Novel anti-FGFR2 antibody

Abstract

The present disclosure relates to anti-FGFR2b antibodies or antigen-binding fragments thereof, nucleic acids encoding same, pharmaceutical compositions comprising same, methods for their production, and uses thereof.

Description

[Technical field]

[0001] The present disclosure relates to the biomedical field, and in particular to an antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, a method for producing same, and uses thereof. [Background technology]

[0002] Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinase receptors that regulate many fundamental biological processes, including embryogenesis, tissue and stem cell maintenance, angiogenesis, and wound healing, through activation of downstream PI3K-AKT and MAPK-ERK pathways (Beenken and Mohammadi, 2009; Katoh and Katoh, 2006; Turner and Grose, 2010). The FGFR family consists of four receptors (FGFR1-FGFR4) that respond to 22 ligands (FGFs) (Korc and Friesel, 2009). Dysregulation of the FGFR2 signaling pathway leads to tumorigenesis and poor prognosis through gene amplification and subsequent protein overexpression (Grose and Dickson, 2005). Splice mutations result in several receptor variants, including two major FGFR2 isoforms, designated FGFR2b and FGFR2c (also called FGFR2IIIb and FGFR2IIIc). In general, the expression of each isoform is restricted to a particular tissue. In particular, the three major ligands of FGFR2b are FGF7, FGF10, and FGF22, which are mostly expressed in epithelial cells, whereas FGFR2c is expressed in mesenchymal tissues (Ornitz et al., 1996; Zhang et al., 2006). In FGFR2-amplified gastric cancer, the FGFR2b isoform predominates.

[0003] Previous studies have shown that FGFR2b is overexpressed in 2.5%–31.1% of gastroesophageal adenocarcinomas (GEA), depending on the antibody and assay used (Ahn et al., 2016; Angal et al., 1993; Nagatsuma et al., 2015). Bemarituzumab (FPA144), a humanized afucosylated immunoglobulin G1 monoclonal antibody specific for FGFR2b, blocks FGFR2b signaling by competitive binding inhibition of FGF, inducing enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) against FGFR2b-overexpressing tumor cells (Xiang et al., 2021). Bemarituzumab has demonstrated remarkable antitumor activity in preclinical studies and good efficacy in phase I clinical trials (Catenacci et al., 2020; Xiang et al., 2021). However, corneal toxicity was reported in 3 of 28 patients (10.7%) who received the high dose (Catenacci et al., 2020). The mechanism of corneal toxicity is hypothesized to be the result of inhibition of FGF10, which is involved in regulating corneal epithelial wound healing (Itoh, 2016). Previous in vivo studies have examined the therapeutic effect of FGF10 in an inflammation-induced rabbit dry eye model and demonstrated the effect of FGF10 in healing corneal epithelial cells (Zheng et al., 2015). There is an increasing need for anti-FGFR2b antibodies that reduce corneal toxicity without affecting antitumor activity.

[0004] In order to develop new anti-FGFR2b antibodies with low corneal toxicity to fulfill unmet clinical needs, the inventors generated a specific antibody against FGFR2b that exhibits high tumor suppression potential while exhibiting low FGF10 inhibition and thus is expected to have fewer clinical adverse events. Summary of the Invention

[0005] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising a heavy chain complementarity determining region 1 (CDR1) having an amino acid sequence at least 80% identical to SEQ ID NO:1, a heavy chain CDR2 having an amino acid sequence at least 80% identical to SEQ ID NO:2, and a heavy chain CDR3 having an amino acid sequence at least 80% identical to SEQ ID NO:3.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof has at least one of the following properties: i) weak inhibition of the binding of FGF10 to FGFR2b; and ii) no detectable binding affinity for FGFR2c;

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:4, a light chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:6.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:7 and a light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO:8.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:8.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO:9 and a light chain having the amino acid sequence of SEQ ID NO:10.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a human IgG constant region. In a preferred embodiment, the constant region is derived from human IgG1.

[0013] In some embodiments, the constant region of an antibody or antigen-binding fragment thereof provided herein contains one or more modifications that enhance antibody-dependent cellular cytotoxicity (ADCC).

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are afucosylated.

[0015] In some embodiments, the antibody or antigen-binding fragment provided herein is a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an F(Fab')2 fragment, an scFv fragment, an Fv fragment, a Fab' fragment, or a domain antibody.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of inhibiting FGF-induced FGFR2 phosphorylation and proliferation of cancer cells.

[0017] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with the above-mentioned antibody or antigen-binding fragment thereof for binding to FGFR2b.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the same epitope on FGFR2b recognized by an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively.

[0019] In another aspect, the disclosure provides a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein.

[0020] In some embodiments, the nucleic acid comprises a heavy chain-encoding nucleic acid having the nucleotide sequence of SEQ ID NO:11 and / or a light chain-encoding nucleic acid having the nucleotide sequence of SEQ ID NO:12.

[0021] In another aspect, the invention provides an expression vector comprising a nucleic acid provided herein.

[0022] In another aspect, the disclosure provides a host cell comprising an expression vector provided herein.

[0023] In another aspect, the disclosure provides a pharmaceutical composition comprising: (a) an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, or an expression vector provided herein; and (b) A pharma- ceutically acceptable carrier.

[0024] In another aspect, the disclosure provides antibody-drug conjugates in which an antibody, or antigen-binding fragment thereof, provided herein is conjugated to one or more conjugate moieties.

[0025] In another aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof provided herein, the method comprising culturing a host cell provided herein under conditions that allow for expression of the antibody or antigen-binding fragment thereof.

[0026] In another aspect, the disclosure provides use of an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein in the manufacture of a medicament for treating a disease or condition associated with abnormal expression of FGFR2b in a subject.

[0027] In another aspect, the disclosure provides a method of inhibiting or reducing FGF-induced proliferation of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein.

[0028] In another aspect, the disclosure provides a method of inhibiting or reducing FGF-induced FGFR2 phosphorylation in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein.

[0029] In another aspect, the present disclosure provides a method of killing tumor cells associated with aberrant expression of FGFR2b and reducing corneal toxicity in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein.

[0030] In another aspect, the disclosure provides a method of treating a disease or condition associated with abnormal expression of FGFR2b in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein.

[0031] In some embodiments, the disease or condition is cancer, and optionally, the cancer is characterized by expressing or overexpressing FGFR2b. In a preferred embodiment, the cancer is characterized by expressing or overexpressing FGFR2b.

[0032] In some embodiments, the disease or condition is one selected from the group consisting of 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, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, and urothelial carcinoma. In a preferred embodiment, the disease or condition is gastric cancer. In a preferred embodiment, the disease or condition is FGFR2-positive gastric cancer. In a preferred embodiment, the disease or condition is FGFR2-amplified gastric cancer.

[0033] In some embodiments, according to any one of the above methods, an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, a host cell provided herein, a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein is administered sequentially or simultaneously with at least one additional therapeutic agent.

[0034] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof provided herein. In a preferred embodiment, the kit according to the present disclosure further comprises instructions for guiding the use of the antibody or antigen-binding fragment thereof of the present disclosure, such as in treating or preventing a disease or condition associated with aberrant expression of FGFR2b in a subject, such as cancer.

[0035] These and other aspects are described in further detail herein. Each aspect presented may include various embodiments presented herein. Thus, each aspect described may include embodiments including any element or combination of elements, and it is anticipated that all combinations of such aspects and embodiments are expressly contemplated. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1. Binding of anti-FGFR2b antibodies to different FGFR2 isoforms. [Figure 1A] FIG. 1A is a diagram showing that HC18 specifically binds to hFGFR2b-expressing CHOK1 cells. [Figure 1B] FIG. 1B shows that HC18 did not bind to hFGFR2c-expressing CHOK1 cells. [Diagram 2] FIG. 2 shows inhibition of FGFR2b binding to its ligand by anti-FGFR2b antibodies. [Figure 2A] FIG. 2A is a diagram showing that HC18 significantly inhibited the binding of FGF7 to FGFR2b. [Figure 2B] FIG. 2B is a diagram showing that HC18 partially inhibited the binding of FGF10 to FGFR2b. [Diagram 3] FIG. 3 shows a surface plasmon response (SPR) sensorgram of an anti-FGFR2b antibody binding to the human FGFR2b antigen. [Figure 4] FIG. 4 shows an ADCC (antibody-dependent cellular cytotoxicity) bioassay using PBMC as effector cell induction by anti-FGFR2b antibody and Ba / F3 cells expressing full-length human FGFR2b target cells (E:T=20:1). [Diagram 5] FIG. 5. Inhibition of FGFR2 phosphorylation induced by FGF7 (FIG. 5A) and FGF10 (FIG. 5B) in SNU-16 cells by anti-FGFR2b antibody and negative control hIgG1. [Figure 6] FIG. 6 shows inhibition of ERK1 / 2 phosphorylation induced by FGF7 (FIG. 6A) and FGF10 (FIG. 6B) in SNU-16 cells by anti-FGFR2b antibody and negative control hIgG1. [Figure 7] FIG. 7. Inhibition of FGF7-induced proliferation of SNU-16 cells by anti-FGFR2b antibody and negative control hIgG1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings generally understood by those skilled in the art. In addition, unless otherwise clearly indicated by the context, singular terms shall include plural terms and vice versa. In consideration of the readability of this application, certain terms are defined below.

[0038] definition As used herein, the indefinite article "a" or "an" is understood to refer to "one or more" of the described or listed components.

[0039] The term "about" as used herein, when applied to a numerical value, refers to a value that is reasonably close to the stated value and within an acceptable error range as determined by one of ordinary skill in the art, which may depend in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" may mean within ±50%, preferably within ±25%, and more preferably within ±10% of the stated reference value. When a specific value is provided in this application, the meaning of "about" should be understood to be within an acceptable error range for that specific value, according to the practice in the art, unless otherwise specified.

[0040] "Antibody" (Ab) includes, but is not limited to, the glycoprotein immunoglobulin (Ig) that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each H chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region of an IgG antibody contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (herein abbreviated as V L The light chain constant region of an IgG antibody comprises one constant domain, C L Includes: VH Area and V L The regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs). H and V L contains three CDRs (light chain CDRs include LCDR1, LCDR2, and LCDR3, heavy chain CDRs include HCDR1, HCDR2, and HCDR3) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. Various methods have been used to define the CDR domains in antibodies, including the Rabat, Chothia, AbM, Contact, and IMGT definitions. Kabat numbering is used as the default in this disclosure unless otherwise stated. The constant region of an antibody may mediate the binding of Ig to various cells of the immune system (e.g., effector cells) and host tissues or factors, such as the first component (Clq) of the classical complement system. Antibodies are assigned to classes based on the amino acid sequence of the constant region of the heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM. Some of the major antibody classes are divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0041] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, and bispecific antibody that binds to a specific antigen. An intact antibody or an antibody fragment having an antigen-binding portion of the antibody can be used. The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from a portion of an intact antibody that includes one or more CDRs, or other antibody fragment that is capable of binding to an antigen but does not include the intact native antibody structure. The term "antibody or antigen-binding fragment thereof" as used herein refers to an intact antibody or an antibody fragment having an antigen-binding portion. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. The antibodies or antigen-binding fragments thereof include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments containing the complementarity determining regions (CDRs), single chain variable fragments (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0042] "Fab fragment" is V L Domain, V H Domain, C L A "F(ab')2 fragment" is a bivalent fragment having two Fab fragments connected by a disulfide bond at the hinge region. An "Fv fragment" is a V fragment derived from a single arm of an antibody. L Domain and V H A "domain antibody (dAb)" is a H Domain or V L A "single-chain variable fragment (scFv)" consists of a V L Area and V HThe term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which is connected by a V domain connected by a linker that is too short to pair the two domains on the same chain. H Domain and V L A diabody comprises a polypeptide chain that is a polypeptide chain that is a polypeptide chain of a diabody, and includes a domain that allows each domain to pair with a complementary domain on another polypeptide chain. If the two polypeptide chains of a diabody are the same, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to produce diabodies or bispecific antibodies with two different antigen-binding sites. Diabodies or bispecific antibodies also refer to artificial antibodies or antigen-binding fragments that have fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be on the same antigen or on two different antigens. Similarly, triabodies, tetrabodies, or other multispecific antibodies are antibodies that include three, four, or multiple polypeptide chains that may be the same or different, thus forming three, four, or multiple antigen-binding sites, each of which may be the same or different.

[0043] The term "human" antibody, as used herein, refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences and, further, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.

[0044] The term "humanized" antibody as used herein refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are left unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not impair the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains the same antigen specificity as the original antibody.

[0045] The term "monoclonal" antibody (mAb), as used herein, refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules which are essentially identical in primary sequence and which display a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0046] The term "chimeric" antibody as used herein refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. In an illustrative example, a chimeric antibody may contain 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, e.g., a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

[0047] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. The binding affinity of the antibodies and antigen-binding fragments provided herein is expressed as K, which represents the ratio of the dissociation rate to the association rate (koff / kon) when the binding between an antigen and an antigen-binding molecule (e.g., an antibody or antigen-binding fragment) reaches equilibrium. D The antigen binding affinity (e.g., K D) can be suitably determined using any suitable method known in the art, including, for example, Biacore, KinExA (KinExA) and flow cytometry.

[0048] As used herein, the term "compete for 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%, at least 90%, or at least 95%). Those skilled in the art will know that they can determine whether a given antibody competes with an antibody of the present disclosure for binding to FGFR2b without undue experimentation.

[0049] The term "epitope" as used herein refers to a specific atom or group of amino acids on an antigen to which an antibody or antigen-binding moiety binds. The minimum size of an epitope may be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be a continuous linear sequence of the primary structure of the antigen, since the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structure of the antigen. CDRs are important for recognizing the epitope of an antigen.

[0050] As used herein, "percent (%) 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. Sequence identity refers to a perfect match between the nucleotides or amino acids of the two sequences being compared. Sequence identity can be determined by those skilled in the art by conventional means, such as the BLAST algorithm.

[0051] As used herein, "antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to in vitro or in vivo cell-mediated cytotoxicity in which non-specific effector cells expressing Fc receptors (FcR) on the effector cell surface (e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils) recognize the Fc region of an antibody bound to a surface antigen on a target cell and actively lyse the target cell. In principle, any effector cell with an activated FcR can be triggered to mediate ADCC. The ADCC activity of an antibody can be measured as described in Example 5 or by any method known to one of skill in the art.

[0052] As used herein, modified antibodies include one or more modifications that "enhance ADCC," meaning that the modified antibody has a higher level of ADCC activity than the ADCC induced by an unmodified antibody. For example, the enhanced ADCC described in the present disclosure is characterized by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, or about 75% higher lysis of FGFR2b-expressing cells. Various methods for enhancing ADCC have been described in the prior art (Liu R, Oldham RJ, Teal E, Beers SA, Cragg MS. Antibodies (Basel). 2020 Nov 17;9(4):64). For example, studies have shown that afucosylated (i.e., fucose-deficient or non-fucosylated) antibodies have increased binding to FcγRIII and increased 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 Appln. Pub. No. 1176195). In some embodiments, the afucosylated antibodies provided herein lack fucose at asparagine 297 (Asn297) of the heavy chain. Asn297 (position 314 of the Eu numbering, or Kabat numbering, of Fc region residues) is a conserved N-linked glycosylation site found in each CH2 domain of the Fc region of the IgG1 isotype of antibodies.

[0053] As used herein, "administering," "administered," or "administration" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. A preferred route for administration of therapeutic antibodies is intravenous (IV) administration. Other routes of administration include subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration (generally by injection), including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, an antibody or antigen-binding fragment thereof according to the present disclosure can be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0054] The term "fibroblast growth factor receptor 2 (FGFR2)" as used herein is also known as CD332 (cluster of differentiation 332), a member of the FGFR family encoded by the FGFR2 gene located on chromosome 10 in humans. FGFRs are highly conserved and share common structural features: an extracellular ligand-binding section composed of distinct Ig-like domains (α isoforms contain all three Ig-like domains D1, D2, and D3, whereas β isoforms contain only two Ig-like domains D2 and D3, but not D1), a transmembrane domain, and an intracellular tyrosine kinase catalytic domain. There are two natural isoforms of FGFR2, FGFR2IIIb and FGFR2IIIc, which are generated by splicing of the third immunoglobulin-like domain (D3). FGFR2IIIb is a high affinity receptor for FGF1 and a specific receptor for members of the KGF family, such as FGF10, FGF22, and especially FGF7. KGF (FGF7) and KGFR (FGFR2IIIb) have been found to be aberrantly expressed in various types of cancer, including pancreatic, gastric, ovarian, and breast cancer (Helsten T, Elkin S, Arthur E, Tomson BN, Carter J, Kurzrock R. Clin Cancer Res. 2016 Jan 1;22(1):259-67).

[0055] As used herein, "abnormal expression of FGFR2b" includes, but is not limited to, FGFR2b mutations, FGFR2b amplifications, FGFR2b fusions, FGFR2 translocations, and FGFR2 overexpression.

[0056] As used herein, "anti-FGFR2b antibody" refers to an antibody that can specifically bind to human or non-human FGFR2b (e.g., the protein disclosed in the UniProt database as UniProtKB-P21802-3, UniProtKB-A0A2K5TL84, UniProtKB-P21803-2). In some embodiments, the anti-FGFR2b antibody is at a concentration of 1×10 -4 Less than M, 1×10-5 Less than M, 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10 Binding affinity (K D In some embodiments, the anti-FGFR2b antibody can specifically bind to FGFR2b with a K of less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. D In a preferred embodiment, the anti-FGFR2b antibody can specifically bind to FGFR2b at a concentration of 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10 Binding affinity (K D ) and specifically binds to FGFR2b.

[0057] The term "cancer" as used herein refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors, which can invade adjacent tissues and metastasize to distant sites in the body through the lymphatic system or bloodstream. The term includes any kind of cancerous growth or tumorigenic process, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. The term "tumor" as used herein refers to cancer cells, e.g., a mass of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as cancers affecting the lung, breast, thyroid, lymphatic, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate cancer and / or testicular cancers, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are designed for the treatment or diagnosis of cancer in a subject. The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues, including respiratory, digestive, genitourinary, testicular, breast, prostate, endocrine, and melanoma. In some embodiments, the cancer is renal or melanoma. Exemplary carcinomas include those forming from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to a cancer arising from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin.

[0058] As used herein, a "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, monkeys, chimpanzees, gorillas, and rodents such as mice, rats, and guinea pigs. In a preferred embodiment, the subject is a human. As used herein, the terms "subject" and "patient" are used interchangeably.

[0059] As used herein, a "vector" refers to a polynucleotide molecule that allows the replication / cloning of a desired nucleic acid fragment contained therein or allows the expression of a protein encoded by such a desired nucleic acid fragment 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 may be operably inserted to effect expression of the protein. An expression vector may include various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may include an origin of replication. A vector can be introduced into a host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0060] As used herein, "host cell" refers to a cell that has been transformed or can be transformed by a nucleic acid sequence and thereby expresses a target gene. Host cells can be prokaryotic cells (e.g., E. coli), eukaryotic cells (e.g., yeast, plants such as tobacco and tomato, and animals such as humans, monkeys, hamsters, rats, mice, or insects), or hybridomas.

[0061] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease, reduces the severity of disease symptoms, increases the frequency and duration of symptom-free periods of the disease, prevents or reduces functional impairment or disability due to disease, or otherwise promotes regression of a disease as evidenced by an improvement in the disease symptoms in said subject. Furthermore, the terms "effective" and "efficacy" with respect to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote regression of a disease in a patient, such as regression of a cancer. Physiological safety refers to an acceptable level of toxicity or other adverse physiological effects (side effects) at the cell, organ, and / or organism level resulting from administration of the drug. The efficacy of a therapeutic agent can be evaluated using a variety of methods known to physicians, such as assaying the activity of the therapeutic agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0062] The term "strong inhibition" as used herein refers to an antibody or antigen-binding fragment thereof of the present disclosure having a relative inhibition rate of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a reference antibody. The term "weak inhibition" refers to an antibody or antigen-binding fragment thereof of the present disclosure having a relative inhibition rate of up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, or up to about 40% compared to a reference antibody. In some embodiments, the anti-FGFR2b reference antibody is FPA144. In some preferred embodiments, the term "strong inhibition" refers to an antibody or antigen-binding fragment thereof of the present disclosure having a relative inhibition rate of at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a reference antibody FPA144. In some preferred embodiments, the term "weak inhibition" refers to an antibody or antigen-binding fragment thereof of the present disclosure having a relative inhibition rate of up to about 55%, up to about 50%, up to about 45%, or up to about 40% compared to the reference antibody FPA144.

[0063] As used herein, the term "treatment" or "treating" a disease or condition includes preventing or alleviating the condition, delaying the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0064] As used herein, the term "pharmaceutical acceptable carrier" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0065] Anti-FGFR2b antibodies and antigen-binding fragments The present disclosure provides a novel anti-FGFR2b antibody (e.g., HC18) or an antigen-binding fragment thereof that specifically binds to FGFR2b, such as human FGFR2b expressed on the surface of a cell, but has no detectable binding affinity to FGFR2c. Furthermore, the antibody or antigen-binding fragment thereof of the present disclosure strongly inhibits the binding of FGF7 to FGFR2b, but weakly inhibits the binding of FGF10 to FGFR2b, thereby improving the balance between safety and efficacy.

[0066] Experiments have shown that the antibodies provided herein significantly block the FGF7-FGFR2b pathway by inhibiting FGF7-induced FGFR2b, ERK1 / 2 phosphorylation, and tumor cell proliferation (e.g., SNU-16 cells), and relatively weakly block the binding of FGF10 to FGFR2b compared with benchmarks (e.g., FPA144), and may have a better safety profile in corneal toxicity. ADCC functional assays have revealed that the antibodies of the present invention induce strong ADCC effects and kill FGFR2b-expressing tumor cells.

[0067] Antibodies or antigen-binding fragments thereof provided herein include: a heavy chain complementarity determining region 1 (CDR1) having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:1, or having up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:1; 2 or 3) amino acid addition, substitution, and / or deletion compared to SEQ ID NO:3; and a heavy chain CDR3 having an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:3 or has a maximum of one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:3.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain CDR1 having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:4, or having up to two (e.g., one or two) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:4; a light chain CDR2 having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6, or having up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:6; and a light chain CDR3 having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6, or having up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:6.

[0069] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3.

[0070] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:4, a light chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:6.

[0071] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises the CDR sequences of HC18 (see Table 1).

[0072] [Table 1]

[0073] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0074] Although CDRs are known to be involved in antigen binding, it has been found that not all six CDRs are essential or invariant. In other words, one or more CDRs in HC18 can be replaced, altered, or modified while still substantially retaining a particular binding affinity to FGFR2b.

[0075] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may contain one or more modifications or substitutions in one or more CDR regions, as shown in Table 1. Such variants retain the specific binding affinity of the parent antibody to FGFR2b, but may have one or more improved properties, such as increased antigen-binding affinity or reduced potential for glycosylation.

[0076] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be modified to remove one or more Asn or Asp hot spots within the CDR regions (or within the variable regions), which may cause degradation of the antibody and, consequently, reduce the stability of the antibody.

[0077] In some embodiments, one or more of the modifications or substitutions are conservative substitutions.

[0078] The antibodies or antigen-binding fragments thereof provided herein further comprise suitable framework region (FR) sequences, so long as the antibodies can specifically bind to FGFR2b. The CDR sequences shown in Table 1 are obtained from mouse antibodies, but can be grafted to any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, etc., using suitable methods known in the art, such as recombinant techniques. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (such as a heavy chain variable region) having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7, or up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:7. and a light chain variable region having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8, or having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO: 8 (i.e., the light chain variable region of HC18). In a preferred embodiment, the substitutions are conservative substitutions.

[0080] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:8.

[0081] The antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a human immunoglobulin constant region, optionally a human IgG constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In some embodiments, the heavy chain constant region comprises an Fc region. In some embodiments, the light chain constant region comprises a Cκ or Cλ. In preferred embodiments, the constant region is derived from human IgG1 (hIgG1). In preferred embodiments, the constant region is a human IgG1 constant region.

[0082] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise at least one heavy chain and / or at least one light chain. In one embodiment, the heavy chain has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:9, or has up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:9 (i.e., the full-length heavy chain sequence of HC18). In one embodiment, the light chain has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10, or has up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO: 10 (i.e., the full-length light chain sequence of HC18). In a preferred embodiment, the substitutions are conservative substitutions.

[0083] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO:9 and a light chain having the amino acid sequence of SEQ ID NO:10.

[0084] Table 2 shows the amino acid and nucleotide sequences of HC18.

[0085] [Table 2]

[0086] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may contain one or more modifications or substitutions in one or more sequences provided herein, but retain specific binding affinity to FGFR2b. To achieve this goal, various methods known in the art can be used. For example, computer software can be used to virtually simulate the binding of an antibody to FGFR2b and identify amino acid residues on the antibody that form the binding interface. Such residues are avoided in substitutions to prevent a decrease in binding affinity, or are targeted for substitution to provide stronger binding.

[0087] As used herein, "conservatively modified variants" or "conservative substitutions" refer to the replacement of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), which often result in changes being made without altering the biological activity of the protein. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Moreover, substitution of structurally and / or functionally similar amino acids is less likely to inhibit biological activity. Various embodiments of antibodies or antigen-binding fragments thereof according to the present disclosure include polypeptide chains having a sequence that includes up to 0 (unaltered), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 or more conservative amino acid substitutions when compared to a specific amino acid sequence disclosed herein, such as SEQ ID NO: 7, 8, 9, or 10. As used herein, the phrase "up to X" conservative amino acid substitutions includes any number of substitutions, including 0 substitutions and up to X substitutions. Such exemplary substitutions are preferably made according to those set forth in the table below.

[0088] [Table 3]

[0089] Functionally conservative variants of the antibodies or antigen-binding fragments thereof according to the present disclosure are also contemplated by the present disclosure. A "functionally conservative variant" is one in which one or more amino acid residues in the protein have been altered without altering the overall conformation and function of the polypeptide, including but not limited to the substitution of amino acids with similar properties.

[0090] The antibodies or antigen-binding fragments thereof provided herein also comprise a constant region capable of inducing effector function. In some embodiments, the constant region comprises one or more modifications that enhance antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibodies or antigen-binding fragments thereof are afucosylated.

[0091] In some embodiments, an afucosylated antibody can increase the effector function (e.g., ADCC) of the antibody or antigen-binding fragment thereof by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to a wild-type antibody or antigen-binding fragment thereof.

[0092] As used herein, "antibody or antigen-binding fragment thereof" refers to an intact antibody or an antibody fragment having an antigen-binding portion. Various types of antibodies or antigen-binding fragments are known in the art and can be developed based on the antigen-binding portion of the anti-FGFR2b antibody (e.g., HC18) provided herein.

[0093] In some embodiments, an antibody or antigen-binding fragment of the disclosure is a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an F(Fab')2 fragment, an scFv fragment, an Fv fragment, a Fab' fragment, or a domain antibody.

[0094] In some embodiments, the antigen-binding fragments provided herein can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a single chain variable fragment (scFv) described herein fused to a CD3ζ transmembrane domain and endodomain. In some embodiments, the chimeric antigen receptor also comprises the intracellular signaling domains of various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to enhance potency. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0095] Also contemplated are antibodies or antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof provided herein (e.g., HC18) for binding to FGFR2b. In one embodiment, such a competing antibody specifically binds to an epitope that is the same as or overlaps with an epitope bound by an antibody or antigen-binding fragment thereof according to the present disclosure. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the same or overlapping epitope on FGFR2b recognized by HC18, which comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to FGFR2b on an epitope that is distinct from FPA144. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the binding interaction between human FGFR2b and anti-FGFR2b antibody HC18 by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. One of ordinary skill in the art will know that, without undue experimentation, it is possible to determine whether a given antibody competes with an antibody of the present disclosure for binding to FGFR2b.

[0096] Antibody characteristics Fibroblast growth factor receptor 2b (FGFR2b) is overexpressed in approximately 2%-30% of GC cases and is associated with poor prognosis. Bemarituzumab is an afucosylated humanized monoclonal antibody that specifically inhibits FGFR2b. In the FIGHT study, bemarituzumab showed beneficial responses in GC, but a high incidence of corneal adverse events was observed. It was hypothesized that FGF10 inhibition may be a mechanism of corneal toxicity by bemarituzumab (Catenacci et al, J Clin Oncol. 2020: 38(21):2418-2426). The present disclosure provides a novel antibody or antigen-binding fragment thereof against FGFR2b that has weak inhibitory activity against FGF10. Thus, the antibody or antigen-binding fragment thereof may be a differentiated antibody therapeutic that may reduce adverse effects in the eye.

[0097] The antibodies or antigen-binding fragments thereof provided herein have at least one of the following properties: 1) specific binding ability to FGFR2b and undetectable binding affinity to FGFR2c; 2) It strongly inhibits the binding of FGF7 to FGFR2b, while weakly inhibiting the binding of FGF10 to FGFR2b.

[0098] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can bind to FGFR2b, thereby blocking the interaction of the receptor with its respective ligand, reducing phosphorylation of FGFR2, reducing phosphorylation of downstream signaling pathways (e.g., the MAPK pathway, the PI3K / AKT1 / MTOR pathway), and / or directly killing cancer cells by ADCC and / or CDC.

[0099] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are administered at a concentration of 1×10 -4 Less than M, 1×10 -5 Less than M, 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9Less than M or 1×10 -10 Binding affinity (K D In some embodiments, K D is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a binding affinity (K D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, or at least or about 200% of the FGFR2b-binding ability compared to FPA144 or an FPA144 analog.

[0100] In a preferred embodiment, the antibodies or antigen-binding fragments thereof provided herein are administered at a concentration of 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1×10 -10 Binding affinity (K D ) and specifically binds to human FGFR2b.

[0101] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0102] ELISA assays or other conventional techniques can be used to measure the inhibition of FGF7 and FGF10 binding to FGFR2b by anti-FGFR2b antibodies. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a relative inhibition rate of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in inhibiting FGF7 binding to FGFR2b compared to a reference antibody (e.g., FPA144). In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a relative inhibition rate of up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, or up to about 40% in inhibiting FGF10 binding to FGFR2b compared to a reference antibody (e.g., FPA144).

[0103] In some preferred embodiments, the antibodies or antigen-binding fragments thereof provided herein have a relative inhibition rate of at least about 85%, at least about 90%, at least about 95%, or at least about 100% in inhibiting binding of FGF7 to FGFR2b, and a relative inhibition rate of up to about 55%, up to about 50%, up to about 45%, or up to about 40% in inhibiting binding of FGF10 to FGFR2b, compared to a reference antibody (e.g., FPA144).

[0104] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can have at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold increase in complement dependent cytotoxicity (CDC) compared to an isotype control antibody.

[0105] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can increase antibody-dependent cell-mediated cytotoxicity (ADCC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold compared to an isotype control antibody.

[0106] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein inhibit FGF-induced FGFR2 phosphorylation and proliferation of cancer cells.

[0107] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce the level of phosphorylation (e.g., FGF7-induced phosphorylation) of FGFR2 in a cell (e.g., an FGFR2b-expressing cell) by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% compared to a non-specific antibody or an isotype control antibody. In some embodiments, after treatment with the antibody or antigen-binding fragment thereof, the percentage of cells containing phosphorylated FGFR2 is less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, less than about 100%, less than about 110%, less than about 120%, less than about 130%, less than about 140%, less than about 150%, or less than about 200% compared to treatment with FPA144 or an FPA144 analog.

[0108] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce the phosphorylation level (e.g., FGF7-induced phosphorylation) of downstream signaling pathways involved in cancer cell proliferation, survival, and / or apoptosis. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce the phosphorylation level of ERK in a cell (e.g., an FGFR2b-expressing cell) by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% compared to a nonspecific antibody or an isotype control antibody. In some embodiments, cells (e.g., FGFR2b-expressing cells) treated with the antibody or antigen-binding fragment thereof have a ratio of phosphorylated ERK1 / 2 in total ERK that is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% compared to cells treated with FPA144 or an FPA144 analog.

[0109] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are less effective at reducing the level of phosphorylation (e.g., FGF10-induced phosphorylation) of FGFR2 in a cell (e.g., an FGFR2b-expressing cell) compared to FPA144 or an FPA144 analog.

[0110] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are less effective at reducing phosphorylation (e.g., FGF10-induced phosphorylation) levels of downstream signaling pathways involved in cancer cell proliferation, survival, and / or apoptosis compared to FPA144 or an FPA144 analog.

[0111] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce FGF7-induced proliferation of cells (e.g., FGFR2b-expressing cells) by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% compared to a non-specific antibody or an isotype control antibody.

[0112] Complex The disclosure provides antibody-drug conjugates in which the antibody or antigen-binding fragment thereof is conjugated to one or more conjugate moieties.

[0113] In some embodiments, one or more of the conjugate moieties is a stabilizing molecule (e.g., a molecule that increases the half-life of an antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Attachment of a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or increase its biological activity in vitro (e.g., when stored in tissue culture or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0114] In some embodiments, one or more of the conjugate moieties is a therapeutic agent. The antibody-drug conjugates comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, cyclophosphamide, and analogs thereof).

[0115] Polynucleotides and methods of production The present invention also provides nucleic acids encoding the antibodies or antigen-binding fragments thereof provided herein.

[0116] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless otherwise specified, the term includes polynucleotides containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular polynucleotide sequence implicitly includes not only the sequence explicitly shown, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, homologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base 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)).

[0117] In some embodiments, the nucleic acid encoding the antibody or antigen-binding fragment thereof comprises a heavy chain-encoding nucleic acid having a nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11, and / or a light chain-encoding nucleic acid having a nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12. DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0118] A nucleic acid encoding an anti-FGFR2b antibody (e.g., containing the sequence of HC18 set forth in SEQ ID NOs: 11 and 12) can be inserted into a vector for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art.

[0119] The antibodies or antigen-binding fragments thereof provided herein can be produced by any method known in the art for the synthesis of proteins (e.g., antibodies), in particular by chemical synthesis or preferably by recombinant expression techniques.

[0120] Recombinant expression of an antibody requires the construction of an expression vector containing a nucleic acid encoding the antibody. Once the nucleic acid encoding the antibody is obtained, a vector for producing the antibody can be produced by recombinant DNA techniques. In the present disclosure, an expression vector is constructed to contain an antibody coding sequence and appropriate transcriptional and translational regulatory elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0121] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody, or antigen-binding fragment thereof, in accordance with this disclosure.

[0122] In one embodiment, a method for producing an antibody or antigen-binding fragment thereof according to the present disclosure comprises culturing a host cell according to the present disclosure under conditions that allow expression of said antibody or antigen-binding fragment thereof, in a preferred embodiment, the method further comprises recovering and / or purifying the obtained antibody or antigen-binding fragment thereof from the host cell and / or culture medium.

[0123] Pharmaceutical ingredients The present disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, or an antibody-drug conjugate provided herein, and one or more pharma- ceutically acceptable carriers. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective antibody or antigen-binding fragment provided herein, and one or more additional components, such as a pharma- ceutically acceptable carrier, vehicle, or medium. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, such as a pharma- ceutically acceptable liquid, gel, or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antibacterial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspending / dispersing agent, a sequestering / chelating agent, a diluent, an adjuvant, an excipient, or a non-toxic auxiliary substance, other components known in the art, or various combinations thereof.

[0124] In some embodiments, the composition comprises a sterile diluent (e.g., sterile water or saline), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent, antibacterial or antifungal agent (such as benzyl alcohol or methylparaben), chlorobutanol, phenol, ascorbic acid and the like, antioxidant (ascorbic acid or sodium bisulfite), chelating agent (such as ethylenediaminetetraacetic acid), buffer (such as acetate, citrate, or phosphate), and isotonic agent such as sugar (e.g., dextrose), polyalcohol (e.g., mannitol or solvitol), or salt (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharma- ceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The preparation of the composition can be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where necessary (e.g., as injectable formulations), proper fluidity can be maintained by the use of a coating such as, for example, lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by the inclusion of agents which delay absorption (e.g., mononucleic acid and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0125] The pharmaceutical composition may be administered by any suitable method known to those skilled in the art, including parenteral and oral routes as described above. In a preferred embodiment, the pharmaceutical composition may be administered by intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, or intrasternal injection.

[0126] The present invention also provides a kit comprising the antibody or antigen-binding fragment thereof provided herein. In a preferred embodiment, the kit according to the present disclosure further comprises instructions for guiding the use of the antibody or antigen-binding fragment thereof of the present disclosure, such as in treating or preventing a disease associated with aberrant expression of FGFR2b in a subject, such as cancer.

[0127] In some embodiments, an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, or an antibody-drug conjugate provided herein, and at least one additional therapeutic agent are administered in the same composition. In some embodiments, an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, an expression vector provided herein, or an antibody-drug conjugate provided herein, and at least one additional therapeutic agent are administered in two different compositions.

[0128] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3 kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine-2,3-dioxygenase 1 (IDO1) (e.g., Epacadostat).

[0129] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of a HER3 inhibitor, an LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an activated Hedgehog signaling pathway inhibitor, and an agent that selectively degrades the estrogen receptor.

[0130] In some embodiments, the additional therapeutic agent is Trabectedin, nab-Paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, Everolimus, Fluoropyrimidine, IFL, Regorafenib, Reolysin, Alimta, Zykadia, Sutent, Temsirolimus, Axitinib, Everolimus, Sorafenib, Votrient, Pazopanib, IM The therapeutic agent may include one or more therapeutic agents selected from the group consisting of A-901, AGS-003, Cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temazolomide, IL-2, IFNa, Vinblastine, Thalomid, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacytidine, Lenalidomide, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate, and Enzastaurin.

[0131] In some embodiments, the additional therapeutic agents may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapeutic agent, a CXCL9 targeted therapeutic agent, a CXCL10 targeted therapeutic agent, a CCL5 targeted therapeutic agent, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0132] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0133] Treatment Methods and Uses The present disclosure provides a method for inhibiting or reducing FGF-induced proliferation of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate, and / or pharmaceutical composition provided herein.

[0134] The present disclosure provides a method for inhibiting or reducing FGF-induced FGFR2 phosphorylation in tumor cells of a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate, and / or pharmaceutical composition provided herein.

[0135] The present disclosure provides a method for killing tumor cells associated with abnormal expression of FGFR2b in a subject and reducing corneal toxicity, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate, and / or pharmaceutical composition provided herein.

[0136] The present disclosure provides a method for treating or preventing a disease or condition associated with abnormal expression of FGFR2b in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate, and / or pharmaceutical composition of the present disclosure.

[0137] Furthermore, the antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions provided herein can be used in the manufacture of medicaments and / or kits for treating or preventing diseases or conditions associated with abnormal expression of FGFR2b in a subject.

[0138] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is cancer characterized by expression or overexpression of FGFR2b. In some embodiments, the disease or condition is cancer associated with FGFR2b mutations, FGFR2b amplifications, FGFR2b fusions, FGFR2 translocations, and / or FGFR2 overexpression.

[0139] In some embodiments, the cancer includes, but is not limited to, 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, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, and urothelial carcinoma. In a preferred embodiment, the cancer is gastric cancer. In a preferred embodiment, the cancer is FGFR2 positive gastric cancer. In a preferred embodiment, the cancer is FGFR2 amplified gastric cancer. Cancer patients can be identified by various methods known in the art.

[0140] A therapeutically effective amount of an antibody or antigen-binding fragment thereof, a nucleic acid, an expression vector, a host cell, an antibody-drug conjugate, or a pharmaceutical composition provided herein can be administered one or more times. In any of the methods described herein, an antibody or antigen-binding fragment thereof, a nucleic acid, an expression vector, a host cell, an antibody-drug conjugate, and / or a pharmaceutical composition provided herein, and optionally at least one additional therapeutic agent, can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0141] In some embodiments, the subject can be administered an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate, and / or pharmaceutical composition provided herein for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of time for treatment using any method in the art for tracking the effectiveness of a diagnosis or treatment (e.g., observation of at least one symptom of cancer). As described herein, a medical professional of ordinary skill in the art can also alter the identity and number (e.g., increase or decrease) of the antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions provided herein (and / or one or more additional therapeutic agents) administered to a subject, and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of the antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions provided herein (and / or at least one other additional therapeutic agent) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0142] In some embodiments, the antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions provided herein are administered sequentially or simultaneously with at least one additional therapeutic agent. In some embodiments, at least one other additional therapeutic agent can be administered to the subject prior to or after administration of the antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions provided herein.

[0143] Data obtained from cell culture assays and animal studies can be used in formulating the appropriate dosage of any agent for use in the subject (e.g., human). A therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein is an amount that treats a disease in a subject and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., human). The efficacy and dosage of an antibody or antigen-binding fragment thereof provided herein can be determined by a medical or veterinary professional using methods known in the art, as well as by observing one or more symptoms of the disease in a subject (e.g., human). Certain factors may affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0144] Exemplary dosage amounts include milligram or microgram amounts of the antibodies or antigen-binding fragments thereof, or antibody-drug conjugates provided herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, or about 1 μg / kg to about 50 μg / kg). These dosage amounts will vary widely, but one of skill in the art will appreciate that therapeutic agents comprising the antibodies and antigen-binding fragments thereof vary in potency, and effective amounts can be determined by methods known in the art. Generally, a relatively low dose is administered initially, and the attending medical or pharmaceutical professional (for therapeutic applications) or researcher (for studies in development stages) can then gradually increase the dose until an appropriate response is obtained. It will further be understood that the specific dosage level for a particular subject will depend on a variety of factors, including the activity of the particular compound used, the subject's age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, and the in vivo half-life of the antibody or antibody fragment. EXAMPLES

[0145] This invention is illustrated by the following examples which should not be construed as limiting.

[0146] Example 1. Preparation of anti-FGFR2b antibody SJL mice (5-6 week old females) were immunized with FGFR2(β)IIIb-Fc and anti-CD25 / CD40 antibodies (anti-CD25 antibody was initially administered at a dose of 100 μg / animal, followed by two final doses of anti-CD40 antibody at 50 μg / animal) intraperitoneally at weekly intervals, with the antigens suspended in MPL / TDM (Sigma-Aldrich). Three days after the final injection, popliteal lymphocyte cells were extracted and fused with Sp2 / 0-Ag14 mouse myeloma cells at a 1:1 ratio using a Hybridune Electrofusion System (Cyto Pulse Sciences). Hybridomas were selected 24 hours later by adding 2×HAT (Sigma). Ten days after fusion, hybridoma culture supernatants were screened by ELISA for their ability to bind to FGFR2IIIb-his but not to FGFR2IIIc-his. The selected monoclonal antibodies were then screened by ELISA for their ability to recognize FGFR2IIIb on CHOK1-FGFR2IIIb transfectants and inhibit FGF7 binding to FGFR2IIIb. The selected hybridomas were cloned twice using limiting dilution. The subcloned hybridomas were sequenced and fused with hFc to generate chimeric antibodies. The fusions yielded a number of anti-FGFR2 chimeric antibodies, including the monoclonal antibody HC18.

[0147] Example 2. FACS analysis of binding specificity of anti-FGFR2b antibodies to cell surface human FGFR2b protein Appropriate cells (1 x 10 5) were washed twice with FACS buffer (PBS + 2% BSA) and resuspended in 100 μL of FACS buffer containing serially diluted (1:5) anti-FGFR2b monoclonal antibodies and incubated at 4° C. for 1 hour. The cells were then washed twice with FACS buffer and bound antibodies were detected by incubation with APC anti-human IgGFc (Biolegend, Cat. No. 410712) for 1 hour at 4° C. After washing twice with FACS buffer, the cells were analyzed on a CytoFLEX (BeckmanCoulter). Aprutumab was also tested as a positive control capable of binding to both FGFR2b and FGFR2c. The results are shown in FIG. 1. The antibodies of the present disclosure showed comparable binding ability compared to the benchmark (FPA144).

[0148] Example 3. Inhibition of binding of FGF7 and FGF10 to FGFR2b by anti-FGFR2b antibodies Inhibition of FGF7 and FGF10 binding to FGFR2b by anti-FGFR2b antibodies was measured by ELISA assay. Each step of each assay was performed by incubating with the appropriate reagent for 1 h at room temperature, except for the initial plate coating step, which was performed overnight at 4°C. Between each step, the plates were washed three times with PBS containing 0.05% Tween 20.

[0149] To examine the ability of anti-FGFR2b monoclonal antibodies to inhibit ligand binding to FGFR2b, plates were coated with 1 μg / mL hFGFR2b-Fc (Kactus, Catalog No. FGR-HM2BB) and then blocked with 2% BSA. The plates were then incubated with human FGF7-his (20 nM, SinoBiological, Catalog No. 10210-H07E) or biotin-FGF10 (5 nM, Kactus, Catalog No. FGF-HE010B) in the presence of serial dilutions (1:5) of anti-FGFR2b monoclonal antibodies, and bound ligand was detected using HRP-anti-6XHis tag (Abcam, Catalog No. ab1187) or streptavidin-protein HRP (Thermo, Catalog No. 21126) and TMB substrate (CellSignaling, Catalog No. 7004P6). The results are shown in Figure 2 and Table 4. Compared to the benchmark, the antibodies of the present disclosure significantly inhibited FGF7 binding to FGFR2b, but only partially inhibited FGF10 binding, suggesting that the antibodies of the present disclosure may have a better safety profile in terms of corneal toxicity.

[0150] [Table 4]

[0151] Example 4. Surface plasmon response (SPR) sensorgram of anti-FGFR2b antibody binding to human FGFR2b antigen Surface plasmon resonance (Octet, Sartorius) was used to measure the binding affinity of anti-FGFR2b monoclonal antibodies to human FGFR2b (FGFR2b-his). Anti-FGFR2b monoclonal antibodies were immobilized on a dextran chip using an amine coupling kit, and 100 mM sodium borate (pH 8.0) containing 100 mM ethylenediamine was used as the blocking reagent. FGFR2b-his protein diluted in HEPES-buffered saline with 0.05% surfactant P20 running buffer was run over the immobilized antibody. The results are shown in Figure 3 and Table 5.

[0152] [Table 5]

[0153] Example 5. ADCC against anti-FGFR2b antibodies To produce afucosylated (AF) monoclonal antibodies, expression plasmids provided by the client were transformed into Escherichia coli (E. coli) and grown at appropriate scales. NucleoBond Xtra Maxi Plus EF kit was used for large-scale plasmid production. Constructs containing the heavy and light chains of each antibody were co-transfected with PEI into FUT8-KO CHO cells. Conditioned media was harvested 9-11 days after transfection. Conditioned media expressing the target antibodies were harvested by centrifugation and filtration, and then loaded onto a protein A affinity column. Purified antibodies were analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0154] Cytotoxic activity was assessed using FACS analysis. Effector cells and human peripheral blood mononuclear cells were obtained from individual human donors (Milestone Biotechnologies) and cultured overnight with 10 ng / mL hIL-2 (PeproTech, 200-02). Target Ba / F3 cells expressing full-length human FGFR2b were labeled with CellTrace Far Red (Thermo, Cat. No. C34564) for 10 min at 37°C, washed twice with RPMI1640 medium (Gibco, Cat. No. A10491-01) containing 10% FBS (Gibco, Cat. No. 10099-141), and plated in a 96-well round-bottom plate at an effector to target cell ratio (20:1). Serially diluted (1:3) anti-FGFR2b monoclonal antibodies were added to the designated rows of the assay plate. After 4 hours of incubation at 37°C, 2μL of propidium iodide staining solution (BD biosciences, Catalog No. 556547) was added to each well to stain dead cells for 10 minutes at room temperature. Cells were directly analyzed by CytoFLEX (Beckmann Coulter). The results are shown in Figure 4. Antibodies of the present disclosure, including HC18 and HC18-AF, induced a strong ADCC response that killed FGFR2b-expressing cells, with afucosylated (AF) antibodies showing a stronger effect.

[0155] Example 6. Inhibition of FGFR2 phosphorylation induced by FGF7 and FGF10 in SNU-16 cells by anti-FGFR2b antibody The effect of anti-FGFR2b antibodies on tumor cell FGFR2 phosphorylation was measured in vitro in SNU-16 cells. Approximately 50,000 SNU-16 cells were seeded onto a 96-well plate in RPMI1640 medium (Gibco, Cat. No. A10491-01) and incubated at 37°C with 5% CO2 for 4 hours. SNU-16 cells were then treated with serially diluted (1:10) anti-FGFR2b monoclonal antibodies for 1 hour. SNU-16 cells were then treated with 30ng / mL FGF7 (R&D, Cat. No. 251-KG-01M) or FGF10 (Kactus, Cat. No. FGF-HE010B) and 20μg / mL heparin (Sigma, Cat. No. H3149-500KU) and incubated at 37°C with 5% CO2 for 5 minutes. To harvest cells for phosphorylated FGFR2 (Tyr653 / 654) detection, the 96-well plate was centrifuged at 300g for 3 minutes. Then, the entire volume in the well was slowly aspirated without disrupting the cells. 50 μL of 1× supplemented lysis buffer (Cisbio, Cat. No. 64FGFR2Y6PEG) was added immediately and incubated at room temperature for at least 30 minutes with shaking according to the manufacturer's preparation protocol. FGFR2 phosphorylation was read by fluorescence emission at two different wavelengths (665 nm and 620 nm) in a compatible HTRF® reader (EnVision, PerkinElmer). The experiment was performed in triplicate. The results are shown in FIG. 5. The antibody of the present disclosure significantly inhibited the phosphorylation of FGFR2 in SNU-16 cells induced by FGF7 and FGF10, especially FGF7.

[0156] Example 7. Inhibition of ERK1 / 2 phosphorylation induced by FGF7 and FGF10 in SNU-16 cells by anti-FGFR2b antibody The effect of anti-FGFR2b antibodies on tumor cell ERK1 / 2 phosphorylation was measured in vitro in SNU-16 cells. Approximately 50,000 SNU-16 cells were seeded onto a 96-well plate in RPMI1640 medium (Gibco, Cat. No. A10491-01) and incubated at 37°C with 5% CO2 for 4 hours. SNU-16 cells were then treated with serially diluted (1:5) anti-FGFR2b monoclonal antibodies for 1 hour. SNU-16 cells were then treated with 30ng / mL FGF7 (R&D, Cat. No. 251-KG-01M) or FGF10 (Kactus, Cat. No. FGF-HE010B) and 20μg / mL heparin (Sigma, Cat. No. H3149-500KU) and incubated at 37°C with 5% CO2 for 15 minutes. To harvest cells for phosphorylated ERK (Thr202 / Tyr204) detection, the 96-well plate was centrifuged at 300g for 3 minutes. Then, the entire volume in the well was slowly aspirated without disrupting the cells. 50 μL of 1× supplemented lysis buffer (Cisbio, Cat. No. 64ERKPEH) was added immediately and incubated at room temperature for at least 30 minutes with shaking according to the manufacturer's preparation protocol. ERK1 / 2 phosphorylation was read by fluorescence emission at two different wavelengths (665 nm and 620 nm) in a compatible HTRF® reader (EnVision, PerkinElmer). Experiments were performed in triplicate. The results are shown in FIG. 6. The antibody of the present disclosure significantly inhibited the phosphorylation of ERK1 / 2 in SNU-16 cells induced by FGF7 and FGF10, especially FGF7.

[0157] Example 8. Inhibition of FGF7-induced proliferation of SNU-16 cells by anti-FGFR2b antibodies The effect of anti-FGFR2b antibody on tumor cell proliferation was measured in vitro in SNU-16 cells. Approximately 10,000 SNU-16 cells were seeded on a 96-well plate in RPMI1640 medium (Gibco, Cat. No. A10491-01), and the cells were serum-starved and incubated overnight at 37°C with 5% CO2 for 16 hours. SNU-16 cells were then treated with serially diluted (1:10) anti-FGFR2b monoclonal antibody for 1 hour. SNU-16 cells were then treated with 50ng / mL FGF7 (R&D, Cat. No. 251-KG-01M) and 5μg / mL heparin (Sigma, Cat. No. H3149-500KU) and incubated at 37°C with 5% CO2 for 4 days. To harvest cells for cell viability assay, the 96-well plate was centrifuged at 300g for 3 minutes. Next, half of the total volume in the well (approximately 100 μL) was slowly aspirated without disrupting the cells. Then, CellTiter-Glo Reagent (100 μL) from CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat. No. G7571) was added according to the manufacturer's preparation protocol. The plate was shaken for 15 minutes at room temperature. During incubation with CellTiter-Glo Reagent, the plate was protected from light. Cell proliferation was read via luminescence on a SpectraMax M5 (Molecular Devices). Experiments were performed in triplicate. The results are shown in FIG. 7. The antibodies of the present disclosure showed equal or greater efficacy than FPA144 in inhibiting the proliferation of SNU-16.

[0158] Example 9. Epitope binning of anti-FGFR2b monoclonal antibodies Epitope binning of anti-FGFR2b antibodies was measured by ELISA assay. Each step of each assay was performed by incubating with the appropriate reagent for 1 h at room temperature, except for the first plate coating step, which was performed overnight at 4° C. Between each step, the plates were washed three times with PBS containing 0.05% Tween 20.

[0159] To determine the epitope binning of anti-FGFR2b monoclonal antibodies, plates were coated with 1 μg / mL HC18 or BMKFPA144, followed by blocking with 2% BSA. Then, plates were incubated with a mixture of 0.1 μg / mL biotin-hFGFR2b-Fc (Kactus, Catalog No. FGR-HM2BB) in the presence of serially diluted (1:5) anti-FGFR2b monoclonal antibodies, and bound antigen was detected with streptavidin-protein HRP (Thermo, Catalog No. 21126) and TMB substrate (Cell Signaling, Catalog No. 7004P6). The results are shown in Table 6. It shows that the antibodies of the present disclosure have different epitope bins compared to FPA144.

[0160] [Table 6]

[0161] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FFFR2b, An antibody or its antigen-binding fragment comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

6.

2. A heavy chain variable region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 7, or a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7, and A light chain variable region having an amino acid sequence at least 80% identical to SEQ ID NO: 8, or a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8, The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a light chain having the amino acid sequence of SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a human IgG, preferably a human IgG1 constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the constant region comprises one or more modifications that enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

6. The antibody or antigen-binding fragment thereof according to claim 4, which is afucosylated.

7. Human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F (Fab') ２ The antibody or antigen-binding fragment thereof according to claim 1, which is a fragment, scFv fragment, Fv fragment, Fab' fragment, or domain antibody.

8. An antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment described in claim 1 for binding to FFFR2b, or An antibody or its antigen-binding fragment that specifically binds to the same epitope on FGFR2b recognized by an antibody or its antigen-binding fragment, which includes a heavy chain variable region and a light chain variable region having the amino acid sequences represented by SEQ ID NOs: 7 and 8, respectively.

9. A nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in claim 1.

10. The nucleic acid according to claim 9, comprising a heavy-chain coding nucleic acid having the nucleotide sequence of SEQ ID NO: 11 and / or a light-chain coding nucleic acid having the nucleotide sequence of SEQ ID NO:

12.

11. An expression vector comprising the nucleic acid described in claim 9.

12. A host cell comprising the expression vector described in claim 11.

13. Pharmaceutical compositions including the following: (a) The antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 9, or the expression vector according to claim 11, and (b) A pharmaceutically acceptable carrier.

14. An antibody-drug conjugate comprising one or more complex portions to which the antibody or antigen-binding fragment thereof described in claim 1 is conjugated.

15. A method for producing an antibody or an antigen-binding fragment thereof as described in claim 1, comprising culturing a host cell as described in claim 12 under conditions that enable the expression of the antibody or the antigen-binding fragment thereof.

16. Use of the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 9, the expression vector according to claim 11, the host cell according to claim 12, the pharmaceutical composition according to claim 13, or the antibody-drug conjugate according to claim 14 in the manufacture of a pharmaceutical product for treating a disease or condition associated with abnormal expression of FGFR2b in a subject.

17. A formulation for inhibiting or reducing FGF-induced proliferation of tumor cells in a target, comprising: an antibody or antigen-binding fragment thereof as described in claim 1; a nucleic acid as described in claim 9; an expression vector as described in claim 11; a host cell as described in claim 12; a pharmaceutical composition as described in claim 13; or a therapeutically effective amount of the antibody-drug conjugate as described in claim 14.

18. A formulation for inhibiting or reducing FGF-induced FFFR2 phosphorylation in a target, comprising: the antibody or antigen-binding fragment thereof according to claim 1; the nucleic acid according to claim 9; the expression vector according to claim 11; the host cell according to claim 12; the pharmaceutical composition according to claim 13; or a therapeutically effective amount of the antibody-drug conjugate according to claim 14.

19. A formulation for killing tumor cells associated with abnormal expression of FGFR2b in a target and reducing corneal toxicity, comprising: the antibody or antigen-binding fragment thereof according to claim 1; the nucleic acid according to claim 9; the expression vector according to claim 11; the host cell according to claim 12; the pharmaceutical composition according to claim 13; or a therapeutically effective amount of the antibody-drug conjugate according to claim 14.

20. A formulation for treating a disease or condition associated with abnormal expression of FGFR2b in a subject, comprising: the antibody or antigen-binding fragment thereof as described in claim 1; the nucleic acid as described in claim 9; the expression vector as described in claim 11; the host cell as described in claim 12; the pharmaceutical composition as described in claim 13; or a therapeutically effective amount of the antibody-drug conjugate as described in claim 14.

21. The use according to claim 16, characterized in that the disease or condition is cancer, and optionally the cancer expresses or overexpresses FGFR2b.

22. The formulation according to claim 20, wherein the disease or condition is cancer, and optionally the cancer expresses or overexpresses FGFR2b.

23. The use according to claim 16, wherein the disease or condition is one selected from the group consisting of 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, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, bile duct cancer, intrahepatic bile duct cancer, and urothelial carcinoma.

24. The preparation according to claim 20, wherein the disease or condition is one selected from the group consisting of 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, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, bile duct cancer, intrahepatic bile duct cancer, and urothelial carcinoma.

25. The use according to claim 16, wherein the disease or condition is gastric cancer.

26. The preparation according to claim 20, wherein the disease or condition is gastric cancer.

27. The use according to claim 16, wherein the disease or condition is FGFR2-positive gastric cancer.

28. The formulation according to claim 20, wherein the disease or condition is FGFR2-positive gastric cancer.

29. The formulation according to claim 17, wherein the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid according to claim 9, the expression vector according to claim 11, the host cell according to claim 12, the pharmaceutical composition according to claim 13, or the antibody-drug conjugate according to claim 14 is administered sequentially or simultaneously with at least one additional therapeutic agent.

30. A kit comprising the antibody or antigen-binding fragment thereof as described in claim 1.

Images