Novel anti-FGFR2b antibody

Abstract

The present disclosure provides anti-FGFR2b antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof.

Description

Technical Field

[0001]

[0001] This disclosure generally relates to novel anti-human FGFR2b antibodies.

Background Art

[0002]

[0002] Fibroblast growth factor receptor (FGFR) is a transmembrane tyrosine kinase encoded by four structurally related genes (FGFR1 - FGFR4). FGFRs are characterized by alternative splicing of their mRNAs, resulting in various isoforms (see Ornitz et al., J. Biol. Chem. 271:15292, 1996; also UniProtKB P21802 and isoforms P21802 - 1 to P21802 - 23 for the sequence of human FGFR2 and its isoforms; UniProtKB P11362 and isoforms P11362 - 1 to P11362 - 21 for the sequence of human FGFR1 and its isoforms). FGFRs have a common structural feature consisting of an extracellular ligand - binding section composed of various Ig - like domains (the α - isoform contains all three Ig - like domains D1, D2, and D3, while the β - isoform contains only two Ig - like domains D2 and D3 and lacks D1), a transmembrane domain, and an intracellular tyrosine kinase catalytic domain. FGF binds to the receptor mainly through regions in D2 and D3 of the receptor. In FGFR1 - FGFR3, all forms contain the first half of D3, and isoforms that contain only the first half of D3 are designated as the IIIa form, while two alternative exons can be utilized in the second half of D3, giving rise to the IIIb and IIIc forms. For example, in FGFR - 1, alternative splicing of the exon encoding the third Ig - like domain produces the FGFR1IIIb or FGFR1IIIc (or precisely FGFR1b and FGFR1c) splice forms, which have distinct ligand - binding preferences. For FGFR2, these forms are designated as FGFR2IIIb and FGFR2IIIc (or precisely FGFR2b and FGFR2c), respectively. FGFR2b is produced only in cells of epithelial origin, and FGFR2c is produced only in mesenchymal cells.The FGFR2b form of FGFR2 is a high-affinity receptor for FGF1 and is a receptor specific for KGF family members (e.g., FGF10, FGF22, and especially FGF7), whereas FGFR2c binds well to both FGF1 and FGF2 but does not bind KGF family members (Miki et al., Proc. Natl. Acad. Sci. USA 89:246, 1992).

[0003]

[0003] FGF upon binding to FGFR mediates various responses in various cell types including proliferation, migration and differentiation, especially during embryonic development (Ornitz et al., J. Biol. Chem. 271:15292, 1996), and in adults is involved in tissue homeostasis and repair. KGF (FGF7) and KGFR (FGFR2IIIb) have been found to be involved in various types of cancers such as pancreatic cancer, gastric cancer, ovarian cancer and breast cancer. FGF7 and FGFR2b are overexpressed in pancreatic cancer (Ishiwata et al., Am. J. Pathol. 153:213, 1998), and their co-expression correlates with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Amplification and overexpression of FGFR2 are strongly associated especially with undifferentiated diffuse-type gastric cancer with poor prognosis, and inhibition of FGFR2 activity by small molecule compounds strongly inhibits the growth of such cancer cells (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006). The FGFR2b ligands FGF1, FGF7 and FGF10 induce proliferation, motility and protection from cell death in EOC cell lines (Steele et al., Growth Factors 24:45, 2006), suggesting that FGFR2b may contribute to the malignant phenotype in ovarian cancer. FGFR2b is highly expressed in about 5% of breast cancers (Finch and Rubin 2006) and mediates the signaling cascade via MAPK and PI3K (Moffa, Tannheimer et al. 2004). Frequently occurring activating FGFR2 mutations (e.g., S252W) have also been found to be associated with various cancers.

[0004]

[0004] Amplification or activation of FGFR1 has been reported in many cancers, including oral squamous cell carcinoma (Freier et al., Oral Oncol. 43(1):60-66, 2007), breast cancer (Turner et al., Cancer Res. 70(5):2085-2094, 2010), esophageal squamous cell carcinoma (Ishizuka et al., Biochem Biophys Res Commun. 296(1):152-155, 2002), ovarian cancer (Gorringe et al., Clin Cancer Res. 13(16):4731-4739, 2007), bladder cancer (Simon et al., Cancer Res. 61(11):4514-4519, 2001), prostate cancer (Edwards et al., Clin Cancer Res. 9(14):5271-5281, 2003), and mainly squamous cell subtype lung cancer (Dutt et al., PLoS One. 6(6):e20351, 2011; Weir et al., Nature. 450(7171):893-898, 2007; Weiss et al., Sci Transl Med. 2(62):62ra93, 2010).

Summary of the Invention

Problems to be Solved by the Invention

[0005]

[0005] There is a great need for novel anti-FGFR2b antibodies. In particular, antibodies that can bind to both FGFR2b and FGFR1b are not reported and are considered non-existent.

Means for Solving the Problems

[0006]

[0004] Throughout this disclosure, the articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "antibody" means one antibody or more than one antibody.

[0007]

[0005] The present disclosure provides novel monoclonal anti-FGFR2b antibodies, their amino acid and nucleotide sequences, and their uses.

[0006] In one aspect, the present disclosure provides an isolated anti-FGFR2b antibody comprising one, two, or three heavy chain complementarity determining region (CDR) sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and / or one, two, or three light chain CDR sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, which is capable of specifically binding to both FGFR2b and FGFR1b. In some embodiments, the antibodies provided herein do not have a detectable binding affinity for FGFR2c.

[0008]

[0007] In some embodiments, the antibodies provided herein comprise the heavy chain CDR3 of SEQ ID NO: 5, and / or the light chain CDR3 of SEQ ID NO: 6. In some embodiments, the antibodies provided herein have a heavy chain variable region (V H ) comprising one, two, or three heavy chain CDR sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and / or a light chain variable region (V L ) comprising one, two, or three light chain CDR sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. In some embodiments, the antibodies provided herein comprise a heavy chain variable region (V H ) comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and / or a light chain variable region (V L ) comprising SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. In some embodiments, the antibodies provided herein comprise a heavy chain variable region (V H ) comprising SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 5, and / or a light chain variable region (V L ) comprising SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.

[0009]

[0008] In some embodiments, the antibodies provided herein include a heavy chain variable region comprising SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16. In some embodiments, the antibodies provided herein include a light chain variable region comprising SEQ ID NO: 10 or SEQ ID NO: 14, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 14. In some embodiments, the antibodies provided herein include a heavy chain variable region comprising SEQ ID NO: 8 and a light chain variable region comprising SEQ ID NO: 10. In some embodiments, the antibodies provided herein include a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO: 14. In some embodiments, the antibodies provided herein include a heavy chain variable region comprising SEQ ID NO: 16 and a light chain variable region comprising SEQ ID NO: 10.

[0010]

[0009] In some embodiments, the antibodies provided herein further include one or more amino acid residue substitutions or modifications that still retain specific binding affinity for FGFR2b and / or FGFR1b. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or in one or more of the V H or V L sequences, or in one or more of the V H or V L sequences, but outside any of the CDR sequences.

[0011]

[0010] In some embodiments, the antibodies provided herein further include an immunoglobulin constant region, optionally a constant region of a human immunoglobulin, preferably a constant region of human IgG, more preferably a constant region of human IgG1.

[0012]

[0011] In some embodiments, the antibodies provided herein, within their constant regions, a) introduce or remove a glycosylation site, b) introduce a free cysteine residue, c) further comprises one or more modifications that enhance binding to activating Fc receptors and / or d) enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0013]

[0012] In some embodiments, the antibodies provided herein are glyco-engineered. In some embodiments, the antibodies provided herein are afucosylated. In some embodiments, the afucosylated antibodies provided herein lack fucose at Asn297. In some embodiments, the glyco-engineered antibodies exhibit enhanced ADCC activity compared to their non-engineered counterparts. In some embodiments, the enhanced ADCC is characterized by lysis of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% of FGFR2b-expressing cells.

[0014]

[0013] In some embodiments, the antibodies provided herein are chimeric antibodies. In some other embodiments, the antibodies provided herein are humanized antibodies.

[0014] In some embodiments, the antibodies provided herein are linked to one or more conjugate moieties. In certain embodiments, the conjugate moieties include a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modifying moiety, or a purification moiety. In some embodiments, the conjugate moieties are covalently linked directly or via a linker.

[0015]

[0015] In another aspect, the disclosure further provides isolated antibodies or antigen-binding fragments thereof that compete with the antibodies described above with respect to binding to FGFR2b and / or FGFR1b.

[0016]

[0016] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody provided herein. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, and its homologous sequences having at least 80% sequence identity to SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. In some embodiments, the homologous sequence encodes the same protein as encoded by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0017]

[0017] In another aspect, the present disclosure provides an expression vector comprising an isolated polynucleotide provided herein.

[0018] In yet another aspect, the present disclosure provides a host cell comprising the expression vector of the present disclosure.

[0018]

[0019] In yet another aspect, the present disclosure provides a method for producing an antibody provided herein. In some embodiments, the method comprises culturing the host cell of the present disclosure under conditions in which the expression vector of the present disclosure is expressed. In some embodiments, the method further comprises purifying the antibody produced by the host cell.

[0019]

[0020] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody provided herein and a pharmaceutically acceptable carrier.

[0021] In another aspect, the present disclosure provides a method for treating an FGFR2b and / or FGFR1b-related disease or condition in a subject, the method comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition of the present disclosure.

[0020]

[0022] In some embodiments, the disease or condition is cancer, and optionally, the cancer is characterized by expressing or overexpressing FGFR2b and / or FGFR1b.

[0023] In some embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration. In some embodiments, the subject is human.

[0021]

[0024] In another aspect, the present disclosure provides a method for detecting the presence or amount of FGFR2b and / or FGFR1b in a sample, the method comprising contacting the sample with an antibody of the present disclosure and determining the presence or amount of FGFR2b and / or FGFR1b in the sample.

[0022]

[0025] In another aspect, the present disclosure provides a method for diagnosing an FGFR2b and / or FGFR1b-related disease or condition in a subject, the method comprising: a) contacting a sample obtained from the subject with an antibody of the present disclosure; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; and c) correlating the presence or amount of FGFR2b and / or FGFR1b with the presence or situation of an FGFR2b and / or FGFR1b-related disease or condition in the subject.

[0023]

[0026] In another aspect, the present disclosure provides a method for prognosing an FGFR2b and / or FGFR1b-related disease or condition in a subject, comprising: a) contacting a sample obtained from the subject with an antibody of the present disclosure; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; and c) correlating the presence or amount of FGFR2b and / or FGFR1b with the subject's potential responsiveness to an FGFR2b and / or FGFR1b antagonist.

[0024]

[0027] In another aspect, the present disclosure provides the use of an antibody of the present disclosure in the manufacture of a medicament for treating a disease or condition that would benefit from modulation of FGFR2b and / or FGFR1b expression in a subject.

[0025]

[0028] In another aspect, the present disclosure provides the use of the antibodies of the present disclosure in the manufacture of a diagnostic reagent for detecting FGFR2b and / or FGFR1b-related diseases or conditions.

[0026]

[0029] In yet another aspect, the present disclosure provides a kit for detecting FGFR2b and / or FGFR1b, comprising the antibody of the present disclosure.

Brief Description of the Drawings

[0027]

Figure 1

[0030] Amino acid sequences of the light chain (A) and heavy chain (B) of the full Ab hu21-26 (designated as "hu21-26" in the figure), with the CDRs underlined.

Figure 2

[0031] Biacore binding Ka, Koff, and affinity KD of Ab 21c, Ab hu21-21, and afhu21-26 (designated as "21c", "hu21-21", and "afhu21-26" respectively in the figure) against human FGFR2b or human FGFR1b, using EPA144 as a control antibody for reference comparison.

Figure 3

[0032] Flow cytometry of dose-dependent binding of chimeric Ab 21c to FGFR2b in KATOIII cells.

Figure 4

[0033] Interspecies binding of Ab 21c to human, cynomolgus monkey, and rat / mouse FGFR2b.

Figure 5

[0034] Binding selectivity of mouse Ab 21 (designated as "21" in the figure) to various family members of human FGFR.

Figure 6

[0035] Inhibition of FGF7-induced cell proliferation of Ba / F3 cells stably transfected with human FGFR2b by Ab 21c, using isotype human IgG1 as a negative control.

Figure 7

[0036] Dose-dependent downregulation of FGFR2b phosphorylation by Ab 21c and downstream target ERK phosphorylation.

Figure 8

[0037] Antibody internalization shown by confocal images of intracellular distribution of mouse Ab 21 after incubation with KATOIII cells for 2 hours and 4 hours at 37°C. Confocal images of intracellular distribution of mouse Ab 21 after incubation with KATOIII cells for 2 hours and 4 hours at 4°C were used as negative baseline controls.

Figure 9

[0038] ADCC activity of defucosylated Ab hu21-26 (designated as "afhu21-26" in the figure) and fucosylated antibody Ab 21c against KATOIII cells.

Figure 10

[0039] In vivo antitumor efficacy of Ab afhu21-26 administered intraperitoneally at 10 mg / kg twice a week in SNU16 gastric cancer xenograft model (A) and LC038 patient-derived xenograft lung cancer model (B). EPA144 was used as a comparison.

Figure 11

[0040] Drug-antibody ratio analysis of unconjugated afhu21-26 (lower curve) and ADC conjugate afhu21-26-MMAE (upper curve) using HIC-HPLC.

Figure 12

[0041] (A) In vivo antitumor efficacy of afhu21-26-MMAE in LC038 patient-derived xenograft lung cancer model and (B) Ab 21c-MMAE, Ab 21c-MMAF in SNU16 gastric cancer xenograft model. Animals were administered intravenously weekly.

Mode for Carrying Out the Invention

[0028]

[0042] The following description of the present disclosure is merely intended to illustrate various embodiments of the present disclosure. Accordingly, the specific modifications discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included herein. All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

[0029]

[0043] Definitions

[0044] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody, and antigen-binding fragments thereof that bind to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain consists of a variable region (V H ) as well as first, second, and third constant regions (C H1 , C H2 , C H3 ), respectively, while mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V Land a constant region. The antibody has a "Y" shape, and the base of the Y consists of the second and third constant regions of two heavy chains joined together via disulfide bonds. Each arm of the Y includes the variable region of a single light chain and the variable region and the first constant region of a single heavy chain joined thereto. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, and heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The CDR boundaries for the antibodies disclosed herein can be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. December 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. December 21-28; 342(6252):877-83 (1989); Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd Edition); chapter 26, 481-514 (2015)). The three CDRs are inserted between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions.Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are further classified into subclasses such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0030]

[0045] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a part of an intact antibody that includes one or more CDRs, or any other antibody fragment that can bind to an antigen but does not include an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab’, F(ab’) 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-chain Fv-Fc antibodies (scFv-Fc), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments are capable of binding to the same antigen to which the parent antibody binds.

[0031]

[0046] "Fab" with respect to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) linked by a disulfide bond to a single heavy chain variable region and the first constant region.

[0032]

[0047] "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[0048] "F(ab’) 2"Fab'" refers to a dimer of Fab. "Fv" with respect to an antibody refers to the smallest fragment of an antibody that possesses the complete antigen-binding site. An Fv fragment consists of a variable region of a single heavy chain bound to a variable region of a single light chain.

[0033]

[0049] "dsFv" refers to an Fv fragment stabilized by a disulfide in which the bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv) 2 " or "(dsFv-dsFv')" consists of three peptide chains: two V L portions each linked by a peptide linker (e.g., a long flexible linker) via a disulfide bond and bound to two V H portions. In some embodiments, dsFv-dsFv' is bispecific, where each disulfide-paired heavy and light chain has a different antigen specificity.

[0034]

[0050] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region that are directly or via a peptide linker sequence bound to each other (Huston JS et al Proc Natl Acad Sci USA, 85:5879 (1988)).

[0035]

[0051] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second and third constant regions of a first heavy chain bound via a disulfide bond to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is involved in various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0036]

[0052] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv bound to the Fc region of an antibody.

[0053] "Camelized single-domain antibody", "heavy-chain antibody", or "HCAb" consists of two V HRefers to an antibody that contains a domain and does not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. December 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4): 277-302 (2001); International Publication No. 94 / 04678; International Publication No. 94 / 25591; US Patent No. 6,005,079). Heavy-chain antibodies are originally derived from the Camelidae family (camels, llamas, and alpacas). Camelized antibodies lack a light chain but have a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428): 446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain of the heavy-chain antibody ("VHH domain") represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13): 3490-8. Epub June 15, 2007 (2007)).

[0037]

[0054] "Nanobody" refers to an antibody fragment consisting of one VH domain and two heavy-chain constant domains, such as CH2 and CH3, derived from the heavy-chain antibody of conventional IgG.

[0055] "Diabody" or "dAb" includes a small antibody fragment having two antigen-binding sites, where the fragment is V L domain bound to V H domain (V H -V L or V L -V Hincluding (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15;90(14):6444-8 (1993); European Patent No. 404097; International Publication No. 93 / 11161). By using linkers that are too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains on another strand, thereby creating two antigen-binding sites. The antigen-binding sites can target the same antigen or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0038]

[0056] In certain embodiments, a "scFv dimer" is a V of one portion H that harmonizes with the V of the other portion L to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes), another V H -V L portion dimerizes with the V H -V L a bivalent diabody or bivalent ScFv (BsFv) containing (linked by a peptide linker). In other embodiments, a "scFv dimer" is a V H1 and V L1 that harmonize, V H2 and V L2 that harmonize, such that the harmonized pairs each have different antigen specificities, V L1 -V H2 (linked by a peptide linker) binds to V H1 -V L2 (similarly linked by a peptide linker) to form a bispecific diabody.

[0039]

[0057] "Domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more V HA domain is covalently shared with a peptide linker to create a bivalent or multivalent domain antibody. The two V H domains of the bivalent domain antibody can target the same antigen or different antigens.

[0040]

[0058] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having a portion of a heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from a different species. By way of example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0041]

[0059] As used herein, the term "humanized" means that an antibody or antigen-binding fragment includes CDRs derived from a non-human animal and FR regions derived from a human, and where applicable, the constant region is derived from a human.

[0042]

[0060] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment having two antigen-binding sites, the term "monovalent" refers to an antibody or antigen-binding fragment having a single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites.

[0043]

[0061] As used herein, a "bispecific" antibody refers to an artificial antibody or antigen-binding fragment having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes can be present on the same antigen or the two epitopes can be present on two different antigens.

[0044]

[0062] Unless otherwise noted, as used herein, the term "FGFR" encompasses any or all of the fibroblast growth factor receptor family members (FGFR1 - FGFR4), and any form of FGFR, such as: 1) natural, untreated FGFR molecules, "full - length" FGFR chains or naturally occurring variants of FGFR, including, for example, allelic variants; 2) any form of FGFR resulting from processing in cells, such as various splicing forms, e.g., FGFR1b, FGFR1c, FGFR2a, FGFR2b, FGFR2c, etc.; or 3) fragments (e.g., truncated forms, extracellular / transmembrane domains) or modified forms (e.g., mutant forms, glycosylated / PEGylated, His - tag / immunofluorescent fusion forms) of FGFR subunits produced by recombinant methods. As used herein, "FGFR" can be derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats).

[0045]

[0063] The terms "FGFR2IIIb" and "FGFR2b" are used interchangeably and refer to the subtype IIIb splicing form of FGFR2. Exemplary sequences of FGFR2b include the Homo sapiens (human) FGFR2b protein (e.g., precursor sequence with signal peptide, Genbank accession number: NP_075259.4), the Rattus norvegicus (rat) FGFR2b protein (e.g., full sequence, Genbank accession number: NP_001103363.1), and the Mus musculus (mouse) FGFR2b protein (e.g., full sequence, Genbank accession number: NP_963895.2).

[0046]

[0064] "FGFR2IIIc" or "FGFR2c" are used interchangeably and refer to the subtype IIIc splice form of FGFR2. Exemplary sequences of FGFR2c include the human FGFR2c protein (e.g., precursor sequence, Genbank accession number: NP_000132.3), the Rattus norvegicus (rat) FGFR2c protein (e.g., full sequence, Genbank accession number: NP_001103362.1), and the Mus musculus (mouse) FGFR2c protein (e.g., full sequence, Genbank accession number: NP_034337.2).

[0047]

[0065] The terms "FGFR1IIIb" and "FGFR1b" are used interchangeably and refer to the subtype IIIb splice form of FGFR1. Exemplary sequences of FGFR1b include the Homo sapiens (human) FGFR1b protein (e.g., precursor sequence with signal peptide, UniProtKB accession number: P11362-19), and the Mus musculus (mouse) FGFR1b protein (e.g., precursor sequence with signal peptide, UniProtKB accession number: P16092-5).

[0048]

[0066] The term "anti-FGFR2b antibody" refers to an antibody capable of specifically binding to FGFR2b. In some embodiments, the anti-FGFR2b antibodies provided herein are capable of specifically binding to both FGFR2b and FGFR1b, but do not bind, or bind poorly to, FGFR2c and FGFR1c (e.g., the binding affinity for FGFR2c or FGFR1c is at least 10-fold lower, or at least 50-fold lower, or at least 100-fold lower, or at least 200-fold lower than the binding affinity for FGFR2b or FGFR1b). In some embodiments, the anti-FGFR2b antibodies provided herein do not have a detectable binding affinity for FGFR2c.

[0049]

[0067] As used herein, the term "specific binding" or "binds specifically" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. The binding affinity of the antibodies and antigen-binding fragments provided herein can be represented by the K D value, and the K D value represents the ratio of the dissociation rate to the association rate (k off / k on ) when the binding between an antigen and an antigen-binding molecule (e.g., an antibody and an antigen-binding fragment) reaches equilibrium. The antigen-binding affinity (e.g., K D ) can be appropriately determined using suitable methods known in the art, including, for example, the Biacore technique (see, e.g., Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, page 2006, 2006), the Kinexa technique (see, e.g., Darling, R.J. et al., Assay Drug Dev. Technol., 2(6):647-657 (2004)), and flow cytometry.

[0050]

[0068] As used herein, the ability to "compete with respect to binding" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human FGFR2b and an anti-FGFR2b antibody) to any detectable extent (e.g., at least 85%, or at least 90%, or at least 95%). Without undue experimentation, it will be understood by those skilled in the art that it is possible to determine whether a given antibody competes with respect to binding to FGFR2b and / or FGFR1b with the antibodies of the present disclosure (e.g., Ab 21, Ab 21c, Ab hu21-21, or Ab hu21-26, defined below).

[0051]

[0069] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds.

[0070] "Conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made among amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), among residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gln), among residues having acidic side chains (e.g., Asp, Glu), among amino acids having basic side chains (e.g., His, Lys, and Arg), or among residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the protein conformation structure and, as a result, the biological activity of the protein can be retained.

[0052]

[0071] As used herein, the terms "homolog" and "homology" are used interchangeably and refer to a nucleic acid sequence (or its complementary strand) or an amino acid sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to another sequence when optimally aligned.

[0053]

[0072] The "percent sequence identity (%)" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences to achieve the maximum number of identical amino acids (or nucleic acids) and introducing gaps as necessary. Conservative substitutions of amino acid residues may or may not be considered to be the same residue. The alignment can be achieved using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, UK), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software for the purpose of determining the amino acid (or nucleic acid) sequence identity percentage. Those skilled in the art may use the default parameters provided by the tools or may customize the parameters regarding the alignment as necessary, for example, by selecting an appropriate algorithm.

[0054]

[0073] An "isolated" substance has been changed from its natural state by human hand. When an "isolated" composition or substance occurs naturally, the "isolated" composition or substance has been changed or removed from, or both, its original environment. For example, a polynucleotide or polypeptide that occurs naturally in a living animal is not "isolated" and the same polynucleotide or polypeptide is "isolated" when it is sufficiently separated from the materials with which it coexists in its natural state so as to exist in a substantially pure state. An "isolated polynucleotide sequence" refers to the sequence of an isolated polynucleotide molecule. In certain embodiments, an "isolated antibody" refers to an antibody having at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% purity as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis, capillary electrophoresis) or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0055]

[0074] "Effector function," as used herein, refers to the biological activity of the Fc region of an antibody due to its binding to its effectors such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis.

[0056]

[0075] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which effector cells expressing an Fc receptor (FcR) recognize an antibody or antigen-binding fragment bound to a target cell and subsequently cause lysis of the target cell. "ADCC activity" refers to the ability of an antibody or antigen-binding fragment bound to a target cell to induce an ADCC reaction as described above.

[0057]

[0076] A "target cell" is a cell to which an antibody containing an Fc region specifically binds via a protein moiety that is generally C-terminal to the Fc region. An "effector cell" is a leukocyte that expresses one or more Fc receptors and performs an effector function. Preferably, the cell expresses at least FcγRIII and performs an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from their natural sources, such as blood or PBMCs, as is known in the art.

[0058]

[0077] As used herein, a "vector" refers to a polynucleotide molecule that enables the replication / cloning of a desired nucleic acid fragment contained therein or enables the expression of a protein encoded by such a desired nucleic acid fragment when introduced into a suitable cell host. Examples of vectors include both cloning vectors and expression vectors. As used herein, the term "expression vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to effect the expression of the protein. An expression vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, selectable elements, and a reporter gene. Further, the vector can contain an origin of replication.

[0059]

[0078] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or expression vector has been introduced.

[0079] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of development of the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, effecting complete or partial regression of the condition, curing the condition, or some combination thereof.

[0060]

[0080] As used herein, a "FGFR2b and / or FGFR1b-related" disease or condition refers to any disease or condition that is sensitive to treatment with an FGFR2b modulator and / or an FGFR1b modulator, or that is associated with the expression or overexpression of FGFR2b and / or FGFR1b. In some embodiments, the FGFR2b and / or FGFR1b-related disease or condition is cancer, optionally cancer that is positive with respect to the expression or elevated expression of FGFR2b and / or FGFR1b.

[0061]

[0081] As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, invasion or metastasis, and includes both solid tumors and non-solid cancers. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. "Non-solid cancer" refers to hematological malignancies such as leukemia, lymphoma, myeloma and other blood malignancies. Examples of cancers or tumors include hematological malignancies (e.g., lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma), oral cancers (e.g., of the lip, tongue or pharynx), tumors in the digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary tract, pancreas, larynx or lungs (small cell or non-small cell) and other respiratory systems, bone, connective tissue, skin (e.g., melanoma), chest, genital organs (fallopian tubes, uterus, cervix, testis, ovary, or prostate), urinary organs (e.g., bladder or kidney), brain and endocrine glands such as the thyroid. In certain embodiments, the cancer is selected from ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), bladder cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck cancer, mesothelioma, melanoma, sarcoma, and brain tumors (e.g., gliomas such as glioblastoma).

[0062]

[0082] The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation and is physiologically compatible with their recipients.

[0063]

[0083] Anti-FGFR2b antibody

[0084] The present disclosure provides an anti-FGFR2b antibody comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of Ab 21. Table 1 shows the CDR sequences of Ab 21. The term "Ab 21" as used herein refers to a mouse monoclonal antibody having the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 14. Ab 21 specifically binds to both FGFR2b and FGFR1b.

[0064]

[0085]

[0065]

Table 1

[0066]

[0086] CDR is known to be involved in antigen binding, but it has been found that not all six CDRs are essential or invariant. In other words, it is possible to replace, alter, or modify one or more CDRs in Ab 21, while substantially retaining the specific binding affinity for FGFR, particularly for FGFR2b and FGFR1b.

[0067]

[0087] In certain embodiments, the anti-FGFR2b antibodies provided herein may include one or more modifications or substitutions in one or more CDR regions as provided in Table 1. Such variants retain the specific binding affinity for FGFR2b and / or FGFR1b of their parental antibodies and may have one or more improvements in properties such as higher antigen binding affinity or reduced likelihood of glycosylation.

[0068]

[0088] In certain embodiments, the anti-FGFR2b antibodies provided herein may be modified to remove one or more Asn or Asp hotspots within the CDR region (or within the variable region). Such Asn and Asp hotspots can lead to antibody degradation and thus reduce antibody stability. Exemplary putative hotspot motifs within the CDR region include Asn-Gly, Asn-Thr, Asn-Ser, Asn-Asn, Asp-Gly, Asp-Thr, Asp-Ser, Asp-Asp, and Asp-His. In certain embodiments, the HCDR2 of Ab 21 is modified to remove the Asn-Gly (NG) hotspot. In certain embodiments, the modified HCDR2 includes SEQ ID NO: 7 (AIYPENRDINYNQKFKG).

[0069]

[0089] In certain embodiments, the anti-FGFR2b antibodies provided herein comprise the heavy chain CDR3 sequence of SEQ ID NO:5 and, optionally, the light chain CDR3 of SEQ ID NO:6. The heavy chain CDR3 region is located at the center of the antigen-binding site and is thus thought to make the most contacts with the antigen and provide the most free energy to the affinity of the antibody for the antigen. Also, the heavy chain CDR3 is thought to be a diverse CDR apart from the group of antigen-binding sites in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302:575-581 (1983)). The diversity in the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45 (2000)) as well as desirable antigen-binding affinities (Schier R, et al. J Mol Biol. 263:551-567 (1996)).

[0070]

[0090] In certain embodiments, the anti-FGFR2b antibodies provided herein further comprise an appropriate framework region (FR) sequence, so long as the antibody can specifically bind to FGFR2b and / or FGFR1b. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but the CDR sequences provided in Table 1 can be grafted onto any appropriate FR sequence of any appropriate species such as mouse, human, rat, rabbit, etc. using any appropriate methods known in the art, especially recombinant techniques and the like.

[0071]

[0091] In certain embodiments, the anti-FGFR2b antibodies provided herein are humanized. Exemplary humanized antibodies provided herein include Ab Hu21-21 and Ab hu21-26.

[0072]

[0092] As used herein, "Ab hu21-21" refers to a humanized antibody based on Ab 21 having the heavy chain variable region of SEQ ID NO:16 and the light chain variable region of SEQ ID NO:10.

[0073]

[0093] As used herein, "Ab hu21-26" refers to a humanized antibody based on Ab 21 having the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 10. The heavy chain variable region sequence of Ab hu21-26 (SEQ ID NO: 8) is identical to the heavy chain variable region sequence of Ab hu21-21 (SEQ ID NO: 16) in other respects except for the G56R mutation that removes the NG hot spot in HCDR2.

[0074]

[0094] In certain embodiments, the anti-FGFR2b antibodies provided herein further comprise an immunoglobulin constant region, optionally a human immunoglobulin, and optionally human IgG. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises C κ or C λ and includes.

[0075]

[0095] In certain embodiments, the anti-FGFR2b antibodies provided herein are chimeric antibodies comprising a mouse variable region and a human constant region. As used herein, "Ab 21c" refers to a chimeric antibody based on Ab 21 comprising the mouse heavy chain variable region of SEQ ID NO: 12 and the mouse light chain variable region of SEQ ID NO: 14, each fused to a human heavy chain constant region and a human light chain constant region.

[0076]

[0096] Tables 2 and 3 show the variable region sequences of exemplary antibodies.

[0097]

[0077]

Table 2

[0078]

[0098]

[0079]

Table 3-1

[0080]

Table 3-2

[0081]

[0099] In certain embodiments, the anti-FGFR2b antibodies provided herein may contain one or more modifications or substitutions in one or more of the variable region sequences provided herein, but retain specific binding affinity for FGFR2b and / or FGFR1b. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequence, FR sequence, or variable region sequence(s) comprises a conservative substitution(s).

[0082] [000100]This object can be achieved using various methods known in the art. For example, a library of antibody variants (e.g., Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for binding affinity to human FGFR2b and / or FGFR1b. With regard to another example, computer software can be used to virtually simulate the binding of an antibody to FGFR2b and / or FGFR1b to identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitutions so as to prevent reduction of binding affinity, or can be targeted for substitution to provide stronger binding.

[0083] [000101]In certain embodiments, the anti-FGFR2b antibodies provided herein contain one or more amino acid residue substitutions in one or more of the CDR sequences within SEQ ID NOs: 1 to 7 and / or one or more FR sequences. In certain embodiments, a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer substitutions are made in the CDR sequence and / or FR sequence.

[0084] [000102]In certain embodiments, the anti-FGFR2b antibody comprises one, two, three, four, five, or six CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence(s) listed in SEQ ID NOs: 1 to 7, while retaining a binding affinity for FGFR2b and / or FGFR1b at a level similar to or even higher than that of its parental antibody.

[0085] [000103]In certain embodiments, the anti-FGFR2b antibody comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence(s) listed in Table 2, while retaining a binding affinity for FGFR2b and / or FGFR1b at a level similar to or even higher than that of its parental antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 2. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (e.g., in the FR).

[0086] [000104]In certain embodiments, the anti-FGFR2b antibodies provided herein comprise a constant region capable of inducing effector functions such as ADCC or CDC. Effector functions such as ADCC and CDC can result in cytotoxicity against cells expressing FGFR and can be evaluated using various assays such as Fc receptor binding assays, C1q binding assays, and cell lysis assays. In certain embodiments, the constant region has the IgG1 isotype known to induce ADCC.

[0087] [000105]In certain embodiments, the anti-FGFR2b antibody comprises one or more modifications in the constant region that confer enhanced ADCC. As used herein, the term "enhanced ADCC" refers to an increase in the number of target cells lysed by a given concentration of antibody in the medium surrounding the target cells over a given time period by the ADCC mechanism as defined above, and / or a reduction in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells over a given time period by the ADCC mechanism.

[0088] [000106]To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as those described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985), U.S. Patent No. 5,821,337; or Bruggemann et al., J Exp Med 166, 1351-1361 (1987) can be performed. Alternatively, a non-radioactive assay method may be used (see, for example, ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Further, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0089] [000107]Various methods related to ADCC enhancement have been described in the prior art. For example, it has been demonstrated that a subset of amino acid residues in the Fc region is involved in the binding to FcγRs such as the following amino acid residues (residues of EU numbering): (1) Lys274 - Arg301 and Tyr407 - Arg416 in the Fc region (Sarmay et al. (1984) Mol. Immunol., 21: 43 - 51 and Gergely et al. (1984) Biochem. Soc. Tans., 12: 739 - 743); (2) Leu234 - Ser239, Asp265 - Glu269, Asn297 - Thr299, and Ala327 - Ile332 (Sondermann et al. (2000) Nature, 406: 267 - 273), and (3) T256, K290, S298, E333, K334, A339 (Shields et al. (2001) J. Biol. Chem., 276: 6591 - 6604; and US Patent Application Publication No. 2004 / 0228856) are involved in the binding to human FcγRIIIA. The amino acid residues listed above can be mutated, for example, in Shields et al. (2001), J Biol Chem 9(2), 6591 - 6604, to enhance the ADCC assay, and the Fc mutants T256A, K290A, S298A, E333A, K334A, and A339T have been proven to enhance the ADCC activity when compared to the native sequence.

[0090] [000108]Alternatively, enhanced ADCC activity can be obtained by manipulating the glycosylated form of the antibody. A number of glycosylated forms have been reported to enhance the ADCC activity of the antibody by enhancing its binding to the Fc receptor of effector cells. Various glycosylated forms include using various saccharides (e.g., lacking one type of sugar such as fucose or having a high level of one type of sugar such as mannose) or some forms of the glycans bound to the antibody having various structures (e.g., various branched structures such as a bifurcated (two - branched), trifurcated (three - branched) or quadrifurcated (four - branched) structure).

[0091] [000109]In certain embodiments, the anti-FGFR2b antibodies provided herein are glycoengineered. A “glycoengineered” antibody or antigen-binding fragment can have an increased or decreased glycosylation level, or both, which is a change in glycosylation pattern when compared to its non-glycoengineered counterpart. In certain embodiments, the glycoengineered antibody exhibits enhanced ADCC activity compared to its non-engineered counterpart. In some embodiments, the enhanced ADCC activity is characterized by lysis of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% of FGFR2b-expressing cells.

[0092] [000110]Antibodies can be glycoengineered by methods known in the art, including any manipulation of the peptide backbone (e.g., modification to the amino acid sequence and / or to the side chain groups of individual amino acids), and / or manipulation of post-translational modifications through the host cell line (e.g., modification to the glycosylation pattern). Methods for altering ADCC activity by manipulating the glycosylation of an antibody have also been described in the art; see, for example, Weikert et al. (1999) Nature Biotech., 17:116-121; Shields R.L. et al. (2002), J. Biol. Chem., 277:26733-26740; Shinkawa et al. (2003), J Biol Chem., 278, 3466-3473; Ferrara et al. (2006), Biotech. Bioeng., 93, 851-861; Yamane-Ohnuki et al. (2004), Biotech Bioeng., 87, 614-622; Niwa et al. (2006), J Immunol Methods 306, 151-160; Shinkawa T. et al., J. Biol. Chem, (2003), 278:3466-3473.

[0093] [000111]In some embodiments, the glycan-engineered antibodies provided herein are defucosylated (i.e., do not contain fucose). Defucosylated (i.e., fucose-deficient or non-fucosylated) antibodies have been shown in several studies to exhibit increased binding to FcγRIII and thus induce higher ADCC activity (Shields et al. (2002) J. Biol. Chem., 277:26733-26740; Shinkawa et al. (2003) J. Biol. Chem., 278:3466-3473; and European Patent Application Publication No. 1176195). In some embodiments, the defucosylated antibodies provided herein lack fucose at asparagine 297 (Asn297) of the heavy chain (based on Kabat numbering). Asn297 is a conserved N-linked glycosylation site found in each CH 2 domain of the Fc region of IgG1 isotype antibodies (Arnold et al., Glycobiology and Medicine, 564:27-43, 2005).

[0094] [000112]In some embodiments, the glycan-engineered antibodies provided herein are characterized by high-mannose glycosylation forms (e.g., mannose e5, mannose 7, 8, 9 glycans). High-mannose glycosylation forms have been demonstrated to enhance ADCC activity (Yu et al. (2012), Landes Bioscience, mAbs 4:4, 475-487).

[0095] [000113]In some embodiments, the antibodies provided herein further comprise one or more modifications within their constant regions to: a) introduce or remove a glycosylation site, b) introduce a free cysteine residue, c) enhance binding to activating Fc receptors, and / or d) enhance ADCC.

[0096] [000114]The anti-FGFR2b antibody or antigen-binding fragment thereof may comprise one or more amino acid residues having side chains to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of the antibody is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) in the antibody sequence. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (with respect to N-linked glycosylation sites) or serine or threonine residues (with respect to O-linked glycosylation sites) present in the antibody sequence is substituted. A new glycosylation site can be created in a similar manner by introducing such a tripeptide sequence or serine or threonine residue.

[0097] [000115]The anti-FGFR2b antibodies provided herein also include variants in which cysteine has been engineered to include one or more introduced free cysteine amino acid residues. The free cysteine residues are not part of a disulfide bridge. Variants in which cysteine has been engineered are useful, for example, for conjugation with cytotoxic and / or imaging compounds, labels, or particularly radioisotopes through maleimide or haloacetyl, for example, at the site of the engineered cysteine. Methods for engineering antibodies to introduce free cysteine residues are known in the art; see, for example, International Publication No. WO 2006 / 034488.

[0098] [000116]The anti-FGFR2b antibodies provided herein also encompass Fc variants that contain one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region. In certain embodiments, the anti-FGFR2b antibody comprises one or more amino acid substitution(s) that improve the pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, which allows them to escape degradation in the transport lysosome and subsequently be trafficked and released from the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art and are described, for example, in Vaughn, D. et al., Structure, 6(1):63-73 (1998); Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0099] [000117]Binding Properties [000118]The anti-FGFR2b antibodies provided herein are capable of specifically binding to FGFR2b and FGFR1b. In certain embodiments, the antibodies provided herein have a -6 Kd of 10 -7 M or less (e.g., 5×10 -7 M or less, 2×10 -7 M or less, 10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 9×10 -10 M or less, 8×10-10 M or less, 7×10 -10 M or less, 6×10 -10 M or less, 5×10 -10 M or less, 4×10 -10 M or less, 3×10 -10 M or less, 2.5×10 -10 M or less, 2×10 -10 M or less, 1.5×10 -10 M or less, 10 -10 M or less, 9×10 -11 M or less, 5×10 -11 M or less, 4×10 -11 M or less, 3×10 -11 M or less, 2×10 -11 M or less, or 10 -11 M or less) of the binding affinity (K D ) specifically binds to human FGFR2b and / or FGFR1b.

[0100] [000119] In some embodiments, the anti-FGFR2b antibody provided herein, when measured by Biacore, is 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, 4×10 -10 M or less, 3×10 -10 M or less, 2×10 -10 M or less, 10 -10 M or less, 5×10 -11 M or less, or 4×10 -11 M or less, 3×10 -11 M or less, 2×10 -11 M or less of the binding affinity (K D ) can specifically bind to human FGFR2b.

[0101] [000120] In some embodiments, the anti-FGFR2b antibody provided herein, when measured by Biacore, is 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10-9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 4×10 -10 Less than M, 3×10 -10 Less than M, 2×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, or 4×10 -11 Less than M, 3×10 -11 Less than M, 2×10 -11 The binding affinity (K D ) less than M can specifically bind to human FGFR1b.

[0102] [000121]In certain embodiments, the anti-FGFR2b antibodies provided herein cross-react with cynomolgus FGFR counterparts, rat FGFR counterparts, and mouse FGFR counterparts.

[0103] [000122]The binding of the antibody to human FGFR2b and / or FGFR1b can also be represented by the "maximum half-effective concentration" (EC 50 ) value, which refers to the concentration of the antibody at which 50% of its maximum effect (e.g., binding or inhibition, etc.) is observed. The EC 50 value can be measured by methods known in the art, such as sandwich assays like ELISA, Western blot, flow cytometry assay, and other binding assays. In certain embodiments, the antibodies provided herein have an EC 50(i.e., 50% binding concentration) specifically binds to human FGFR2b and / or FGFR1b. In certain embodiments, the antibodies provided herein have an EC of 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.8 nM or less, 0.5 nM or less, or 0.3 nM or less by flow cytometry 50 (i.e., 50% binding concentration) specifically binds to human FGFR2b and / or FGFR1b.

[0104] [000123]In certain embodiments, the antibodies provided herein are also capable of specifically binding to cynomolgus FGFR2b and / or FGFR1b and / or rat / mouse FGFR2b and / or FGFR1b. In certain embodiments, the binding affinity of the antibodies provided herein for human FGFR2b and / or FGFR1b is similar to the binding affinity for rat / mouse FGFR2b and / or FGFR1b.

[0105] [000124]In certain embodiments, the antibodies provided herein have a specific binding affinity for human FGFR2b and / or FGFR1b that is sufficient to provide for diagnostic and / or therapeutic use.

[0106] [000125]In certain embodiments, the antibodies provided herein block the binding of human FGFR2b and / or FGFR1b to its ligand, thereby providing a biological activity including, for example, inhibition of the proliferation of FGFR2b and / or FGFR1b-expressing cells.

[0107] [000126]The growth inhibitory effect can be represented by the "50% growth inhibitory concentration" (GI 50 ), which refers to the concentration of the antibody at which 50% of its maximum growth inhibitory effect is observed. GI 50The value can be measured by methods known in the art, such as the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay (see that described in U.S. Patent No. 5,185,450), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (see Berridge et al Biotechnol Annu Rev. 2005;11:127-52), the alamar blue assay (see that described in U.S. Patent No. 5,501,959) and any other method as described in the Assay Guidance Manual (edited by Sittampalam et al 2004). In certain embodiments, the antibodies provided herein, when measured by MTS, have a 50% growth inhibitory concentration (GI 50 ) of 15 nM or less, 14 nM or less, 13 nM or less, 12 nM or less, 11 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 2 nM or less or 1 nM or less and are capable of inhibiting the proliferation of cells expressing human FGFR2b on their surfaces.

[0108] [000127] Antigen-binding fragment [000128] The present disclosure also provides antigen-binding fragments that can specifically bind to FGFR2b and / or FGFR1b. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-FGFR2b antibodies provided herein, including, for example, CDRs and variable sequences of the exemplary antibodies shown in SEQ ID NO: 1 to SEQ ID NO: 7 and Table 2, and various mutants thereof containing modifications or substitutions.

[0109] [000129] In certain embodiments, the anti-FGFR2b antigen-binding fragments provided herein are camelized single-domain antibodies, diabodies, single-chain Fv fragments (scFv), scFv dimers, BsFv, dsFv, (dsFv) 2 , dsFv-dsFv’, Fv fragments, Fab, Fab’, F(ab’) 2is a bispecific antibody, ds diabody, nanobody, domain antibody, single domain antibody, or bivalent domain antibody.

[0110] [000130] Various techniques can be used for the production of such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as Escherichia coli (E. coli) (for example, with respect to Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (for example, with respect to ScFv), and chemical coupling of two Fab’-SH fragments to form F(ab’) 2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0111] [000131] In certain embodiments, the antigen-binding fragment is an scFv. The generation of scFv is described, for example, in International Publication No. WO 93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. The ScFv can be fused to an effector protein at either the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, Borrebaeck).

[0112] [000132] Conjugate [000133]In some embodiments, the anti-FGFR2b antibody further comprises a conjugate moiety. The conjugate moiety can be linked to the antibodies provided herein. The conjugate moiety is a non-proteinaceous or digestible moiety that can be attached to the antibody. A variety of conjugate moieties are contemplated to be linked to the antibodies provided herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). The conjugate moiety can be linked to the antibody by covalent bond, affinity binding, intercalation, coordination bond, complex formation, association, mixing or addition, among other ways.

[0113] [000134]In certain embodiments, the anti-FGFR2b antibody is linked to one or more conjugates via a linker. In certain embodiments, the linker is a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker. In certain embodiments, the linker is a lysosome-cleavable dipeptide, such as valine-citrulline (vc).

[0114] [000135]The conjugate moiety can be a therapeutic agent (e.g., a cytotoxic agent), a radioisotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), a pharmacokinetic modifying moiety, or a purification moiety (e.g., magnetic beads or nanoparticles).

[0115] [000136]Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or β-D-galactosidase), radioisotopes, luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0116] [000137]Examples of radioisotopes include 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P and other lanthanides may be included. Antibodies labeled with radioisotopes are useful in receptor target imaging experiments.

[0117] [000138]In certain embodiments, the pharmacokinetic modifying moiety can be a clearance modifier that aids in increasing the half-life of the antibody. Examples of such can include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol, etc. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers bound to the antibody can vary and if more than one polymer is bound, the more than one polymer can be the same molecule or different molecules.

[0118] [000139]In certain embodiments, the conjugate moiety can be a purification moiety such as magnetic beads or nanoparticles. [000140]Antibody-drug conjugate [000141]In certain embodiments, the conjugate provided herein is an antibody-drug conjugate (ADC) comprising any of the above anti-FGFR2b antibodies conjugated to a cytotoxic agent. In other words, the conjugate moiety comprises a cytotoxic agent.

[0119] [000142]ADCs can be useful, for example, for the local delivery of cytotoxic agents in the treatment of cancer. This allows for the targeted delivery of the cytotoxic agent to the tumor and its intracellular accumulation therein, which can be particularly useful when systemic administration of these unconjugated cytotoxic agents results in unacceptable levels of toxicity to normal cells and tumor cells are sought to be eliminated (Baldwin et al., (1986), Lancet, pp. 603-05; Thorpe, (1985), Monoclonal Antibodies, 84; Pinchera et al. (eds.), Biological And Clinical Applications, pp. 475-506; Syrigos and Epenetos (1999), Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; and U.S. Patent No. 4,975,278).

[0120] [000143]A "cytotoxic agent" can be any agent that is harmful to, or can damage, or kill cancer cells. In certain embodiments, the cytotoxic agent is optionally a chemotherapeutic agent (e.g., a growth inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anti-cancer drug), a toxin, or a highly reactive radioisotope.

[0121] [000144]Examples of cytotoxic agents include, for example, diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin, abrin, modeccin, alpha-sarcin, Aleurites fordii, proteins, dianthin proteins, Phytolaca americana proteins (PARI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crocin, saponaria officinalis inhibitors, gelonin, restrictocin, phenomycin, enomycin, and macromolecular bacterial toxins and plant toxins such as trichothecenes (see, for example, WO 93 / 21232). Such macromolecular toxins can be conjugated to the antibodies provided herein using methods known in the art, as described by Vitetta et al. (1987) Science 238:1098.

[0122] [000145]Cytotoxic agents also include geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), mantanoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res.(pages 53:3336 - 3342), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vindesine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6 - mercaptopurine, 6 - thioguanine, cytarabine, 5 - fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis - dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine and vinblastine), calicheamicin, maytansinoids, dolastatin, auristatins (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), trichothecene, and CC1065, and small molecule toxins and chemotherapeutic agents such as their derivatives having cytotoxic activity may be. Such toxins may be small molecule toxins and chemotherapeutic agents such as their derivatives having cytotoxic activity. Such toxins can be conjugated to the antibodies provided herein using methods known in the art, as described, for example, in U.S. Patent No. 7,964,566; Kline, T. et al., Pharmaceutical Research, 32(11):3480 - 3493.

[0123] [000146]The cytotoxic agent may also be a highly radioactive isotope. As an example, At211 、 I 131 、 I 125 、 Y 90 、 Re 186 、 Sm 153 、 Bi 212 、 P 32 、 Pb 212 and radioisotopes of Lu. Methods for conjugation of radioisotopes to antibodies are known in the art, for example, via appropriate ligand reagents (see, e.g., WO 94 / 11026; Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., eds. Wiley-Interscience, New York, N.Y., Pubs. (1991)). Ligand reagents have chelate ligands that can bind, chelate, or otherwise combine in other situations with the radioisotope metal and have functional groups that are reactive with the thiol of cysteine of the antibody or antigen-binding fragment. Exemplary chelate ligands include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex.).

[0124] [000147]In certain embodiments, the antibody is conjugated to the conjugate moiety via a linker, such as a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0125] [000148]Exemplary bifunctional linkers include, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0126] [000149]In certain embodiments, the linker is cleavable under specific physiological conditions, thereby promoting the release of the cytotoxic agent in the cell. For example, the linker can be an acid-labile linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020). In some embodiments, the linker can include amino acid residues such as dipeptides, tripeptides, tetrapeptides, or pentapeptides. The amino acid residues in the linker can be natural amino acid residues or non-natural amino acid residues. Examples of such linkers include valine-citrulline (ve or val-cit), alanine-phenylalanine (af or ala-phe), glycine-valine-citrulline (gly-val-cit), glycine-glycine-glycine (gly-gly-gly), valine-citrulline-p-aminobenzylcarbonyl ("vc-PAB"). The amino acid linker components can be designed and optimized based on their selectivity for enzymatic cleavage by specific enzymes such as tumor-associated proteases, cathepsin B, cathepsin C, and cathepsin D, or plasmin proteases.

[0127] [000150]In certain embodiments, in the ADCs provided herein, the antibody (or antigen-binding fragment) is conjugated to one or more cytotoxic agents at an antibody:agent ratio of about 1 to about 20, about 1 to about 6, about 1 to about 3, about 1 to about 2, about 1 to about 1, about 2 to about 5, or about 3 to about 4.

[0128] [000151]The ADCs provided herein can be prepared by any suitable method known in the art. In certain embodiments, the nucleophilic group of the antibody is first reacted with a bifunctional linker reagent and then linked to the cytotoxic agent, or vice versa, i.e., first reacting the nucleophilic group of the cytotoxic agent with the bifunctional linker and then linking it to the antibody.

[0129] [000152]In certain embodiments, the cytotoxic agent may contain (or may be modified to contain) a thiol-reactive functional group that can react with the cysteine thiol of the free cysteine of the antibodies provided herein. Exemplary thiol-reactive functional groups include, for example, maleimide, iodoacetamide, pyridyldisulfide, haloacetyl, succinimidyl ester (e.g., NHS, N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, or phosphoramidite (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0130] [000153]After reacting the cytotoxic agent or the antibody with a linker reagent, they may be conjugated to form an ADC. For example, the N-hydroxysuccinimidyl ester (NHS) of the cytotoxic agent may be carried out, isolated, purified, and / or characterized, or the N-hydroxysuccinimidyl ester (NHS) of the cytotoxic agent may be formed in situ and reacted with the nucleophilic group of the antibody.

[0131] [000154]In some embodiments, the cytotoxic agent and the antibody can be linked by in-situ activation and reaction to form the ADC in one step. In another example, the antibody can be indirectly conjugated to a second conjugate that is conjugated to avidin after being conjugated to biotin.

[0132] [000155]In certain embodiments, the conjugate moiety is randomly attached to specific types of surface-exposed amino acid residues in the antibody, such as cysteine residues or lysine residues.

[0133] [000156]In certain embodiments, the conjugate moiety is attached to a defined site to provide an ADC population with high homogeneity and batch-to-batch consistency with respect to the drug-to-antibody ratio (DAR) and the binding site. In certain embodiments, the conjugate moiety is attached to a defined site in the antibody molecule via a natural amino acid, unnatural amino acid, short peptide tag, or Asn297 glycan. For example, conjugation can occur at specific sites outside of the epitope-binding portion.

[0134] [000157]Site-specific conjugation can be achieved by substituting natural amino acids at specific sites of an antibody with amino acids such as cysteine to which a drug moiety can be conjugated, or by introducing amino acids such as cysteine to which a drug moiety can be conjugated before / after a specific site of the antibody (see, for example, Stimmel et al. (2000), JBC, 275(39):30445-30450; Junutula et al. (2008), Nature Biotechnology, 26(8):925-932; and WO 2006 / 065533). Alternatively, site-specific conjugation can be achieved by engineering antibodies to contain unnatural amino acids (e.g., p-acetylphenylalanine (pAcF), N6-((2-azidoethoxy)carbonyl)-L-lysine, p-azidomethyl-L-phenylalanine (pAMF), and selenocysteine (Sec)) at specific sites in their heavy and / or light chains, as described by Axup et al. ((2012), Proc Natl Acad Sci USA. 109(40):16101-16116), where the unnatural amino acids provide the additional advantage that orthogonal chemistry can be designed to link linker reagents and drugs. Exemplary specific sites useful in two of the above-described site-specific conjugation methods (e.g., light chain V205, heavy chain A114, S239, H274, Q295, S396, etc.) are described in many prior arts, such as Strop et al. (2013), Chemistry & Biology, 20, 161-167; Qun Zhou (2017), Biomedicines, 5, 64; Dimasi et al. (2017), Mol.Pharm., 14, 1501-1516; WO 2013 / 093809 and WO 2011 / 005481).Another site-specific ADC conjugation method is glycan-mediated conjugation, where instead of coupling a relatively hydrophobic cytotoxic agent to the amino acid backbone of the antibody, the drug-linker can be conjugated to an Asn297 glycan (e.g., fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, sialic acid) located in the CH2 domain. Also, attempts have been made to introduce a unique short-chain peptide tag (e.g., LLQG, LPETG, LCxPxR) into the antibody via a specific site (e.g., a site in the N-terminal or C-terminal region), followed by functionalizing a specific amino acid in the peptide tag to enable coupling to a drug-linker (Strop et al. (2013), Chemistry & Biology, 20, 161-167; Beerli et al. (2015), PLoS ONE, 10, e0131177; Wu et al. (2009), Proc. Natl. Acad. Sci. 106, 3000-3005; Rabuka (2012), Nat. Protoc. 7, 1052-1067).

[0135] [000158] Polynucleotides and recombinant methods [000159] The present disclosure provides an isolated polynucleotide encoding the anti-FGFR2b antibody provided herein.

[0136] [000160] As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specifically limited, the term encompasses polynucleotides containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly recited sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0137] [000161] In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences such as those set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and / or its homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and / or its variants having only degenerate substitutions, and encodes the variable regions of the exemplary antibodies provided herein. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody). The coding DNA can also be obtained by synthetic methods.

[0138] [000162]An isolated polynucleotide encoding an anti-FGFR2b antibody (e.g., comprising a sequence as shown in Table 3) can be inserted into a vector for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. Many vectors are available. Generally, as vector components, one or more of the following can be mentioned, but are not limited to: signal sequence, origin of replication, one or more marker genes, enhancer element, promoter (e.g., SV40, CMV, EF-1α), and transcription termination sequence. The vector can also include materials that facilitate its entry into cells, including but not limited to virus particles, liposomes, or protein coatings.

[0139] [000163]The present disclosure provides a vector (e.g., a cloning vector or an expression vector) containing a nucleic acid sequence provided herein encoding an antibody, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as expression vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Exemplary plasmids include pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0140] [000164]A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Host cells suitable for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or the higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as the genus Escherichia, for example, Escherichia coli, the genus Enterobacter, the genus Erwinia, the genus Klebsiella, the genus Proteus, the genus Salmonella, for example, Salmonella typhimurium, the genus Serratia, for example, Serratia marcescens, and the genus Shigella, as well as the genus Bacilli, for example, Bacillus subtilis and Bacillus licheniformis, the genus Pseudomonas, for example, Pseudomonas aeruginosa, and the genus Streptomyces.

[0141] [000165]In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding anti-FGFR2b antibodies. Saccharomyces cerevisiae or common baker's yeast is most commonly used among lower eukaryotic host microorganisms. However, fission yeast (Schizosaccharomyces pombe); for example, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (European Patent No. 402,226); Pichia pastoris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and numerous other genera, species, and strains of filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are generally available and useful herein.

[0142] [000166]The host cells suitable for the expression of the antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains and variants, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (fall armyworm), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm) have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used, as the viruses herein according to the present invention, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0143] [000167]However, the most interest has focused on vertebrate cells, and the growth of vertebrate cells in culture (tissue culture) has become an everyday procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse myeloma cell lines (NS0, Galfre and Milstein (1981), Methods in Enzymology, 73:3-46; Sp2 / 0-Ag14, ATCC CRL-1581), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cell is a mammalian cultured cell such as a CHO cell, a BHK cell, or an NS0 cell.

[0144] [000168]In some embodiments, the host cell is capable of producing a glycan-engineered antibody. For example, the host cell line can provide the required glycosylation machinery during post-translational modification. Examples of such host cell lines include those having altered (increased or decreased) activities of glycosylation-related enzymes such as glucosaminyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), glucosyltransferase (e.g., β(1,4)-galactosyltransferase (GT)), sialyltransferase (e.g., α(2,3)-sialyltransferase (ST)), mannosidase (e.g., α-mannosidase II (ManII)), fucosyltransferase (e.g., alpha-1,6-fucosyltransferase gene (FUT8), (1,3) fucosyltransferase), prokaryotic GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), GDP-fucose transporter (GFT), etc., either naturally or through genetic engineering, but are not limited thereto.

[0145] [000169]In some embodiments, the host cell is characterized by the absence of functional FUT8, overexpression of heterologous GnTIII, expression of prokaryotic GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), or absence of functional GFT. FUT8 knockout host cell lines are fucosylation-deficient and produce defucosylated antibodies. Overexpression of GnTIII in the host cell line (see, e.g., the Glycart technology by Roche) results in the formation of a split, non-fucosylated glycosylation form of the antibody. Expression of RMD (as seen, e.g., in the GlymaxX® system of ProBioGen AG) inhibits de novo fucose biosynthesis, and as a result, the antibodies produced by such host cell lines also exhibit reduced fucosylation. GFT knockout in the CHO cell line (see, e.g., the technology by Beijing Mabworks Biotech) blocks both the de novo fucose biosynthesis pathway and the fucose salvage biosynthesis pathway, resulting in reduced fucosylation.

[0146] [000170]The host cells are transformed with the above-described expression or cloning vector for anti-FGFR2b antibody production and cultured in a conventional nutrient medium modified to introduce a promoter, select a transformant, or amplify a gene encoding a desired sequence as needed. In another embodiment, the antibody can be produced by homologous recombination known in the art.

[0147] [000171]The host cells used to produce the antibodies provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. Further, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; International Publication No. 90 / 03430; International Publication No. 87 / 00195; or U.S. Patent Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium chloride, magnesium chloride, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the millimolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. The culture conditions, such as temperature, pH, etc., are those previously used when using the host cells selected for expression and will be apparent to those skilled in the art.

[0148] [000172]When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. When the antibody is produced intracellularly, as a first step, particulate debris, either of the host cell or lysed fragments, is removed, for example, by centrifugation or ultracentrifugation. Carter et al., Bio / Technology 10:163 - 167 (1992), describe procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, a protease inhibitor such as PMSF may be included in any of the foregoing steps, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0149] [000173]Anti - FGFR2b antibodies prepared from cells can be purified, for example, using hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE - cellulose ion - exchange chromatography, ammonium sulfate precipitation, salting - out, and affinity chromatography, and affinity chromatography is the preferred purification technique.

[0150] [000174]In certain embodiments, Protein A immobilized on a solid phase is used for the immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0151] [000175]After any optional pre-purification step(s), a mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably carried out at a low salt concentration (e.g., about 0-0.025 M salt).

[0152] [000176]Pharmaceutical Compositions [000177]The present disclosure further provides a pharmaceutical composition comprising an anti-FGFR2b antibody provided herein and one or more pharmaceutically acceptable carriers.

[0153] [000178]Examples of pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, blocking or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0154] [000179]Suitable components include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. When disclosed herein, by including one or more antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment and conjugate as provided herein, oxidation of the antibody or antigen-binding fragment is reduced. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing the quality retention period. Accordingly, in certain embodiments, a composition comprising one or more antibodies as disclosed herein and one or more antioxidants such as methionine is provided. Further, a method of preventing oxidation of an antibody or antigen-binding fragment as provided herein, a method of extending the quality retention period of an antibody or antigen-binding fragment as provided herein, and / or a method of improving the efficacy of an antibody or antigen-binding fragment as provided herein is provided by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.

[0155] [000180]For further explanation, as pharmaceutically acceptable carriers, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; fixed oils of plant origin, non-aqueous vehicles such as cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents at bacteriostatic or fungistatic concentrations; tonicity agents such as sodium chloride or dextrose; buffer solutions such as phosphate or citrate buffer solutions; antioxidants such as sodium bisulfite; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as Polysorbate 80 (TWEEN-80); sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid may be mentioned. Antibacterial agents used as carriers containing phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride can be added to the pharmaceutical composition in a multiple-dose container. Suitable excipients may include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0156] [000181]The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release preparation, or powder. As oral preparations, standard carriers of pharmaceutical grade such as mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc. may be mentioned.

[0157] [000182]In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as, for example, liquid solutions, suspensions, emulsions, or solid forms suitable for generating liquid solutions, suspensions, or emulsions. As preparations for injection, there may be mentioned sterile and / or pyrogen-free solutions that are immediately injectable, sterile dry soluble products such as lyophilized powders that are combined with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions that are immediately injectable, sterile dry insoluble products that are combined with a vehicle immediately prior to use, and sterile and / or pyrogen-free emulsions. The solution can be either aqueous or non-aqueous.

[0158] [000183]In certain embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial or syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0159] [000184]In certain embodiments, the sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or the reconstitution solution prepared therefrom or other pharmacological components. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, at a substantially neutral pH in one embodiment. Subsequently, sterile filtration of the solution under standard conditions known to those of skill in the art, followed by lyophilization, provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of the anti-FGFR2b antibody or composition thereof. Overfilling the vial by a small amount (e.g., about 10%) above the amount required for a dose or set of doses is acceptable to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder may be stored under suitable conditions, e.g., at about 4°C to room temperature.

[0160] [000185]Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, a suitable carrier of sterile and / or pyrogen-free water or other liquid is added to the lyophilized powder. The exact amount depends on the selected treatment being administered and can be determined empirically.

[0161] [000186]Method of Use [000187]The present disclosure provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment as provided herein, thereby treating or preventing an FGFR2b and / or FGFR1b-related condition or disorder. In some embodiments, the FGFR2b and / or FGFR1b-related condition or disorder is cancer, and optionally, the cancer is characterized by expressing or overexpressing FGFR2b and / or FGFR1b.

[0162] [000188]Examples of cancers include, but are not limited to, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck cancer, mesothelioma, melanoma, sarcoma, and brain tumors (e.g., gliomas such as glioblastoma), and hematological malignancies.

[0163] [000189]In some embodiments, the FGFR2b and / or FGFR1b-related condition or disorder is cancer characterized by expressing or overexpressing FGFR2b and / or FGFR1b.

[0164] [000190]Expression or overexpression of FGFR2b and / or FGFR1b can be determined by assessing an increase in the level of FGFR in a biological sample (e.g., a sample derived from cancer cells or tissue, or a sample derived from tumor infiltrating immune cells) from a subject in a diagnostic or prognostic assay. A variety of methods can be used. For example, a diagnostic or prognostic assay can be used to assess the expression level of FGFR2b and / or FGFR1b present on the surface of cells (e.g., via immunohistochemical assay; IHC). Alternatively, or in addition, for example, the level of FGFR-encoding nucleic acid in cells can be measured via polymerase chain reaction (PCR) techniques such as fluorescence in situ hybridization (FISH, see International Publication No. WO 98 / 45479, published in October 1998), Southern blotting, or real-time quantitative PCR (RT-PCR) (Methods 132:73-80 (1990)). In addition to the above assays, various in vivo assays are available to those of skill in the art. For example, cells in a patient's body can be exposed to an antibody optionally labeled with a detectable label, e.g., a radioisotope, and the binding of the antibody to the cells in the patient can be evaluated, e.g., by external scanning with respect to radioactivity, or by analyzing a biopsy taken from the patient that has been pre-exposed to the antibody.

[0165] [000191]A therapeutically effective amount of an antibody or antigen-binding fragment as provided herein depends, for example, on various factors known in the art such as the subject's body weight, age, medical history, current drug therapy, health status and the potential for cross-reactivity, allergy, sensitivity and adverse side effects, as well as the route of administration and the degree of disease onset. The dosage can be proportionally reduced or increased by a person of skill in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0166] [000192]In certain embodiments, an antibody or antigen-binding fragment as provided herein can be administered at a therapeutically effective dosage of from about 0.01 mg / kg to about 100 mg / kg. In some of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg / kg or less, and in some of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage of administration can vary during the course of treatment. For example, in certain embodiments, the dosage of the initial administration may be higher than the dosage of subsequent administrations. In certain embodiments, the dosage of administration can vary over the course of treatment in response to the subject's response.

[0167] [000193]The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0168] [000194]The antibodies disclosed herein can be administered by any route known in the art, such as, for example, parenterally (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection including intravenous infusion, intramuscular injection or intradermal injection) or non-parenterally (e.g., orally, intranasally, intraocularly, sublingually, rectally or topically).

[0169] [000195]In some embodiments, the antibodies disclosed herein can be administered alone or in combination with one or more additional therapeutic modalities or therapeutic agents. For example, the antibodies disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anti-cancer drug.

[0170] [000196]In some of these embodiments, an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment that is administered "in combination with" another therapeutic agent need not be administered simultaneously with the agent or in the same composition as the agent. An antibody or antigen-binding fragment that is administered before or after another agent is considered to be administered "in combination with" the agent when the phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. Where possible, additional therapeutic agents that are administered in combination with an antibody disclosed herein are administered according to the schedule set forth in the product information sheet for the additional therapeutic agent or according to Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or according to protocols well known in the art.

[0171] [000197]The present disclosure further provides methods of using anti-FGFR2b antibodies. [000198]In some embodiments, the present disclosure provides a method of detecting the presence or amount of FGFR2b and / or FGFR1b in a sample, the method comprising contacting the sample with an antibody and determining the presence or amount of FGFR2b and / or FGFR1b in the sample.

[0172] [000199]In some embodiments, the present disclosure provides a method for diagnosing FGFR2b and / or FGFR-1b related diseases or conditions in a subject, comprising: a) contacting a sample obtained from the subject with an antibody provided herein; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; and c) correlating the presence or amount of FGFR2b and / or FGFR1b with the presence or status of FGFR2b and / or FGFR1b related diseases or conditions in the subject.

[0173] [000200]In some embodiments, the present disclosure provides a method for prognosticating FGFR2b and / or FGFR1b related diseases or conditions in a subject, comprising: a) contacting a sample obtained from the subject with an antibody provided herein; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; and c) correlating the presence or amount of FGFR2b and / or FGFR1b with the potential responsiveness of the subject to an FGFR2b and / or FGFR1b antagonist.

[0174] [000201]In some embodiments, the present disclosure provides a kit comprising an antibody provided herein conjugated, optionally, to a detectable moiety. The kit can be useful for detecting FGFR2b and / or FGFR1b or diagnosing FGFR2b and / or FGFR1b related diseases.

[0175] [000202]In some embodiments, the present disclosure provides the use of an antibody provided herein in the manufacture of a diagnostic / prognostic reagent for diagnosing / prognosticating FGFR2b and / or FGFR1b related diseases or conditions, and in the manufacture of a medicament for treating diseases or conditions that would benefit from modulation of FGFR2b and / or FGFR1b expression in a subject.

[0176] [000203]The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments that fall within the scope of the invention. One of ordinary skill in the art may develop equivalent compositions, materials, and methods without exercising the inventive faculty and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while still remaining within the limits of the invention. It is the intention of the inventors that such variations be encompassed within the scope of the invention.

Examples

[0177] Example 1 Cells and Reagents [000204]The human gastric cancer cell lines KATO III and SNU16, which express FGFR2b, and Ba / F3 cells (pre-B lymphocytes) were purchased from the American Type Culture Collection (ATCC). The human esophageal cancer cell line KYSE180 was donated by Peking University. The above-mentioned human cell lines were cultured according to the recommendations of the suppliers. Human tumor tissues were obtained from Zhongshan Hospital (China) with the patients' consent in accordance with the regulations and used to develop a xenograft model LC038 derived from a human lung cancer patient.

[0178] [000205]To establish a cell-based assay for antibody screening during the antibody generation period, Ba / F3 cells were engineered to express FGFR2b or FGFR2c. Ba / F3 cells were transfected with plasmids encoding the 2b or 2c isoform of human FGFR2. After selection with G418, single clones with high expression of FGFR2b or FGFR2c were isolated.

[0179] [000206] The beta-isoform of human FGFR2b (IgD2 and IgD3 domains) was expressed as an immunoadhesin by fusing the extracellular domain (the "ECD domain") residues 65-267 of FGFR2b (Genbank accession number NP_001138391) to the human Fc region (residues 100-330) in a DNA plasmid. The protein was expressed by transfecting human 293F cells (Invitrogen) and purified from the culture medium using a protein A / G column.

[0180] [000207] The cDNA of cynomolgus (cyno) FGFR2b ECD domain was cloned from cyno skin mRNA by standard techniques, and amino acids 1-253 were fused to mouse Fc to create cyno FGFR2b-Fc for expression. The ECD domain residues of human (hu) FGFR2b (65-267 of NP_001138391) or rat FGFR2b (56-308 of NP_001103363.1) fused to mouse Fc were also expressed. The rat and mouse FGFR2b ECDs are identical.

[0181] [000208] Human Fc fusion proteins of other human FGFR family members, including recombinant FGFR1b-Fc, FGFR1c-Fc, FGFR2c, FGFR1c-Fc, FGFR3b-Fc, FGFR3c-Fc, and FGFR4-Fc proteins, were all purchased from R&D Systems. The alpha-isoform of FGFR2b-Fc and FGF were also purchased from R&D Systems. Heparin was obtained from Sigma-Aldrich (Sigma, #H3149-500KU-9). PBMCs were purchased from AllCell (#LP180322).

[0182] [000209] The clinical-stage anti-human FGFR2b-specific antibody FPA144 was expressed according to International Publication No. WO 2015 / 017600 A1, which is a related patent application. Example 2 Generation of Anti-FGFR Monoclonal Abs [000210]Balb / c or SJL mice were immunized intraperitoneally with human FGFR2b (beta)-Fc in CFA / IFA at an initial dose of 50 μg / mouse, followed by 25 μg / mouse, or an initial dose of 10 μg / mouse, followed by 5 μg / mouse. Serum titers against human FGFR2b-Fc or human FGFR2c-Fc were determined by ELISA. Four days after the final injection, popliteal lymphoid cells were harvested and fused with mouse myeloma cells. Ten days after fusion, hybridoma culture supernatants were screened by ELISA first for binding of FGFR2b (beta)-Fc to NC-Fc (Fc fragment as negative control). Hybridomas having antibodies that bound to FGFR2b (beta)-Fc but not to NC-Fc were selected. Hybridomas passing the primary screening were subjected to a secondary screening panel including binding to BaF3 / FGFR-2b cells and BaF3 / FGFR-2c by FACS, blocking of FGF ligand binding, and cell killing. Several positive clones including the clone designated Ab21 were thus selected. The isotype of the monoclonal antibodies produced by these selected clones was determined using isotype-specific antibodies.

[0183] Example 3 Humanization of Ab 21 [000211]The heavy and light chain variable (VH, VL) region sequences of Ab 21 were determined using standard RACE techniques. Total RNA was extracted from the selected hybridoma cell line. Subsequently, full-length first-strand cDNA containing the 5'-end was generated and amplified by PCR using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) or Gene Racer kit (Invitrogen) according to the manufacturer's instructions. The PCR products were isolated, purified, then TA cloned and sequenced.

[0184] [000212]Next, the chimeric antibody Ab 21c was generated with the V H and V LIt was generated by grafting onto human Fc. Then, using standard methods of molecular biology, the humanization of Ab 21 was designed, constructed, and expressed. Briefly, the CDRs of mouse Ab 21 were grafted onto a human acceptor framework. Subsequently, amino acid residues derived from the mouse antibody were substituted with human framework amino acid residues at framework positions where a computer model suggested significant contact with the CDRs (including M69L, A93T, and R94S of the heavy chain using Kabat numbering, and none in the light chain). This provided a humanized antibody of Ab 21 called Ab hu21-21. The amino acid Asn-Gly (NG) in CDR2 of the heavy chain of Ab hu21-21 was further substituted with the amino acid Asn-Arg (NR) to provide a variant called Ab hu21-26. The CDR region sequences and variable region sequences of the heavy and / or light chains of Ab21, Ab 21c, Ab hu21-21, and Ab hu21-26 are shown in Tables 1 to 3 above.

[0185] [000213] The amino acid sequences of the fully mature Ab hu21-26 light and heavy chains with human IgG1 are shown in Figure 1. Example 4 Defucosylation and Glycan Analysis of Ab hu21-26 [000214] To generate a defucosylated version of Ab hu21-26 (referred to as "afhu21-26" here, where the prefix "af" is an abbreviation for "defucosylated"), 1,6-fucosyltransferase knockout (FUT8- / -) CHOK1 cells (Wuxi Biologics, Shanghai, China) were used as the host cell line to produce an antibody without fucose (i.e., a defucosylated antibody). An expression vector containing the nucleotide sequences encoding the heavy chain (HC) and light chain (LC) of the Ab hu21-26 monoclonal antibody with a human IgG1 constant Fc was transiently transfected into FUT8- / - CHOK1 to produce the defucosylated antibody.

[0186] [000215] afhu21-26 was purified by protein A and SEC-HPLC, dialyzed, exchanged into the formulation buffer, and stored at -80 °C. [000216] Glycan analysis of the produced afhu21-26 was performed using LC-MS. Briefly, 10 μg of the antibody in 10 μL was digested with 1 μL of 12 units / μL of IdeS enzyme (Genovis AB) at 37 °C for 1 hour. Then, 37.5 μL of 8 M guanidine hydrochloride, 2.5 μL of 1 M Tris-HCl, and 1 μL of 1 M DTT were sequentially added, mixed, and incubated at 10 - 30 °C for 30 minutes. The fully reduced antibody was separated by reversed-phase HPLC. Subsequently, the glycan profile was analyzed using LC-MS. The mass of each peak was determined and used to identify each glycan, and the results are shown in Table 4 below.

[0187] [000217]

[0188]

Table 4

[0189] [000218] As a result, as shown in Table 5, G0F, G1F, and G2F were undetectable, demonstrating that the antibody was almost 100% defucosylated. [000219]

[0190]

Table 5

[0191] Example 5 Binding characteristics of the antibody [000220]The binding of the antibody to the human FGFR2b or human FGFR1b antigen was determined by surface plasmon resonance (Biacore). Briefly, a CM5 sensor chip (GE Healthcare Life Sciences) was first activated by injection of a 1:1 fresh mixture of 50 mM N-hydroxysuccinamide (NHS):200 mM ECD domain for 4 minutes. Next, hFGFR2b-Fc or hFGFR1b-Fc was immobilized on the activated CM5 center chip using an amine coupling kit (GE Healthcare Life Sciences) and 1 M ethanolamine as a blocking reagent. Antigen proteins of approximately 20 - 30 response units (RU, where 1 RU represents the binding of 1 pg of protein per square millimeter) were captured.

[0192] [000221]The antibody was diluted in HBS-EP+ running buffer (GE Healthcare Life Sciences) (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) and injected at a series of concentrations (0, 6.25, 12.5, 25, 50, 100, 150, 200 nM), and surface regeneration of the CM5 sensor chip was included in each running cycle. The association constant and dissociation constant were calculated using Biacore T200 evaluation software (version 1.0). As shown in Figure 2, Ab 21c (chimeric) and its humanized mutants Ab hu21-21 and Ab hu21-26 showed strong binding affinity for human FGFR2b with KD values in the range of 220 - 489 pM, which is better than that of the antibody FPA144 used as a positive control. Furthermore, Ab 21c was distinguishable from the antibody FPA144 with respect to FGFR1b binding. Ab 21c binds strongly to human FGFR1b with a KD value of 3.69 nM, as compared to the very weak binding of the antibody FPA144 to human FGFR1b with a KD value of 225 nM. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 showed specific binding to human FGFR1b as well (data not shown).

[0193] [000222]To confirm that the selected antibody can bind to the endogenous form of FGFR2b on the cell membrane, flow cytometry was performed using KATOIII cells expressing FGFR2b. All antibodies were prepared in PBS buffer with 10% donkey serum (Jackson Immunogen #017-000-121). 500,000 KATOIII cells were incubated with 100 μl of anti-FGFR2b antibodies at various concentrations for 60 minutes at 4°C. The cells were washed twice and incubated in 100 μl of 10 μg / ml secondary IgG-ALexa488 antibody (Jackson Immunogen #709546149) for 30 minutes at 4°C in the dark. The cells were washed three times, resuspended in wash buffer, and analyzed by flow cytometer. FACS data showed that Ab 21c strongly binds to KATOIII cells at an EC 50 value of approximately 8 nM, as shown in Figure 3. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 showed specific binding to KATOIII cells as well (data not shown).

[0194] [000223]The cross-species binding of Ab 21c to recombinant cyno, rat / mouse, and human FGFR2b-Fc proteins was performed using ELISA. Briefly, 96-well ELISA plates were coated overnight with approximately 100 μl / well of recombinant human FGFR2b-Fc, recombinant rat / mouse FGFR2b-Fc, or recombinant cyno FGFR2b-Fc protein at 0.1 μg / ml in PBS. Next, the plates were blocked in 2% BSA in PBS with 0.05% Tween 20 and incubated with the antibody samples for 60 minutes at room temperature, followed by washing twice in 1×TBST (Cell Signaling Technology, #9997), and then incubated with anti-human IgG HRP (horseradish peroxidase) conjugate for 60 minutes at room temperature. HRP activity was detected using the tetramethylbenzidine substrate (Cell Signaling Technology, #7004), and the reaction was stopped using the stop solution (Cell Signaling Technology, #7002). The plates were read at 450 nm. As shown in Figure 4, no significant difference was seen in the binding EC 50 for Ab 21c to FGFR2b of different species. Ab 21c had the highest binding affinity for rat / mouse FGFR2b, followed by human FGFR2b, and then cyno FGFR2b. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 also showed specific binding to FGFR2b of various species (data not shown).

[0195] [000224]Similarly, the binding specificities of Ab 21 to various FGFR family members, FGFR1b, FGFR3c, FGFR3b, and FGFR4, were characterized using an ELISA assay. The data are shown in Figure 5. According to the results of the ELISA analysis, Ab 21 specifically binds to FGFR2b and FGFR1b, which is consistent with the data shown in Figure 2, and Ab 21 does not bind to any other FGFR family members. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 showed specific binding to FGFR2b and FGFR1b in the ELISA analysis, but did not show specific binding to any other FGFR family members (data not shown).

[0196] Example 6 In vitro inhibitory activity [000225]The inhibitory activity of the antibodies against ligand-induced cell proliferation was performed in FGFR2b-engineered Ba / F3 cell clones (Ba / F3-FGFR2b). Cells were seeded in 96-well plates at 30,000 cells / well in RPMI1640 medium containing 10% fetal bovine serum and recombinant human FGF protein (10 ng / mL) in the presence of heparin (10 μg / ml). After overnight incubation, various concentrations of anti-FGFR2b antibodies were added to the assay plates and incubated for an additional 72 hours. After 72 hours of incubation, 20 μl of CellTiter Aqueous One Solution reagent was added to each well and the plates were incubated at room temperature for 2 hours. To measure the absorbance, 25 μl of 10% SDS was added to each well to stop the reaction. Absorbance was measured at 490 nm and 650 nm (reference wavelength) using a Tecan Spark 20M. Ab 21c can potently inhibit FGF7-induced BaF3 cell proliferation with a GI50 of approximately 10 nM. This inhibitory activity data of Ab-21c was processed using Prism and the graph is shown in Figure 6. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 showed similar potent inhibition of FGF7-induced BaF3 cell proliferation (data not shown).

[0197] [000226]The inhibition of the FGFR2b signaling pathway by antibodies was examined. SNU16 cells were grown in RPMI medium with 10% FBS, and then seeded at 30,000 cells / well and starved overnight in serum-free RPMI / 0.1% BSA. Next, the cells were collected by scraping, washed once in cold PBS, and then lysed in 2× SDS lysis buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol and 1× protease and phosphatase inhibitor (Pierce)). Subsequently, the lysate was boiled at 100 °C for 10 minutes. Protein concentration was detected by BCA protein assay kit (Pierce), equal amounts of protein were loaded onto SDS-PAGE gels, and then the proteins were transferred to nitrocellulose membranes using iBolt (Invitrogen), and then subjected to Western blotting analysis for phosphorylation of FGFR2 and its downstream gene ERK. As shown in Figure 7, Ab 21c treatment resulted in down-regulation of phosphorylated FGFR2 and phosphorylated ERK in a dose-dependent manner in SNU16 cells. Similar to Ab 21c, both Ab hu21-21 and Ab hu21-26 showed similar down-regulation of phosphorylated FGFR2 and phosphorylated ERK (data not shown).

[0198] [000227]The internalization of the antibodies was detected by confocal microscopy. Briefly, on the day of confocal microscopy, the cells were collected using enzyme-free dissociation solution, and 5×10 5They were prepared at the density of individual cells. The cells were washed and resuspended in 100 μl of blocking buffer (10% donkey serum) at 4°C for 30 minutes. Next, the cells were washed twice and incubated with 100 μl of 10 μg / ml Ab 21 at 4°C for 60 minutes. The cells were washed and divided into multiple aliquots in vials, some of which were kept at 4°C and the rest at 37°C. At the indicated time points, one vial was taken out from 4°C and one vial was taken out from 37°C. After washing and resuspension, the cells were fixed, subsequently blocked with 100 μl of 10% donkey serum, then washed and incubated with 10 μg / ml donkey anti-human IgG-Alexa488 at 4°C in the dark for 30 minutes. The cells were washed and resuspended, 5 μl of the cell suspension was spread on a slide glass (for the purpose of obtaining monolayer cells), dried on the hot surface at 55°C, treated with 5 μl of 1×DAPI, and then sealed with a coverslip. Next, confocal images were taken. As shown in Figure 8, at 4°C where internal translocation cannot occur, Ab 21 was only observed on the cell surface. After being subjected to the 37°C condition that allows internal translocation for 2 hours or 4 hours, the antibody was found inside the cells (indicated by arrows), suggesting that antibody internal translocation occurred. Similar to Ab 21, Ab 21c, Ab hu21-21, and Ab hu21-26 all similarly induced antibody internal translocation in cells (data not shown).

[0199] [000228]An in vitro assay was performed to determine the ADCC activity of the antibody. The ADCC assay was performed using primary NK cells isolated from human PBMC (AllCells, CAT#PB0004F) by the EasySep™ Human NK Cell Isolation Kit (Stemcell, #17955) as effector cells at an effector-to-target (E / T) cell ratio of 8:1. Human PBMC were thawed in RPMI1640 containing 10% FBS + 10 mM HEPES + 1 mM sodium pyruvate and FACS assay was performed the next day. Target cells KATOIII were stained with the cell marker CFSE-FITC (Invitrogen, #C34554) for 30 minutes and then incubated at 37°C for 5 hours in the presence of effector and antibody. Next, the cells were stained with the viability marker Viability staining-APC-Cy7 (BD, #565388). Cytotoxic lysis was determined by FACS by gating cells that were positive for both CFSE staining and viability marker staining. The data are shown in Figure 9. Afhu21-26 showed very good ADCC activity when compared to Ab 21c, and defucosylation was shown to improve the ADCC activity of Ab 21c in both the maximum lysis percentage and EC 50 50. Similar results were also obtained for afhu21-21 as well.

[0200] Example 7 In vivo antitumor activity of the antibody in a tumor mouse model [000229]Immunodeficient nude mice were purchased from VitaRiver. All animal studies were approved by the IACUC and conducted in accordance with institutional and local regulatory requirements.

[0201] [000230]A xenograft (CDX) mouse model derived from the SNU16 human gastric cancer cell line was established by first culturing the cells in vitro and then mixing them with 50% Matrigel / mouse and subcutaneously inoculating the cells into the dorsal flank of the mouse at 1×10 7 6 cells / 200 μl, and the xenograft tumor was 300 - 500 mm3 Once they reached the size of 3 , they were excised, cut into fragments of the same size, and subcutaneously (s.c.) transplanted into a new group of nude mice. The LC038 patient-derived xenograft (PDX) mouse model was established in a similar manner. Briefly, surgically removed tissue from the patient (F0) was cut into fragments of the same size and subcutaneously transplanted into immunodeficient nude mice within 2 hours after surgery (F1 mice). When the xenograft tumors reached 400 - 600 mm 3 in size, they were excised, cut into fragments, and transplanted into nude mice for passage, which was F2, and the same procedure was followed thereafter.

[0202] [000231]Tumor nodules were measured two-dimensionally using calipers, and tumor volume was calculated using the following formula: Tumor volume = (length × width 2 ) × 0.52. When the tumor volume reached 150 - 250 mm 3 , tumor-bearing mice were randomly selected for the treatment group. Subsequently, the mice were treated once / twice a week from the day after random selection with either an isotype control (i.e., IgG1) or a test antibody (i.e., FPA144, afhu21 - 26). The tumor volume and body weight of the mice were measured twice a week, and the raw data were recorded. Tumor growth inhibition from the start of treatment was evaluated by comparing the average change in tumor volume between the control group and the treatment group. The calculation was based on the geometric mean or arithmetic mean of the relative tumor volume (RTV) in each group. RTV was calculated by dividing the tumor volume on the treatment day by the initial tumor volume.

[0203] [000232]The in vivo tumor growth curves of SNU16 cells and LC038 PDX cells using afhu21 - 26 or antibody FPA144 treatment are shown in Figures 10A and 10B, respectively. In both models, Afhu21 - 26 showed better antitumor activity than antibody FPA144. Similar results were also obtained with afhu21 - 21.

[0204] Example 8 Production and Characterization of Antibody - Drug Conjugates (ADCs) [000233]To 3 ml of 10 mg / ml Ab 21c in PBS, fresh TCEP was added at a TCEP / Ab molar ratio of 2.2. After incubation in a 37 °C water bath for 120 minutes, 1 / 10 (v / v) N,N-dimethylacetamide (DMA) was added to the antibody solution. Subsequently, 10 mM mc-vc-MMAE in DMA (mc = maleimidocaproyl; vc = valine-citrulline linker) was added to the Ab solution at an MMAE to antibody molar ratio of 6. After keeping the solution at room temperature overnight, a PD-10 column was equilibrated with 25 ml of 1×PBS. Next, the conjugation mixture was loaded onto the PD-10 column to purify the ADC. The concentration of the ADC was measured by Nanodrop. SEC-HPLC and HIC-HPLC were used for quality control analysis of ADC aggregation and drug-antibody ratio (DAR), respectively. The average DAR was calculated according to standard methods. ADC conjugates 21c-MMAF and afhu21-26-MMAE were generated similarly. The HIC-HPLC histogram of afhu21-26-MMAE is shown in Figure 11. The calculated DAR was 3.76, which is similar to that of the approved ADC drug brentuximab vedotin.

[0205] [000234]The antitumor activities of the ADCs in various tumor xenograft models such as the LC038 PDX model and SNU16 xenografts were evaluated. Treatments with afHu21-26-MMAE, 21c-MMAF, and 21c-MMAE all induced tumor regression in both tumor models (Figures 12A and 12B).

Claims

**Claim 1** a) Heavy chain complementarity determining regions (CDRs) 1, 2, and 3, which are amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, and, Light chain complementarity determining regions (CDRs) 1, 2, and 3, which are amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively, or, b) Heavy chain complementarity determining regions (CDRs) 1, 2, and 3, which are amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 5, respectively, and, Light chain complementarity determining regions (CDRs) 1, 2, and 3, which are amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively, An isolated antibody comprising, and capable of specifically binding to both FGFR2b and FGFR1b. **Claim 2** The antibody according to claim 1, which does not have a detectable binding affinity for FGFR2c. **Claim 3** A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16, or, A homologous sequence thereof having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16, wherein the homologous sequence has substitutions, insertions, or deletions in regions outside the CDRs, The antibody according to claim 1 or 2. **Claim 4** A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 14, or, A homologous sequence thereof having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 14, wherein the homologous sequence has substitutions, insertions, or deletions in regions outside the CDRs, The antibody according to any one of claims 1 to 3. **Claim 5** a) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 10, b) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14, or, c) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 10 The antibody according to any one of claims 1 to 4, comprising. **Claim 6** Further comprising one or more amino acid residue substitutions or modifications that still retain the specific binding affinity for FGFR2b and / or FGFR1b, The substitution or modification is present in one or more of the VH or VL sequences, but outside of any of the CDR sequences, An antibody according to any one of claims 1 to 5. **Claim 7** An antibody according to any one of claims 1 to 6, further comprising an immunoglobulin constant region, optionally a constant region of a human immunoglobulin, or optionally a constant region of human IgG. **Claim 8** The constant region is a) introducing or removing a glycosylation site, b) introducing a free cysteine residue, c) enhancing binding to an activating Fc receptor, and / or d) enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) An antibody according to claim 7, comprising one or more modifications. **Claim 9** An antibody according to claim 8, which has been subjected to glycan engineering. **Claim 10** An antibody according to claim 9, wherein the glycan-engineered antibody exhibits enhanced ADCC activity compared to its non-engineered counterpart. **Claim 11** An antibody according to claim 10, which is defucosylated. **Claim 12** An antibody according to claim 11, which lacks fucose at Asn297. **Claim 13** An antibody according to claim 12, wherein the enhanced ADCC is characterized by lysis of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% of FGFR2b-expressing cells. **Claim 14** An antibody according to any one of claims 1 to 13, which is a chimeric antibody or a humanized antibody. **Claim 15** Camelized single-domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ２ , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') ２ , ds diabody, nanobody, domain antibody, or bivalent domain antibody, the antibody according to any one of claims 1 to 14 **Claim 16** The antibody according to any one of claims 1 to 15, which is capable of specifically binding to human FGFR2b with a K －９ value of 1×10 Ｄ M or less as measured by Biacore. **Claim 17** The antibody according to any one of claims 1 to 16, which is capable of specifically binding to human FGFR1b with a K －９ value of 5×10 Ｄ M or less as measured by Biacore. **Claim 18** The antibody according to any one of claims 1 to 17, which is capable of specifically binding to human FGFR2b expressed on the cell surface with an EC measured by flow cytometry of 10 nM or less. ５０ when. **Claim 19** An antibody according to any one of claims 1 to 18, which is capable of specifically binding to human FGFR2b, cynomolgus monkey FGFR2b, rat FGFR2b, and mouse FGFR2b. **Claim 20** Specifically binds to human FGFR2b expressed on the cell surface and has a 50% growth inhibitory concentration (GI ５０ ) of 15 nM or less when measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric assay, the antibody according to any one of claims 1 to 19, which is capable of inhibiting the proliferation of said cells. **Claim 21** An antibody according to any one of claims 1 to 20, which is linked to one or more conjugate moieties. **Claim 22** An antibody according to claim 21, wherein the conjugate moiety comprises a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modification moiety, or a purification moiety. **Claim 23** An antibody according to claim 22, wherein the therapeutic agent comprises a cytotoxic agent. **Claim 24** An antibody according to claim 22 or 23, wherein the conjugate moiety is covalently bound directly or via a linker. **Claim 25** The antibody according to claim 24, wherein the linker is a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, a thioether linker, and optionally, the linker is a dipeptide cleavable in lysosomes, for example, valine-citrulline (vc).

26. The antibody according to any one of claims 21 to 25, wherein the conjugate moiety is randomly bound to a specific type of surface-exposed amino acid residue, and optionally, the specific residue is a cysteine residue or a lysine residue.

27. The antibody according to any one of claims 21 to 26, wherein the conjugate moiety is bound to a clearly defined site in the antibody molecule via a natural amino acid, a non-natural amino acid, a short-chain peptide tag, or Asn297 glycan.

28. An isolated polynucleotide encoding the antibody according to any one of claims 1 to 27.

29. The isolated polynucleotide according to claim 28, comprising a nucleotide sequence selected from the group consisting of its homologous sequences having at least 80% sequence identity to the sequences shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, wherein the homologous sequence encodes the same protein as encoded by the sequence shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO:

17.

30. An expression vector comprising the isolated polynucleotide according to claim 28 or 29.

31. A host cell comprising the expression vector according to claim 30.

32. The host cell according to claim 31, capable of producing a glycan-engineered antibody or antigen-binding fragment.

33. The host cell according to claim 31, characterized by a lack of functional alpha-1,6-fucosyltransferase (FUT8), overexpression of heterologous beta1,4-N-acetylglucosaminyltransferase III (GnTIII), expression of prokaryotic GDP-6-deoxy-D-lyxo-4-hexulose reductase, or a lack of functional GDP-fucose transporter (GFT).

34. A method for producing an antibody according to any one of claims 1 to 27, comprising culturing a host cell according to any one of claims 31 to 33 under conditions under which the expression vector according to claim 30 is expressed.

35. The method according to claim 34, further comprising a step of purifying the antibody produced by the host cell.

36. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

37. A pharmaceutical composition for treating an FGFR2b and / or FGFR1b-related disease or condition in a subject, comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 27.

38. The pharmaceutical composition according to claim 37, wherein the disease or condition is cancer, and optionally, the cancer is characterized by expressing or overexpressing FGFR2b and / or FGFR1b.

39. The pharmaceutical composition according to claim 38, wherein the cancer is ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, renal cell carcinoma, head and neck cancer, mesothelioma, melanoma, sarcoma, and brain tumor.

40. The pharmaceutical composition according to any one of claims 37 to 39, for oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

41. The pharmaceutical composition according to any one of claims 37 to 40, wherein the subject is human.

42. A method for detecting the presence or amount of FGFR2b and / or FGFR1b in a sample, comprising contacting the sample with an antibody according to any one of claims 1 to 27 and determining the presence or amount of FGFR2b and / or FGFR1b in the sample.

43. A method for providing an indicator for diagnosing an FGFR2b and / or FGFR1b-related disease or condition in a subject, a) contacting a sample obtained from the subject with an antibody according to any one of claims 1 to 27; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; c) correlating the presence or amount of FGFR2b and / or FGFR1b with the presence or situation of an FGFR2b and / or FGFR1b-related disease or condition in the subject comprising.

44. A method for providing an indicator for prognosticating an FGFR2b and / or FGFR1b-related disease or condition in a subject, a) contacting a sample obtained from a subject with an antibody according to any one of claims 1 to 27; b) determining the presence or amount of FGFR2b and / or FGFR1b in the sample; c) correlating the presence or amount of FGFR2b and / or FGFR1b with the potential responsiveness of the subject to an FGFR2b and / or FGFR1b antagonist A method comprising:

45. Use of an antibody according to any one of claims 1 to 27 in the manufacture of a medicament for treating an FGFR2b and / or FGFR1b-related disease or condition in a subject in need of treatment.

46. Use of an antibody according to any one of claims 1 to 27 in the manufacture of a diagnostic reagent for detecting an FGFR2b and / or FGFR1b-related disease or condition.

47. A kit for detecting FGFR2b and / or FGFR1b, comprising an antibody according to any one of claims 1 to 27.

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