EP4642801A1 - Anti-fgfr2b-antikörper und verwendungen davon - Google Patents

Anti-fgfr2b-antikörper und verwendungen davon

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Publication number
EP4642801A1
EP4642801A1 EP23910752.7A EP23910752A EP4642801A1 EP 4642801 A1 EP4642801 A1 EP 4642801A1 EP 23910752 A EP23910752 A EP 23910752A EP 4642801 A1 EP4642801 A1 EP 4642801A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
acid sequence
seq
set forth
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23910752.7A
Other languages
English (en)
French (fr)
Inventor
Xueming Qian
Yi Gu
Hongjun Li
Zhe SUN
Shijie ZHOU
Mangmang LI
Huanhuan GUO
Xinlai YAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Transcenta Therapeutics Co Ltd
Original Assignee
Suzhou Transcenta Therapeutics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Transcenta Therapeutics Co Ltd filed Critical Suzhou Transcenta Therapeutics Co Ltd
Publication of EP4642801A1 publication Critical patent/EP4642801A1/de
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an antibody, and in particular to an anti-FGFR2B antibody and antigen-binding fragment thereof, and method for preparing the antibody and use same for treating or preventing FGFR2B-related diseases or conditions.
  • Targeted therapies interfering with oncogenic driver alterations have achieved great success in chronic myeloid leukaemia (CML) with BCR-ABL fusions, melanoma with BRAF V600E mutations, lung cancer with EGFR mutations, breast cancer with HER2 amplification, and the like.
  • CML chronic myeloid leukaemia
  • types of cancers with specific driver gene alterations are limited (Francavilla C, O'Brien CS. Fibroblast growth factor receptor signalling dysregulation and targeting in breast cancer. Open Biol. 2022 Feb; 12 (2) : 210373. ) .
  • the development of novel therapeutics targeting other cancer driver alterations is extremely urgent to improve patients’ prognosis.
  • Receptor Tyrosine Kinases are single-pass transmembrane proteins, whose overexpression is associated with breast and other cancers and decreased disease-free survival (Templeton AJ, Diez-Gonzalez L, Ace O, Vera-Badillo F, Seruga B, Jordán J, Amir E, Pandiella A, A. Prognostic relevance of receptor tyrosine kinase expression in breast cancer: a meta-analysis. Cancer Treat Rev. 2014 Oct; 40 (9) : 1048-55, and Butti R, Das S, Gunasekaran VP, Yadav AS, Kumar D, Kundu GC.
  • RTKs Receptor tyrosine kinases
  • MAPK mitogen-activated protein kinase
  • JAK Janus kinase
  • STAT signal transducer and activator of transcription
  • PLC ⁇ phospholipase C gamma
  • PI3–K phosphoinositide 3-kinase
  • Fibroblast Growth Factor Receptors and their isoforms are known to be one kind of RTK.
  • dimerization of the FGFR kinase domain induces the phosphorylation of tyrosine (Y) residues leading to full receptor activation and phosphorylation and recruitment of adaptor proteins.
  • Y tyrosine residues
  • the FGFRs are characterized by multiple alternative splicing of their mRNAs, leading to a variety of isoforms (Ornitz et al, J. Biol. Chem.
  • FGFR2 plays a role in epithelial-mesenchymal interactions (Finch et al, Dev. Dyn. 203 : 223, 1995) , e.g., mediating the affections from the tumor microenvironment to facilitate increasing developmental abnormalities or cancer progression.
  • KGF FGF7
  • KGFR FGFR2IIIb
  • Amplification and overexpression of FGFR2 is associated with the undifferentiated, diffuse type of gastric cancer, which has a particularly poor prognosis (Kunii et al, Cancer Res. 68: 2340, 2008; Nakamura et al, Gastroenterol. 131: 1530, 2006) .
  • the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, and methods for preparing and using same, including a method for treating FGFR2B-related diseases or conditions.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three selected from HCDR1, HCDR2 and HCDR3 of a heavy chain variable region (VH) , wherein the amino acid sequence of the VH is as set forth in any one of SEQ ID NOs: 43-49.
  • VH heavy chain variable region
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three selected from LCDR1, LCDR2 and LCDR3 of a light chain variable region (VL) , wherein the amino acid sequence of the VL is as set forth in any one of SEQ ID NOs: 50-56.
  • VL light chain variable region
  • the present invention provides an isolated FGFR2B antibody or antigen-binding fragment thereof, which comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the VH is as set forth in any one of SEQ ID NOs: 43-49, and the amino acid sequence of the VL is as set forth in any one of SEQ ID NOs: 50-56.
  • VH heavy chain variable region
  • VL light chain variable region
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3; wherein the VH and VL are selected from:
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 43
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 50
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 46
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 53
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 48
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 55
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 49 and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 56.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of heavy chain complementary determining regions (HCDRs) , HCDR1, HCDR2 and HCDR3, wherein:
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 1
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 or 7
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 or 8
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 9
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 10
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 11;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 12
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 13
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 17
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 18;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 19
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 21.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of light chain complementary determining regions (LCDRs) , LCDR1, LCDR2 and LCDR3, wherein:
  • LCDRs light chain complementary determining regions
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 22
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 23
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 24;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 25
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 26
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 27;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 28
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 29
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 30;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 31
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 32
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 33;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 34
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 35
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 36;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 37
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 38
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 39;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 40
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 41
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain complementary determining regions (HCDRs) , HCDR1, HCDR2 and HCDR3, and light chain complementary determining region s (LCDRs) , LCDR1, LCDR2 and LCDR3, wherein:
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 1
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 22
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 23
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 24;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 4
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 5
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 25
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 26
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 27;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 7
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 8
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 28
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 29
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 30;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 9
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 10
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 11
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 31
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 32
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 33;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 12
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 13
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 34
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 35
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 36;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 17
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 18, and the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 37
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 38
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 39; or
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 19
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20
  • the HCDR3 comprises the amino acid sequence SEQ ID NO: 21
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 40
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 41
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 42.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any of the combinations listed in Table 1 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) , wherein the VH comprises the amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 43-49.
  • VH heavy chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain variable region (VL) , wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 50-56.
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 44, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 45, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 52.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 46, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 54.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 48, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 55.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 49, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 56.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) as set forth in any of the combinations listed in Table 2 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 57
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 64.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 58
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 65.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 59
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 66
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 60
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 62
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 69.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 63
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 70.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a Fc region.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc ⁇ receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) .
  • said one or more amino acids are mutated at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439, numbered according to the EU numbering system.
  • said one or more amino acids are mutated at the following positions: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339 and P396, numbered according to the EU numbering system.
  • said one or more amino acids which are mutated are selected from the following substitutions: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L, numbered according to the EU numbering system.
  • said amino acids which are mutated comprise the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) , numbered according to the EU numbering system.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises the Fc region with the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) , numbered according to the EU numbering system.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises the Fc region with one or more following groups of substitutions:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC) as set forth in any of the combinations listed in Table 3 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a constant region, which is hypofucosylated or afucosylated.
  • the isolated antibody or antigen-binding fragment thereof provided by the present invention binds to FGFR2b but does not bind to FGFR2c.
  • the anti-FGFR2B antibody provided by the present invention is a monoclonal antibody.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which is a full-length antibody, a single-domain antibody (such as a VHH) , a Fab, a Fab’, a Fab’-SH, a (Fab’) 2, a single-chain antibody (such as a scFv) , a Fv, or a dAb (domain antibody) .
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an Fc region.
  • the amino acid sequence of the Fc region is identical to the sequence of the Fc region of human IgG1, IgG2 or IgG4, or is a variant thereof.
  • the present invention provides an anti-FGFR2B antibody, which is antagonistic, comprising the CDRs of the antibody provided by the present invention.
  • the antagonistic anti-FGFR2B antibody comprises an Fc region variant, which variant enhances the effector functions of the antibody.
  • the effector function is ADCC.
  • the antagonistic anti-FGFR2B antibody comprises an Fc region variant, which variant is human IgG1 VLPYLL.
  • the antagonistic anti-FGFR2B antibody comprises an Fc region variant, which variant is hypofucosylated or afucosylated human IgG1 or human IgG4.
  • the antibody of the present invention has one or more of the following properties:
  • EC 50 value of less than 100 nM, such as less than 50 nM, such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM or 5 nM, wherein preferably the EC 50 value is measured using FACS assay;
  • the present invention provides an isolated nucleic acid, which encodes any of the antibodies or fragment thereof provided by the present invention, wherein preferably, the nucleic acid encodes the heavy chain or the light chain, or the heavy chain variable region or the light chain variable region of the antibody of the present invention.
  • the nucleic acid further comprises a signal peptide coding sequence.
  • the present invention provides a recombinant vector or an expression vector, comprising one or more nucleic acids provided by the present invention, wherein the vector is suitable for recombinant production of any antibody or antigen-binding fragment thereof provided by the present invention.
  • the vector is an expression vector.
  • the present invention provides a host cell, comprising one or more nucleic acids, or recombinant vectors or expression vectors provided by the present invention.
  • the present invention provides a method of producing an anti-FGFR2B antibody or an antigen binding fragment thereof comprising culturing a host cell comprising an expression vector encoding the antibody or antigen binding fragment in culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding FGFR2B, and optionally recovering the expressed antibody or fragment from the host cell.
  • the present invention provides immunoconjugates targeting FGFR2b or pharmaceutically acceptable salts or solvates thereof, comprising the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention conjugated to a payload.
  • the payload is a drug, such as a cytotoxic agent.
  • the present invention provides a pharmaceutical composition, comprising the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the host cell provided by the present invention, and optionally comprising at least one pharmaceutically acceptable auxiliary substance, such as a pharmaceutical carrier or a pharmaceutical excipient.
  • a pharmaceutical composition comprising the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the host cell provided by the present invention, and optionally comprising at least one pharmaceutically acceptable auxiliary substance, such as a pharmaceutical carrier or a pharmaceutical excipient.
  • the present invention provides a combination of medicaments, comprising the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the host cell provided by the present invention, and one or more additional therapeutic agents.
  • the present invention also provides the use of the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector, or the host cell provided by the present invention, in the preparation of medicaments for treating FGFR2B-related diseases or conditions.
  • the present invention also provides a method of killing the FGFR2B-positive cancer cells in vitro or in vivo, wherein the method comprises contacting the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the pharmaceutical composition or the combination of medicaments provided by the present invention to the cell population comprising the FGFR2B-positive cancer cells, or administering the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the pharmaceutical composition or the combination of medicaments provided by the present invention into the subject in need thereof.
  • the present invention also provides the use of the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the pharmaceutical composition or the combination of medicaments of the present invention in the preparation of medicaments for treating cancers, which is preferably selected from breast cancer (e.g., triple negative breast cancer) gastric cancer, GEJ cancer, esophageal cancer, lung cancer (e.g., squamous NSCLC) , ovarian cancer, endometrial, cervical cancer, colorectal cancer, cholangiocarcinoma and pancreatic cancer.
  • breast cancer e.g., triple negative breast cancer
  • GEJ cancer esophageal cancer
  • lung cancer e.g., squamous NSCLC
  • ovarian cancer endometrial
  • cervical cancer colorectal cancer
  • cholangiocarcinoma cholangiocarcinoma
  • pancreatic cancer pancreatic cancer.
  • the present invention also provides the anti-FGFR2B antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the pharmaceutical composition or the combination of medicaments for use in the treatment or prophylaxis of cancers, which is preferably selected from breast cancer (e.g., triple negative breast cancer) , gastric cancer, GEJ cancer, esophageal cancer, lung cancer (e.g., squamous NSCLC) , ovarian cancer, endometrial, cervical cancer, colorectal cancer, cholangiocarcinoma and pancreatic cancer.
  • breast cancer e.g., triple negative breast cancer
  • gastric cancer GEJ cancer
  • esophageal cancer e.g., sophageal cancer
  • lung cancer e.g., squamous NSCLC
  • ovarian cancer endometrial, cervical cancer, colorectal cancer, cholangiocarcinoma and pancreatic cancer.
  • the present invention provides a method for treating or preventing an FGFR2B-related disease or condition, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof, or the nucleic acid, the vector, the host cell, or the pharmaceutical composition or combination of medicaments comprising same provided by the present invention.
  • the FGFR2B-related disease or condition is cancers, such as breast cancer or gastric cancer.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention can also be combined with other therapeutic agents or procedures, for treating or preventing FGFR2B-related diseases or conditions.
  • the present invention also provides a method for detecting FGFR2B in a sample by using the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention.
  • the method can be used to diagnose/detect FGFR2B-related diseases or conditions.
  • the present invention also encompasses any combinations of any embodiments described herein. Any embodiments or any combinations thereof described herein are applicable to any and all anti-FGFR2B antibodies or fragments, methods and uses thereof of the present invention described herein.
  • Figure 1 shows the epitope binning of the humanized anti-FGFR2B mAbs of the present invention.
  • Figure 2 shows ADCC of chimeric anti-FGFR2B mAbs of the present invention on KATO-III cells.
  • Figure 3 shows the binding specificity of humanized anti-FGFR2B mAbs of the present invention, which are tested for binding to other FGFR family members.
  • Figure 4 shows the species cross-reactivity of the humanized anti-FGFR2B mAbs of the present invention.
  • Figure 5 shows the ELISA assay for blocking of humanized mAb to the interaction between FGFR2b and FGF7.
  • Figure 6 shows the Flow cytometry for binding of FGFR2b antibodies to 293T_hFGFR2b (A) and 293T_hFGFR2c (B) cells
  • Figure 7 shows the ADCC reporter bioactivities of humanized lead FGFR2b antibodies targeting to KATO-III cells and KYSE-180 cells.
  • Figure 8 shows the inhibition of the FGF7-induced proliferation of MCF7 cells by several FGFR2b antibodies.
  • the MCF7 cells were incubated in serum-free medium and were either left untreated (medium) or treated with hIgG_isotype or several FGFR2b antibodies at 30 ⁇ g/mL for 72 in the absence or presence of FGF7 (25 ng/mL) .
  • Cell proliferation was assessed by CellTiter- luminescent cell viability assay.
  • Figure 9 shows the HTRF assay to detect the phosphorylation of FGFR2 and ERK1/2 proteins induced by FGF7 or FGF10 in SNU-16 cells.
  • A-B FGF7 mediated phosphorylation of FGFR2 (A) and ERK1/2 (B) were inhibited by several FGFR2b antibodies.
  • C-D FGF10 mediated phosphorylation of FGFR2 (C) and ERK1/2 (D) were inhibited by several FGFR2b antibodies.
  • Figure 10 shows the Human PBMCs mediated ADCC activities of several FGFR2b antibodies targeting KATO-III cells.
  • A Primary ADCC effect mediated by the donor with Fc ⁇ RIIIA genotype was 158V/V.
  • B Primary ADCC effect mediated by the donor with Fc ⁇ RIIIA genotype was 158V/F.
  • Figure 11 shows the plasma concentration-time curves.
  • the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, having a unique CDR sequence, having high affinity and specificity for binding to human FGFR2B, and preferably mediating ADCC more effectively.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention can be used alone or in combination with other therapies for the treatment of diseases or conditions, such as cancers, inflammation or autoimmune diseases.
  • the present invention will be implemented using conventional techniques in molecular biology (including recombinant techniques) , microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
  • the term “about” means a value or an integer within an acceptable error range for the particular value or integer as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” can refer to within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” can refer to a range of up to 5%, 10%or 20% (i.e., ⁇ 5%, ⁇ 10%or ⁇ 20%) .
  • the term “comprise” or “include” means to include the mentioned elements, integers, or steps, but does not exclude any other elements, integers, or steps.
  • the term “comprise” or “include” unless otherwise indicated, encompasses “consisting of” the mentioned elements, integers or steps.
  • an antibody variable region “comprising” a specific sequence it is also intended to encompass an antibody variable region consisting of the specific sequence.
  • FGFR2B belongs to the FGFR family, which comprises four members (FGFR1-4) , and their cognate ligands, fibroblast growth factors (FGFs) , comprise a 22-member family (FGF1-14 and FGF16-23) .
  • FGFs fibroblast growth factors
  • the term refers to any natural FGFR2B from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise stated.
  • the term encompasses a “full length” , unprocessed FGFR2B and any form of FGFR2B or any fragment thereof due to processing in the cell.
  • FGFR2B refers to a full length FGFR2B from human, or a fragment thereof (such as the mature fragment lacking a signal peptide) .
  • a human FGFR2B refers to a mature FGFR2B identical to the amino acid sequence as set forth under the accession number Uniprot#P21802 (amino acid residues 1-21 being the leader peptide) , or a fragment thereof (such as an extracellular domain comprising AA 22-377 thereof) .
  • the term also covers a fusion protein comprising FGFR2B or a fragment thereof (such as an extracellular domain thereof) , such as a fusion protein comprising a human FGFR2B extracellular domain and an Fc region.
  • FGFR2B ligand or “FGF7/10” as used herein refers to the native ligand of FGFR2B, or a functional variant thereof.
  • antibody broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any antigen binding fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art and non-limiting embodiments are discussed below.
  • antibody as used herein may refer to any form of antibody having a desirable bioactivity.
  • a monoclonal antibody including a full-length monoclonal antibody
  • a polyclonal antibody including a multispecific antibody (such as a bispecific antibody)
  • a humanized antibody including a fully human antibody, a chimeric antibody, a CrossMab antibody, or a camelized single-domain antibody.
  • affinity refers to the strength of the sum of all 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 the intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen) .
  • the affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K D ) .
  • K D dissociation constant
  • the term "specific binding” or “specifically binding” in reference to the interaction of an antibody, a binding protein, or a peptide with a second chemical species, means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical species.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody recognizes and binds to a specific protein structure rather than to proteins generally.
  • the specific binding protein binds to the corresponding antigen with a K D of 10 nM or lower, for instance, 1 nM or lower.
  • k on (also "Kon” , “kon” ) , as used herein, is intended to refer to the on-rate constant for association of a binding protein (e.g., an antibody) to an antigen to form an association complex, e.g., antibody/antigen complex, as is known in the art.
  • the “k on” also is known by the terms “association rate constant” , or “ka” , as used interchangeably herein. This value indicates the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen as is shown by the equation below:
  • k off (also "Koff” , “koff” ) , as used herein, is intended to refer to the off-rate constant for dissociation, or "dissociation rate constant” , of a binding protein (e.g., an antibody) from an association complex (e.g., an antibody/antigen complex) as is known in the art.
  • This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below:
  • K D (also "K d " ) , as used herein, is intended to refer to the "equilibrium dissociation constant” , and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (k off ) by the association rate constant (k on ) .
  • the association rate constant (k on ) , the dissociation rate constant (k off ) , and the equilibrium dissociation constant (K D ) are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
  • a (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden) .
  • Biolayer interferometry (BLI) using, e.g., the RED96 system (Pall FortéBio LLC) is another affinity assay technique.
  • a (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, Idaho) can also be used.
  • antagonists include antibodies capable of inhibiting and/or blocking FGFR2B-mediated biological signal transduction activity.
  • an FGFR2B antagonist antibody inhibits or suppresses the signal transduction pathway triggered by FGFR2B, and/or inhibits or reduces an FGFR2B-mediated cellular response such as cancer cell proliferation or cancer cell survival, for example by blocking the binding of FGFR2B to an FGFR2B ligand or substantially reducing the binding of FGFR2B to an FGFR2B ligand.
  • FGFR2B-related disease or condition refers to a non-physiological state related to the expression or function or activity of FGFR2B, or to the activity of FGFR2B-mediated signal transduction, including but not limited to cancers, inflammation and autoimmune diseases. In some preferable embodiments, the diseases will benefit from the blocking of FGFR2B-mediated signal transduction.
  • immune response and “immune reaction” are used interchangeably herein and refer to the action of, such as lymphocytes, antigen presenting cells, phagocytes and granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complements) that results in the selective damage, destruction or elimination of invading pathogens, cells or tissues infected with pathogens, cancer cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues, from the human body.
  • the FGFR2B antibody antagonist of the present invention inhibits or reduces immune reaction, for example, reduces immune rejection in graft-versus-host diseases.
  • the FGFR2B antibody agonist of the present invention enhances an anti-tumor immune reaction.
  • signal transduction refers to a biochemical causal relationship generally initiated by a protein-protein interaction such as binding of FGF7/10 (ligand) to FGFR2B (receptor) , resulting in the transmission of a signal from one part of a cell to another part of a cell.
  • the transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that cause the signal transduction.
  • the penultimate process generally includes nuclear events, thereby causing changes in gene expression.
  • activity and “biological activity” or the terms “biological property” and “biological feature” as used herein are used interchangeably herein and include, but not limited to, epitope/antigen affinity and specificity, ability to neutralize or antagonize an FGFR2B activity in vivo or in vitro, ability to enhance or activate FGFR2B in vivo or in vitro, IC 50 that blocks the binding of FGFR2B to FGF7/10, IC 50 that blocks FGFR2B-FGF7/10-mediated cell proliferation, in vivo stability of an antibody and immunogenicity of an antibody.
  • antibodies known in the art include, for example, (species) cross-reactivity (i.e., cross-reacting generally with non-human homologs of targeted peptides, or with other proteins or tissues) , and the ability to maintain high levels of antibody expression in mammalian cells.
  • the properties or features mentioned above can be observed, determined or evaluated using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasmon resonance assay, in vitro or in vivo neutralization assay, receptor binding, production and/or secretion of cytokines or growth factors, signal transduction and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other sources) .
  • the terms “whole antibody” , “full-length antibody” and “intact antibody” are used interchangeably herein and refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of 3 domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • the VH region and VL region can be further divided into hypervariable regions (termed complementary determining region (CDR) ) , interspersed with more conservative regions (termed framework region (FR) ) .
  • CDR region or “CDR” is a region in an antibody variable domain, which is hypervariable in sequence and forms a structurally defined loop ( “hypervariable loop” ) and/or contains antigen-contacting residues ( “antigen contact sites” ) .
  • CDR is mainly responsible for binding to epitopes.
  • CDRs of heavy chain and light chain are generally called CDR1, CDR2 and CDR3, which are numbered sequentially from the N-terminus.
  • the CDRs located in an antibody heavy chain variable domain are called HCDR1, HCDR2 and HCDR3 respectively, while the CDRs located in the antibody light chain variable domain are called LCDR1, LCDR2 and LCDR3 respectively.
  • Each VH or VL consists of three CDRs and 4 FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Constant regions are not directly involved in the binding of an antibody to an antigen, but show multiple effector functions.
  • the accurate amino acid sequence boundary of each CDR can be determined by using any one of the various well known schemes or a combination thereof, including, for example: Chothia scheme (Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins” , Journal of Molecular Biology, 196, 901-917 (1987) ) ; Kabat scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • the boundary of the CDRs of the anti-FGFR2B antibody in the present invention can be determined according to any schemes or a combination thereof in the art and manual evaluation.
  • the light chains of antibodies can be assigned to one of two types (referred to as kappa ( ⁇ ) and lambda ( ⁇ ) ) based on the amino acid sequence of the constant domain thereof.
  • the heavy chains of antibodies can be divided into 5 major different classes according to the amino acid sequence of the heavy chain constant region thereof: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • an “antibody in the form of IgG” means the heavy chain constant region of the antibody is of an IgG form.
  • an antibody in the form of IgG2 means that the heavy chain constant region thereof is of IgG2 isotype.
  • antigen-binding fragment of an antibody as used herein includes fragments or derivatives of the antibody.
  • the antigen-binding fragment includes at least one fragment (such as one or more CDRs) of the antigen-binding region or variable region of the antibody, and maintains at least some of the binding properties of the antibody.
  • an antigen-binding fragment include, but are not limited to Fab, Fab', F (ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., sc-Fv) ; and nanobodies and multispecific antibodies formed from antibody fragments.
  • the binding fragments or derivatives When the antigen-binding activity is expressed in molar concentration, the binding fragments or derivatives generally maintain at least 10%of the antigen-binding activity of the antibody from which they are derived. Preferably, the binding fragments or derivatives maintain at least 20%, 50%, 70%, 80%, 90%, 95%or 100%or more of the antigen binding activity of the antibody from which they are derived.
  • an antibody or antigen-binding fragment thereof may include a conservative or non-conservative amino acid substitution that does not significantly change its biological activity (referred to as “conserved variant” or “functionally conserved variant” of the antibody) .
  • a conservative substitution is from the exemplary conservative substitution residues shown in Table A below, and preferably, the preferred conservative amino acid substitution residues shown in Table A.
  • An epitope is a region of an antigen that is bound by an antibody.
  • An epitope can be formed from contiguous amino acids or non-continuous amino acids juxtaposed by tertiary folding of a protein.
  • an isolated anti-FGFR2B antibody or antigen-binding fragment thereof refers to the purified state of the anti-FGFR2B antibody or antigen-binding fragment thereof.
  • isolated may mean that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars or other substances, such as cell debris and growth medium.
  • isolated is not intended to mean the complete absence of such substances, or the absence of water, buffer or salt unless they are present in an amount that substantially interferes with the experimental or therapeutic application of the antibodies described herein.
  • the isolated antibody or antigen-binding fragment has a purity of greater than 95%, greater than 96%, greater than 97%, greater than 98%or greater than 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) .
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include different antibodies directed against different epitopes (or specific for different epitopes) .
  • the modifier “monoclonal” indicates the feature of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring any particular method to produce the antibody.
  • chimeric antibody refers to an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domain is obtained from the antibody of an experimental animal such as a rodent, while the constant domain sequence is obtained from a human antibody, so that the obtained chimeric antibody is less likely to induce adverse immune response in human subjects than the antibody from the experimental animal.
  • humanized antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • the humanized antibody comprises at least one, and generally two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin, and all or substantially all of the framework (FR) regions correspond to those of human immunoglobulin.
  • the humanized antibody can optionally comprise at least a portion of a constant region (for example, the Fc region) derived from a human immunoglobulin.
  • amino acid mutations can be introduced into humanized antibodies (e.g., variable domains, framework regions, and/or constant regions (if present) ) , for example, to improve certain properties of the antibodies; such antibody forms still fall within the scope of the “humanized antibody” of the present invention.
  • an antibody may have a sugar chain found in the cells for producing the antibody.
  • the antibody when produced in mice, in mouse cells, or in hybridomas derived from mouse cells, the antibody may contain a mouse sugar chain.
  • the antibody when produced in rats, in rat cells, or in hybridomas derived from rat cells, the antibody may contain a rat sugar chain.
  • Fc region as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the native-sequence Fc region covers a variety of naturally occurring immunoglobulin Fc sequences, such as various Ig subtypes and allogeneic Fc regions thereof (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu. 2014.00520) .
  • the Fc region of the human IgG heavy chain extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.
  • lysine at the C-terminus (Lys447) of Fc region may or may not be present.
  • amino acid residues in Fc region or constant region are numbered in accordance with the EU numbering system, also referred to EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Fc region variant and “variant Fc region” as used herein are used interchangeably herein, and refer to an Fc region polypeptide comprising an amino acid modification (s) relative to a native sequence Fc region.
  • the Fc region variants of the present invention are defined according to the amino acid modifications that compose them.
  • L235V refers to an Fc region variant with the substitution of leucine with valine at position 235 relative to the parent polypeptide, where the number is in accordance with EU index.
  • the modifications can be an addition, deletion or substitution.
  • Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids.
  • the variant may contain non-natural amino acids.
  • Fc receptor or “FcR” as used herein describes a receptor that binds to an antibody Fc region.
  • FcR is a native sequence human FcR.
  • FcR is Fc ⁇ R (agamma receptor) , including receptors of Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, and also including allelic variants and alternative splicing forms of those receptors.
  • Fc ⁇ RII includes Fc ⁇ RIIA (an “activating receptor” ) and Fc ⁇ RIIB (an “inhibitory receptor” ) , which have similar amino acid sequences and differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Annu. Rev. Immunol. 15: 203-234 (1997) ) .
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • FcR see, e.g., Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) ; Capel et al., Immunomethods 4: 25-34 (1994) ; and de Haas et al., J. Lab. Clin. Med.
  • FcR Fc receptor
  • FcRn neonatal receptor
  • the in vivo binding to human FcRn and the serum half-life of a polypeptide with a high binding affinity for a human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing a human FcRn, or in primates administered with a polypeptide with a variant Fc region.
  • WO 2000/42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. See, also, for example, Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) .
  • auxiliary substance refers to a diluent, an adjuvant (e.g., Freund’s adjuvant (complete and incomplete) ) , a pharmaceutical excipient, a pharmaceutical carrier or a stabilizer, etc., which is administered with an active substance.
  • an adjuvant e.g., Freund’s adjuvant (complete and incomplete)
  • a pharmaceutical excipient e.g., a pharmaceutical carrier or a stabilizer, etc.
  • composition refers to such a composition that exists in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administrated.
  • therapeutic agent covers any substances that are effective in the prevention or treatment of related diseases, such as cancers.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical small molecule drugs that are useful in the treatment of cancers or immune system diseases.
  • small molecule drug refers to a compound with a low molecular weight that can regulate biological processes.
  • a “small molecule” is defined as a molecule with a molecular weight smaller than 10 kD, typically smaller than 2 kD and preferably smaller than 1 kD.
  • the small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules containing a radioactive atom, synthetic molecules, peptide mimics and antibody mimics. As therapeutic agents, small molecules are better able to penetrate cell membranes than large molecules, are less susceptible to degradation, and are less likely to trigger an immune response.
  • an immunomodulator refers to a natural or synthetic active agent or drug that modulates (e.g., suppresses or enhances) an immune response.
  • the immune response can be a humoral response or a cellular response.
  • an immunomodulator includes an immunosuppressant that inhibits an immune response, for example, an immunosuppressant that beneficially inhibits an immune response in inflammation and autoimmune diseases.
  • an immunomodulator includes an active agent or a drug that enhances an immune response, for example, an active agent or a drug that beneficially enhances an anticancer immune response in cancer treatment.
  • cancer refers to or describe physiological disorders in mammals, generally characterized by unregulated cell growth. This definition includes benign and malignant tumors and dormant tumors or micrometastasis.
  • the “cancer” includes, but is not limited to, solid tumors and blood cancers. Examples of various cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia.
  • isolated nucleic acid means a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by human intervention, is not associated with all or a portion of the polynucleotides with which it is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
  • polynucleotide e.g., of genomic, cDNA, or synthetic origin, or some combination thereof
  • vector refers to any recombinant polynucleotide constructs that can be used for the purpose of transformation (i.e. introduction of heterologous DNA into host cells) .
  • plasmid a circular double stranded DNA loop, into which an additional DNA segment can be ligated.
  • viral vector a viral vector, in which an additional DNA segment can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • some vectors can guide the expression of genes that are operably linked.
  • Such vectors are referred to as “expression vector” herein.
  • the expression vector refers to the nucleic acid that can replicate and express a target gene when the vector is transformed, transfected or transduced into a host cell.
  • the expression vector comprises one or more phenotypic selectable markers and origins of replication to ensure vector maintenance and provide amplification in the host if necessary.
  • Transformation refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
  • the term "recombinant host cell” is intended to refer to a cell into which exogenous DNA has been introduced.
  • the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in US Patent No. 7,262,028, for example.
  • Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life.
  • eukaryotic cells include protist, fungal, plant and animal cells.
  • host cells include but are not limited to the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, Jurkat, COS, NS0, SP2 and PER. C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, bovine, chicken, amphibians, reptiles, etc.
  • therapeutically effective amount refers to the amount of the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention that effectively prevents or improves the symptoms of one or more diseases or conditions or the development of the diseases or conditions when given to cells, tissues or subjects alone or in combination with other therapeutic drugs.
  • therapeutically effective dose also refers to the amount of the antibody or antigen-binding fragment thereof that is sufficient to result in improvement of the symptoms, such as the amount to treat, cure, prevent or improve related medical conditions or to increase the speed of treatment, cure, prevention or improvement of such conditions.
  • the active ingredient alone is administered to an individual, the therapeutically effective dose refers only to the ingredient.
  • the therapeutically effective dose refers to the total amount of active ingredients contributing to therapeutic effects, regardless of administration in combination, in sequence or at the same time.
  • the effective amount of the therapeutic agent will result in an improvement in the diagnostic criteria or parameter by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.
  • to treat” or “treating” or “treatment” includes 1) therapeutic measures, which cure, alleviate and relieve the symptoms of a diagnosed pathological condition or disease and/or stop the progression of the diagnosed pathological condition or disease, and 2) preventive or prophylactic measures, which prevent and/or slow the development of a pathological condition or disease. Therefore, the subject receiving the treatment includes an individual who has suffered from the disease, an individual who is prone to suffer from the disease, and an individual who wants to prevent the disease.
  • the present invention relates to the treatment of a disease or condition. In some other embodiments, the present invention relates to the prevention of a disease or condition.
  • the “treatment” of a disease or condition refers to the improvement of the disease or condition (i.e., alleviating or preventing or reducing the progression of the disease or at least one of its clinical symptoms) .
  • “treatment” refers to relieving or improving at least one body parameter, including those physical parameters that may not be discernible by the patient.
  • “treatment” refers to the regulation of a disease or condition physically (e.g., stabilization of a discernible symptom) , physiologically (e.g., stabilization of a physical parameter) , or both.
  • prevention of a disease or condition includes inhibition of the occurrence or development of the disease or condition or the symptom of a particular disease or condition.
  • a subject with a family history of cancer is a candidate for a prophylactic regimen.
  • prevention refers to administration of drugs to a subject prior to the onset of conditions or symptoms of cancer, in particular, in a subject at risk of cancer.
  • an individual patient is considered to have been successfully treated if the individual shows one or more of the following: the number of cancer cells is decreased or cancer cells disappear completely; tumor size is decreased; infiltration of cancer cells into peripheral organs is inhibited or absent, including, for example, the spread of cancer cells to soft tissues and bones; tumor metastasis is inhibited or absent; tumor growth is inhibited or absent; one or more symptoms associated with the specific cancer are relieved; incidence and mortality are reduced; the quality of life is improved; the tumor incidence, frequency or tumorigenicity is reduced; the number or frequency of cancer stem cells in tumor is reduced; tumor cells are differentiated into a non-tumorigenic state; or a combination of some of the effects.
  • “Inhibition of tumor growth” refers to any mechanism by which tumor cell growth can be inhibited.
  • tumor cell growth is inhibited by delaying tumor cell proliferation.
  • tumor cell growth is inhibited by stopping tumor cell proliferation.
  • tumor cell growth is inhibited by killing tumor cells.
  • tumor cell growth is inhibited by inducing tumor cell apoptosis.
  • tumor cell growth is inhibited by inducing tumor cell differentiation.
  • tumor cell growth is inhibited by depriving tumor cells of nutrients.
  • tumor cell growth is inhibited by preventing tumor cell migration.
  • tumor cell growth is inhibited by preventing tumor cell invasion.
  • sequence identity refers to the degree of identity of sequences based on one by one nucleotide or amino acid comparing in the comparison window.
  • the “ (percentage) sequence identity” can be calculated as follows: comparing the two optimally aligned sequences in the comparison window, determining the number of positions with the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size) , and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acid base e.g., A, T, C, G, I
  • amino acid residue e.g., Al
  • Optimal alignment for purposes of determining the percentage of sequence identity can be achieved in various ways known in the art, for example, using publicly available computer softwares such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art is able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences or the target sequence area being compared.
  • the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment in accordance with the Kabat numbering scheme.
  • the antibody of the present invention can be produced by any suitable method for producing an antibody.
  • Any suitable form of FGFR2B can be used as an immunogen (antigen) to produce antibodies.
  • any FGFR2B variant or fragment thereof can be used as an immunogen.
  • hybridoma cells producing murine monoclonal anti-human FGFR2B antibodies may be produced by methods well-known in the art. These methods include but are not limited to the hybridoma technique originally developed by Kohler et al., (1975) (Nature 256: 495-497) .
  • mouse spleen cells are isolated and fused with mouse myeloma cell line by PEG or electrofusion.
  • hybridoma cells secreting an antibody with a FGFR2B binding activity are screened.
  • the DNA sequences of the immunoglobulin variable regions from hybridoma cells of the present invention can be detected by the method based on a degenerate primer PCR.
  • Antibodies from rodents may cause undesired antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use causes an immune response against therapeutic antibodies in humans. This kind of immune response will at least lead to the loss of therapeutic efficacy, and in severe cases, lead to potentially lethal allergic reaction.
  • One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which a mouse variable region is fused with a human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43) . However, the retention of intact rodent variable regions in chimeric antibodies may still cause harmful immunogenicity in patients.
  • CDR from a rodent variable region into a human framework has been used to further minimize a rodent sequence.
  • murine CDR regions can be inserted into a human germline framework using a method known in the art. See Winter et al., U.S. Patent No. 5,225,539 and Queen et al., U.S. patent US 5,530,101; US 5,585,089; US 5,693,762 and US 6,180,370.
  • the accurate amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined by using any one of many well known schemes such as Kabat, Chothia, AbM, Contact or North. It should be noted that the boundary of CDR of the variable region of the same antibody obtained by different definition systems may be different. That is, the CDR sequences of the variable region of the same antibody defined by different assignment systems are different. Therefore, when it comes to defining an antibody with a specific CDR sequence as defined in the present invention, the scope of the antibody also covers an antibody, the variable region sequence of which comprises the specific CDR sequence, but with a designated CDR boundary different from the one specified in the present invention for the specific CDR sequence, due to the application of different schemes (such as different definition systems or combinations thereof) .
  • Antibodies with different specificities have different binding sites for different antigens.
  • CDR is different from antibody to antibody, only a limited number of amino acid positions in CDR are directly involved in antigen binding.
  • the minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM and North schemes to provide a “smallest binding unit” for antigen binding.
  • the smallest binding unit can be a subset of CDR residues.
  • the residues of the rest of the CDR sequence can be determined according to the structure and protein folding of the antibody. Therefore, the present invention also contemplates any variants of the CDR presented herein.
  • the amino acid residues of the smallest binding unit remain unchanged, while the other CDR residues as defined according to Kabat or IMGT can be replaced by conservative amino acid residues.
  • the present invention also provides antibodies with altered effector function (s) .
  • effector functions refer to those biological activities attributable to Fc region of an antibody, which vary with the antibody class.
  • the effector functions of antibodies include, for example, but are not limited to: C1q binding and complement dependent cytotoxicity (CDC) ; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; recruitment of immune cells; and antibody cross-linking mediated by the binding of a Fc region to a FcR receptor on a cell surface.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • phagocytosis phagocytosis
  • recruitment of immune cells and antibody cross-linking mediated by the binding of a Fc region to a FcR receptor on a cell surface.
  • a suitable antibody Fc region sequence can be selected according to needs, such as whether it is desired to recruit the immune system to kill target cells, or induce the cross-linking of the antibody by interacting with FcR.
  • the Fc region of the antibody can be selected or further modified to provide enhanced binding to activated Fc ⁇ R receptors and/or complement to promote, for example, ADCC or CDC effector functions.
  • an Fc region can be selected or mutated so that the antibody containing it selectively binds to one or more Fc receptors, while the binding to another one or more FcRs is reduced or eliminated, so as to achieve the adjustment of antibody effector functions, such as enhancing the cross-linking of antibodies while changing the intensity of ADCC activity.
  • one or more amino acid modifications can be introduced into the Fc region of the antibody provided by the present invention to produce Fc region variants.
  • the Fc region variant can comprise a human Fc region sequence (such as the Fc region of human IgG1, IgG2, IgG3, or IgG4) comprising amino acid modifications (such as substitutions) at one or more amino acid positions.
  • a number of modifications to human IgG1 to enhance or reduce its binding to Fc ⁇ R and enhance or reduce the corresponding function are summarized in article of Bruhns and published in Immunol Rev. 2015 Nov; 268 (1) : 25-51, page 44.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three selected from HCDR1, HCDR2 and HCDR3 of a heavy chain variable region (VH) , wherein the amino acid sequence of the VH is as set forth in any one of SEQ ID NOs: 43-49.
  • VH heavy chain variable region
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three selected from LCDR1, LCDR2 and LCDR3 of a light chain variable region (VL) , wherein the amino acid sequence of the VL is as set forth in any one of SEQ ID NOs: 50-56.
  • VL light chain variable region
  • the present invention provides an isolated FGFR2B antibody or antigen-binding fragment thereof, which comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the VH is as set forth in any one of SEQ ID NOs: 43-49, and the amino acid sequence of the VL is as set forth in any one of SEQ ID NOs: 50-56.
  • VH heavy chain variable region
  • VL light chain variable region
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3; wherein the VH and VL are selected from:
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 43
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 50
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 46
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 53
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 48
  • VL comprising the amino acid sequence as set forth in SEQ ID NO: 55
  • VH comprising the amino acid sequence as set forth in SEQ ID NO: 49 and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 56.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of heavy chain complementary determining regions (HCDRs) , HCDR1, HCDR2 and HCDR3, wherein:
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 1 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 or 7 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 or 8 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 9 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 10 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 11 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 12 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 13 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 17 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 18 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith; or
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 19 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 21 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith.
  • the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of light chain complementary determining regions (LCDRs) , LCDR1, LCDR2 and LCDR3, wherein:
  • LCDRs light chain complementary determining regions
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 22 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 23 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 24 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 25 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 26 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 27 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 28 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 29 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 30 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 31 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 32 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 33 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 34 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 35 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 36 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith;
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 37 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 38 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 39 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith; or
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 40 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 41 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 42 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain complementary determining regions (HCDRs) , HCDR1, HCDR2 and HCDR3, and light chain complementary determining region s (LCDRs) , LCDR1, LCDR2 and LCDR3, wherein:
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 1 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 22 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 23 and optionally further
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 25 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 26 and optionally further
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 7 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 8 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 28 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 29 and optionally further
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 9 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 10 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 11 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 31 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 32 and optionally further
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 12 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 13 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 14 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 34 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 35 and optional
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 16 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 17 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 18 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 37 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 38 and optionally further
  • the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 19 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 20 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the HCDR3 comprises the amino acid sequence SEQ ID NO: 21 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 40 and optionally further has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared therewith
  • the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 41 and optionally further has at least 1
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any of the combinations listed in Table 1 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) , wherein the VH comprises the amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 43-49.
  • VH heavy chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain variable region (VL) , wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 50-56.
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 50.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 44, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 51.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 45, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 52.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 46, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 53.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 47, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 54.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 48, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 55.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 49, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 56.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) as set forth in any of the combinations listed in Table 2 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 57
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 64.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 58
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 65.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 59
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 66
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 60
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 62
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 69.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC)
  • the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 63
  • the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 70.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a Fc region.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc ⁇ receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) .
  • said one or more amino acids are mutated at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439, numbered according to the EU numbering system.
  • said one or more amino acids are mutated at the following positions: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339 and P396, numbered according to the EU numbering system.
  • said one or more amino acids which are mutated are selected from the following substitutions: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L, numbered according to the EU numbering system.
  • said amino acids which are mutated comprise the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) , numbered according to the EU numbering system.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises the Fc region with the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) , numbered according to the EU numbering system.
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC) as set forth in any of the combinations listed in Table 3 below:
  • the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an constant region, which is hypofucosylated or afucosylated.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes of the present invention occur in regions outside CDRs (for example, in FRs) . More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region. In some embodiments, the amino acid changes occur in a heavy chain constant region and/or a light chain constant region.
  • the antibodies of the present invention comprising amino acid changes have comparable or similar properties to the specific antibodies disclosed herein.
  • the anti-FGFR2B antibody of the present invention includes post-translational modifications to CDRs, light chain variable regions, heavy chain variable regions, light chains, or heavy chains.
  • the anti-FGFR2B antibody provided by the present invention is a full-length antibody, a single-domain antibody such as a VHH, a Fab, a Fab’, a Fab’-SH, a (Fab’) 2, a single-chain antibody such as a scFv, a Fv, a dAb (domain antibody) or a bis (multi) specific antibody.
  • a single-domain antibody such as a VHH, a Fab, a Fab’, a Fab’-SH, a (Fab’) 2
  • a single-chain antibody such as a scFv, a Fv, a dAb (domain antibody) or a bis (multi) specific antibody.
  • the anti-FGFR2B antibody provided by the present invention is an antibody in the form of any IgG isotype, such as an antibody in the form of IgG1, IgG2, IgG3 or IgG4.
  • the antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody.
  • the addition or deletion of the glycosylation sites of an antibody can be conveniently achieved by changing the amino acid sequence so as to produce or remove one or more glycosylation sites.
  • Glycosylation can be changed, for example, to increase affinity of the antibody for the “antigen” .
  • Such carbohydrate modification can be accomplished, for example, by changing one or more glycosylation sites within the antibody sequence.
  • one or more amino acid substitutions can be made, which results in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at this site. This aglycosylation can increase affinity of the antibody for the antigen.
  • cysteine engineered antibody e.g., “thioMAb”
  • cysteine residues of the antibody are substituted with cysteine residues.
  • the antibody provided herein may be further modified to comprise additional non-protein moieties that are known in the art and readily available.
  • a moiety suitable for antibody derivatization includes, but is not limited to, a water soluble polymer.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , ethylene glycol/propylene glycol co-polymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dialkane, poly-1, 3, 6-trialkane, ethylene/maleic anhydride co-polymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol
  • the antibody of the present invention has one or more of the following properties:
  • EC 50 value of less than 100 nM, such as less than 50 nM, such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM or 5 nM, wherein preferably the EC 50 value is measured using FACS assay;
  • the FGFR2B antibody of the present invention is an antagonist antibody, comprising an Fc region that binds to FcR (e.g., Fc ⁇ R) , such as a human IgG1, IgG2, or IgG4 Fc region or a variant thereof (e.g., IgG4 with S228P) , preferably a human IgG1 Fc region or a variant thereof.
  • FcR e.g., Fc ⁇ R
  • the variant preferably has a binding affinity for Fc ⁇ R comparable to or stronger than that of the parent Fc region (e.g., a native-sequence Fc region) .
  • the antibody comprises a human IgG1 or IgG4 Fc region sequence identical to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 74 or 75, or comprises a human IgG1 or IgG4 Fc region variant having at least 95%, 96%, 97%or 99%identity to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 74 or 75, or having no more than 10, 5 or 1-3 amino acid changes relative to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 74 or 75.
  • the antibody comprises an Fc region variant, wherein, for example, relative to a parent Fc region (e.g., a native-sequence Fc region) , the binding affinity of the Fc region variant for FC ⁇ R is enhanced by at least 10%, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%or higher.
  • the antibody of the present invention comprising an Fc region variant has enhanced Fc ⁇ R-mediated effector functions relative to the corresponding antibody comprising a parent Fc region (e.g., a native-sequence Fc region) .
  • the Fc region of the antibody comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) .
  • the antibody comprises a human IgG1 Fc region sequence identity to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 21, or comprises a human IgG1 Fc region variant having at least 95%, 96%, 97%, 98%or 99%identity to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 21, or having no more than 10, 5 or 1-3 amino acid changes relative to the Fc region sequence of the constant region sequence as set forth in SEQ ID NO: 21, and comprises a mutation (s) that reduces the binding affinity of the Fc region to Fc ⁇ R, preferably L235V, F243L, R292P, Y300L, and P396L substitutions.
  • the FGFR2B antagonist antibody of the present invention has one or more of the following properties:
  • binding to human FGFR2B with a high affinity such as with a K D value of less than 10 nM, more preferably less than 5 nM, wherein preferably the K D value is measured using surface plasmon resonance assay;
  • binding to human FGFR2B expressed on the surface of cells with a high affinity, such as with a EC50 value of less than 10 nM, more preferably less than 5 nM, wherein preferably the EC50 value is measured using FACS assay;
  • the present invention relates to a host cell comprising one or more expression vectors and a method for producing any antibody or antigen-binding fragment thereof of the present invention, wherein the method comprises culturing the host cell, purifying and recovering the antibody or antigen-binding fragment.
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) .
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) .
  • the present invention provides a nucleic acid encoding any of the above anti-FGFR2B antibodies or antigen-binding fragments thereof.
  • the present invention provides a nucleic acid encoding a segment comprising a heavy chain, a light chain, a variable region or a complementarity determining region described herein.
  • the nucleic acid encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 17 or 18.
  • the nucleic acid encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 19 or 20.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector.
  • the choice of an expression vector depends on the host cell in which the vector is intended to be expressed.
  • an expression vector comprises a promoter and other regulatory sequence (e.g., enhancer) operably linked to a nucleic acid encoding an anti-FGFR2B antibody or antigen-binding fragment thereof.
  • the expression vector further comprises a sequence encoding the antibody constant region.
  • the present invention provides host cells for expressing the recombinant antibodies of the present invention, including prokaryotic or eukaryotic cells.
  • Escherichia coli is a prokaryotic host that can be used to clone and express the nucleic acid of the present invention.
  • Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas.
  • expression vectors can also be prepared, which generally comprise expression control sequences (for example, an origin of replication) that are compatible with the host cells.
  • mammalian host cells are used to express and produce the anti-FGFR2B antibody polypeptides of the present invention.
  • they may be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines with exogenous expression vectors, including normal human cells, or immortalized animal or human cells.
  • suitable host cell lines capable of secreting intact immunoglobulin have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells and hybridomas.
  • the present invention provides a method for preparing an anti-FGFR2B antibody, wherein the method comprises introducing an expression vector into a mammalian host cell, and allowing the antibody to be expressed in the host cell by culturing the host cell for a sufficient period of time, or more preferably, secreting the antibody into the medium in which the host cell is grown to produce the antibody.
  • Standard protein purification methods can be used to recover antibodies from the culture medium.
  • the antibody molecule prepared as described herein can be purified by known available techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to a person skilled in the art.
  • the purity of the antibody molecule of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.
  • Antibodies expressed by different cell lines or expressed in transgenic animals are likely to have different glycosylation types from each other.
  • all antibodies encoding by the nucleic acids provided herein or comprising the amino acid sequences provided herein are parts of the present invention, regardless of the glycosylation types of the antibodies.
  • the present invention relates to immunoconjugates comprising any anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention, conjugated to a payload.
  • the immunoconjugates comprises one or more drugs (e.g., cytotoxic agents, small molecule compounds, immune stimulators, and the like) or markers as the payload.
  • the physical/chemical properties and/or biological activities of the anti-FGFR2B antibody provided herein can be identified, screened, or characterized by a variety of assays known in the art.
  • the antigen-binding activity of the antibody of the present invention is tested, for example, by a known method such as ELISA and Western blot. Methods known in the art can be used to determine binding to FGFR2B, and exemplary methods are disclosed herein.
  • the present invention also provides an assay method for identifying anti-FGFR2B antibodies with a desired biological activity.
  • the biological activities can include, for example, binding to FGFR2B (for example, binding to human FGFR2B) , increasing FGFR2B-mediated signal transduction (e.g., increasing NF ⁇ B-mediated transcription) , enhancing T effector cell function (e.g., by increasing effector T cell proliferation and/or increasing cytokine production (e.g., gamma interferon) by effector T cells) , etc.
  • Antibodies having such biological activities in vivo and/or in vitro are also provided.
  • the antibodies of the present invention are tested for such biological activities.
  • Cells for use in any of the above-mentioned in vitro assay methods include cell lines that naturally express FGFR2B or are engineered to express FGFR2B, such as tumor cell lines. Such cells also include cell lines that normally do not express FGFR2B and which which are transfected with DNA encoding the FGFR2B to express FGFR2B.
  • immunoconjugates or immune fusions of the present invention can be used, in place of or in addition to the anti-FGFR2B antibody, to perform any of the above-mentioned assay methods.
  • the pharmaceutical composition of the present invention may include the antibody of the present invention and a pharmaceutically acceptable auxiliary substance.
  • the pharmaceutical composition of the present invention can be included in a pharmaceutical kit.
  • the pharmaceutical composition of the present invention can be included in a kit, such as a diagnostic kit.
  • a “pharmaceutical carrier” includes any and all solvents, dispersion medium, isotonic agents, absorption delaying agents, etc., that are physiologically compatible.
  • Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. It is also possible to use saline solutions, aqueous dextrose and glycerol solutions as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, diol, water, ethanol, etc.
  • the composition may also contain a small amount of a wetting agent or an emulsifier, or a pH buffering agent.
  • compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release agents, etc.
  • Oral formulation can comprise standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, etc.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more monoclonal antibodies binding to FGFR2B or antigen-binding fragments thereof, or nucleic acids, vectors or host cells, or immunoconjugates or immune fusions.
  • the anti-FGFR2B antibody provided by the present invention, or antigen-binding fragment thereof, the nucleic acid, the vector or the host cell thereof, or the immunoconjugate, or the immune fusion in a pharmaceutical composition can be formulated with suitable pharmaceutical carriers, excipients and another co-administrated agent suitable used in a pharmaceutical preparation, so as to provide improved transfer, delivery, tolerance, etc.
  • the pharmaceutical preparation comprising the anti-FGFR2B antibody described herein can be prepared by mixing the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention having the desired degree of purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of aqueous solutions or lyophilized preparations.
  • exemplary lyophilized antibody preparations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody preparations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter describing a preparation comprising a histidine-acetate buffering agent.
  • compositions or preparations of the present invention may also comprise one or more other active ingredients that are required for the treatment of specific diseases, preferably those active ingredients with complementary activities that do not adversely affect each other.
  • other therapeutic agents are also included.
  • the other therapeutic agents are chemotherapeutic agents, radio therapeutic agents, cytokines, vaccines, other antibodies, immunomodulators or other biomacromolecular drugs.
  • the pharmaceutical composition of the present invention may also comprise a nucleic acid encoding the anti-FGFR2B antibody or an antigen-binding fragment thereof.
  • the present invention provides a method for preventing, diagnosing or treating FGFR2B-related diseases or conditions.
  • the method comprises administering to a patient in need thereof an effective amount of the anti-FGFR2B antibody, or an antigen-binding fragment thereof, or an immunoconjugate or an immune fusion or a pharmaceutical composition comprising same, or a nucleic acid, a vector or a host cell described herein.
  • the present invention provides the use of an anti-FGFR2B antibody or antigen-binding fragment thereof, or an immunoconjugate or an immune fusion or a pharmaceutical composition comprising same, in the manufacture or preparation of drugs for the prevention or treatment of FGFR2B-related diseases or conditions in subjects.
  • the anti-FGFR2B antibodies provided by the present invention, and antigen-binding fragments thereof, and a pharmaceutical composition comprising same can be used as a therapeutic agent to prevent or treat FGFR2B-related diseases or conditions in a subject.
  • the anti-FGFR2B antibodies and antigen-binding fragments thereof disclosed in the present invention, and pharmaceutical compositions or immunoconjugates or immune fusions comprising same, or the nucleic acids, vectors or host cells described herein can be administered.
  • the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent or procedure.
  • the therapeutic agents are, for example, chemotherapeutic agents, radio therapeutic agents, cytokines, vaccines, other antibodies, immunomodulators or other biomacromolecular drugs.
  • the therapeutic procedures include surgery; and radiation therapy, local irradiation or focus irradiation, etc.
  • the above-mentioned combination therapy includes combined administration (in which two or more therapeutic agents are contained in the same or separate preparations) and separate administration, wherein the administration of the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention may occur prior to, simultaneously with, or after administration of additional therapeutic agent and/or adjuvant and/or procedure.
  • the FGFR2B-related diseases or conditions of the present invention refer to diseases or conditions related to abnormal FGFR2B expression, activity and/or signal transduction in a subject, including but not limited to cancers, inflammation and autoimmune diseases.
  • the nucleic acid (level or content) encoding FGFR2B is increased, or FGFR2B expression is increased, or protein level or activity of FGFR2B is increased, or signal transduction mediated by FGFR2B is increased.
  • the nucleic acid (level or content) encoding FGFR2B is reduced, or FGFR2B expression is decreased, or protein level or activity of FGFR2B is decreased, or signal transduction mediated by FGFR2B is decreased.
  • the treatment of the diseases or conditions will benefit from inhibiting FGFR2B at the nucleic acid or protein level, or from blocking the binding of FGFR2B to its ligand or inhibiting FGFR2B-mediated signal transduction.
  • the treatment of the diseases or conditions will benefit from increasing FGFR2B at the nucleic acid or protein level, or benefit from enhancing FGFR2B-mediated signal transduction.
  • FGFR2B-related diseases or conditions are cancers.
  • the cancers include, but are not limited to, solid tumors, breast cancer, urothelial cancer, melanoma, kidney cancer, ovarian cancer, head and neck cancer, stomach cancer, liver cancer, small-cell lung cancer, non-small cell lung cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, prostate cancer, lymphatic leukemia and sarcoma.
  • the antibody for preventing, diagnosing or treating FGFR2B-related cancers is an FGFR2B agonist.
  • FGFR2B-related diseases or conditions are inflammation and/or autoimmune diseases.
  • FGFR2B-related inflammation and/or autoimmune diseases are selected from idiopathic dermatitis, rheumatoid arthritis, asthma (e.g., allergic asthma) , COPD, autoimmune uveitis, multiple sclerosis, lupus (such as systemic lupus erythematosus) , ulcerative colitis, scleroderma, and graft-versus-host disease (GVHD) .
  • the antibody for treating or preventing FGFR2B-related inflammation and/or autoimmune diseases is an FGFR2B antagonist.
  • the subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or having a risk of suffering from a disease described herein) .
  • the subject suffers from or has a risk of suffering from a disease described herein (e.g., cancer) .
  • the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy.
  • the antibody or antigen-binding fragment (as well as the immunoconjugates, compositions, pharmaceutical compositions, preparations, combination of medicaments and kits comprising the same) of the present invention may be administered in any suitable manner, including oral, parenteral, intrapulmonary and intranasal administration, and, if topical treatment is needed, it can be administered intralesionally.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term.
  • Various administration regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.
  • the antibody or antigen-binding fragment (as well as the immunoconjugates, compositions, pharmaceutical compositions, preparations, combination of medicaments and kits comprising the same) of the present invention will be formulated and administered in a manner consistent with good medical practice.
  • the factors considered in this case include the specific disease being treated, the specific mammal being treated, the clinical condition of an individual patient, the cause of a disease, the delivery site of a drug, the administration mode, the dosing schedule, and other factors known to practitioners.
  • the antibody is formulated with one or more agents currently used to prevent or treat the disease. The effective amount of these other agents depends on the amount of antibody present in the preparation, the condition to be treated, or the therapeutic mode, and other factors discussed above.
  • the antibody or antigen-binding fragment (as well as the immunoconjugates, compositions, pharmaceutical compositions, preparations, combination of medicaments and kits comprising the same) of the present invention when used alone or in combination with one or more additional therapeutic agents will be administrated in a suitable dosage depending on the type of diseases to be treated, the type of antibodies, the severity and course of the disease, whether the antibody is for the purpose of prevention or treatment, the previous treatment, the patient’s clinical history and response to the antibody and the judgment of the attending physician.
  • the antibody is appropriately administered to the patient at one time or over a series of treatments.
  • any anti-FGFR2B antibody or antigen-binding fragment thereof provided herein can be used to detect the presence of FGFR2B in a biological sample.
  • detection when used herein includes quantitative or qualitative detection.
  • the biological sample is blood, serum, or other liquid samples of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from hyperproliferative or cancerous lesion related lesions.
  • the antibody or antigen-binding fragment thereof of the present invention can be used to diagnose FGFR2B-related diseases or conditions, such as cancers, for example to evaluate (e.g., monitor) the treatment or progression of the diseases described herein, and diagnosis and/or staging thereof in an individual.
  • a labeled anti-FGFR2B antibody or antigen-binding fragment thereof is provided.
  • Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) , and moieties that are indirectly detectable, such as enzymes or ligands, for example, by enzymatic reactions or intermolecular interactions.
  • a kit for diagnosing FGFR2B-related diseases which kit comprises the antibody or antigen-binding fragment thereof of the present invention.
  • the sample is obtained prior to treatment with the anti-FGFR2B antibody or antigen-binding fragment thereof. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.
  • the present invention includes any combinations of specific embodiments described herein. It should be understood that although the specific content and examples are described to illustrate the preferred embodiments of the present invention, these are merely illustrative and used as examples. The present invention further covers embodiments modified on the basis of the preferred embodiments of the present invention that are obvious to a person skilled in the art. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety.
  • Anti-FGFR2b monoclonal antibodies were generated by conventional hybridoma fusion technology.
  • the mAbs with FGFR2b binding specificity in enzyme-linked immunosorbent assay (ELISA) were selected for further characterization.
  • the DNA encoding full-length human FGFR2b isoform (Accession Number in Uniprot P21802-2) was inserted into pcDNA3.1 (+) vector (Synbio Technologies) by seamless cloning, the construct was confirmed by DNA sequencing. Large-scale DNA was prepared by using Plasmid Maxiprep System from Qiagen for immunizing.
  • mice were immunized with 100 ⁇ g pcDNA3.1-human FGFR2b vector prepared above via intramuscular injection, and boosted with 10 ⁇ g recombinant human FGFR2b_extracelluar domain (ECD) fused with a human Fc moiety at their C-termini (Sino Biological, human FGFR2b-Fc, Cat: 16485-H02H) via intramuscular injection three times.
  • the antibody immune response was monitored by a FGFR2b-specific ELISA.
  • Ten days after serum screening, the mice with the highest anti-FGFR2b antibody serum titers were boosted via intravenous injection with 10 ⁇ g of FGFR2b-Fc.
  • FGFR2b-specific antibodies were selected and the supernatants of the murine hybridoma clones, and the hybridoma cell lines that produce FGFR2b-specific antibodies, which only bind to FGFR2b not to FGFR2c, were selected.
  • ELISA and flow cytometry assay indicated that several antibodies, including 55D6, 38D4, 39C2, 35B11, 52E2, 61B7 and 30C7, exhibited strong affinity towards human FGFR2-IIIb and showed no detectable binding to the human FGFR2c. No binding of these antibodies to human FGFR1, FGFR3c FGFR3b or FGFR4 was detected. Interestingly, all these FGFR2b-specific antibodies blocked the interaction between FGF7 and FGFR2b as determined by ELISA.
  • the lead antibodies with desired profile were selected for V-gene cloning.
  • the sequences of the murine anti-human FGFR2b light chain and heavy chain variable regions were obtained by the polymerase chain reaction (PCR) amplification technique operated according to Wang, Z. et al., 2000 (Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′to 5′exonuclease activity. J. Immunol. Methods 233, 167–177) .
  • RNA from the positive hybridoma cell was isolated by using the MiniBest Universal RNA Extraction Kit (TaKaRa) , and the cDNA was synthesized by using 1st Strand cDNA Synthesis Kit (TaKaRa) with Oligo (dT) primer.
  • the variable regions of mouse IgG gene were amplified by PCR by using primers of different isotypes for heavy chain variable region and Kappa chain primers for light chain variable region.
  • the PCR products were subcloned into a TA cloning vector. For each variable gene construct, more than 10 single colonies were used for DNA sequencing by Synbio Technologies (Suzhou, China) .
  • the amino acid sequences of Vh and Vk were derived from the DNA sequencing results.
  • Seven antibodies including 55D6, 38D4, 39C2, 35B11, 52E2, 61B7 and 30C7, were selected as lead antibodies to generate chimeric antibodies with human IgG1 as constant region, and their SEQ were listed in the Table 4.
  • cDNAs of the variable regions of heavy chain and light chain were synthesized and fused with the sequences of the constant region of human IgG1with and human kappa.
  • the Fc domain of these chimeric mAb was engineered with L235V, F243L, R292P, Y300L, and P396L (VLPYLL) .
  • the signal peptide sequences (MEFGLSWVFLVALFRGVQC and MDMRVPAQLLGLLLLWLRGARC) were added at N-terminal of heavy chain and light chain, respectively.
  • the resulting chimeric antibody genes were cloned into an expression vector. Large-scale DNA was prepared by using Plasmid Maxi-prep System from Qiagen.
  • ExpiFectamine TM CHO Reagent from Invitrogen according to the manufacturer’s protocol.
  • ExpiCHO-S cells in ExpiCHO Expression Medium at 5-6x10 6 cell /ml were transfected with equal amount of heavy chain vector and light chain vector DNA at a final concentration of 0.8 ⁇ g/ml by using ExpiFectamine TM CHO Reagent.
  • the plasmid DNA or ExpiFectamine TM CHO Reagent were diluted with cold OptiPRO TM medium, and then mixed by swirling tube and/or inversion.
  • the ExpiFectamine TM CHO/plasmid DNA mixtures were incubated at room temperature for 1-5 minutes, and then slowly transferred into a shaker flask with cells.
  • the transfecting cells were incubated at 37°C with a humidified atmosphere of 5%CO 2 on an orbital shaker (125rpm shaking speed) .
  • 18 to 22 hours after transfection ExpiCHO TM Feed was added, and the conditioned medium was harvested on day 10.
  • the supernatant was centrifuged at 4,000 rpm for 20 minutes and then filtered through 0.22um filtration capsule to remove cell debris. The filtered supernatant was loaded onto a pre-equilibrated Protein-A affinity column.
  • Protein-A resin was washed with equilibration buffer (PBS) , and 25 mM citrate (pH3.5) was then used to elute the antibody.
  • the purified antibody solution was adjusted to pH 6.0-7.0 by using 1M Tris-base (pH 9.0) .
  • the endotoxin was controlled below 1EU/mg.
  • the purified antibody was characterized by SDS-PAGE.
  • Bema_VLPYLL An anti-human FGFR2b specific antibody, Bema_VLPYLL, (VH and VL sequences are from patent WO2015/017600A1, FIVE PRIME THERAPEUTICS) , was also expressed as positive control, and the Fc is human IgG1_VLPYLL.
  • Epitope characterization of chimeric mAbs and Bema_VLPYLL were performed by using BLI.
  • 100 nM human FGFR2b-biotion protein (Katcus, Cat : FGF-HM4ABB) was loaded onto SA biosensor. After washing step, the biosensor was dipped into 1 st antibodies solution to associate 1st antibody for 90 s, the signal of bind is observed obsviously.
  • the bisosensor was washed with KD buffer, followed by incubate with the 2 nd antibody, and this signal can indicate whether the epitopes of the two antibodies compete.
  • the ADCC reporter bioassay a bioluminescent reporter-gene assay for quantifying the biological activity of the antibody via Fc ⁇ RIIIa-mediated pathway activation, was used to evaluate the ADCC activity of FGFR2b antibodies.
  • the Jurkat-NFAT Luc-Fc ⁇ RIIIa-V176 cells (Jurkat cells (Shanghai Institutes for Biological Sciences, Cat#SCSP-513) were transfected with PGL4.30-Luc /NFAT-RE/Hygro plasmid (Promega) , and then screened with hygromycin.
  • the cell line Jurkat-nfat-Luc was stably expressed.
  • the sequence of Fc ⁇ RIIIA-V176 (SEQ ID NO: 37) was constructed into the vector pVitro-neo (InvivoGen) to obtain the plasmid pVitro-neo-pcDNa3.1-Fc ⁇ RIIIA-V176.
  • the obtained cell lines were transfected with pVitro-neo-Fc ⁇ riiA-V176 plasmid, and the stably expressed cell line Jurkat-nFAT-Luc-Fc ⁇ RIIIA-V176 was screened with antibiotic G418.
  • Jurkat cells (Shanghai Institutes for Biological Sciences, Cat# SCSP-513) were transfected with PGL4.30-Luc /NFAT-RE/Hygro plasmid (Promega) , and then screened with hygromycin.
  • the cell line Jurkat-nfat-Luc was stably expressed.
  • the sequence of Fc ⁇ RIIIA-V176 (SEQ ID NO: 37) was constructed into the vector pVitro-neo (InvivoGen) to obtain the plasmid pVitro-neo-pcDNa3.1-Fc ⁇ RIIIA-V176.
  • the obtained cell lines were transfected with pVitro-neo-Fc ⁇ RIIIA-V176 plasmid, and the stably expressed cell line Jurkat-nFAT-Luc-Fc ⁇ RIIIA-V176 was screened with antibiotic G418. ) were used as the effector cells.
  • the effector cells were maintained in RPMI-1640 medium supplemented with 10%FBS, 100 ⁇ g/mL hygromycin, 250 ⁇ g/mL G418, 1 mM sodium pyruvate and 0.1 mM MEM non-essential amino acids.
  • Human FGFR2 gene-amplificated cancer cell lines (KATO-III cells and KYSE-180 cells) were used as the target cells in the ADCC reporter bioassay.
  • KATO-III a gastric cancer cell line
  • KYSE-180 a esophageal squamous cell carcinoma cell line
  • the target cells were plated in a 96-well white bottom assay plate at 20,000 cells per well, followed by incubation with serial dilutions of antibodies.
  • the ADCC reporter bioassay here was used for confirming the ADCC activity of the chimeric mAbs respectively.
  • 55D6, 38D4, 39C2, 35B11, 52E2, 61B7 and 30C7 presented effective ADCC activities against KATO-III cells (FGFR2b amplification and high overexpression) .
  • a humanization process enabling the generation of highly optimized mAb consists of five steps.
  • the first step is to select acceptor human frameworks (FR) appropriate for their antigen binding activity, immunogenicity, expression, stability, and pharmacokinetics, and the selected germlines include IGHV1-46*01 (for VHs of 55D6, 39C2 and 38D4) , IGHV1-69-2*02 (for VH of 35B11) , IGHV1-2*02 (for VH of 52E2) , IGKV3-11*01 (for 55D6 VL) , IGKV1-39*01 (for VLs of 39C2, 38D4 and 52E2) , and IGKV2-30*02 (for 35B11 VL) .
  • acceptor human frameworks FR
  • the second step is to generate CDR-grafted mAb by graft the CDRs of murine antibody to the FRs of human antibody descripted in the first step, then simulate three-dimensional (3D) Fv structure model of parental murine antibody and the CDR-grafted mAb, finnaly, according to the guidance of the structure, the amino acids in the frame region close to the CDR region, located at the interface of the VH and VL and inside the structure, are backmutated into the corresponding amino acids in the FR of murine antibody.
  • the third step is to prepare expression vectors for multiple versions of humanized antibodies.
  • the fourth step is to express and purify the humanized antibodies.
  • the last step is to multidimensionally evaluate the humanized antibodies.
  • humanized antibodies of 55D6, 38D4, 39C2, 35B11, 52E2, and named as Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 accordingly.
  • the constant region of humanized mAbs are human IgG1 and Kappa.
  • these humanized antibodies were expressed as low-fucose version by adding fucose analog 2-Deoxy-2-fluoro-L-fucose (Biosynth, W-203582) in the expression medium.
  • Bema constant region is human IgG1 and kappa
  • Bema constant region is human IgG1 and kappa
  • the VH and VL sequences of humanized mAbs are list in the following table.
  • Dissociation of antigen for the measurement of koff 180s.
  • concentrations of antigen diluted with 1 ⁇ Kinetics Buffer were used, including 100 nM, 33.3 nM, 11.1 nM and 0 nM. Baseline and dissociation steps were carried out in 1 ⁇ Kinetics Buffer. The ratio of koff to kon determined the KD.
  • the Biosensors were regenerated for 5s in Regeneration Buffer (10 mM Glycine-HCL, pH 1.7) , followed by neutralization for 5s in Neutralization Buffer (1 ⁇ PBS, pH 7.4, 0.02%Tween 20, 0.1%BSA) . This process repeated 3 times.
  • Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 binds human FGFR2b with high affinity, and the KD of these humanized mAbs are better than that of Bema.
  • Example 7 Binding specificity of humanized mAbs to human FGFR2b
  • Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 specifically binds to FGFR2b, and they don’ t bind to any other FGFR family members (See figure 3) .
  • Example 8 Species cross-reactivity of humanized mAbs
  • Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 not only bind to human FGFR2b, but also cross-bind to mouse, rat and cynomologus FGFR2b.
  • mouse models can be used to evaluate the efficacy of these antibodies, and rats and monkeys can be used to evaluate the preclinical toxicology of these humanized antibodies (See figure 4) .
  • hFGFR2b-Fc (Sino Biological, cat#16485-H02H, lot#LC13JL2910) was coated to the ELISA plate and incubated at 4°C overnight. The plate was washed three times and was blocked at 37°Cfor 1h.
  • the plate was washed and 50 ⁇ l diluted humanized mAb and 50 ⁇ l biotin- labeled FGF7-Fc (prepared in-house, FGF7-Fc was expressed and purified, then it was biotin-labeled according to the method of EZ-Link TM Sulfo-NHS-LC-Biotin Kit, ThermoFisher, Catalog number: A39257) added into each well, and incubate at 37°Cfor 1h. The plates were washed 6 times followed by addition of 100 ul/well of 1: 5000 HRP-conjugated streptavidin (Abcam, cat#ab7403, lot#GR3259274-11) .
  • the blockade of all the 5 humanized mAbs to the interaction between FGFR2b and FGF10 was determined by a BLI method. Breifly, FGFR2b-biotion was loaded onto SA biosensor, then incubate with tested antibodies or isotype control, after that the sensor was dipped into the FGF10 in solution. The 5 humanized mAbs also block the interaction between FGFR2b and FGF10, the data is not shown here.
  • Example 10 Flow cytometry binding analysis of FGFR2b antibodies on HEK293T cells stably expressing human FGFR2b and FGFR2c
  • the HEK293T cells (Cell Bank of Chinese Academy of Sciences, Cat: GNHu44) were stably transfected with the expression vectors that respectively express human FGFR2-IIIb (293T-hFGFR2b) (Accession Number in Uniprot, P21802-3) and human FGFR2-IIIc (293T-hFGFR2c) (Accession Number in Uniprot, P21802) .
  • 293T-hFGFR2b/2c cells were maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum (FBS) , 1 ⁇ Penicillin-streptomycin, and 1 ⁇ g/mL puromycin.
  • FBS fetal bovine serum
  • the cells were washed and seeded in 96-well plates at 5 ⁇ 10 4 cells per well in the cold washing buffer (1 ⁇ PBS containing 2%FBS) , and then incubated with several antibody dilutions at 4°C for 60 min. After twice washing by the cold washing buffer, 0.5 ⁇ g/mL FITC conjugated goat pAb anti-human IgG1 (Abcam, cat#ab98623, lot#GR3319406) was immediately added to the cells with, and incubated at 4°C for 30 min. After twice washing by the cold washing buffer, cells were re-suspended with 120 ⁇ L cold PBS and analyzed with flow cytometry.
  • MFI median fluorescence intensity
  • Example 11 ADCC reporter bioassay of humanized mAbs
  • the ADCC reporter bioassay descripted in example 3 was also used to determine the ADCC activity humanized FGFR2b antibodies. All the humanized FGFR2b antibodies showed strong ADCC activity against KATO-III cells (ATCC, Cat#HTB-103) and KYSE-180 cells (FGFR2b low expression) (Cobioer, Cat: CBP60456) , with higher maximum RLU value of Hu52E2 than Bema and other FGFR2b antibodies (See figure 7) .
  • Example 12 Inhibition of FGF7-induced proliferation of MCF7 cells by FGFR2b antibodies
  • MCF7 cells (Cobioer, Cat: CBP60380) , derived from the patient suffered triple negative breast cancer (TNBC) , were maintained by EMEM culture medium containing 10%FBS and 1 ⁇ Penicillin-streptomycin. The cells were seeded in 96-well plates at 10,000 cells per well in complete growth medium and cultured overnight to allow for adherence. Cells were then incubated in serum-free medium for 24 hours, followed by treatment with 30 ⁇ g/mL of human IgG isotype or several FGFR2b antibodies in the absence or presence of FGF7 (25 ng/mL) for 72 hours. Cell proliferation was assessed by luminescent cell viability assay supplied by Promega (Cat. no. G7571) .
  • Example 13 HTRF assay to detect the phosphorylation of FGFR2 and ERK1/2 proteins induced by FGF7 or FGF10 in SNU-16 cells
  • FGFR2b is a receptor tyrosine kinase involved in cell survival, proliferation, migration, and angiogenesis mainly via RAS-MAPK signal pathway.
  • HTRF cell-based fluorescence resonance energy transfer
  • SNU-16 cells were cultured in RPMI-1640 culture medium with 10%FBS and 1 ⁇ Penicillin-streptomycin until the confluence reached 90%.
  • Cells were plated into 96-well plates with PRMI-1640 without FBS medium and incubated overnight at 37°C, and followed by treatment with 15 ⁇ g/mL or 0.15 ⁇ g/mL of human IgG isotype or several FGFR2b antibodies for 1.5 hours. Then the cells were treated with the ligand induced complex containing 30ng/ml FGF7/10 and 20ug/ml heparin for 5 minutes at 37°C. After the activation, cells were immediately incubated by lysis buffer for at least 30 minutes at room temperature under shaking.
  • Example 14 PBMCs mediated ADCC activity of FGFR2b antibodies targeting to KATO-III cells
  • KATO-III cells ATCC, Cat#HTB-103 were maintained in IMDM culture medium containing 20%FBS and 1 ⁇ Penicillin-streptomycin, and freshly isolated PBMCs from healthy donors were obtained from SailyBio. lnc. (Shanghai, China) .
  • Primay ADCC assays were conducted using effector cells from 2 independent donors on two different days. ADCC assay testing was performed using freshly isolated human PBMCs as effector cells at effector to target (E/T) cell ratio of 40: 1.
  • the PK profile of anti-FGFR2b lead molecules and Bema as benchmark was characterized and compared head-to-head in Sprague Dawley (SD) rats following a single i. v. administration at 30 mg/kg.
  • 18 female rats were randomly assigned into 6 groups (3 animals/group) and administered with either 30 mg/kg Bema, or Hu39C2, Hu55D6, Hu52E2, Hu35B11, Hu38D4 once via i. v. slow bolus injection at a dose volume of 10 mL/kg.
  • Plasma across groups were harvested at pre-dose (0 min) and 30 min, 2 h, 8 h, 24 h, 48 h, D4, D7, D10, D14, D21, D28 post dosing and measured by partially validated ELISA assay for PK analysis with detection range from 0.156 to 20 ng/mL.
  • Microplate wells are pre-coated with Human FGFR2b protein, His Tag (Acrobiosystems, FGB-H5223) . After blocking, standard (STD) , quality control (QC) samples, matrix blank sample and the test samples are added to the wells.
  • the biotin mouse anti-human IgG4 (BD Pharmingen TM , 555879) is added to the microplate wells and followed by Streptavidin labeled with horseradish peroxidase (HRP) . Tetramethylbenzidine (TMB) is added to the microplate wells and colorimetric signal (blue) was developed in the presence of HRP. When color developed, stop solution is added to each well to stop the reaction. The optical density (OD) is measured using a microplate reader set to 450nm and 620nm. The conversion of optical density (OD) values for QC and test samples into concentration is performed by comparison to a concurrently analyzed standard curve regressed with a 4-parameter logistic model. mean, and mean plasma concentration-time curves were depicted in Figure 11.
  • the related PK parameters (Table 7) were calculated and assessed by non-compartment analysis (NCA) using Phoenix software.
  • mice were treated with isotype control or humanized FGFR2b antibodies at a dose of 10 mg/kg, twice a week for 4 weeks by i.p. injection. Animals were sacrificed at the end of the study with CO2 inhalation. Tumor size and volume were measured twice a week. Results were analyzed using Prism GraphPad and expressed as mean ⁇ S.E.M.
  • TGI Tumor Growth Inhibition

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