressed. Cancers containing FGFR2IIIc had 2-fold, 3-fold, 5-fold, or 1-fold higher expression levels than normalized FGFR2IIIc expression levels. or 10-fold higher normalized levels of FGFR2IIIb. In some embodiments, the expression levels are normalized to GUSB. In this study, cancers overexpress FGFR2IIIb but do not contain FGFR2 gene amplification. In some embodiments, the gastric cancer comprises FGFR2 gene amplification. In one embodiment, the gastric cancer comprising FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, the gastric cancer containing FGFR2 amplification expresses FGFR2II. In some embodiments, FGFR2IIIb is overexpressed relative to FGFR1c. Gastric cancers containing FR2 amplification showed a 2-fold increase in FGFR2IIIc expression compared with normalized FGFR2IIIc expression levels. expressing FGFR2IIIb at normalized levels that are 3-fold, 5-fold, or 10-fold higher. In some embodiments, the expression levels are normalized to GUSB. In some embodiments, the gastric cancer overexpresses FGFR2IIIb but not the FGFR2 gene. In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression.

[0166] FGFR2IIIb gene amplification was confirmed by in situ hybridization (ISH). It may be determined by any suitable method in the art, including but not limited to In some embodiments, the FGFR2 amplification is FGFR2:CEN10 (chromosome 1 0 centromere) ratio >3.

[0167] FGFR2IIIb mRNA overexpression includes methods including quantitative PCR (qPCR) may be determined by any suitable method in the art, including but not limited to. The term "FGFR2IIIb mRNA overexpression" refers to increased FGFR2IIIb mRNA Regardless of the cause of the level (i.e., increased transcription and / or decreased mRNA degradation), Whether the increased levels are caused by: The increased levels of FGFR2IIIb mRNA were means.

[0168] FGFR2IIIb protein overexpression can be detected by antibody-based methods such as immunohistochemistry (IHC). It can be determined by any suitable method in the art, including but not limited to, In some embodiments, IHC staining may be performed according to methods known in the art. The term "FGFR2IIIb protein overexpression" refers to increased FGFR2IIIb expression. Regardless of the cause of GFR2IIIb protein levels (i.e., increased translation) by reduced degradation of proteins and / or proteins, by other mechanisms, or by a combination of mechanisms. Increased levels of FGFR2IIIb (whether or not this results in an increase in FGFR2IIIb levels) In some embodiments, the expression level of a protein by IHC is increased. 1+, 2+, or 3+ staining of tumor cells indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2I In some embodiments, IHC staining is performed as described in Example 6. The results are scored accordingly.

[0169] Pharmaceutical Compositions In various embodiments, the compositions comprising the afucosylated antibodies are prepared in a wide variety of pharmaceutically acceptable carriers. The compound is provided in a formulation together with an acceptable carrier (see, e.g., Gennaro, Remington: The Science and d Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (200 3); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed. , Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutic al Excipients, 3 rd ed., Pharmaceutical Press (2000). Vehicle, adjuvant A variety of pharmaceutically acceptable carriers are available, including acetone, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, various pharmaceutically acceptable adjuvants, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include saline, dextrose, water, glycerol, alcohol, ethanol, and combinations thereof.

[0170] In various embodiments, a composition comprising an afucosylated anti-FGFR2IIIb antibody is, e.g., For example, vegetable oils or other oils, synthetic aliphatic glycerides, higher fatty acids, or propylene glycol. by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents such as ethanol; If desired, conventionally used agents such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives may be added. It may also be formulated for injection, including subcutaneous administration, with suitable additives. Pressurized acceptable propellants such as dichlorofluoromethane, propane, nitrogen, etc. The composition may be formulated for inhalation using an agent. It may be formulated into sustained release microcapsules with biodegradable or non-biodegradable polymers, etc. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid polymers. Exemplary non-biodegradable formulations include polyglycerol fatty acid esters. A specific method for this purpose is described, for example, in EP 1 125 584 A1. It is being done.

[0171] Administration route In various embodiments, the afucosylated anti-FGFR2IIIb antibody is administered orally, intra-arterially, Parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transoral It has been administered in vivo by various routes, including dermally and intrathecally, or by implantation or inhalation. The subject compositions may be formulated in solid, semi-solid, liquid, or gas form; Forms include tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalation, and aerosols. Suitable formulations and routes of administration depend on the intended application. may be selected.

[0172] The pharmaceutical composition may be used to treat stomach cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer. A therapeutically effective amount is typically administered in an amount effective for treating or preventing cancer, such as steroids. the weight of the subject being treated, his or her physical condition or health status, and the severity of the health condition being treated or the age of the subject being treated. Usually, afucosylated anti-FGFR2III b The antibody is administered in an amount of about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the afucosylated anti-FGFR2IIIb antibody may be It may be administered in an amount of about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the anti-FGFR2IIIb antibody is administered at a dose of about 100 μg / In some embodiments, the amount of riboflavin administered may be in the range of about 10 mg / kg body weight to about 10 mg / kg body weight. Therefore, the afucosylated anti-FGFR2IIIb antibody is administered at a dose of approximately 100 μg / kg body weight. In some embodiments, afco may be administered in an amount of from about 20 mg / kg body weight. The silyl anti-FGFR2IIIb antibody is administered at a dose of about 0.5 mg / kg body weight to about 20 mg / kg body weight. It may be administered in amounts of g / kg body weight.

[0173] The afucosylated anti-FGFR2IIIb antibody composition is administered to a subject in need thereof. The frequency of administration may depend on the condition being treated, the age of the subject being treated, the condition being treated, The severity of the condition, the general health condition of the subject to be treated, etc., should be taken into consideration when making such a determination. In some embodiments, the afucosylated anti-FGFR2IIIb antibody An effective dose is administered to a subject one or more times. The effective dose of the anti-FGFR2IIIb antibody may be administered once a month, more than twice a month, for example, twice a month. In another embodiment, the afucosylated antibody is administered to a subject once a month or once every three months. The effective dose of the FGFR2IIIb antibody may be administered, for example, less than once a month, for example, once every two weeks. or weekly to the subject. In some embodiments, the afucosylated anti-FGF antibody is administered to the subject at least once. The effective dose of the R2IIIb antibody may be administered for at least 1 month, at least 6 months, or at least Multiple doses may be administered over a one year period.

[0174] Combination Therapy The afucosylated anti-FGFR2IIIb antibody may be administered alone or in combination with other treatment modalities. It may be administered before other treatment methods, such as surgery, chemotherapy, or radiation therapy. In some embodiments, the afco Afucosylated anti-FGFR2IIIb antibodies are administered in combination with other anti-cancer drugs. Non-limiting examples of anti-cancer drugs that may be administered with the anti-FGFR2IIIb antibody include platinum-containing drugs ( For example, cisplatin, oxaliplatin, and carboplatin), paclitaxel (T AXOL®), an albumin-engineered nanoparticle formulation of paclitaxel (ABRAXA NE®), docetaxel (TAXOTERE®), gemcitabine ( GEMZAR®), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), epirubicin (ELLENCE®), PHARMORUBICIN (registered trademark), FOLFOX (an orally administered orally in combination with 5-FU) Xaliplatin), FOLFIRI (a combination of leucovorin, 5-FU, and irinotecan) leucovorin, fluorouracil (5-FU, EFUDEX®), mycobacterial Tomycin C (MITOZYTREX™, MUTAMYCIN®, and Doxorubicin hydrochloride (Adriamycin PFS, Adriamycin RDF, RUBEX In some embodiments, the afucosylated anti-FGFR2 III In some embodiments, the antibody is administered in combination with paclitaxel. The silyl anti-FGFR2IIIb antibody is administered in combination with cisplatin and / or 5-FU. In some embodiments, the afucosylated anti-FGFR2IIIb antibody is In some embodiments, the afucosylated Anti-FGFR2IIIb antibodies are used in combination with FOLFOX (oxaliplatin, 5-FU, and leukotriene). It is administered in combination with borolin.

[0175] Kits / Products The present invention provides kits, medicaments, compositions, and units for any of the methods described herein. Includes dosage forms.

[0176] The kit of the present invention comprises an afucosylated anti-FGFR2IIIb antibody (or a unit dose form, and In some embodiments, the container comprises one or more additional containers containing the product. A predetermined amount of a composition containing an afucosylated anti-FGFR2IIIb antibody, with or without a drug. In some embodiments, a unit dose is provided that contains the composition. is provided in a disposable, pre-filled syringe for injection. The compositions contained in the unit dose may include buffers such as saline, sucrose, etc.; phosphate, etc. and / or may be formulated within a stable and effective pH range. In an embodiment, the composition may be reconstituted by the addition of a suitable liquid, such as, for example, sterile water. In some embodiments, the composition may be provided as a lyophilized powder. one or more substances that inhibit protein aggregation, including but not limited to, sugars and arginine In some embodiments, the compositions of the present invention contain heparin and / or protease inhibitors. Contains theoglycan.

[0177] In some embodiments, the kits of the present invention are used in accordance with any of the methods described herein. For example, cancer (e.g., stomach cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or The kit further comprises instructions for use in the treatment of cancer (e.g., esophageal cancer). The instructions accompanying the kit of the present invention are typically written on a label or are instructions provided on the package insert (e.g., on a sheet of paper included in the kit), but are machine readable. Readable instructions (e.g., instructions stored on magnetic or optical disks) are also acceptable. In one embodiment, the kit further comprises another therapeutic agent.

[0178] The kits of the present invention are suitably packaged. Suitable packaging may include vials, bottles, jars, etc. Examples include, but are not limited to, flexible packaging (e.g., sealed Mylar or plastic bags). Kits may optionally provide additional components, such as buffers and instructions. The present application therefore relates to vials (e.g., sealed vials), bottles, jars, flexible We also provide products including packaging. [Example]

[0179] The examples set forth below are intended to be merely illustrative of the present invention and in no way limiting thereof. The examples below should not be construed as limiting all or any of the examples. It is not intended to illustrate the only examples that have been performed. Although care has been taken to ensure accuracy (e.g., amounts, temperatures, etc.), Some experimental error and deviation should be accounted for. ) is parts by weight, molecular weight is weight average molecular weight, temperature is temperature in degrees Celsius , and pressure is at or near atmospheric pressure.

[0180] Example 1: Production of afucosylated anti-FGFR2IIIb antibody Construction of expression vectors for heavy chain (HC) and light chain (LC) of monoclonal antibody αFGFR2b The nucleotide sequence encoding the nucleotide sequence was obtained using GS GeneEx according to the manufacturer's instructions. and cloned into the pression System (Lonza, Basel, Switzerland). The system produces a double gene vector containing expression cassettes for both the light and heavy chains. (DGV) was created.

[0181] Selection of host cell lines Afucosylated monoclonal antibody αFGFR2bA (αFGFR2b The "A" after "afucosylated" indicates "afcosylated" and is produced using Potassium iodide as a host cell. elligent® CHOK1SV cells (BioWa / Lonza, Allendale, NJ) were selected. Potelligent® CHOK1SV cells were selected for the FUT8 gene. (α1,6-fucosyltransferase) and therefore fucose-free It produces antibodies (afcosylated antibodies).

[0182] Construction of a stable cell line for the production of afucosylated αFGR2bA antibody:αFGFR2 b Expression vectors containing nucleotide sequences encoding antibody heavy and light chains are prepared according to the manufacturer's instructions. According to the manufacturer's instructions, Potelligent® was used by electroportation. CHOK1SV cells were transfected. Electroported cells were Approximately 10,000 cells / 50 μl / well in a 96-well plate, without L-glutamine The next day, the cells were seeded in 67 μM methionine sulfoximine ( CH CHO selection medium containing MSX, SIGMA cat#M5379, St. Louis, MO) was added at 150 μl / u. Cell growth and clone formation were analyzed using an IN Cell Analyzer 2000 (IN Cells were monitored using a Cell Analyzer 2000 (GE Healthcare, Piscataway, NJ).

[0183] After 4-6 weeks, surviving colonies were cultured using XL665-conjugated Protein A and Cryptate-conjugated polyclonal rabbit IgG (Cisbio, Bedford, MA) was used. A standard curve was generated for purified αFGFR2b antibody using a homogeneous Homogenous Time Resolved Fluorescence (HTRF)-based assay , and the expression of αFGFR2bA antibody was screened. 24-well plate, centrifugation Tube, shake flask, bench-scale bioreactor, and platform products Through a series of incremental production processes, including a platform production process The clones with the highest expression were then expanded. Only the subset was subjected to the next step. The final producing clones were selected based on the protein production titer. - for evaluation of cell growth characteristics, product quality, stability, and scalability in bioreactors The final production strain was selected based on its expression level of approximately 3.5 g / L for αFGFR2bA. Fucosylation of αFGFR2bA in the final production cell line The absence of was confirmed using normal phase HPLC (N-HPLC) chromatography.

[0184] The αFGFR2bA antibody was purified by column chromatography and purified by ultrafiltration. The purified material was concentrated and purified by diafiltration using purification buffer (20 mM histidine, 1 50 mM L-arginine, 0.01% polysorbate 20, pH 6.0). Antibodies were stored below -70°C.

[0185] Glycan analysis of αFGFR2bA revealed the release of glycans from the protein, anthranil This is carried out by labeling glycans with 2-amino-2-hydroxybenzoic acid (2-AA) and purifying the labeled glycans. Normal phase HPLC with fluorescence detection separates the glycans and determines the relative abundance of each glycan in the antibody. FIG. 7 shows the Potelligent® CHOK αFGFR2b derived from the 1SV cell line and αFGFR produced by CHOK1SV (A) Potelligent 2b has two different glycan distributions. The glycan distribution of αFGFR2b derived from the CHOK1SV cell line was (B) αFG derived from the CHOK1SV cell line, which lacks fucose ("G0"). The glycan distribution of FR2b indicates that the antibody contains fucose ("G0F").

[0186] Glycan peaks obtained from normal-phase HPLC separation were detected using two orthogonal methods. First, αFGF produced by Potelligent® CHOK1SV R2b was labeled and separated by normal-phase HPLC. After fluorescence detection, the glycans The mass of each peak was detected and assigned to each glycoprotein. The results are shown in Table 2. [Table 2]

[0187] The masses of each fucosylated form of the glycan (G0F, G1F, and G2F) were also calculated by Potell αFGFR2b produced by igent® CHOK1SV Figure 8 shows the G0, G1, G2, and G3 phases typically observed in antibodies. Skimming of G0F, G1F, G2F, and mannose-5 (or Man-5) glycan structures A system diagram is shown.

[0188] The peaks shown by the HPLC assay were also obtained from glycans purchased from Prozyme. A standard was used, and the standard and the Potelligent® CHOK1SV cell line and Conservation time between the αFGFR2 profiles obtained from both the αFGFR2 and CHOK1SV cell lines These standards were G0, G0F, Man5, G1, and G 1F and G2F were detected.

[0189] The results obtained by HPLC assay and characterization data indicate that Potellige Fucosylation of αFGFR2 from nt® CHOK1SV cell line The deficiency was confirmed.

[0190] Example 2: Binding affinity of afucosylated anti-FGFR2b antibodies αFGFR2bA and αFGFR2bF for Fc gamma RIIIA (V158) The binding affinity (the "F" after αFGFR2b indicates "fucosylation") was determined by surface plasmon resonance imaging (SPIR). The amine coupling was determined by ion resonance assay. kit (GE Healthcare Life Sciences, Piscataway, NJ) and 1 as a blocking agent. 100 mM sodium borate buffer (pH 7.5) containing 0.05 mM ethylenediamine (Sigma) 8.0) (Sigma, St. Louis, MO) was used to covalently couple the dextran chip. Approximately 600-800RU of αFGFR2bA and αFGFR2bF were separated into flow cells and Flow cytometer derivatized with Protein A (Pierce) served as a control. Fc gamma RIIIA (V158) (R&D Systems, Minneapolis, MN) in HBS-P+ running buffer (Biacore, GE Healthcare, Piscataway, NJ). It was diluted and prepared at five concentrations (0 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, and and 1000 nM) were injected in duplicate. The binding constants, dissociation constants, and affinities for the 1:1 binding sites were calculated using Biacore T200 Evaluation Software. The affinity constants for αFGFR2bF binding were calculated using the Biacore model. The results were determined using the T200 Evaluation Software steady state affinity model. Shown in 3. [Table 3]

[0191] As shown in Table 3, Fc gamma RIIIA (V158) inhibited αFGFR2bF. It bound to αFGFR2bA with 20-fold higher affinity than to αFGFR2bA.

[0192] Example 3: ADCC activity of afucosylated anti-FGFR2b antibodies To determine the ADCC activity of αFGFR2bA versus αFGFR2bF antibodies, In vitro assays were performed. Ba / F3 FGFR2IIIb-expressing target cells were as follows: Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Ba / F3 cells obtained from Zellkulturen GmbH (DSMZ, cat# ACC300, Braunschweig, Germany) rmany) was supplemented with 10% fetal bovine serum (Mediatech, cat# 35-010-CV), 1 ng / mL mouse I L-3 (Peprotech, cat# 213-13, Rocky Hill, NJ), 1 x BME (Invitrogen, cat# 104 7574, Grand Island, NY), and 1× penicillin-streptomycin (Mediatech cat# The cells were maintained in RPMI (Mediatech, cat# 10-041-CV, Manassas, VA) supplemented with 30-002-Cl. Ba / F3 cells were cultured using Cell Line Nucleofector® Kit V (Lonza, cat# VCA-1003 ) according to the manufacturer's protocol, FGFR2IIIb, pBNew-hFGF The pBNew vector was transfected with an expression vector expressing R2b. Chicken β-actin intron and ampicillin and puromycin growth selection genes The transfected cells were allowed to grow for 3 days. The cells were incubated in full growth media and treated with 2 μg / mL of puromycin. Puromycin (InVivoGen, cat # ant-pr-1, San Diego, CA) was used. Selection was maintained throughout the culture. To generate individual stable clones, cells were The cells were seeded at a density of 1 cell per 3 wells. Fluorescence-activated cell sorting (FACS) was used to identify clones with the highest FGFR2IIIb expression levels. was used to select the

[0193] MFM-223 cells were obtained from the Health Protection Agency (UK). OCUM-2M cells were obtained from Osaka City University (Osaka, Japan), and KATO III cells were obtained from ATCC (Rockville, MD). All cells were prepared according to standard methods. Freshly isolated PMBCs from healthy donors were cultured using AllCell s (Emeryville, CA).

[0194] ADCC assays were performed using effector cells obtained on three separate days from three independent donors. The ADCC assay was performed using freshly isolated human PBMCs. were used as effector cells at an effector to target (E / T) cell ratio of 25:1. Target cells were incubated for 16 h in the presence of effectors and increasing antibody concentrations. The ADCC assay was performed on HERC-positive SKOV-3 target cells. EPTIN® and RITUXIN® against Raji target cells The antibody was evaluated using two positive control antibodies: a fully human IgG1 negative control antibody and a A positive control antibody (Eureka Therapeutics, Catalog ET901, Emeryville, CA) was used to Cytotoxicity was determined according to the manufacturer's instructions (CytoTox Non Quantification of LDH release according to Radioactive Cytotoxicity Assay (Promega, Madison, WI). It was decided by.

[0195] Maximum lysis was determined in the presence of 5% Triton X-100, and spontaneous release was determined by the non- Percentage of specific lysis was determined as the ratio of maximal lysis minus spontaneous release. As a percentage, it was calculated as follows: (Experimental value - spontaneous release) / (Maximum value - spontaneous release) x 100 = Specific lysis (%)

[0196] The results for Ba / F3 cells are shown in Figure 1. The afucosylated αFGFR2bA antibody In FGFR2IIIb-expressing Ba / F3 cells, fucosylated αFGFR2bF antibody Induced highly specific lysis in OCUM-2M, MFM223, and KATO III cells In this study, the maximum specific lysis of the antibody was observed in both OCUM-2M and MFM 223 cells. (data not shown), but αFGFR2bA was lower than αFGFR2bF. Furthermore, αFGFR2b showed higher ADCC activity than FGFR2IIIc expression. In current Ba / F3 cells, it shows little or no ADCC activity. See Figure 1A.

[0197] Example 4: Afucosylated anti-FGFR2b antibody in gastric and breast cancer xenograft models activity From Charles River Laboratories (Wilmington, MA), 6 weeks Female CB17 SCID mice were purchased and allowed to acclimate for one week before the start of the study. The human gastric cancer cell line OCUM-2M or breast cancer cell line was used as a tumor model. -2M was purchased from Osaka City University (OCU, Osaka, Japan), and MFM-223 is Culture Collections (Public Health England) (98050130, HPA Cells were purchased from the Culture Collections, Salisbury, UK. Cells were cultured in complete growth medium for up to three times. OCUM-2M and MFM-223 cells were passaged in vitro and expanded for transplantation. They were cultured in Dulbecco's minimum essential medium (DMEM) and minimal essential medium (MEM), respectively. All media contain 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin solution were added. The cells were incubated in a humidified atmosphere of 5% CO The cells were incubated at 37°C under ambient conditions.

[0198] When the cultured cells reached 85-90% confluence, the cells were harvested and 50% maturated. Chilled Ca containing Rigel (BD BioSciences, San Jose, CA) 2+ and Mg 2+ Phosphate-free OCUM-2M cells were resuspended in buffered saline (PBS) at 5x10 5 cell / 1 MFM-223 cells were subcutaneously implanted into the right flank of the mice at 5x the volume of 1000 μl / mouse. 10 5 The cells were implanted subcutaneously into the right flank of the mouse at 100 μl per mouse, and the concentration was 0.72 mg 90-day release 17-β estradiol pellets (Innovative Research of America, Sarasota, FL) were inoculated subcutaneously into the right flank. Mice were monitored twice weekly for tumor growth after cell implantation. Tumor size was monitored using the formula: tumor size (mm 3 ) = (Width (mm) x Length (m m) 2 ) / 2. MFM-223 tumors were 80 mm 3 When the mouse reaches The patients were assigned to groups, randomized (n=10), and treatment was initiated. Regarding tumors, the average size of the tumors was 100 mm 3 Anti-FGFR2b was used when Monotherapy was initiated and tumors reached an average size of 250 mm 3 Combination therapy is initiated when It was.

[0199] Anti-FGFR2IIIb humanized antibody (afucosylated, αFGFR2bA; fucosylated, α FGFR2bF) or albumin as a negative control, as shown in the figure legends. The chemotherapy drugs were administered intraperitoneally at doses of 1 to 10 mg / kg twice a week. 12 mg / kg for taxel and 2.3 mg for fluorouracil (5-FU) 1 mg / kg and cisplatin 33 mg / kg, administered intraperitoneally twice weekly. After the start of the study, tumor size was measured twice a week in each mouse. The length and width of each tumor were The tumor volume was measured using a caliper and calculated according to the formula above. is 2000mm 3 Mice were euthanized when the tumor volume exceeded 100 μg / ml or when the tumor became excessively necrotic. made him do so.

[0200] Comparisons of resulting tumor volumes were determined to be statistically significant when P<0.05. P values are from an independent, two-tailed t-test of calculated tumor volumes on the last day tumors were measured. was calculated using

[0201] Figure 2 shows the results of the OCUM-2M human gastric cancer xenograft model with FGFR2 amplification. αFGFR2bA and αFGFR2bA at 10 mg / kg (A and B) and 3 mg / kg (C and D). The efficacy of αFGFR2bF is shown. (A) At 10 mg / kg, fucosylation and afucosylation were significantly reduced. Both afucosylated αFGF and αFGF-1 induced direct tumor regression. R2bA induced a more durable response than fucosylated αFGFR2bF. (B) Afucosylated αFGFR2bA has a higher final O CUM-2M tumor size was significantly reduced. Statistical significance was determined by two-tailed unpaired t-test. (C) At a dose of 3 mg / kg, afucosylated αFGFR2bA was In fact, afucosylated αFGFR2bF reduced tumor growth compared to afucosylated αFGFR2bF. 2bA showed higher tumor regression and durable responses compared with fucosylated αFGFR2bF. (Note: One animal was removed from the αFGFR2bF group at approximately 42 days. This resulted in a shift in the curve. (D) Compared to fucosylated αFGFR2bF Afucosylated αFGFR2bA at 3 mg / kg reduced final OCUM-2M tumor size Statistical significance was determined by two-tailed unpaired t-test.

[0202] Figure 3 shows dose-dependent tumor inhibition by αFGFR2bA. (A) Mean tumor size is about 100mm 3 When the OCUM-2M xenografts reached 100 μg / ml, SCID mice bearing subcutaneous OCUM-2M xenografts were cultured. were treated with 1, 1.5, 2, 3, or 5 mg / kg afucosylated αFGFR2bA. All doses of afucosylated αFGFR2bA inhibited tumor growth, but the highest inhibition was and sustained responses were observed at 3 and 5 mg / kg, and at 2, 1.5, and 1 mg / kg αF. (B) Afucosylated αFGFR2bA ultimately suppressed growth. It reduced OCUM-2M tumor size, with higher doses showing more potent growth inhibition. Tumor growth inhibition was statistically significant for all doses (5 mg / kg, p<0. 0001;3mg / kg, p<0.0001;2mg / kg, p<0.0001;1.5 mg / kg, p=0.0003; 1 mg / kg, p=0.0013). Statistical significance was Determined by two-tailed unpaired t-test.

[0203] Figure 4 shows the afucosylated αFGFR2b in the OCUM-2M gastric cancer xenograft model. The results show that administration of A enhances the antitumor activity of paclitaxel. (A) The mean tumor size was approximately 285mm 3 When the patient reached 18.5°C, SCID mice bearing subcutaneous OCUM-2M xenografts were Afucosylated αFGFR2bA (5 mg / kg), paclitaxel (12 mg / kg), or a combination of the two. and paclitaxel alone. (B) αFGFR2bA / paclitaxel combination therapy reduced tumor size. No significant difference was observed between clitaxel (p=0.0005) and αFGFR2bA (p=0.0009) alone. Statistical significance: was determined by two-tailed unpaired t test.

[0204] Figure 5 shows the afucosylated αFGFR2b in the OCUM-2M gastric cancer xenograft model. The results show that administration of cisplatin / 5-FU enhances the antitumor activity of cisplatin / 5-FU. (A) Mean tumor size The size is approximately 260 mm 3 When the OCUM-2M xenografts were obtained, SCID mice were transplanted with the OCUM-2M xenografts. The mice received afucosylated αFGFR2bA (5 mg / kg), fluorouracil (5-FU ) and cisplatin (2.3 and 33 mg / kg, respectively), or a combination of these three The patients were treated with 5-FU / cisplatin in combination with afucosylated αFGFR2bA. This resulted in a significant reduction in tumor size compared with either αFGFR2bA or 5-FU / cisplatin alone. (B) αFGFR2bA / 5-FU / cisplatin combination therapy Significantly reduced final OCUM-2M tumor size compared to 5-FU / cisplatin (p=0.0003). The further reduction in tumor size with αFGFR2bA compared to αFGFR2bA alone was significant, There was no statistical significance. Statistical significance was determined by a two-tailed unpaired t-test.

[0205] Figure 6 shows the efficacy of αFGFR2bA in the MFM-223 human breast cancer xenograft model. (A) The MFM-223 human breast cancer cell line, which harbors FGFR2 amplification, was shown to be a marker of inflammatory bowel disease in SCID mice. The tumors were implanted subcutaneously in mice, with an average tumor size of approximately 80 mm. 3 When the afucosylated αF GFR2bA therapy was initiated. Afucosylated αFGFR2bA (5 mg / kg) significantly increased MFM-223 tumor growth compared to albumin control (5 mg / kg) (B) Afucosylated αFGFR2bA reduced the αFGFR2b level compared to the albumin control. This significantly reduced the final MFM-223 tumor size (p=0.0008). Statistical significance was determined by two-tailed unpaired t-test.

[0206] Example 5: Afucosylated anti-FGFR2b antibody has higher affinity than fucosylated anti-FGFR2b antibody Mediates ADCC. To determine the ADCC activity of αFGFR2bA versus αFGFR2bF antibodies, In vitro assays were performed. Ba / F3 FGFR2IIIb-expressing target cells were used as described above. OCUM-2M cells were obtained from Osaka City University (Osaka, Japan). All cells were cultured using standard methods. Freshly isolated P cells from healthy donors were MBCs were obtained from AllCells (Emeryville, CA).

[0207] The ADCC assay uses freshly isolated human PBMCs as effector cells. The effector-to-target (E / T) cell ratio was 25:1. The cells were incubated for 16 hours in the presence of increasing concentrations of antibody. All conditions were performed in triplicate. Cytotoxicity was determined according to the manufacturer's recommendations. of LDH release according to Promega's CytoTox Non-Radioactive Cytotoxicity Assay. For OCUM-2M cells, maximum lysis was determined by 5% Triton X-10. Specific lysis was determined in the presence of X-100 and spontaneous release was determined in the absence of antibody. The percentage of maximum dissolution minus spontaneous release is given as follows: Specific lysis (SRS) was calculated as follows: (experimental value - spontaneous release) / (maximum value - spontaneous release) x 100 = specific lysis (SRS) %) EC50 values were obtained using GraphPad software curve-fitting analysis.

[0208] The results for Ba / F3 cells are shown in Figure 9. The afucosylated αFGFR2bA antibody In FGFR2IIIb-expressing Ba / F3 cells, fucosylated αFGFR2bF antibody It showed high titer (higher ADCC activity at low concentration). Similar results for all the samples are shown in Figure 10. Table 4 shows the results for two different samples shown in Figures 9 and 10. Afucosylated anti-FGFRb antibody compared to fucosylated FGFR2b antibody in cell lines The fold increase in body titer is shown. [Table 4]

[0209] Example 6: Immunohistochemical detection of FGFR2IIIb protein in tumor tissues The mouse variable region of GAL-FR21 (see U.S. Pat. No. 8,101,723) and mouse A murine αFGFR2b antibody containing the IgG2a constant region was isolated from formalin-fixed paraffin-embedded It was used to detect FGFR2IIIb protein in FFPE gastric cancer tissue. FFPE tumor tissue was washed with a series of xylenes and then washed with a series of decreasing concentrations of xylene. The paraffin was removed by washing with ethanol, and the sections were rehydrated in water for 5 minutes. The sections were then mounted on slides. The tissue sections were immersed in 0.1 mM citrate buffer (pH 6.0) for 15 minutes. The tissue sections were then cooled and placed in 3% H2O2 at room temperature for 5 minutes. The cells were then placed in TBST (0.05M Tris, 0.15M NaCl, 0.0 5% Tween 20) twice and then incubated in blocking buffer (2.5 mL) for 30 minutes at room temperature. The tissue sections were then blocked with TBST containing 100% normal horse serum. The cells were incubated with 5 μg / ml αFGFR2b antibody in a buffer at room temperature for 30 minutes. Incubation times ranging from 30 minutes to 2 hours yielded similar staining intensities. Following three washes with ST buffer, tissue sections were washed with blocking buffer (Vector La Ready-to-use (RTU) biotin was diluted in 1000 mg of ... Tissue sections were incubated with monovalent horse secondary antibody (10 μg / ml) for 30 minutes at room temperature. The cells were washed twice with TBST buffer and then incubated with streptavidin-horseradish paraffin. Incubate with oxidase (HRP; Vector Laboratories, Cat. No. PK-7200) at room temperature for 30 minutes. Detection was performed using 3',3'-diaminobenzidine (DAB) substrate (Vector Laboratories). (Cat. No. PK-4105) according to the manufacturer's instructions for 10 minutes at room temperature. Tissue sections were then prepared using hematoxylin according to the manufacturer's instructions. The tissue sections were stained with graded concentrations of ethanol and xylene. The sections were dehydrated by washing with ethanol and then covered with a coverslip.

[0210] A staining concentration of 0.1 to 5 mg / ml of αFGFR2b antibody was used for protein detection. It was particularly effective.

[0211] Staining was scored as follows: 0 No staining with αFGFR2b antibody is observed. Weak staining with αFGFR2b antibody is observed in 1+ samples. No staining is observed in corresponding sections stained with a control antibody. 2+ αFGFR2b antibody-specific membrane-cytoplasmic staining was more widespread in sections be observed. 3+ with a distinct membrane distribution in at least one subset of stained tumor cells Strong and specific staining with αFGFR2b antibody is observed.

[0212] FIG. 11 shows exemplary 0 (A), 1+ (B, C), 2+ (D), and 3+ (E) tumor tissues. In Figure 11A, ductal cells and scattered tumor cells were stained with αFGFR2b antibody. In Figure 11B, which shows a stromal gastric tumor sample, the tumor cells The alveoli (arrows) show some staining with the αFGFR2b antibody, but the surrounding ductal cells In Figure 11C, which shows a diffuse gastric tumor sample, tumor cells (arrows) show no staining. Shows some staining with FGFR2b antibody, but no staining of surrounding stromal cells (arrowheads) In Figure 11D, tumor cells (arrows) were not detected using the αFGFR2b antibody. In Figure 11E, the stromal cells show some staining, while the surrounding stromal cells (arrowheads) show no staining. Tumor cells (arrows) show high membrane-distributed staining with the αFGFR2b antibody, but there is no peripheral staining. Stromal cells (arrowheads) show no staining. In Figure 11F, tumor cells (arrows) express αFGF High membrane / cytoplasmic staining with R2b antibody, but no staining in surrounding interstitial cells (arrowheads) Not shown. [Table 5] TIFF2025118617000006.tif226167

Claims

1. A composition comprising multiple anti-FGFR2IIIb antibodies, each anti-FGFR2IIIb antibody comprises a heavy chain and a light chain variable region; Heavy chain variable region (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 (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; at least 95% of the anti-FGFR2IIIb antibody in the composition is afucosylated; composition.

2. The composition of claim 1 , wherein the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO:

4.

3. The composition of claim 1 or 2, wherein the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

5.

4. 2. The composition of claim 1, wherein the heavy chain variable region domain comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable region domain comprises the amino acid sequence of SEQ ID NO:

5.

5. The composition of any one of claims 1 to 4, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

2.

6. The composition of any one of claims 1 to 5, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

3.

7. 2. The composition of claim 1, wherein 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. 1. A composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies, A composition, wherein the anti-FGFR2IIIb antibody 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.

9. The composition according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.

10. The composition according to any one of claims 1 to 9, wherein the antibody is a chimeric antibody.

11. The composition according to any one of claims 1 to 10, wherein the antibody is a humanized antibody.

12. The composition of any one of claims 1 to 11, wherein the antibody lacks fucose at Asn297.

13. The composition of any one of claims 1 to 12, wherein the antibody comprises a kappa light chain constant region.

14. The composition of any one of claims 1 to 13, wherein the antibody comprises an IgG1 heavy chain constant region.

15. The composition of any one of claims 1 to 14, wherein the afucosylated antibody has enhanced in vitro ADCC activity compared to a fucosylated anti-FGFR2IIIb antibody having the same amino acid sequence.

16. 16. The composition of claim 15, wherein the afucosylated anti-FGFR2IIIb antibody causes 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 the specific lysis caused by the fucosylated anti-FGFR2IIIb antibody.

17. 17. The composition of claim 15 or 16, wherein the ADCC activity is determined using Ba / F3 cells expressing FGFR2IIIb as target cells and isolated human PBMCs as effector cells.

18. The composition of any one of claims 1 to 17, wherein the afucosylated antibody has enhanced affinity for Fc gamma RIIIA compared to a fucosylated anti-FGFR2IIIb antibody having the same amino acid sequence.

19. 19. The composition of claim 18, wherein the afucosylated anti-FGFR2IIIb antibody binds to 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 greater affinity than the fucosylated anti-FGFR2IIIb antibody.

20. 20. The composition of claims 18 and 19, wherein the affinity for Fc gamma RIIIA is determined by surface plasmon resonance.

21. The composition of any one of claims 18 to 20, wherein the Fc gamma RIIIA is selected from Fc gamma RIIIA(V158) and Fc gamma RIIIA(F158).

22. 22. The composition of claim 21, wherein the Fc gamma RIIIA is Fc gamma RIIIA(V158).

23. The composition of any one of claims 1 to 22, wherein the afucosylated anti-FGFR2IIIb antibody binds to FGFR2IIIb but not to FGFR2IIIc.

24. A host cell comprising a nucleic acid encoding the anti-FGFR2IIIb antibody of any one of claims 1 to 23, wherein the host cell lacks a functional alpha-1,6-fucosyltransferase (FUT8) gene.

25. 25. The host cell of claim 24, which is a CHO cell.

26. A method for producing an afucosylated anti-FGFR2 IIIb antibody, comprising culturing a host cell described in claim 24 or 25 under conditions suitable for expressing a nucleic acid encoding the anti-FGFR2 IIIb antibody.

27. 26. A method for producing an afucosylated anti-FGFR2 IIIb antibody, comprising culturing a host cell according to claim 24 or 25 under conditions suitable for producing an afucosylated anti-FGFR2 IIIb antibody.

28. 28. The method of claim 26 or 27, further comprising recovering the anti-FGFR2IIIb antibody produced by the host cell.

29. The method of any one of claims 26 to 28, wherein at least 95% of the anti-FGFR2IIIb antibodies produced by the host cell are afucosylated.

30. 30. The method of claim 29, wherein fucose is not detected in the anti-FGFR2IIIb antibody produced by the host cell.

31. 31. The method of claim 29 or 30, wherein fucose is detected by a method comprising HPLC, capillary electrophoresis, or MALDI-TOF mass spectrometry.

32. A pharmaceutical composition comprising the composition of any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

33. 33. A method of treating cancer in an individual comprising administering to the individual having cancer an effective amount of the pharmaceutical composition of claim 32.

34. 34. The method of claim 33, wherein the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.

35. 35. The method of claim 34, wherein the cancer is gastric cancer.

36. The method of any one of claims 33 to 35, wherein the cancer overexpresses FGFR2IIIb.

37. 37. The method of claim 36, wherein FGFR2IIIb expression is determined by immunohistochemistry (IHC).

38. 38. The method of claim 36 or 37, wherein the cancer does not involve FGFR2 gene amplification.

39. The method of any one of claims 33 to 37, wherein the cancer comprises FGFR2 gene amplification.

40. 40. The method of any one of claims 33-39, further comprising 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.

41. 41. The method of claim 40, wherein the platinum agent is selected from cisplatin, oxaliplatin, and carboplatin.

42. 42. The method of claim 41, wherein the additional therapeutic agent is paclitaxel.

43. 42. The method of claim 41, wherein the additional therapeutic agent is cisplatin and / or 5-FU.

44. 33. Use of the pharmaceutical composition of claim 32 for treating cancer in an individual in need thereof.

45. 45. The use of claim 44, wherein the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer.

46. 46. The use of claim 45, wherein the cancer is gastric cancer.

47. The use according to any one of claims 44 to 46, wherein the cancer overexpresses FGFR2IIIb.

48. 48. The use according to claim 47, wherein FGFR2IIIb expression is determined by immunohistochemistry (IHC).

49. 49. The use of claim 47 or 48, wherein the cancer does not involve FGFR2 gene amplification.

50. The use according to any one of claims 42 to 48, wherein the cancer comprises FGFR2 gene amplification.

51. A pharmaceutical composition for treating cancer in an individual, comprising an effective amount of the anti-FGFR2IIIb antibody of any one of claims 1 to 23, and a pharmaceutically acceptable carrier.

52. 52. The pharmaceutical composition of claim 51, wherein the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.

53. 53. The pharmaceutical composition of claim 52, wherein the cancer is gastric cancer.

54. 54. The pharmaceutical composition of any one of claims 51 to 53, wherein the cancer overexpresses FGFR2IIIb.

55. 55. The pharmaceutical composition of claim 54, wherein FGFR2IIIb expression is determined by immunohistochemistry (IHC).

56. 56. The pharmaceutical composition of claim 54 or 55, wherein the cancer does not involve FGFR2 gene amplification.

57. The pharmaceutical composition of any one of claims 51 to 54, wherein the cancer comprises FGFR2 gene amplification.

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