US20150050273A1 - Afucosylated anti-fgfr2iiib antibodies - Google Patents

Afucosylated anti-fgfr2iiib antibodies Download PDF

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US20150050273A1
US20150050273A1 US14/447,751 US201414447751A US2015050273A1 US 20150050273 A1 US20150050273 A1 US 20150050273A1 US 201414447751 A US201414447751 A US 201414447751A US 2015050273 A1 US2015050273 A1 US 2015050273A1
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fgfr2iiib
antibody
antibodies
amino acid
afucosylated
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Thomas Harding
Kristen Pierce
Namrata Patil
Thomas Brennan
Julie Hambleton
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Five Prime Therapeutics Inc
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Five Prime Therapeutics Inc
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Assigned to FIVE PRIME THERAPEUTICS, INC. reassignment FIVE PRIME THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PATIL, NAMRATA, HARDING, THOMAS, BRENNAN, THOMAS, PIERCE, KRISTEN, HAMBLETON, JULIE
Publication of US20150050273A1 publication Critical patent/US20150050273A1/en
Priority to US15/166,798 priority patent/US10172937B2/en
Priority to US16/181,784 priority patent/US11235059B2/en
Priority to US17/554,423 priority patent/US20220168416A1/en
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    • A—HUMAN NECESSITIES
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K31/33—Heterocyclic compounds
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/507—Comprising a combination of two or more separate antibodies
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Afucosylated anti-FGFR2IIIb antibodies are provided.
  • the fibroblast growth factor (FGF) family members bind to four known tyrosine kinase receptors, fibroblast growth factor receptors 1-4 (FGFR1-4) and their isoforms, with the various FGFs binding the different FGFRs to varying extents (Zhang et al., J. Biol. Chem. 281:15694, 2006).
  • a protein sequence of human FGFR2 is provided in, e.g., GenBank Locus AF487553.
  • Each FGFR consists of an extracellular domain (ECD) comprising three immunoglobulin (Ig)-like domains (D1, D2 and D3), a single transmembrane helix, and an intracellular catalytic kinase domain (Mohammadi et al., Cytokine Growth Factor Revs, 16:107, 2005).
  • ECD extracellular domain
  • Ig immunoglobulin
  • D1 and D2 immunoglobulin-like domains
  • AB intracellular catalytic kinase domain
  • the region containing D1 and AB is believed to be involved in autoinhibition of the receptor, which is relieved by binding to ligand.
  • FGFs bind to the receptors primarily through regions in D2 and D3 of the receptors.
  • the FGFRs are characterized by multiple alternative splicing of their mRNAs, leading to a variety of isoforms (Ornitz et al., J. Biol. Chem. 271:15292, 1996; see also Swiss-Prot P21802 and isoforms P21802-1 to -20 for sequences of FGFR2 and its isoforms). Notably, there are forms containing all three Ig domains ( ⁇ isoform) or only the two Ig domains D2 and D3 domains without D1 ( ⁇ isoform). In FGFR1-FGFR3, all forms contain the first half of D3 denoted IIIa, but two alternative exons can be utilized for the second half of D3, leading to IIIb and IIIc forms.
  • FGFR2IIIb and FGFR2IIIc are respectively denoted FGFR2IIIb and FGFR2IIIc (or just FGFR2b and FGFR2c); the corresponding beta forms are denoted FGFR2(beta)IIIb and FGFR2(beta)IIIc.
  • the FGFR2IIIb form of FGFR2 (also denoted K-sam-II) is a high affinity receptor for both FGF1 and KGF family members (FGF7, FGF10, and FGF22)
  • FGFR2IIIc also denoted K-sam-I
  • FGFR2IIIb is the only receptor for KGF family members (Ornitz et al., 1996, op. cit.) and is therefore also designated KGFR.
  • FGFRs and their isoforms are differentially expressed in various tissues.
  • FGFR2IIIb (and the Mb forms of FGFR1 and FGFR3) is expressed in epithelial tissues, while FGFRIIIc is expressed in mesenchymal tissues (Duan et al., J. Biol. Chem. 267:16076, 1992; Ornitz et al., 1996, op. cit.).
  • Certain of the FGF ligands of these receptors have an opposite pattern of expression.
  • KGF subfamily members including FGF7 (KGF), FGF 10, and FGF22, bind only to FGFRIIIb (Zhang et al., op.
  • FGFR2 plays a role in epithelial-mesynchymal interactions (Finch et al., Dev. Dyn. 203:223, 1995), so it is not surprising that knock-out of FGFR2IIIb in mice leads to severe embryonic defects and lethality (De Moerlooze et al., Development 127:483, 2000).
  • KGF (FGF7) and KGFR (FGFR2IIIb) are overexpressed in many pancreatic cancers (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and their coexpression correlates with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007).
  • Somatic mutations of the FGFR2 gene were found in 12% of a large panel of endometrial (uterine) carcinomas, and in several tested cases were required for tumor cell survival (Dutt et al., Proc. Natl. Acad. Sci. USA 105:8713, 2008).
  • FGFR2 mutation In two tumors the FGFR2 mutation was found to be the same S252W substitution associated with Apert syndrome. Amplification and overexpression of FGFR2 is associated with the undifferentiated, diffuse type of gastric cancer, which has a particularly poor prognosis, and inhibition of the FGFR2 activity by small molecule compounds potently inhibited proliferation of such cancer cells (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006).
  • an anti-FGFR2IIIb antibody wherein the heavy chain variable region comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein the antibody is afucosylated.
  • compositions comprising a plurality of anti-FGFR2IIIb antibodies are provided, wherein the heavy chain variable region of each anti-FGFR2IIIb antibody in the composition comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region of each anti-FGFR2IIIb antibody in the composition comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein at least 95% of the antibodies in the composition are afucosylated.
  • the composition may be a supernatant from an antibody-producing cell line. In some embodiments, the composition may be a buffered composition.
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 3.
  • 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.
  • a composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies is provided, wherein the antibodies compete for binding to FGFR2IIIb with an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5.
  • the antibodies are monoclonal antibodies. In some embodiments, the antibodies are chimeric antibodies. In some embodiments, the antibodies are humanized antibodies. In any of the embodiments described herein, the antibodies may comprise a ⁇ light chain constant region. In any of the embodiments described herein, the antibodies may comprise an IgG1 heavy chain constant region.
  • the antibodies have enhanced ADCC activity in vitro compared to fucosylated anti-FGFR2IIIb antibodies having the same amino acid sequence.
  • the afucosylated anti-FGFR2IIIb antibodies cause specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with fucosylated anti-FGFR2IIIb antibodies.
  • ADCC activity is determined using Ba/F3 cells expressing FGFR2IIIb as target cells and isolated human PBMCs as effector cells.
  • the antibodies have enhanced affinity for Fc gamma RIIIA compared to fucosylated anti-FGFR2IIIb antibodies having the same amino acid sequence.
  • the afucosylated anti-FGFR2IIIb antibodies bind 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 fucosylated anti-FGFR2IIIb antibodies.
  • affinity for Fc gamma RIIIA is determined using surface plasmon resonance.
  • Fc gamma RIIIA is selected from Fc gamma RIIIA(V158) and Fc gamma RIIIA(F158). In some embodiments, Fc gamma RIIIA is Fc gamma RIIIA(V158).
  • the antibodies may bind FGFR2IIIb but not FGFR2IIIc.
  • a composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies comprises at least 95% afucosylated antibodies.
  • a composition comprising a plurality of afucosylated anti-FGFR2IIIb antibodies may have undetectable fucosylation.
  • the presence of fucose may be determined by a method comprising high performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.
  • host cells are provided that comprise nucleic acid encoding an anti-FGFR2IIIb antibody described herein, wherein the host cell lacks a functional alpha-1,6-fucosyltransferase gene (FUT8) gene.
  • the host cell is a CHO cell.
  • a method comprises culturing a host cell under conditions suitable for expressing nucleic acid encoding the anti-FGFR2IIIb antibody, wherein the host cell lacks a functional alpha-1,6-fucosyltransferase gene (FUT8) gene.
  • a method for making afucosylated anti-FGFR2IIIb antibodies comprises culturing the host cell under conditions suitable for producing the afucosylated anti-FGFR2IIIb antibody.
  • the method further comprises recovering the anti-FGFR2IIIb antibody produced by the host cell.
  • less than 5% of the anti-FGFR2IIIb antibodies produced by the host cell comprise fucose. In some embodiments, at least 95% of the anti-FGFR2IIIb antibodies produced by the host cell lack fucose (i.e., are afucosylated). In some embodiments, fucose is undetectable in the anti-FGFR2IIIb antibodies produced by the host cell. In some embodiments, presence of fucose is detected by a method comprising HPLC, capillary electrophoresis, or MALDI-TOF mass spectrometry.
  • compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-FGFR2IIIb antibodies described herein and a pharmaceutically acceptable carrier.
  • a method comprises administering an effective amount of a pharmaceutical composition comprising afucosylated anti-FGFR2IIIb antibodies described herein and a pharmaceutically acceptable carrier.
  • the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.
  • the cancer is gastric cancer.
  • the cancer comprises an FGFR2 gene amplification.
  • FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10 centromere) ratio of >3.
  • the cancer overexpresses FGFR2IIIb.
  • a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc.
  • a cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression.
  • the expression levels are normalized to GUSB.
  • the cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • expression or overexpression of FGFR2IIIb is determined by IHC.
  • 1+, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, 2+ or 3+ staining in tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, the IHC staining is scored as described in Example 6.
  • a method of treating cancer further comprises administering at least one additional therapeutic agent selected from a platinum agent, paclitaxel, ABRAXANE®, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C, and doxorubicin hydrochloride.
  • the platinum agent is selected from cisplatin, oxaliplatin, and carboplatin.
  • a method of treating cancer further comprises administering paclitaxel.
  • a method of treating cancer further comprises administering cisplatin and/or 5-FU.
  • a pharmaceutical composition comprising afucosylated anti-FGFR2IIIb antibodies described herein and a pharmaceutically acceptable carrier are provided.
  • such use is for treating cancer in an individual with cancer.
  • the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer.
  • the cancer is gastric cancer.
  • the cancer comprises an FGFR2 gene amplification.
  • FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10 centromere) ratio of >3.
  • the cancer overexpresses FGFR2IIIb.
  • a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc.
  • a cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression.
  • the expression levels are normalized to GUSB.
  • the cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • expression or overexpression of FGFR2IIIb is determined by IHC.
  • 1+, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, 2+ or 3+ staining in tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, the IHC staining is scored as described in Example 6.
  • compositions for treating cancer comprising afucosylated anti-FGFR2IIIb antibodies described herein and a pharmaceutically acceptable carrier.
  • the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer.
  • the cancer is gastric cancer.
  • the cancer comprises an FGFR2 gene amplification.
  • FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10 centromere) ratio of >3.
  • the cancer overexpresses FGFR2IIIb.
  • a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc.
  • a cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression.
  • the expression levels are normalized to GUSB.
  • the cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • expression or overexpression of FGFR2IIIb is determined by IHC.
  • 1+, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, 2+ or 3+ staining in tumor cells by IHC indicates FGFR2IIIb overexpression. In some embodiments, the IHC staining is scored as described in Example 6.
  • FIG. 1A to 1C shows ADCC activity of afucosylated ⁇ FGFR2bA and fucosylated ⁇ FGFR2bF against FGFR2IIIb-expressing Ba/F3 cells, as discussed in Example 3.
  • ⁇ FGFR2bF/FGFR2b indicates that fucosylated ⁇ FGFR2bF antibody was tested against FGFR2IIIb-expressing Ba/F3 target cells.
  • FIG. 2A to 2D shows efficacy of afucosylated ⁇ FGFR2bA and fucosylated ⁇ FGFR2bF in an OCUM-2M gastric cancer xenograft model, at (A and B) 10 mg/kg and (C and D) 3 mg/kg, as discussed in Example 4.
  • FIGS. 3A and 3B shows dose-dependent efficacy of afucosylated ⁇ FGFR2bA in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.
  • FIGS. 4A and 4B shows efficacy of combination therapy with afucosylated ⁇ FGFR2bA and paclitaxel in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.
  • FIGS. 5A and 5B shows efficacy of combination therapy with afucosylated ⁇ FGFR2bA and 5-FU/cisplatin in an OCUM-2M gastric cancer xenograft model, as discussed in Example 4.
  • FIGS. 6A and 6B show efficacy of afucosylated ⁇ FGFR2bA in a MFM-223 breast cancer xenograft model, as discussed in Example 4.
  • FIG. 7 shows the glycan profile of ⁇ FGFR2b antibody produced in (A) Potelligent® CHOK1SV cells and (B) CHOK1SV cells, as described in Example 1.
  • FIG. 8 shows schematic diagrams of N-linked glycans typically found in antibodies.
  • FIG. 9 shows ADCC of Ba/F3 FGF2b cells with increasing concentrations of ⁇ FGFR2bA or ⁇ FGFR2bF. Assays were performed with normal human PBMC at an E:T ratio of 25:1, as described in Example 5. Data are plotted as LDH release.
  • FIG. 10 shows ADCC of OCUM-2M cells with increasing concentrations of ⁇ FGFR2bA or ⁇ FGFR2bF. Assays were performed with normal human PBMC at an E:T ratio of 25:1. As described in Example 5. Data are plotted as percent specific lysis.
  • FIG. 11A to 11F shows detection of FGFR2IIIb in tumor tissue samples using immunohistochemistry, as described in Example 6.
  • Afucosylated antibodies that bind FGFR2IIIb are provided.
  • afucosylated antibody heavy chains and light chains that are capable of forming antibodies that bind FGFR2IIIb are also provided.
  • afucosylated antibodies, heavy chains, and light chains comprising one or more particular hypervariable regions (HVRs) are provided.
  • afucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity relative to fucosylated anti-FGFR2IIIb antibodies.
  • afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA relative to fucosylated anti-FGFR2IIIb antibodies.
  • afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA(V158) relative to fucosylated anti-FGFR2IIIb antibodies. In some embodiments, afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA(F158) relative to fucosylated anti-FGFR2IIIb antibodies. In some embodiments, afucosylated anti-FGFR2IIIb antibodies do not bind to FGFR2IIIc.
  • Polynucleotides encoding antibodies that bind FGFR2IIIb are provided. Polynucleotides encoding antibody heavy chains or lights chains are also provided. Host cells that express afucosylated anti-FGFR2IIIb antibodies are provided. Methods of treatment using afucosylated antibodies to FGFR2IIIb are provided. Such methods include, but are not limited to, methods of treating cancer, such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.
  • cancer such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.
  • nucleic acid molecule refers to a polymer of nucleotides.
  • polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • FGFR2IIIb or “FGFR2b” are used interchangeably to refer to the fibroblast growth factor receptor 2 IIIb splice form.
  • An exemplary human FGFR2IIIb is shown in GenBank Accession No. NP — 075259.4, dated Jul. 7, 2013.
  • a nonlimiting exemplary mature human FGFR2IIIb amino acid sequence is shown in SEQ ID NO: 1.
  • FGFR2IIIc or “FGFR2c” are used interchangeably to refer to the fibroblast growth factor receptor 2 IIIc splice form.
  • An exemplary human FGFR2IIIc is shown in GenBank Accession No. NP — 000132.3, dated Jul. 7, 2013.
  • a nonlimiting exemplary mature FGFR2IIIc amino acid sequence is shown in SEQ ID NO: 12.
  • epitope refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein containing antibody binding regions binds.
  • a target molecule e.g., an antigen, such as a protein, nucleic acid, carbohydrate or lipid
  • an antigen-binding molecule e.g., an antibody, antibody fragment, or scaffold protein containing antibody binding regions binds.
  • Epitopes often consist of a chemically active surface grouping of molecules such as amino acids, polypeptides or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed both from contiguous or juxtaposed noncontiguous residues (e.g., amino acids, nucleotides, sugars, lipid moiety) of the target molecule.
  • Epitopes formed from contiguous residues typically are retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding typically are lost on treatment with denaturing solvents.
  • An epitope may include but not limited to at least 3, at least 5 or 8-10 residues (e.g., amino acids or nucleotides). In some examples an epitope is less than 20 residues (e.g., amino acids or nucleotides) in length, less than 15 residues or less than 12 residues.
  • Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • a “nonlinear epitope” or “conformational epitope” comprises noncontiguous polypeptides, amino acids and/or sugars within the antigenic protein to which an antibody specific to the epitope binds.
  • a “linear epitope” comprises contiguous polypeptides, amino acids and/or sugars within the antigenic protein to which an antibody specific to the epitope binds.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′) 2 .
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.
  • heavy chain variable region refers to a region comprising heavy chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3.
  • a heavy chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4.
  • heavy chain constant region refers to a region comprising at least three heavy chain constant domains, C H 1, C H 2, and C H 3.
  • Nonlimiting exemplary heavy chain constant regions include ⁇ , ⁇ , and ⁇ .
  • Nonlimiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody comprising ⁇ constant region is an IgM antibody
  • an antibody comprising an ⁇ constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising a ⁇ 1 constant region), IgG2 (comprising a ⁇ 2 constant region), IgG3 (comprising a ⁇ 3 constant region), and IgG4 (comprising a ⁇ 4 constant region) antibodies;
  • IgA antibodies include, but are not limited to, IgA1 (comprising an ⁇ 1 constant region) and IgA2 (comprising an ⁇ 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.
  • heavy chain refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
  • a heavy chain comprises at least a portion of a heavy chain constant region.
  • full-length heavy chain refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.
  • light chain variable region refers to a region comprising light chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3.
  • a light chain variable region also comprises an FR1 and/or an FR4.
  • light chain constant region refers to a region comprising a light chain constant domain, C L .
  • Nonlimiting exemplary light chain constant regions include ⁇ and ⁇ .
  • light chain refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence.
  • a light chain comprises at least a portion of a light chain constant region.
  • full-length light chain refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”).
  • native four-chain antibodies comprise six HVRs; three in the V H (H1, H2, H3), and three in the V L (L1, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).
  • Exemplary CDRs CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3.
  • hypervariable regions HVRs
  • CDRs complementarity determining regions
  • an “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (V L ) framework or a heavy chain variable domain (V H ) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 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.
  • the V L acceptor human framework is identical in sequence to the V L human immunoglobulin framework sequence or human consensus framework sequence.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K d ). Affinity can be measured by common methods known in the art, including those described herein.
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) and/or complementarity determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • CDRs complementarity determining regions
  • a “chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.).
  • a chimeric antibody comprises at least one mouse variable region and at least one human constant region.
  • a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.
  • a “humanized antibody” refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region.
  • a humanized antibody comprises at least one human constant region or fragment thereof.
  • a humanized antibody is an Fab, an scFv, a (Fab′) 2 , etc.
  • HVR-grafted antibody refers to a humanized antibody in which one or more hypervariable regions (HVRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • effector functions include Fc receptor binding; Clq binding; CDC; ADCC; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays.
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • an Fc ⁇ R is a native human FcR.
  • an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of those receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Fc receptor or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, 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); WO 2004/92219 (Hinton et al.).
  • “Effector functions” refer to biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least Fc ⁇ RIII and perform ADCC effector function(s). Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes e.g., monocytes
  • macrophages cytotoxic T cells
  • neutrophils neutrophils.
  • the effector cells may be isolated from a native source, e.g., from blood.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. NK cells, neutrophils, and macrophages
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed.
  • Useful effector cells for such assays include PBMC and NK cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci . ( USA ) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, e.g., in U.S. Pat. No. 7,923,538, and U.S. Pat. No. 7,994,290.
  • an antibody having an “enhanced ADCC activity” refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect, and when the amounts of such antibody and parent antibody used in the assay are essentially the same.
  • the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated.
  • ADCC activity will be determined using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated.
  • an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (V 158). In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (F158).
  • An antibody with “altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity and/or ADCC activity compared to a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect.
  • An antibody that “displays increased binding” to an FcR binds at least one FcR with better affinity than the parent antibody.
  • An antibody that “displays decreased binding” to an FcR binds at least one FcR with lower affinity than a parent antibody.
  • Such antibodies that display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0-20% binding to the FcR compared to a native sequence IgG Fc region.
  • Enhanced affinity for Fc gamma RIIIA refers to an antibody that has greater affinity for Fc gamma RIIIA (also referred to, in some instances, as CD16a) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect.
  • the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. Any suitable method for determining affinity for Fc gamma RIIIA may be used.
  • affinity for Fc gamma RIIIA is determined by a method described herein.
  • an antibody with enhanced affinity for Fc gamma RIIIA has enhanced ADCC activity.
  • an antibody with enhanced affinity for Fc gamma RIIIA has enhanced affinity for Fc gamma RIIIA(V158). In some embodiments, an antibody with enhanced affinity for Fc gamma RIIIA has enhanced affinity for Fc gamma RIIIA(F158).
  • “Afucosylated” antibody or an antibody “lacking fucose” refers to an IgG1 or IgG3 isotype antibody that lacks fucose in its constant region glycosylation. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylated biantennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues. In some embodiments, an afucosylated antibody lacks fucose at Asn297. These structures are designated as G0, G1 ( ⁇ 1,6 or ⁇ 1,3) or G2 glycan residues, depending on the amount of terminal Gal residues. See, e.g., Raju, T.
  • CHO type glycosylation of antibody Fc is described, e.g., in Routier, F. H., Glycoconjugate J. 14: 201-207 (1997).
  • at least 85% of a batch of antibodies recombinantly expressed in non glycomodified CHO host cells are fucosylated at Asn297.
  • the antibodies are considered to be afucosylated if ⁇ 5% of the antibodies in the composition comprise fucose at Asn297.
  • Methods of measuring fucose include any methods known in the art, including the methods described herein.
  • fucose is detected by the method described in Example 1. In some embodiments, fucose is undetectable in a composition comprising a plurality of afucosylated antibodies. In some embodiments, an afucosylated antibody has enhanced ADCC activity. In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RIIIA In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RIIIA(V158). In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RIIIA(F158).
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen.
  • a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
  • Antibodies with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described, e.g., in U.S. Pat. No. 6,194,551 B1, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290 and WO 1999/51642. See also, e.g., Idusogie et al., J. Immunol. 164: 4178-4184 (2000).
  • substantially similar denotes a sufficiently high degree of similarity between two or more numeric values such that one of skill in the art would consider the difference between the two or more values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said value.
  • the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25%, or 50%.
  • the phrase “substantially reduced,” or “substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values.
  • the two substantially different numeric values differ by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • leader sequence and “signal sequence” are used interchangeably to refer to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell.
  • a leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein.
  • Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached.
  • a “native sequence” polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide found in nature.
  • a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any mammal.
  • Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term “native sequence” polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.
  • a polypeptide “variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide.
  • a variant will have at least about 80% amino acid sequence identity.
  • a variant will have at least about 90% amino acid sequence identity.
  • a variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.
  • Percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid.
  • Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions”. More substantial changes are provided in Table 1 under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • vector is used to describe a polynucleotide that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell.
  • a vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that may be used in colorimetric assays, e.g., ⁇ -galactosidase).
  • expression vector refers to a vector that is used to express a polypeptide of interest in a host cell.
  • a “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells may be prokaryotic cells or eukaryotic cells.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced.
  • a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
  • a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.
  • mammals including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder.
  • a “disease” or “disorder” refers to a condition where treatment is needed.
  • cancer refers to a malignant proliferative disorder associated with uncontrolled cell proliferation, unrestrained cell growth, and decreased cell death via apoptosis.
  • Nonlimiting exemplary cancers include gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and esophageal cancer.
  • a cancer comprises an FGFR2 gene amplification.
  • FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10 centromere) ratio of >3.
  • a cancer comprising an FGFR2 gene amplification overexpresses FGFR2IIIb.
  • a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc.
  • a cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression.
  • the expression levels are normalized to GUSB.
  • a cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • a gastric cancer comprises an FGFR2 gene amplification.
  • a gastric cancer comprising an FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, a gastric cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, a gastric cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, the expression levels are normalized to GUSB. In some embodiments, a gastric cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification. In some embodiments, overexpression is mRNA overexpression. In some embodiments, overexpression is protein overexpression.
  • tumor is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth.
  • a tumor may be benign, pre-malignant, or malignant; malignant tumor cells are cancerous.
  • Tumor cells may be solid tumor cells or leukemic tumor cells.
  • tumor growth is used herein to refer to proliferation or growth by a cell or cells that comprise a tumor that leads to a corresponding increase in the size of the tumor.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • Treatment covers any administration or application of a therapeutic for disease in a mammal, including a human.
  • beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
  • treatment is a reduction of pathological consequence of a proliferative disease. The methods of the invention contemplate any one or more of these aspects of treatment.
  • treating includes any or all of: inhibiting growth of tumor cells or cancer cells, inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
  • inhibitors refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • a “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
  • a reference may be obtained from a healthy and/or non-diseased sample.
  • a reference may be obtained from an untreated sample.
  • a reference is obtained from a non-diseased on non-treated sample of a subject individual.
  • a reference is obtained from one or more healthy individuals who are not the subject or patient.
  • “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
  • an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount may be delivered in one or more administrations.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • Such formulations may be sterile.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • a “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.
  • the term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent.
  • the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.
  • administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
  • administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.
  • conjunction with refers to administration of one treatment modality in addition to another treatment modality.
  • in conjunction with refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein.
  • the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
  • the invention provides an afucosylated antibody directed against FGFR2IIIb.
  • Afucosylated anti-FGFR2IIIb antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, and antibodies comprising the heavy chain and/or light chain HVRs (e.g. CDRs) discussed herein.
  • the invention provides isolated afucosylated antibodies that bind to FGFR2IIIb.
  • an afucosylated anti-FGFR2IIIb antibody modulates FGFR2IIIb activity.
  • an afucosylated anti-FGFR2IIIb antibody has enhanced ADCC activity.
  • an afucosylated anti-FGFR2IIIb antibody has enhanced affinity for Fc gamma RIIIA In some embodiments, an afucosylated anti-FGFR2IIIb antibody has enhanced affinity for Fc gamma RIIIA(V158). In some embodiments, an afucosylated anti-FGFR2IIIb antibody has enhanced affinity for Fc gamma RIIIA(F158).
  • the anti-FGFR2IIIb antibody designated “ ⁇ FGFR2b” described herein in the Examples and the sequence listing is intended to have the same amino acid sequence as antibody HuGAL-FR21 in U.S. Pat. No. 8,101,723 B2, issued Jan. 24, 2012.
  • U.S. Pat. No. 8,101,723 B2 is specifically incorporated herein by reference for any purpose, and in particular, FIGS. 13 and 14 of U.S. Pat. No. 8,101,723 B2, which show the amino acid sequences of the variable regions and full-length mature antibody chains of HuGAL-FR21, are incorporated by reference herein for any purpose.
  • the HVR sequences of antibody HuGAL-FR21 which are underlined in FIG. 13 of U.S. Pat. No. 8,101,723 B2, are specifically incorporated by reference herein for any purpose.
  • the invention provides an afucosylated anti-FGFR2IIIb antibody comprising at least one, two, three, four, five, or six HVRs (e.g., CDRs) selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6
  • HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7
  • HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8
  • HVR-L1 comprising the amino acid
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an afucosylated anti-FGFR2IIIb antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region.
  • an afucosylated anti-FGFR2IIIb antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 3.
  • the invention provides an afucosylated anti-FGFR2IIIb antibody comprising six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the afucosylated anti-FGFR2IIIb antibody comprises the six HVRs as described above and binds to FGFR2IIIb. In some embodiments, the afucosylated anti-FGFR2IIIb antibody comprises the six HVRs as described above, binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the afucosylated anti-FGFRIIIb antibody does not bind to FGFR2IIIc.
  • the invention provides an afucosylated anti-FGFR2IIIb antibody that competes with an anti-FGFR2IIIb antibody comprising six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the invention provides an afucosylated antibody comprising at least one, at least two, or all three V H HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.
  • the invention provides an afucosylated antibody comprising at least one, at least two, or all three V L HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an afucosylated antibody of the invention comprises (a) a V H domain comprising at least one, at least two, or all three V H HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 8; and (b) a V L domain comprising at least one, at least two, or all three V L HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable domain (V H ) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.
  • V H heavy chain variable domain
  • a V H sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 4.
  • substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody comprises the V H sequence in SEQ ID NO: 5, including post-translational modifications of that sequence.
  • the V H comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.
  • an afucosylated anti-FGFR2IIIb antibody comprising a light chain variable domain (V L ) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.
  • V L light chain variable domain
  • a V L sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 5.
  • the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody comprises the V L sequence in SEQ ID NO: 4, including post-translational modifications of that sequence.
  • the V L comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain variable domain (V H ) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain (V L ) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.
  • V H heavy chain variable domain
  • V L light chain variable domain
  • a V H sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence
  • a V L sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 4.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 5.
  • substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody comprises the V H sequence in SEQ ID NO: 4 and the V L sequence of SEQ ID NO: 5, including post-translational modifications of one or both sequence.
  • the V H comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the V L comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an afucosylated anti-FGFR2IIIb antibody comprising a V H as in any of the embodiments provided above, and a V L as in any of the embodiments provided above.
  • the antibody comprises the V H and V L sequences in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, including post-translational modifications of those sequences.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an afucosylated anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2.
  • substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody heavy chain comprises the V H sequence in SEQ ID NO: 2, including post-translational modifications of that sequence.
  • the heavy chain comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.
  • an afucosylated anti-FGFR2IIIb antibody comprising a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3.
  • the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody light chain comprises the V L sequence in SEQ ID NO: 3, including post-translational modifications of that sequence.
  • the light chain comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an afucosylated anti-FGFR2IIIb antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb. In certain embodiments, such anti-FGFR2IIIb antibody retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2IIIb antibody comprising that sequence retains the ability to bind to FGFR2IIIb.
  • an anti-FGFR2IIIb antibody comprising that sequence retains the ability to selectively bind to FGFR2IIIb without binding to FGFR2IIIc.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3.
  • substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
  • the afucosylated anti-FGFR2IIIb antibody heavy chain comprises the V H sequence in SEQ ID NO: 2, including post-translational modifications of that sequence and the afucosylated anti-FGFR2IIIb antibody light chain comprises the V L sequence in SEQ ID NO: 3, including post-translational modifications of that sequence.
  • the heavy chain comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.
  • an antibody such as an afucosylated antibody, provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • Nonlimiting exemplary afucsoylated chimeric antibodies include chimeric antibodies comprising heavy chain HVR1, HVR2, and HVR3, and/or light chain HVR1, HVR2, and HVR3 sequences described herein.
  • the afucosylated chimeric anti-FGFR2IIIb antibody comprises the variable regions described above and binds to FGFR2IIIb.
  • the afucosylated chimeric anti-FGFR2IIIb antibody comprises the variable regions described above, binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments, the afucosylated chimeric anti-FGFRIIIb antibody does not bind to FGFR2IIIc.
  • an afucosylated chimeric anti-FGFR2IIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical SEQ ID NO: 4, wherein the antibody binds FGFR2IIIb.
  • an afucosylated chimeric anti-FGFR2IIIb antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, wherein the antibody binds FGFR2IIIb.
  • an afucosylated chimeric anti-FGFR2IIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5; wherein the antibody binds FGFR2IIIb.
  • Exemplary afucosylated chimeric anti-FGFR2IIIb antibodies also include chimeric antibodies that compete for binding to FGFR2IIIb with an antibody or fragment thereof described herein.
  • a chimeric anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.
  • the antibody competes for binding to FGFR2IIIb, but does not bind FGFR2IIIc.
  • a chimeric antibody described herein comprises one or more human constant regions.
  • the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from ⁇ and ⁇ .
  • a chimeric antibody described herein comprises a human IgG constant region.
  • a chimeric antibody described herein comprises a human IgG4 heavy chain constant region.
  • a chimeric antibody described herein comprises a human IgG4 constant region and a human ⁇ light chain.
  • effector function may depend on the particular method of treatment intended for an antibody.
  • a chimeric anti-FGFR2IIIb antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected.
  • a chimeric anti-FGFR2IIIb antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.
  • afucosylated humanized antibodies that bind FGFR2IIIb are provided.
  • Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic.
  • HAMA human anti-mouse antibody
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, 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 (see, e.g., Almagro and Fransson, (2008) Front. Biosci.
  • Nonlimiting exemplary humanized antibodies include ⁇ FGFR2b, described herein.
  • Nonlimiting exemplary afucosylated humanized antibodies include ⁇ FGFR2bA, described herein, which has the same amino acid sequences as ⁇ FGFR2b comprising fucose (also referred to as ⁇ FGFR2bF).
  • Nonlimiting exemplary afucosylated humanized antibodies also include antibodies comprising a heavy chain variable region of ⁇ FGFR2b and/or a light chain variable region of ⁇ FGFR2b.
  • Nonlimiting exemplary afucosylated humanized antibodies include antibodies comprising a heavy chain variable region of SEQ ID NO: 4 and/or a light chain variable region of SEQ ID NO: 5.
  • Exemplary humanized antibodies also include, but are not limited to, humanized antibodies comprising heavy chain HVR1, HVR2, and HVR3, and/or light chain HVR1, HVR2, and HVR3 of ⁇ FGFR2b.
  • the humanized anti-FGFR2IIIb antibody comprises the HVRs described above (i.e., (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11) and binds to FGFR2IIIb.
  • the humanized anti-FGFR2IIIb antibody comprises the HVRs described above, binds to FGFR2IIIb and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)).
  • Fc gamma RIIIA such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)
  • the afucosylated humanized anti-FGFRIIIb antibody does not bind to FGFR2IIIc.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises heavy chain HVR1, HVR2, and HVR3 and/or a light chain HVR1, HVR2, and HVR3 of ⁇ FGFR2b.
  • Nonlimiting exemplary afucosylated humanized anti-FGFR2IIIb antibodies include antibodies comprising sets of heavy chain HVR1, HVR2, and HVR3 set forth in SEQ ID NOs: 6, 7, and 8.
  • Nonlimiting exemplary afucosylated humanized anti-FGFR2IIIb antibodies also include antibodies comprising sets of light chain HVR1, HVR2, and HVR3 set forth in SEQ ID NOs: 9, 10, and 11.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, and wherein the antibody binds FGFR2IIIb.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, wherein the antibody binds FGFR2IIIb.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5; wherein the antibody binds FGFR2IIIb.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises at least one of the HVRs discussed herein. That is, in some embodiments, an afucosylated humanized anti-FGFR2IIIb antibody comprises at least one HVR selected from a heavy chain HVR1 discussed herein, a heavy chain HVR2 discussed herein, a heavy chain HVR3 discussed herein, a light chain HVR1 discussed herein, a light chain HVR2 discussed herein, and a light chain HVR3 discussed herein.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises at least one mutated HVR based on a HVR discussed herein, wherein the mutated HVR comprises 1, 2, 3, or 4 amino acid substitutions relative to the HVR discussed herein.
  • one or more of the amino acid substitutions are conservative amino acid substitutions.
  • One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular HVR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the antibody comprising the mutated HVR.
  • Exemplary afucosylated humanized anti-FGFR2IIIb antibodies also include antibodies that compete for binding to FGFR2IIIb with an antibody or fragment thereof described herein.
  • a humanized anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with ⁇ FGFR2b.
  • an afucosylated humanized anti-FGFR2IIIb antibody is provided that competes for binding to FGFR2IIIb with ⁇ FGFR2b and has at least one activity selected from enhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)).
  • the afucosylated humanized anti-FGFRIIIb antibody does not bind to FGFR2IIIc.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprises one or more human constant regions.
  • the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from ⁇ and ⁇ .
  • an afucosylated humanized antibody described herein comprises a human IgG constant region.
  • an afucosylated humanized anti-FGFR2IIIb antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected.
  • an afucosylated humanized antibody described herein comprises a human IgG1 constant region.
  • an afucosylated humanized antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated.
  • an afucosylated human antibody described herein comprises a human IgG1 constant region and a human ⁇ light chain.
  • an afucosylated humanized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 3.
  • an afucosylated humanized antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 2, and any post-translational modifications, and a light chain consisting the amino acid sequence of SEQ ID NO: 3, and any post-translational modifications.
  • an afucosylated antibody described herein comprises one or more human constant regions.
  • the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from ⁇ and ⁇ .
  • an afucosylated antibody described herein comprises a human IgG constant region.
  • an afucosylated anti-FGFR2IIIb antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected.
  • an afucosylated antibody described herein comprises a human IgG1 constant region.
  • an afucosylated antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated.
  • an afucosylated antibody described herein comprises a human IgG 1 constant region and a human ⁇ light chain.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference.
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • an antibody of the invention comprises a variant Fc region has at least one amino acid substitution compared to the Fc region of a wild-type IgG or a wild-type antibody.
  • the variant Fc region has two or more amino acid substitutions in the Fc region of the wild-type antibody.
  • the variant Fc region has three or more amino acid substitutions in the Fc region of the wild-type antibody.
  • the variant Fc region has at least one, two or three or more Fc region amino acid substitutions described herein.
  • the variant Fc region herein will possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent antibody.
  • the variant Fc region herein will possess at least about 90% homology with a native sequence Fc region and/or with an Fc region of a parent antibody. In certain embodiments, the variant Fc region herein will possess at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibodies with certain improved properties.
  • antibodies having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region (i.e., afucosylated antibodies).
  • the amount of fucose in a composition comprising a plurality of such antibodies may be from 0% to about 5%.
  • a composition comprising a plurality of such antibodies comprises at least 95% afucosylated antibodies.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures).
  • Nonlimiting exemplary methods of detecting fucose in an antibody include MALDI-TOF mass spectrometry (see, e.g., WO 2008/077546), HPLC measurement of released fluorescently labeled oligosaccharides (see, e.g., Schneider et al., “N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection,” Agilent Technologies, Inc. (2012); Lines, J. Pharm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
  • Asn297 is found in the sequence QY N ST, and is in bold and underlined in the Table of Sequences shown below, SEQ ID NO: 2. Fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos.
  • Antibodies are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibodies may have improved CDC function. Such antibodies are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • Antibodies are also provided with amino-terminal leader extensions.
  • one or more amino acid residues of the amino-terminal leader sequence are present at the amino-terminus of any one or more heavy or light chains of an antibody.
  • An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of an antibody.
  • human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice, in humans, or in non-human primates to which the polypeptides with a variant Fc region are administered. See also, e.g., Petkova et al. International Immunology 18(12):1759-1769 (2006).
  • an afucosylated antibody mediates ADCC in the presence of human effector cells more effectively than a parent antibody that comprises fucose
  • ADCC activity may be determined using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated.
  • the “K D ,” “K d ,” “Kd” or “Kd value” of the antibody is measured by using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at ⁇ 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CMS, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions.
  • CMS carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml ( ⁇ 0.2 ⁇ M) before injection at a flow rate of 5 ⁇ L/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, serial dilutions of polypeptide, e.g., full length antibody, are injected in PBS with 0.05% TWEEN-20® surfactant (PBST) at 25° C. at a flow rate of approximately 25 ⁇ L/min.
  • PBST TWEEN-20® surfactant
  • association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio k off /k on . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).
  • an “on-rate,” “rate of association,” “association rate,” or “k on ” of the antibody can also be determined as described above using a BIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc., Piscataway, N.J.).
  • the difference between said two values is substantially the same, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.
  • the difference between said two values is substantially different, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.
  • a leader sequence from a heterologous protein may be desirable.
  • employing heterologous leader sequences may be advantageous in that a resulting mature polypeptide may remain unaltered as the leader sequence is removed in the ER during the secretion process.
  • the addition of a heterologous leader sequence may be required to express and secrete some proteins.
  • leader sequence sequences are described, e.g., in the online Leader sequence Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.
  • afucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity in vitro and/or in vivo. In some embodiments, afucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity in vitro. In some embodiments, ADCC activity in vitro is determined by a method described herein, e.g., in Example 3. Briefly, FGFR2IIIb-expressing cells are contacted with freshly isolated human PBMCs at a ratio of 25:1 effector (PBMCs) to target cells, in the presence of fucosylated antibody or afucosylated antibody. In some embodiments, Ba/F3 cells that express FGFR2IIIb are used as target cells.
  • PBMCs effector
  • cytotoxicity is determined by quantifying LDH release using CytoTox Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.).
  • maximal lysis is determined using 5% Triton X-100 and spontaneous release is determined in the absence of antibody.
  • an afucosylated anti-FGFR2IIIb antibody having enhanced ADCC activity results in specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with the same amount of a fucosylated antibody, at at least one concentration of antibody tested.
  • an afucosylated anti-FGFR2IIIb antibody having enhanced ADCC activity results in specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with a fucosylated antibody, where each antibody is at a concentration of between 0.01 and 1 ⁇ g/ml and the target cells are Ba/F3 cells expressing FGFR2IIIb.
  • the antibodies are tested at a concentration of 0.01 ⁇ g/ml, 0.1 ⁇ g/ml, or 1 ⁇ g/ml.
  • afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA In some embodiments, afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA(V158). In some embodiments, afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIA(F158).
  • antibody affinity for Fc gamma RIIIA is determined using surface plasmon resonance, e.g., as described herein in Example 2, and/or as follows, which is described with reference to Fc gamma RIIIA(V158), but which is also suitable for determining affinity for Fc gamma RIIIA(F158).
  • fucosylated or afucosylated anti-FGFR2IIIb antibody is captured on a protein A-coated dextran chip.
  • Fc gamma RIIIA (V158) (available from, e.g., R&D Systems) is injected at various concentrations.
  • the association constant, dissociation constant, and affinity of Fc gamma RIIIA (V158) for fucosylated and afucosylated anti-FGFR2IIIb antibody may be determined, e.g., using software provided with the surface plasmon resonance system (for example, Biacore T200 Evaluation Software 1:1 binding model).
  • an afucosylated anti-FGFR2IIIb antibody with enhanced affinity for Fc gamma RIIIA 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 a fucosylated anti-FGFR2IIIb antibody.
  • an afucosylated anti-FGFR2IIIb antibody binds to Fc gamma RIIIA (V158) with an affinity (K D ) of 9.2 nM and a fucosylated anti-FGFR2IIIb antibody binds to Fc gamma RIIIA (V158) with an affinity (K D ) of 207 nM
  • nucleic acid molecules comprising polynucleotides that encode one or more chains of anti-FGFR2IIIb antibodies are provided.
  • a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti-FGFR2IIIb antibody.
  • a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an anti-FGFR2IIIb antibody.
  • a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
  • the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides.
  • a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
  • a polynucleotide encoding a heavy chain or light chain of an anti-FGFR2IIIb antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain.
  • the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
  • Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
  • a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
  • Vectors comprising polynucleotides that encode anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains are provided. Vectors comprising polynucleotides that encode anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
  • a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain.
  • the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
  • a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain.
  • the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts).
  • a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells.
  • a mass ratio of between 1:1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Frog. 20:880-889 (2004).
  • anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
  • exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells.
  • anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the anti-FGFR2IIIb heavy chains and/or anti-FGFR2IIIb light chains.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc.
  • Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001).
  • Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
  • afucosylated anti-FGFR2IIIb antibodies are produced in cells capable of producing afucosylated antibodies, such as Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as cell lines lacking a functional alpha-1,6-fucosyltransferase gene, FUT8, e.g., knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.
  • afucosylated anti-FGFR2IIIb antibodies are produced in CHO cells lacking a functional FUT8 gene. In some embodiments, afucosylated anti-FGFR2IIIb antibodies are produced in Potelligent® CHOK1SV cells (BioWa/Lonza, Allendale, N.J.).
  • Anti-FGFR2IIIb antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the FGFR2IIIb ECD and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an anti-FGFR2IIIb antibody. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.
  • an anti-FGFR2IIIb antibody is produced in a cell-free system.
  • a cell-free system Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).
  • Antibodies of the invention, and compositions comprising antibodies of the invention are provided for use in methods of treatment for humans or animals.
  • Methods of treating disease comprising administering afucosylated anti-FGFR2IIIb antibodies are also provided.
  • Nonlimiting exemplary diseases that can be treated with afucosylated anti-FGFR2IIIb antibodies include, but are not limited to cancer.
  • methods of treating cancer are provided, comprising administering an afucosylated anti-FGFR2IIIb antibody.
  • the cancer is selected from gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer.
  • methods of treating gastric cancer are provided, comprising administering an afucosylated anti-FGFR2IIIb antibody.
  • a cancer comprises an FGFR2 gene amplification. In some embodiments, a cancer comprising an FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, a cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression. In some embodiments, the expression levels are normalized to GUSB.
  • a cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • a gastric cancer comprises an FGFR2 gene amplification.
  • a gastric cancer comprising an FGFR2 gene amplification overexpresses FGFR2IIIb.
  • a gastric cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc.
  • a gastric cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalized level that is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalized level of FGFR2IIIc expression.
  • the expression levels are normalized to GUSB.
  • a gastric cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.
  • overexpression is mRNA overexpression.
  • overexpression is protein overexpression.
  • FGFR2IIIb gene amplification may be determined by any suitable method in the art, including but not limited to, in situ hybridization (ISH).
  • FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10 centromere) ratio of >3.
  • FGFR2IIIb mRNA overexpression may be determined by any suitable method in the art, including but not limited to, methods comprising quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • the term “FGFR2IIIb mRNA overexpression” means elevated levels of FGFR2IIIb mRNA, regardless of the cause of such elevated levels (i.e., whether the elevated levels are a result of increased transcription and/or decreased degradation of mRNA, other mechanism, or a combination of mechanisms).
  • FGFR2IIIb protein overexpression may be determined by any suitable method in the art, including but not limited to, antibody-based methods such as immunohistochemistry (IHC).
  • IHC staining is scored according to methods in the art.
  • the term “FGFR2IIIb protein overexpression” means elevated levels of FGFR2IIIb protein, regardless of the cause of such elevated levels (i.e., whether the elevated levels are a result of increased translation and/or decreased degradation of protein, other mechanism, or a combination of mechanisms).
  • 1+, 2+, or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression.
  • 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression.
  • the IHC staining is scored as described in Example 6.
  • compositions comprising afucosylated anti-FGFR2IIIb antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000)).
  • Various pharmaceutically acceptable carriers which include vehicles, adjuvants, and diluents, are available.
  • Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • compositions comprising afucosylated anti-FGFR2IIIb antibodies may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers.
  • a non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid polymer.
  • a non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1 125 584 A1.
  • afucosylated anti-FGFR2IIIb antibodies may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation.
  • the subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
  • the appropriate formulation and route of administration may be selected according to the intended application.
  • compositions are administered in an amount effective for treatment or prophylaxis of cancer, such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer.
  • the therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.
  • afucosylated anti-FGFR2IIIb antibodies may be administered in an amount in the range of about 10 ⁇ g/kg body weight to about 100 mg/kg body weight per dose.
  • afucosylated anti-FGFR2IIIb antibodies may be administered in an amount in the range of about 50 ⁇ g/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, anti-FGFR2IIIb antibodies may be administered in an amount in the range of about 100 ⁇ g/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, afucosylated anti-FGFR2IIIb antibodies may be administered in an amount in the range of about 100 ⁇ g/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, afucosylated anti-FGFR2IIIb antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the afucosylated anti-FGFR2IIIb antibody compositions may be administered as needed to subjects. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.
  • an effective dose of an afucosylated anti-FGFR2IIIb antibody is administered to a subject one or more times.
  • an effective dose of an afucosylated anti-FGFR2IIIb antibody is administered to the subject once a month, more than once a month, such as, for example, every two months or every three months.
  • an effective dose of an afucosylated anti-FGFR2IIIb antibody is administered less than once a month, such as, for example, every two weeks or every week.
  • An effective dose of an afucosylated anti-FGFR2IIIb antibody is administered to the subject at least once.
  • the effective dose of an afucosylated anti-FGFR2IIIb antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.
  • Afucosylated anti-FGFR2IIIb antibodies may be administered alone or with other modes of treatment. They may be provided before, substantially contemporaneous with, or after other modes of treatment, for example, surgery, chemotherapy, or radiation therapy. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is administered in conjunction with another anti-cancer agent.
  • Nonlimiting exemplary anti-cancer agents that may be administered with an afucosylated anti-FGFR2IIIb antibody include platinum agents (such as cisplatin, oxaliplatin, and carboplatin), paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE®), docetaxel (TAXOTERE®), gemcitabine (GEMZAR®), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), epirubicin (ELLENCE®, PHARMORUBICIN®), FOLFOX (oxaliplatin combined with 5-FU and leucovorin), FOLFIRI (combination of leucovorin, 5-FU and irinotecan), leucovorin, fluorouracil (5-FU, EFUDEX®), mitomycin C (MITOZYTREXTM, MUTAMYCIN®), and doxorubicin hydrochloride (A
  • an afucosylated anti-FGFR2IIIb antibody is administered in conjunction with paclitaxel. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is administered in conjunction with cisplatin and/or 5-FU. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is administered in conjunction with cisplatin and 5-FU. In some embodiments, an afucosylated anti-FGFR2IIIb antibody is administered in conjunction with FOLFOX (oxaliplatin, 5-FU, and leucovorin).
  • FOLFOX oxaliplatin, 5-FU, and leucovorin
  • the invention also provides kits, medicines, compositions, and unit dosage forms for use in any of the methods described herein.
  • Kits of the invention include one or more containers comprising an afucosylated anti-FGFR2IIIb antibody (or unit dosage forms and/or articles of manufacture).
  • a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising an afucosylated anti-FGFR2IIIb antibody, with or without one or more additional agents.
  • such a unit dosage is supplied in single-use prefilled syringe for injection.
  • the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range.
  • the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water.
  • the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine.
  • a composition of the invention comprises heparin and/or a proteoglycan.
  • kits of the invention further comprise instructions for use in the treatment of, e.g., cancer (such as gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, or esophageal cancer) in accordance with any of the methods described herein.
  • the kit may further comprise a description of selection an individual suitable or treatment.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the kit further comprises another therapeutic agent.
  • kits of the invention are in suitable packaging.
  • suitable packaging include, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • Nucleotide sequences encoding the heavy chain (HC) and light chain (LC) of monoclonal antibody ⁇ FGFR2b were cloned into the GS Gene Expression System (Lonza, Basel, Switzerland) according to the manufacturer's instructions. The system generates a double gene vector (DGV) containing the expression cassettes for both the light chain and the heavy chain.
  • HC heavy chain
  • LC light chain
  • ⁇ FGFR2bA afucosylated monoclonal antibody ⁇ FGFR2bA
  • the Potelligent® CHOK1SV cells BioWa/Lonza, Allendale, N.J.
  • Potelligent® CHOK1SV cells lack the FUT8 gene ( ⁇ 1,6-fucosyltransferase) and therefore produce fucose-free antibodies (afucosylated antibodies).
  • the expression vector comprising the nucleotide sequences encoding ⁇ FGFR2b antibody heavy chain and light chain described above transfected into Potelligent® CHOK1SV cells by electroporation according to the manufacturer's instructions. Electroporated cells were seeded into a 96-well plate at about 10,000 cells/50 ⁇ l/well in CD CHO medium without L-glutamine. Selective CD CHO medium containing 67 ⁇ M L-methionine sulfoximine (MSX, SIGMA cat#M5379, St. Louis, Mo.) was added the next day at 150 ⁇ l/well. Cell growth and clone formation was monitored with an IN Cell Analyzer 2000 (GE Healthcare, Piscataway, N.J.).
  • ⁇ FGFR2bA antibody After 4-6 weeks, the surviving colonies were screened for expression of ⁇ FGFR2bA antibody using a Homogenous Time Resolved Fluorescence (HTRF) based assay against standard curves generated with purified ⁇ FGFR2b antibody, using XL665 conjugated protein A and cryptate conjugated polyclonal rabbit IgG (Cisbio, Bedford, Mass.).
  • HTRF Homogenous Time Resolved Fluorescence
  • the highest expressing clones were expanded through a series of increasing-scale production processes, including 24-well plates, spin tubes, shake flasks, bench scale bioreactors, and finally a platform production process. At each step, only a subset comprising the highest-expressing clones were taken on to the next process.
  • the final production clone was selected based on the evaluation of protein product titers, cell growth characteristics, product quality, stability, as well as scalability in bioreactors.
  • the final production line had an expression level of about 3.5 g/L for ⁇ FGFR2bA. Lack of fucosylation in ⁇ FGFR2bA produced in the final production cell line was confirmed using normal phase HLPC (N-HPLC) chromatography.
  • ⁇ FGFR2bA antibody was purified by column chromatography and ultrafiltration to concentrate the purified material, then diafiltration to exchange into formulation buffer (20 mM histidine, 150 mM L-arginine, 0.01% polysorbate 20, pH 6.0). Antibody was stored below ⁇ 70° C.
  • FIG. 7 shows that ⁇ FGFR2b derived from the Potelligent® CHOK1SV cell line and ⁇ FGFR2b produced from CHOK1SV had two different glycan distributions.
  • A Glycan distribution of ⁇ FGFR2b derived from the Potelligent® CHOK1SV cell line shows that the antibody lacks fucose (“G0”).
  • B Glycan distribution of ⁇ FGFR2b derived from the CHOK1SV cell line shows that the antibody contains fucose (“G0F”).
  • Glycan peaks from the normal phase HPLC separation were identified using two orthogonal methods. First, the Potelligent® CHOK1SV produced ⁇ FGFR2b were labeled and separated using the normal phase HPLC method. After the fluorescent detection, the glycans were passed through a QTrap mass spectrometer. The mass of each peak was determined and used to positively identify each glycan, and is shown in Table 2.
  • FIG. 8 shows schematic diagrams of the G0, G1, G2, G0F, G1F, G2F, and mannose-5 (or Man-5) glycan structures typically observed in antibodies.
  • the peak identities from the HPLC assay were also confirmed using glycan standards purchased from Prozyme and matching the retention time between the standards and the ⁇ FGFR2 profiles from both the Potelligent® CHOK1SV cell line and the CHOK1SV cell line. These standards were able to identify G0, G0F, Man5, G1, G1F, and G2F.
  • the binding affinities of ⁇ FGFR2bA and ⁇ FGFR2bF were determined by surface plasmon resonance. Briefly, Protein A was covalently attached to a dextran chip using the Amine Coupling Kit (GE Healthcare Life Sciences, Piscataway, N.J.) and 100 mM ethylenediamine (Sigma) in 100 mM sodium borate buffer, pH 8.0 (Sigma, St. Louis, Mo.) as the blocking reagent.
  • Fc gamma RIIIA(V158) bound ⁇ FGFR2bA with more than 20-fold greater affinity than it bound ⁇ FGFR2bF.
  • Ba/F3 FGFR2IIIb-expressing target cells were produced as follows. Ba/F3 cells acquired from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ, cat# ACC300, Braunschweig, Germany) were maintained in RPMI (Mediatech, cat#10-041-CV, Manassas, Va.), supplemented with 10% fetal bovine serum (Mediatech, cat#35-010-CV), 1 ng/mL murine IL-3 (Peprotech, cat#213-13, Rocky Hill, N.J.), 1 ⁇ BME (Invitrogen, cat#1047574, Grand Island, N.Y.), and 1 ⁇ Penicillin-streptomycin (Mediatech cat#30-002-C1).
  • the Ba/F3 cells were transfected with an expression vector that expresses FGFR2IIIb, pBNew-hFGFR2b, using Cell Line Nucleofector® Kit V (Lonza, cat# VCA-1003) following the manufacturer's protocol.
  • pBNew vector contains a CAG promoter with a chicken ⁇ -actin intron and ampicillin and puromycin growth selection genes.
  • Transfected cells were incubated in full growth media for 3 days then treated with 2 ⁇ g/mL of puromycin (InVivoGen, cat # ant-pr-1, San Diego, Calif.). Puromycin selection was maintained throughout culturing. To generate individual stable clones, cells were plated at a density of 1 cell per 3 wells. Fluorescent activated cell sorting (FACS) with an anti-FGFR2IIIb antibody was used to select the clones with the highest level of FGFR2IIIb expression.
  • FACS Fluorescent activated cell sorting
  • 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 cultured using standard methods. Freshly isolated PMBC from healthy donors were obtained from AllCells, Emeryville, Calif.
  • ADCC assays were conducted using effector cells from 3 independent donors on 3 different days. ADCC assay testing was performed using freshly isolated human PBMCs as effector cells at an effector to target (E/T) cell ratio of 25:1. The target cells were incubated for 16 hours in the presence of effectors and increasing concentrations of antibody. The ADCC assay was validated using 2 positive control antibodies, HERCEPTIN® on SKOV-3 target cells and RITUXIN® on Raji target cells. A fully human IgG1 negative control antibody (Eureka Therapeutics, Catalog ET901, Emeryville, Calif.) was used for non-specific cell cytotoxicity. Cytotoxicity was determined by quantifying LDH release as per the manufacturer's instructions (CytoTox Non Radioactive Cytotoxicity Assay, Promega, Madison, Wis.).
  • FIG. 1 The results for the Ba/F3 cells are shown in FIG. 1 .
  • Afucosylated ⁇ FGFR2bA antibody induced greater specific lysis than fucosylated ⁇ FGFR2bF antibody in FGFR2IIIb-expressing Ba/F3 cells.
  • ⁇ FGFR2bA showed greater ADCC activity at lower concentrations than ⁇ FGFR2bF, although maximal specific lysis of the antibodies was comparable in OCUM-2M and MFM 223 cells (data not shown).
  • ⁇ FGFR2b showed little to no ADCC activity in FGFR2IIIc-expressing Ba/F3 cells. See FIG. 1A .
  • OCUM-2M Human gastric carcinoma cell line OCUM-2M or breast carcinoma cell line MFM-223 were used as the tumor models.
  • OCUM-2M was purchased from Public University Corporation Osaka City University (OCU, Osaka, Japan), and MFM-223 was purchased from Culture Collections, Public Health England (98050130, HPA Culture Collections, Salisbury, UK). The cells were cultured for up to three passages in complete growth medium to expand for implantation.
  • DMEM Dulbecco's Minimum Essential Medium
  • MEM Minimum Essential Medium
  • FBS Fetal Bovine Serum
  • Penicillin-Streptomycin solution 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine, and Penicillin-Streptomycin solution.
  • Cells were grown at 37° C. in a humidified atmosphere with 5% CO 2 .
  • OCUM-2M cells were implanted subcutaneously over the right flank of the mice at 5 ⁇ 10 5 cells/100 ⁇ l/mouse.
  • MFM-223 cells were implanted subcutaneously over the right flank of the mice at 5 ⁇ 10 5 cells/100 ⁇ l/mouse, and 0.72 mg 90-day release 17- ⁇ estradiol pellets (Innovative Research of America, Sarasota, Fla.) were inoculated subcutaneously into the right flank.
  • OCUM-2M tumors single therapy with anti-FGFR2b was initiated once tumors reached an average size of 100 mm 3 , and combination therapy was initiated once tumors reached an average size of 250 mm 3 .
  • Anti-FGFR2IIIb humanized antibody (Afucosylated, ⁇ FGFR2bA; Fucosylated, ⁇ FGFR2bF) or albumin as a negative control was administered at doses ranging from 1 to 10 mg/kg via intraperitoneal injection twice per week as specified in the figure legends.
  • Chemotherapeutic agents were administered via intraperitoneal injection twice per week at 12 mg/kg for paclitaxel, 2.3 mg/kg for fluorouracil (5-FU), and 33 mg/kg for cisplatin.
  • tumor sizes were measured in each mouse twice weekly. The length and width of each tumor was measured using calipers and the tumor size calculated according to the formula above. Mice were euthanized when the subcutaneous tumor volumes exceeded 2000 mm 3 or when the tumors became excessively necrotic.
  • FIG. 2 shows efficacy of ⁇ FGFR2bA and ⁇ FGFR2bF at (A and B) 10 mg/kg and (C and D) 3 mg/kg in an OCUM-2M human gastric cancer xenograft model with FGFR2 amplification.
  • A At 10 mg/kg, both fucosylated and afucosylated antibody induced immediate tumor regression. However, afucosylated ⁇ FGFR2bA induced a more durable response than fucosylated ⁇ FGFR2bF.
  • FIG. 3 shows dose-dependent tumor inhibition by ⁇ FGFR2bA.
  • A SCID mice bearing subcutaneous OCUM-2M xenografts were treated with 1, 1.5, 2, 3, or 5 mg/kg afucosylated ⁇ FGFR2bA when average tumor size reached approximately 100 mm 3 . Although all doses of afucosylated ⁇ FGFR2bA inhibited tumor growth, the greatest suppression and durable response were observed with 3 and 5 mg/kg, with reduced growth suppression observed with 2, 1.5, and 1 mg/kg ⁇ FGFR2bA.
  • B Afucosylated ⁇ FGFR2bA reduced final OCUM-2M tumor size, with higher doses showing more potent growth suppression.
  • FIG. 4 shows enhancement of paclitaxel anti-tumor activity in an OCUM-2M gastric cancer xenograft model by administration with afucosylated ⁇ FGFR2bA.
  • A SCID mice bearing subcutaneous OCUM-2M xenografts were treated with afucosylated ⁇ FGFR2bA (5 mg/kg), paclitaxel (12 mg/kg), or a combination of the two when average tumor size reached approximately 285 mm 3 .
  • Combining afucosylated ⁇ FGFR2bA with paclitaxel reduced tumor size compared to either ⁇ FGFR2bA or paclitaxel alone.
  • FIG. 5 shows enhancement of cisplatin/5-FU anti-tumor activity in an OCUM-2M gastric cancer xenograft model by administration with afucosylated ⁇ FGFR2bA.
  • A SCID mice bearing subcutaneous OCUM-2M xenografts were treated with afucosylated ⁇ FGFR2bA (5 mg/kg), fluorouracil (5-FU) plus cisplatin (2.3 and 33 mg/kg, respectively), or a combination of the three when average tumor size reached approximately 260 mm 3 .
  • Combining afucosylated ⁇ FGFR2bA with 5-FU/cisplatin reduced tumor size compared to either ⁇ FGFR2bA or 5-FU/cisplatin alone.
  • FIG. 6 shows efficacy of ⁇ FGFR2bA in an MFM-223 human breast cancer xenograft model.
  • Afucosylated ⁇ FGFR2bA (5 mg/kg) dramatically reduced growth of MFM-223 tumors compared to albumin control (5 mg/kg).
  • ADCC assays were conducted using freshly isolated human PBMCs as effector cells at an effector to target (E:T) cell ratio of 25:1. The target cells were incubated for 16 hours in the presence of effectors and increasing concentrations of antibody. All conditions were tested in triplicate. Cytotoxicity was determined by quantifying LDH release as per the manufacturer's recommendations (Promega's CytoTox Non-Radioactive Cytotoxicity Assay). For the OCUM-2M cells, maximal lysis was determined in the presence of 5% Triton X-100, and spontaneous release was determined in the absence of antibody.
  • GraphPad software curve-fitting analysis was used to obtain EC50 values.
  • FIG. 9 The results for the Ba/F3 cells are shown in FIG. 9 .
  • Afucosylated ⁇ FGFR2bA antibody showed higher potency (greater ADCC activity at lower concentrations) than fucosylated ⁇ FGFR2bF antibody in FGFR2IIIb-expressing Ba/F3 cells.
  • Similar results are shown for OCUM-2M cells in FIG. 10 .
  • Table 4 shows the potency fold increase of afucosylated anti-FGFRb antibody compared to fucosylated anti-FGFR2b antibody in the two different cell lines shown in FIGS. 9 and 10 .
  • Murine ⁇ FGFR2b antibody comprising the murine variable regions of GAL-FR21 (see U.S. Pat. No. 8,101,723 B2) and a murine IgG2a constant region was used to detect FGFR2IIIb protein in formalin-fixed paraffin embedded (FFPE) gastric tumor tissue.
  • FFPE tumor tissue as de-paraffinized with successive washes of xylenes and decreasing concentrations of ethanol, and rehydrated with water for five minutes. Slide-mounted tissue sections were immersed in 0.1 mM citrate buffer (pH 6.0) heated to 95°-99° C. for 15 minutes.
  • Tissue sections were cooled and contacted with 3% H 2 O 2 for 5 minutes at room temperature, and then washed twice in TBST (0.05M Tris, 0.15M NaCl, 0.05% Tween 20) and blocked in blocking buffer (2.5% normal horse serum in TBST) for 30 minutes at room temperature. Tissue sections were then incubated with 5 ⁇ g/ml ⁇ FGFR2b antibody in blocking buffer for 30 minutes at room temperature. Incubation times between 30 minutes and 2 hours produced similar staining intensities.
  • tissue sections were incubated with ready-to-use (RTU) biotinylated secondary horse anti-mouse antibody (10 ⁇ g/ml diluted in blocking buffer (Vector Laboratories, Burlingame, Calif.; Cat. No. PK-7200) for 30 minutes at room temperature.
  • RTU ready-to-use
  • Tissue sections were washed twice with TBST buffer, and the incubated with RTU streptavidin-horse radish peroxidase (HRP; Vector Laboratories, Cat. No. PK-7200) for 30 minutes at room temperature.
  • Detection was performed using 3′,3′-diaminobenzidine (DAB) substrate (Vector Laboratories, Cat. No.
  • Tissue sections were then counter-stained with hematoxylin (Dako North America, Carpinteria, Calif., Cat. No. K-8008) per manufacturer's instructions. Tissue sections were dehydrated using successive washes with increasing concentrations of ethanol and xylenes, and then covered with a coverslip.
  • Staining concentrations of 0.1 to 5 mg/ml ⁇ FGFR2b antibody were found to be particularly effective for protein detection.
  • the staining was scored as follows:
  • FIG. 11 shows exemplary 0 (A), 1+ (B, C), 2+ (D), and 3+ (E, F) staining of tumor tissues.
  • ductal cells and interspersed tumor cells show an absence of staining using the ⁇ FGFR2b antibody.
  • FIG. 11B which shows an interstitial type gastric tumor sample
  • tumor cells (arrows) show some staining using the ⁇ FGFR2b antibody
  • surrounding ductal cells show no staining.
  • FIG. 11C which shows a diffuse-type gastric tumor sample
  • tumor cells (arrows) show some staining using the ⁇ FGFR2b antibody
  • surrounding stromal cells show no staining.
  • tumor cells (arrows) show intermediate staining using the ⁇ FGFR2b antibody, while surrounding stromal cells (arrowheads) show no staining.
  • tumor cells (arrows) show high membrane-localized staining using the ⁇ FGFR2b antibody, while surrounding stromal cells (arrowheads) show no staining.
  • tumor cells (arrows) show high membrane/cytoplasmic staining using the ⁇ FGFR2b antibody, while surrounding stromal cells (arrowheads) show no staining

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