Novel anti-FGFR2 antibody

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b, and a preparation method and use thereof. Background Art

[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, by activating 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 to FGFR4) corresponding to 22 ligands (FGFs) (Korc and Friesel, 2009). Dysregulation of the FGFR2 signaling pathway can lead to gene amplification and protein overexpression, resulting in tumorigenesis and poor prognosis (Grose and Dickson, 2005). Splicing variations produce several receptor variants, including two major FGFR2 isoforms, called FGFR2b and FGFR2c (also known as FGFR2IIIb and FGFR2IIIc). The expression of each isoform is usually restricted to specific tissues. Specifically, the three major ligands of FGFR2b are FGF7, FGF10, and FGF22, which are usually expressed in epithelial cells, while FGFR2c is expressed in mesenchymal tissues (Ornitz et al., 1996; Zhang et al., 2006). In FGFR2-amplified gastric cancer, the FGFR2b isoform is dominant.

[0003] In previous studies, FGFR2b was overexpressed in 2.5%-31.1% of gastroesophageal adenocarcinoma (GEA), depending on the antibody and detection method used (Ahn et al., 2016; Angal et al., 1993; Nagatsuma et al., 2015). Bemarituzumab (FPA144) is a FGFR2b-specific humanized afucosylated immunoglobulin G1 monoclonal antibody that blocks FGFR2b signaling by competitively binding to inhibit FGF and induces enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) against FGFR2b-overexpressing tumor cells (Xiang et al., 2021). Bemarituzumab has shown significant anti-tumor activity in preclinical trials and has shown positive efficacy in phase I clinical trials (Catenacci et al., 2020; Xiang et al., 2021). However, 3 of 28 patients (10.7%) reported corneal toxicity when receiving high doses (Catenacci et al., 2020). The mechanism of corneal toxicity is thought to be due to the inhibition of FGF10, which is involved in regulating corneal epithelial wound healing (Itoh, 2016). In previous in vivo studies, the therapeutic effect of FGF10 was examined in an inflammation-induced rabbit dry eye model, suggesting a role for FGF10 in corneal epithelial cell healing (Zheng et al., 2015). Anti-FGFR2b antibodies that reduce corneal toxicity without affecting antitumor activity are urgently needed.

[0004] To develop a novel anti-FGFR2b antibody with low corneal toxicity to meet unmet clinical needs, we generated a specific antibody against FGFR2b that exhibited significant tumor inhibitory ability but low FGF10 inhibition, implying low clinical adverse events. Summary of the invention

[0005] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b, comprising: a heavy chain complementary 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:

[0007] i) The inhibitory effect on the binding of FGF10 to FGFR2b is weak;

[0008] ii) Undetectable binding affinity to FGFR2c.

[0009] 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.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0011] 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.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:8.

[0013] 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.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region, optionally a constant region of a human immunoglobulin, or optionally a constant region of human IgG. In a preferred embodiment, the constant region is derived from human IgG1.

[0015] In some embodiments, the constant region of the antibodies or antigen-binding fragments thereof provided herein comprises one or more modifications that enhance antibody-dependent cellular cytotoxicity (ADCC).

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are afucosylated.

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(Fab')2 fragments, scFv fragments, Fv fragments, Fab' fragments or domain antibodies.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of inhibiting FGFR2 phosphorylation and FGF-induced cancer cell proliferation.

[0019] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes for binding to FGFR2b with the antibody or antigen-binding fragment thereof as described above.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the same epitope on FGFR2b that is 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 shown in SEQ ID NOs: 7 and 8, respectively.

[0021] In another aspect, the disclosure provides nucleic acids encoding the antibodies or antigen-binding fragments thereof provided herein.

[0022] In some embodiments, the nucleic acid includes: 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.

[0023] In another aspect, the present disclosure provides an expression vector comprising a nucleic acid provided herein.

[0024] In another aspect, the present disclosure provides a host cell comprising an expression vector provided herein.

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0026] (a) an antibody or antigen-binding fragment thereof provided herein, a nucleic acid provided herein, or an expression vector provided herein, and

[0027] (b) a pharmaceutically acceptable carrier.

[0028] In another aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof provided herein linked to one or more conjugated moieties.

[0029] In another aspect, the present 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 expression of the antibody or antigen-binding fragment thereof.

[0030] In another aspect, the present 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 for the preparation of a medicament for treating a disease or disorder associated with abnormal expression of FGFR2b in a subject.

[0031] In another aspect, the present disclosure provides a method for inhibiting or reducing FGF-induced tumor cell proliferation 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, or a pharmaceutical composition provided herein, or an antibody-drug conjugate provided herein.

[0032] In another aspect, the present 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.

[0033] In another aspect, the present disclosure provides a method for killing tumor cells associated with abnormal expression of FGFR2b and reducing corneal toxicity 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.

[0034] In another aspect, the present disclosure provides a method for treating 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 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.

[0035] In some embodiments, the disease or disorder as described above is cancer, and optionally the cancer is characterized by expression or overexpression of FGFR2b. In preferred embodiments, the cancer is characterized by expression or overexpression of FGFR2b.

[0036] Figure 1 Binding of anti-FGFR2b antibodies to different FGFR2 isoforms. Figure 1A HC18 was shown to bind specifically to CHOK1 cells expressing hFGFR2b. Figure 1B HC18 was shown not to bind to CHOK1 cells expressing hFGFR2c.

[0037] FIG2 Inhibition of the binding of FGFR2b to its ligand by anti-FGFR2b antibodies. Figure 2A It showed that HC18 significantly inhibited the binding of FGF7 to FGFR2b. Figure 2B It was shown that HC18 partially inhibited the binding of FGF10 to FGFR2b.

[0038] On the other hand, the present disclosure provides a kit comprising an antibody or its antigen-binding fragment provided herein. In a preferred embodiment, the kit according to the present disclosure also includes instructions for instructing the use of the antibody or its antigen-binding fragment of the present disclosure, such as for treating or preventing a disease or condition associated with abnormal expression of FGFR2b in a subject, such as cancer.

[0039] These and other aspects will be described in more detail herein. Each aspect provided may include the multiple embodiments provided herein. Therefore, it is contemplated that each aspect described may include each embodiment involving an element or combination of elements, and the combination of all such aspects and embodiments is explicitly contemplated. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 5 Effects of anti-FGFR2b antibody and negative control hIgG1 on FGF7 ( Figure 5A ) and FGF10( Figure 5B )-induced inhibition of FGFR2 phosphorylation.

[0041] Figure 5 Effects of anti-FGFR2b antibody and negative control hIgG1 on FGF7 ( Figure 5A ) and FGF10( Figure 5B )-induced inhibition of FGFR2 phosphorylation.

[0042] Figure 3 Surface plasmon response (SPR) sensorgrams of anti-FGFR2b antibody binding to human FGFR2b antigen.

[0043] Figure 4 ADCC (antibody-dependent cellular cytotoxicity) bioassay was performed using PBMCs induced with anti-FGFR2b antibody as effector cells and Ba / F3 cells expressing full-length human FGFR2b as target cells (E:T=20:1).

[0044] Figure 5 Effects of anti-FGFR2b antibody and negative control hIgG1 on FGF7 ( Figure 5A ) and FGF10( Figure 5B )-induced inhibition of FGFR2 phosphorylation.

[0045] Figure 6 Effects of anti-FGFR2b antibody and negative control hIgG1 on FGF7 ( Fig. 6A ) and FGF10( Figure 6B )-induced inhibition of ERK1 / 2 phosphorylation.

[0046] Figure 7 Inhibition of FGF7-induced proliferation of SNU-16 cells by anti-FGFR2b antibody and negative control hIgG1. DETAILED DESCRIPTION

[0047] Unless otherwise defined herein, the scientific and technical terms used herein should have the meanings commonly understood by those skilled in the art. In addition, unless the context clearly indicates otherwise, singular terms are intended to include plural terms, and vice versa. In order to facilitate reading of this application, some terms are defined below.

[0048] definition

[0049] As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any stated or listed components.

[0050] As used herein, the term "about" when used for numerical values ​​refers to a value that is reasonably close to the value and within an acceptable error range determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean a range of plus or minus 50% of the reference value, preferably a range of plus or minus 25%, or more preferably a range of plus or minus 10%. When a specific numerical value is provided in the application, the meaning of "about", unless otherwise stated, should be understood as within an acceptable error range for the specific numerical value according to the practice in the art.

[0051] "Antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin (Ig) that specifically binds to 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 (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region of IgG Ab contains three constant domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL ) and the light chain constant region. The light chain constant region of IgG Ab contains a constant domain, C L . V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V LIt comprises 3 CDRs (the light chain CDR comprises LCDR1, LCDR2 and LCDR3, and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3) and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. A variety of methods have been used to describe the CDR domains within Ab, including Rabat, Chothia, AbM, contact and IMGT definitions. Unless otherwise specified, the present disclosure uses Kabat numbering by default. The constant region of Ab can mediate the binding of Ig to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies are classified according to the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM. Several major antibody classes are divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0052] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody that binds to a specific antigen. A complete antibody or an antibody fragment having an antigen-binding portion of an antibody can be used. The term "antigen-binding fragment" as used herein refers to an antibody fragment formed by a portion of a complete antibody, which contains one or more CDRs; or any other antibody fragment that can bind to an antigen but does not contain a complete natural antibody structure. The term "antibody or its antigen-binding fragment" as used herein refers to a complete antibody or antibody fragment having an antigen-binding portion. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antibodies or their antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), fragments containing complementary determining regions (CDRs), single-chain variable fragments (scFv), chimeric antibodies, double antibodies, three antibodies, four antibodies, and polypeptides containing at least a portion of an immunoglobulin, which are sufficient to confer specific antigen binding to the polypeptide.

[0053] A "Fab fragment" is a fragment having a V L 、V H , 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" has the V from a single arm of an antibody. L and V H Domain. A "domain antibody (dAb)" consists of V H or V LA "single-chain variable fragment (scFv)" is an antibody in which V L and V H The V domains are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the length of the linker is sufficient to allow the protein chain to form a monovalent antigen binding site. The term "diabody" is a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a V domain connected by a linker. H and V L Domain, the linker is too short for the two domains on the same chain to pair, thereby allowing each domain to pair with a complementary domain on another polypeptide chain. If the two polypeptide chains of the diabody are identical, 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 from two different monoclonal antibodies and are able to bind to two different epitopes. The two epitopes may be present on the same antigen or on two different antigens. Similarly, tribodies, tetrabodies or other multispecific antibodies are antibodies comprising three, four or more polypeptide chains that may be the same or different, thereby forming three, four or more antigen-binding sites that may be the same or different, respectively.

[0054] The term "human" Ab used herein refers to an Ab with a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the Ab contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.

[0055] The term "humanized" Ab used herein refers to an Ab in which some, most or all of the amino acids outside the non-human Ab CDR domains are replaced by the corresponding amino acids derived from human immunoglobulin. In one embodiment of a humanized form of Ab, some, most or all of the amino acids outside the CDR domains have been replaced by amino acids from human immunoglobulin, while some, most or all of the amino acids in one or more CDR regions remain unchanged. As long as the ability of the Ab to bind a specific antigen is not affected, a small amount of addition, deletion, insertion, substitution or modification of amino acids is allowed. "Humanized" Ab retains an antigenic specificity similar to the original Ab.

[0056] The term "monoclonal" Ab (mAb) used herein refers to a non-naturally occurring Ab molecule preparation of a single molecule composition, i.e., an Ab molecule whose primary sequence is substantially identical and exhibits a single binding specificity and affinity for a specific epitope. MAbs can be produced by hybridoma, recombination, transgenic or other techniques known to those skilled in the art.

[0057] The term "chimeric" Ab used herein refers to an Ab in which the variable region is derived from one species and the constant region is derived from another species, such as an Ab in which the variable region is derived from a mouse Ab and the constant region is derived from a human Ab. In an exemplary embodiment, a chimeric antibody may comprise a constant region from a human and a variable region from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0058] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as a binding reaction between an antibody and an antigen. The binding affinity of the antibodies and antigen-binding fragments provided herein can be expressed as K. D The value represents the ratio of the off-rate to the on-rate (koff / kon) when the binding between the antigen and the antigen-binding molecule (e.g., antibody and antigen-binding fragment) reaches equilibrium. D ) can be suitably determined using appropriate methods known in the art (including, for example, Biacore technology, Kinexa technology and flow cytometry).

[0059] The term "competitive binding" as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human FGFR2b and anti-FGFR2b antibodies) to any detectable extent (e.g., at least 85%, or at least 90%, or at least 95%). Those skilled in the art will recognize that it is not necessary to overexperiment to determine whether a given antibody competes with an antibody of the present disclosure for binding to FGFR 2b.

[0060] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody or antigen binding portion binds. The minimum size of an epitope can be about three, four, five, six or seven amino acids, but these amino acids do not need to be located in a continuous linear sequence of the primary structure of the antigen, as the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen. CDRs are important for identifying antigenic epitopes.

[0061] As used herein, "percent (%) identity" of 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 alignment of the sequences. Sequence identity refers to an exact match between the nucleotides or amino acids of the two sequences being compared. Sequence identity can be determined by conventional methods by those skilled in the art, such as the BLAST algorithm.

[0062] As used herein, "antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to in vitro or in vivo cell-mediated cytotoxicity in which nonspecific effector cells (e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils) expressing Fc receptors (FcRs) on the surface of effector cells recognize the Fc region of an Ab 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 Ab can be measured as described in Example 5, or by any method known to those skilled in the art.

[0063] As used herein, the modified Ab includes one or more modifications of "enhancing ADCC", which means that the ADCC activity level of the modified Ab is higher than the ADCC induced by the unmodified Ab. For example, the enhanced ADCC described in the present disclosure is characterized in that the lysis of FGFR2b expressing cells is 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%. Various methods for enhancing ADCC have been described in the prior art (Liu R, Oldham RJ, Teal E, Beers SA, Cragg MS. Antibodies (Basel). 2020Nov 17; 9 (4): 64). For example, studies have shown that afucosylated (i.e., fucose-deficient or non-fucosylated) antibodies exhibit increased binding to FcγRIII, thereby stimulating higher ADCC activity (Shields et al. (2002) J. Biol. Chem., 277: 26733-26740; Shinkawa et al. (2003) J. Biol. Chem., 278: 3466-3473; and European Patent Application Publication No. 1176195). In some embodiments, the afucosylated antibodies provided herein lack fucose at heavy chain asparagine 297 (Asn297). Asn297 (Eu numbering of Fc region residues; or position 314 in Kabat numbering) is a conserved N-linked glycosylation site found in each CH2 domain of the Fc region of IgG1 isotype antibodies.

[0064] As used herein, "administering", "applying" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. The preferred route of administration for 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 by injection or infusion. The phrase "parenteral administration" used herein refers to a mode of administration other than intestinal and topical administration, usually administered by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibody or antigen-binding fragment thereof according to the present disclosure can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. For example, administration may also be performed once, multiple times, and / or over one or more extended periods of time.

[0065] The term "fibroblast growth factor receptor 2 (FGFR2)" used herein, also referred to as CD332 (differentiation cluster 332), is a member of the FGFR family and is encoded by the FGFR2 gene located on chromosome 10 in humans. FGFR is highly conserved, has common structural features, and is composed of an extracellular ligand binding portion (the extracellular ligand binding portion is composed of different Ig-like domains; the α isotype contains all three Ig-like domains D1, D2 and D3; the β isotype contains only two Ig-like domains D2 and D3 domains, but does not contain D1), a transmembrane domain, and an intracellular tyrosine kinase catalytic domain. FGFR2 has two naturally occurring isotypes, FGFR2IIIb and FGFR2IIIc, which are spliced ​​by the third immunoglobulin-like domain (D3). FGFR2IIIb is a high-affinity receptor for FGF1 and a specific receptor for KGF family members (e.g., FGF10, FGF22, especially FGF7). KGF (FGF7) and KGFR (FGFR2IIIb) were found to be abnormally expressed in a variety of cancers, such as pancreatic cancer, gastric cancer, ovarian cancer 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).

[0066] As used herein, "abnormal expression of FGFR2b" includes, but is not limited to, FGFR2b mutation, FGFR2b amplification, FGFR2b fusion, FGFR2 translocation, and FGFR2 overexpression.

[0067] As used herein, "anti-FGFR2b antibody" refers to an antibody that can specifically bind to human or non-human FGFR2b (e.g., proteins disclosed as UniProtKB-P21802-3, UniProtKB-A0A2K5TL84, UniProtKB-P21803-2 in the UniProt database). In some embodiments, the anti-FGFR2b antibody can specifically bind to FGFR2b with a binding affinity (K D ) is less than 1x 10 -4 M, less than 1x 10 -5 M, less than 1x 10 -6 M, less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M, or less than 1x 10 -10 M. In some embodiments, the anti-FGFR2b antibody can specifically bind to FGFR2b, and its K D In a preferred embodiment, the anti-FGFR2b antibody specifically binds to FGFR2b with a binding affinity (K D ) is less than 1x 10 - 7 M, less than 1x 10 -8 M, less than 1x 10 -9 M, or less than 1x 10 -10 M.

[0068] The term "cancer" as used herein refers to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can lead to the formation of malignant tumors, which invade adjacent tissues and may also be transferred to distant parts of the body through the lymphatic system or blood. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasion stage. The term "tumor" as used herein refers to cancer cells, such as a large number of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroid, lymph, gastrointestinal tract and urogenital tract, and adenocarcinomas, including malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers and esophageal cancers. In some embodiments, antibodies or antigen-binding fragments thereof provided herein are designed to treat or diagnose cancer in subjects. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, it includes a malignant tumor composed of cancer tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer that is derived from glandular tissue or tumor cells to form a recognizable glandular structure. The term "sarcoma" is recognized and refers to a malignant tumor derived from a mesenchyme.

[0069] As used herein, "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. The terms "subject" and "patient" are used interchangeably herein.

[0070] "Vector" used herein refers to a polynucleotide molecule that can replicate / clone the desired nucleic acid fragment contained therein or can express a protein encoded by such a desired nucleic acid fragment introduced into an appropriate cell host. Examples of vectors include cloning vectors and expression vectors. The term "expression vector" used herein refers to a vector that can operably insert a polynucleotide encoding a protein to achieve expression of the protein. An expression vector may contain a variety of elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may contain a replication origin. The vector can be introduced into a host cell by methods known in the art, such as electroporation, chemical transfection (such as DEAE-dextran), transformation, transfection, and infection and / or transduction (such as with a recombinant virus). Non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0071] As used herein, "host cell" refers to a cell that has been transformed or can be transformed by a nucleic acid sequence to express a target gene. The host cell can be a prokaryotic cell (e.g., Escherichia coli), a eukaryotic cell (e.g., yeast, plants including tobacco and tomatoes, animals including humans, monkeys, hamsters, rats, mice, or insects), or a hybridoma.

[0072] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of a drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, can protect a subject from the onset of disease or promote regression of a subject's disease, wherein the promotion of disease regression is manifested as a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, the prevention or alleviation of damage or disability caused by disease affliction, or the alleviation of disease symptoms. In addition, the terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote regression of disease (e.g., regression of cancer) in a patient. Physiological safety refers to an acceptable level of toxicity or other adverse physiological effects (side effects) produced at the cellular, organ, and / or organismal level after administration of a drug. The efficacy of a therapeutic agent can be evaluated using a variety of methods known to professionals, such as evaluation of human subjects during clinical trials, evaluation in animal model systems that predict human efficacy, or detection of the activity of a drug by in vitro assays.

[0073] The term "effective inhibition" as used herein refers to an antibody or antigen-binding fragment thereof disclosed herein 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%, at least about 100% compared to a reference antibody. The term "weak inhibition" refers to an antibody or antigen-binding fragment thereof disclosed herein having a relative inhibition rate of at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40% compared to a reference antibody. In some embodiments, the anti-FGFR2b reference antibody is FPA144. In some preferred embodiments, the term "effective inhibition" refers to an antibody or antigen-binding fragment thereof disclosed herein having a relative inhibition rate of at least about 85%, at least about 90%, at least about 95%, at least about 100% compared to the reference antibody FPA144. In some preferred embodiments, the term "weak inhibition" means that the antibody or antigen-binding fragment thereof of the present disclosure has a relative inhibition rate of at most about 55%, at most about 50%, at most about 45%, or at most about 40% compared to the reference antibody FPA144.

[0074] As used herein, the term "treating" or "treatment" of a disease or disorder as used herein includes preventing or alleviating the disorder, slowing the onset or development of the disorder, reducing the risk of developing the disorder, preventing or delaying the development of symptoms associated with the disorder, alleviating or stopping symptoms associated with the disorder, causing complete or partial regression of the disorder, curing the disorder, or some combination thereof.

[0075] The term "pharmaceutically acceptable carrier" as used herein means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients contained in the formulation and physiologically compatible with the recipient thereof.

[0076] Anti-FGFR2b antibodies and antigen-binding fragments

[0077] The present disclosure provides novel anti-FGFR2b antibodies (e.g., HC18) or antigen-binding fragments thereof, which specifically bind to FGFR2b, such as human FGFR2b expressed on the cell surface, but do not have detectable binding affinity to FGFR2c. In addition, the antibodies or antigen-binding fragments thereof of the present disclosure mediate effective inhibition of FGF7 binding to FGFR2b, but weak inhibition of FGF10 binding to FGFR2b, which provides an improved safety / effectiveness balance.

[0078] Experiments show that the antibodies provided herein exhibit significant blocking of the FGF7-FGFR2b pathway and tumor cell proliferation (e.g., SNU-16 cells) by inhibiting FGF7-induced FGFR2b and ERK1 / 2 phosphorylation, while the blocking effect on the binding of FGF10 to FGFR2b is relatively weak compared to the benchmark (e.g., FPA144), suggesting that it may have better safety in terms of corneal toxicity. ADCC functional assays show that the antibodies disclosed herein induce strong ADCC effects and kill tumor cells expressing FGFR2b.

[0079] The antibodies or antigen-binding fragments thereof provided herein comprise: a heavy chain complementary determining region 1 (CDR1) 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: 1, or having up to 1 amino acid addition, substitution and / or deletion compared to SEQ ID NO: 1; a heavy chain CDR2 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: 2, or having up to 3 (e.g., 1, 2 or 3) amino acid additions, substitutions and / or deletions compared to SEQ ID NO: 2; 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 having up to 1 amino acid addition, substitution and / or deletion compared to SEQ ID NO: 4. A heavy chain CDR3 having an amino acid sequence with at most 1 amino acid addition, substitution and / or deletion compared to NO:3.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain CDR1 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:4, or having up to 2 (e.g., 1 or 2) amino acid additions, substitutions and / or deletions compared to SEQ ID NO:4; a light chain CDR2 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:5, or having up to 1 amino acid addition, substitution and / or deletion compared to SEQ ID NO:5; and a light 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:6, or having up to 1 amino acid addition, substitution and / or deletion compared to SEQ ID NO:7. A light chain CDR3 having an amino acid sequence with at most 1 amino acid addition, substitution and / or deletion compared to NO:6.

[0081] 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.

[0082] 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.

[0083] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises the CDR sequences of HC18 (see Table 1).

[0084] Table 1 CDR sequences of HC18.

[0085]

[0086] In a preferred embodiment, the antibody or its antigen-binding fragment comprises: a heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 3; a light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 6.

[0087] It is known that CDR is responsible for antigen binding, however, it has been found that not all six CDRs are indispensable or unchangeable. In other words, one or more CDRs in HC18 can be replaced or changed or modified, but the specific binding affinity to FGFR2b is basically retained.

[0088] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may comprise one or more modifications or substitutions in one or more CDR regions provided in Table 1. Such variants retain the specific binding affinity of their parent antibodies to FGFR2b, but may have improvements in one or more properties, such as higher antigen binding affinity or reduced glycosylation potential.

[0089] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can be modified to remove one or more Asn or Asp hot spots in the CDR region (or in the variable region). Such Asn and Asp hot spots can lead to antibody degradation, thereby reducing the stability of the antibody.

[0090] In some embodiments, one or more modifications or substitutions are conservative substitutions.

[0091] The antibodies or their antigen-binding fragments provided herein also include suitable framework region (FR) sequences, as long as the antibody can specifically bind to FGFR2b. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but they can be transplanted to any suitable FR sequence of any suitable species (e.g., mouse, human, rat, rabbit, etc.) using suitable methods known in the art (e.g., recombinant techniques). In some embodiments, the antibodies or their antigen-binding fragments provided herein are humanized.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity with SEQ ID NO: 7 (i.e., the heavy chain variable region of HC18), 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: 7; the light chain variable region having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity with SEQ ID NO: 8 (i.e., the light chain variable region of HC18), or having at least 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: NO: 8 has 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 the amino acid sequence. In a preferred embodiment, the substitution is a conservative substitution.

[0093] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:8.

[0094] 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 and / or a light chain constant region. The heavy chain constant region comprises a CH1, a hinge region, and / or a 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 C κ or C λIn a preferred embodiment, the constant region is derived from human IgG1 (hIgG1). In a preferred embodiment, the constant region is that of human IgG1.

[0095] 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 at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity with SEQ ID NO: 9 (i.e., the full-length heavy chain sequence of HC18), 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. 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 (i.e., the full-length light chain sequence of HC18), 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. In a preferred embodiment, the substitution is a conservative substitution.

[0096] 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.

[0097] Table 2 shows the amino acid sequence and nucleotide sequence of HC18.

[0098] Table 2 Amino acid sequence and nucleotide sequence of HC18.

[0099]

[0100]

[0101]

[0102] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may include one or more modifications or substitutions in one or more sequences provided herein, but still retain a specific binding affinity to FGFR2b. Various methods known in the art can be used to achieve this purpose. For example, computer software can be used to virtually simulate the combination of antibodies and FGFR2b, and identify the amino acid residues that form a binding interface on the antibody. Such residues can be avoided in the substitution to prevent a reduction in binding affinity, or substitutions can be performed in a targeted manner to provide a stronger combination.

[0103] As used herein, "conservatively modified variants" or "conservative substitutions" refer to substitutions of amino acids in proteins with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can generally be made without changing the biological activity of the protein. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of polypeptides will not significantly change biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). In addition, substitutions of amino acids with similar structure and / or function are unlikely to destroy biological activity. Various embodiments of antibodies or antigen-binding fragments thereof according to the present disclosure include polypeptide chains whose sequences include up to 0 (no change), 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 compared to specific amino acid sequences disclosed herein (e.g., SEQ ID NO: 7, 8, 9 or 10). As used herein, the phrase "up to X" conservative amino acid substitutions includes 0 substitutions and any number of substitutions up to X (including X) substitutions. Such exemplary substitutions are preferably performed as shown in the following table:

[0104] Table 3 Exemplary conservative amino acid substitutions

[0105] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0106] The present disclosure also contemplates functional conservative variants of the antibodies or antigen-binding fragments thereof according to the present disclosure. A "functional conservative variant" is a variant in which one or more amino acid residues in a protein are changed, but the overall conformation and function of the polypeptide are not changed, including but not limited to replacing an amino acid with an amino acid having similar properties.

[0107] The antibodies or antigen-binding fragments thereof provided herein also comprise a constant region capable of inducing effector functions. 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.

[0108] In some embodiments, the 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 the wild-type antibody or antigen-binding fragment thereof.

[0109] As described above, "antibody or its antigen-binding fragment" used herein refers to a complete antibody or an antibody fragment with 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.

[0110] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(Fab')2 fragments, scFv fragments, Fv fragments, Fab' fragments or domain antibodies.

[0111] In some embodiments, the antigen binding fragment provided herein can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) as described herein, fused with CD3-zeta transmembrane and intracellular domains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness. Therefore, in one aspect, the present disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0112] 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 are also contemplated. In one embodiment, such a competing antibody specifically binds to an epitope identical or overlapping to 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, the HC18 comprising a heavy chain variable region and a light chain variable region having amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to FGFR2b at an epitope different from the epitope bound by FPA144. In some embodiments, the antibody or antigen-binding fragment 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 skilled in the art will recognize that it is not necessary to overexperiment to determine whether a given antibody competes with an antibody of the present disclosure for binding to FGFR2b.

[0113] Antibody characteristics

[0114] Fibroblast growth factor receptor 2b (FGFR2b) is overexpressed in about 2-30% of GC and is associated with a poor prognosis. Bemarituzumab is a non-fucosylated humanized monoclonal antibody that specifically inhibits FGFR2b. In the FIGHT trial, bemarituzumab showed a beneficial response in GC, but it seemed to cause high corneal adverse events. It is hypothesized that FGF10 inhibition may be the mechanism by which bemarituzumab causes corneal toxicity (Catenacci et al., J Clin Oncol. 2020: 38 (21): 2418-2426). The present disclosure provides a novel antibody or antigen-binding fragment thereof for FGFR2b, which has weak inhibitory activity against FGF10. Therefore, the antibody or antigen-binding fragment thereof may represent a different antibody therapy with the potential to reduce eye side effects.

[0115] The antibodies or antigen-binding fragments thereof provided herein have at least one of the following characteristics:

[0116] It has specific binding ability to FGFR2b and has undetectable binding affinity to FGFR2c.

[0117] It has a potent inhibitory effect on the binding of FGF7 to FGFR2b, but a weak inhibitory effect on the binding of FGF10 to FGFR2b.

[0118] 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 the phosphorylation of FGFR2; reducing the phosphorylation of downstream signaling pathways (e.g., MAPK pathway, PI3K / AKT1 / MTOR pathway); and / or directly killing cancer cells through ADCC and / or CDC.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein specifically bind to human FGFR2b with a binding affinity (K D ) is less than 1x 10 -4 M, less than 1x 10 -5 M, less than 1x 10 -6 M, less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M, or less than 1x 10 -10 M. In some embodiments, K D Less than 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a binding affinity (K) comparable to that of a positive control antibody (e.g., FPA144). D ) specifically binds to human FGFR2b, e.g., as measured by surface plasmon resonance. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a FGFR2b binding capacity of 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%, at least or about 200% compared to FPA144 or an FPA144 analog.

[0120] In a preferred embodiment, the antibodies or antigen-binding fragments thereof provided herein specifically bind to human FGFR2b with a binding affinity (K D ) is less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M or less than 1x 10 -10 M.

[0121] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0122] ELISA assay or other common techniques can be used to measure the inhibition of anti-FGFR2b antibodies to FGF7 and FGF10 combined with FGFR2b. In some embodiments, provided herein is an antibody or its antigen-binding fragment compared with a reference antibody (e.g., FPA144), with 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% of the relative inhibition rate of the inhibitory effect of FGF7 combined with FGFR2b. In some embodiments, provided herein is an antibody or its antigen-binding fragment compared with a reference antibody (e.g., FPA144), with at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45% or at most about 40% of the relative inhibition rate of the inhibitory effect of FGF10 combined with FGFR2b.

[0123] 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% of the inhibitory effect on the binding of FGF7 to FGFR2b, and a relative inhibition rate of at most about 55%, at most about 50%, at most about 45%, or at most about 40% of the inhibitory effect on the binding of FGF10 to FGFR2b, compared to a reference antibody (e.g., FPA144).

[0124] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can increase complement dependent cytotoxicity (CDC) 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.

[0125] 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.

[0126] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein inhibit FGF-induced FGFR2 phosphorylation and cancer cell proliferation.

[0127] In some embodiments, compared to a non-specific antibody or isotype control antibody, the antibody or its antigen-binding fragment provided herein reduces the phosphorylation (e.g., phosphorylation induced by FGF7) level of FGFR2 in a cell (e.g., a cell expressing FGFR2b) to 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%. In some embodiments, after treatment with the antibody or its antigen-binding fragment, the proportion of cells containing phosphorylated FGFR2 is less than or about 50%, less than or about 60%, less than or about 70%, less than or about 80%, less than or about 90%, less than or about 100%, less than or about 110%, less than or about 120%, less than or about 130%, less than or about 140%, less than or about 150%, less than or about 200% compared to after treatment with FPA144 or FPA144 analogs.

[0128] In some embodiments, provided herein is an antibody or its antigen-binding fragment reducing the phosphorylation (for example, phosphorylation induced by FGF7) level of the downstream signal transduction pathways involved in cancer cell proliferation, survival and / or apoptosis. In some embodiments, compared with non-specific antibodies, isotype control antibodies, provided herein is an antibody or its antigen-binding fragment reducing the phosphorylation level of ERK in cells (for example, cells expressing FGFR2b) to 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%. In some embodiments, cells treated with the antibody or antigen-binding fragment thereof (e.g., cells expressing FGFR2b) have a ratio of phosphorylated ERK1 / 2 of less than or about 90%, less than or about 80%, less than or about 70%, less than or about 60%, less than or about 50%, less than or about 40%, less than or about 30%, less than or about 20%, less than or about 10%, less than or about 5% of total ERK compared to cells treated with FPA144 or an FPA144 analog.

[0129] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a weaker effect of reducing the level of phosphorylation (eg, FGF10-induced phosphorylation) of FGFR2 in cells (eg, cells expressing FGFR2b) than FPA144 or an FPA144 analog.

[0130] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a weaker effect of reducing the phosphorylation level (e.g., FGF10-induced phosphorylation) of downstream signaling pathways involved in cancer cell proliferation, survival and / or apoptosis than FPA144 or FPA144 analogs.

[0131] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce FGF7-induced cell (e.g., cell expressing FGFR2b) proliferation to 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, an isotype control antibody.

[0132] Conjugate

[0133] The present disclosure provides antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof linked to one or more conjugated moieties.

[0134] In some embodiments, one or more conjugated moieties are stabilizing molecules (e.g., molecules that increase 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). Conjugated stabilizing molecules can increase the half-life of an antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity.

[0135] In some embodiments, one or more conjugated moieties are therapeutic agents. Antibody-drug conjugates comprising antibodies or antigen-binding fragments thereof can covalently or non-covalently bind therapeutic agents. In some embodiments, therapeutic agents are cytotoxic agents or cytostatic agents (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, ​​diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, cyclophosphamide and the like).

[0136] Polynucleotides and methods for producing the same

[0137] The present disclosure also provides nucleic acids encoding the antibodies or antigen-binding fragments thereof provided herein.

[0138] The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses polynucleotides containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) 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)).

[0139] 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 methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Encoding DNA can also be obtained by synthetic methods.

[0140] Nucleic acids encoding anti-FGFR2b antibodies (eg, including the HC18 sequence shown in SEQ ID NOs: 11 and 12) can be inserted into vectors for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art.

[0141] The antibodies or antigen-binding fragments thereof provided herein can be produced by any protein (eg, antibody) synthesis method known in the art, in particular by chemical synthesis or preferably by recombinant expression technology.

[0142] The recombinant expression of antibodies requires the construction of expression vectors containing nucleic acids encoding the antibodies. Once the nucleic acids encoding the antibodies are obtained, vectors for producing the antibodies can be produced by recombinant DNA techniques. In the present disclosure, expression vectors are constructed to contain antibody coding sequences and appropriate transcription and translation regulatory elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0143] The antibody or antigen-binding fragment thereof according to the present disclosure can be produced by transferring the expression vector into a host cell by conventional techniques and then culturing the transfected cells by conventional techniques.

[0144] In one embodiment, the 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 the 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.

[0145] Pharmaceutical composition

[0146] The present disclosure provides a pharmaceutical composition, which includes an antibody or its antigen-binding fragment provided herein, a nucleic acid provided herein, an expression vector provided herein, or an antibody-drug conjugate provided herein, and one or more pharmaceutically acceptable carriers. In a preferred embodiment, the pharmaceutical composition includes a therapeutically effective amount of an antibody or its antigen-binding fragment provided herein, and one or more additional ingredients, such as a pharmaceutically acceptable carrier, carrier or medium. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, which may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous carrier, a non-aqueous carrier, an antibacterial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspension / dispersant, a masking agent or a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art, or various combinations thereof.

[0147] In some embodiments, the composition can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent (e.g., benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), an antioxidant (e.g., ascorbic acid or sodium bisulfite), a chelating agent (e.g., ethylenediaminetetraacetic acid), a buffer (e.g., acetate, citrate or phosphate) and an isotonic agent, for example, a sugar (e.g., glucose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition can be formulated and packaged in an ampoule, a disposable syringe or a multiple dose vial. Where necessary (e.g., in an injectable preparation), appropriate fluidity can be maintained by using a coating (e.g., lecithin) or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by adding agents that delay absorption (e.g., aluminum monostearate 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.).

[0148] The pharmaceutical composition can be administered by any suitable method known to those skilled in the art, such as parenteral and non-parenteral methods as described above. In a preferred embodiment, the pharmaceutical composition can be administered by intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural or intrasternal injection.

[0149] The present invention also provides a kit comprising an antibody or antigen-binding fragment thereof provided herein. In a preferred embodiment, the kit according to the present disclosure also comprises instructions for guiding the use of the antibody or antigen-binding fragment thereof disclosed herein, such as for treating or preventing a disease associated with abnormal expression of FGFR2b in a subject, such as cancer.

[0150] 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.

[0151] In some embodiments, the additional therapeutic agent may include 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).

[0152] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated Hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0153] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrien t), pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0154] In some embodiments, the additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0155] 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.

[0156] Treatment methods and uses

[0157] The present disclosure provides a method for inhibiting or reducing FGF-induced tumor cell proliferation 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.

[0158] 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.

[0159] The present disclosure provides a method for killing tumor cells associated with abnormal expression of FGFR2b and reducing corneal toxicity 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.

[0160] The present disclosure also 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 the antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate and / or pharmaceutical composition of the present disclosure.

[0161] 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 to prepare drugs and / or kits, which are, for example, used to treat or prevent a disease or condition associated with abnormal expression of FGFR2b in a subject.

[0162] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a cancer characterized by expressing or overexpressing FGFR2b. In some embodiments, the disease or condition is a cancer with FGFR2b mutation, FGFR2b amplification, FGFR2b fusion, FGFR2 translocation and / or FGFR2 overexpression.

[0163] In some embodiments, 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, bile duct cancer, intrahepatic bile duct carcinoma 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 gastric cancer amplified by FGFR2. Various methods known in the art can be utilized to identify patients suffering from cancer.

[0164] The therapeutically effective amount of the antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, antibody-drug conjugate or pharmaceutical composition provided herein can be administered once or multiple times. In any method described herein, the antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, antibody-drug conjugate and / or pharmaceutical composition provided herein 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), and optionally, at least one additional therapeutic agent.

[0165] 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 over an extended period of time (e.g., 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). Skilled medical professionals can determine the length of the treatment period using any method in the art for diagnosing or tracking the effects of treatment (e.g., observing at least one cancer symptom). As described herein, a skilled medical professional can also change the identity and amount (e.g., increase or decrease) of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate and / or pharmaceutical composition provided herein (and / or one or more additional therapeutic agents) administered to a subject, and can also adjust (e.g., increase or decrease) the dose or frequency of administration of an antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, antibody-drug conjugate and / or pharmaceutical composition provided herein (and / or at least one other additional therapeutic agent) to a subject based on an assessment of the therapeutic effect (e.g., using any of the methods described herein and known in the art).

[0166] 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 may be administered to a subject before 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.

[0167] The data obtained from cell culture experiments and animal studies can be used to formulate the appropriate dosage of any given agent for a subject (e.g., a person). The therapeutically effective amount of an antibody or its antigen-binding fragment provided herein is an amount for treating a disease in a subject, reducing the severity, frequency, and / or duration of one or more symptoms of a disease in a subject (e.g., a person). The effectiveness and dosage of an antibody or its antigen-binding fragment provided herein can be determined by a health care professional or a veterinary professional using methods known in the art, or can be determined by observing the symptoms of one or more diseases in a subject (e.g., a person). Certain factors may affect the dosage and time required for the effective treatment of a subject (e.g., the severity of a disease or condition, previous treatment, the general health status and / or age of the subject, and the presence of other diseases).

[0168] Exemplary dosages include milligrams or micrograms of antibodies or antigen-binding fragments thereof provided herein or antibody-drug conjugates 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 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). Although these dosages cover a wide range, it will be appreciated by those of ordinary skill in the art that the efficacy of therapeutic agents (including antibodies and antigen-binding fragments thereof) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, and then the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (while still working in the development phase) can subsequently gradually increase the dose until an appropriate response is obtained. Furthermore, it will be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, time of administration, route of administration, rate of excretion and half-life of the antibody or antibody fragment in vivo.

[0169] Example

[0170] The invention will now be illustrated by the following examples which should not be construed as limiting.

[0171] Example 1 Preparation of anti-FGFR2b antibodies

[0172] SJL mice (5-6 week old females) were immunized every other week by intraperitoneal injection of FGFR2 (beta) IIIb-Fc and anti-CD25 / CD40 antibodies (initial anti-CD25 antibody, then a dose of 100 μg / animal, then anti-CD40 antibody, and the last 2 doses of 50 μg / animal), and the antigen was suspended in MPL / TDM (Sigma-Aldrich). Three days after the last injection, popliteal lymphoid cells were extracted and fused with Sp2 / 0-Ag14 mouse myeloma cells at a ratio of 1:1 using the Hybrimune electrofusion system (Cyto Pulse Sciences). Hybridomas were screened by adding 2x HAT (Sigma) 24 hours later. Ten days after fusion, the hybridoma culture supernatant was screened for the ability to bind to FGFR2IIIb-his but not to FGFR2IIIc-his using ELISA. The selected mAb was then screened by ELISA for its ability to recognize FGFR2IIIb on the CHOK1-FGFR2IIIb transfectant and block the binding of FGF7 to FGFR2IIIb. The selected hybridoma was then cloned twice using the limiting dilution technique. The subcloned hybridomas were sequenced and chimeric antibodies were generated by fusion with hFc. Many anti-FGFR2 chimeric antibodies were obtained from the fusion, including mAb HC18.

[0173] Example 2 FACS analysis of binding specificity of anti-FGFR2b antibodies to human FGFR2b protein on cell surface

[0174] Appropriate cells (1×10 5 ) were washed twice in FACS buffer (PBS + 2% BSA) and resuspended in 100 μl of FACS buffer containing serial dilutions (1:5) of anti-FGFR2b mAb and incubated at 4°C for 1 hour. The cells were then washed twice in FACS buffer and the bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, catalog #410712) for 1 hour at 4°C. After washing twice in FACS buffer, the cells were analyzed on Cytoflex (Beckmann Coulter). Aprutumab was also tested as a positive control, which can bind to FGFR2b and FGFR2c. The results are shown in Figure 1. Compared to the benchmark (FPA144), the antibodies of the present disclosure showed comparable binding ability.

[0175] Example 3. Anti-FGFR2b antibodies inhibit the binding of FGF7 and FGF10 to FGFR2b

[0176] The 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 reagents for 1 hour at room temperature, except for the initial plate coating step which was performed overnight at 4°C. Between each step, the plates were washed 3 times in PBS containing 0.05% Tween 20.

[0177] In order to determine the ability of anti-FGFR2b mAb blocking ligand to bind to FGFR2b, 1 μg / ml hFGFR2b-Fc (Kactus, catalog number #FGR-HM2BB) was coated with plates, and then blocked with 2% BSA. Next, plates were incubated together with human FGF7-his (20nM, Sino Biological, catalog number #10210-H07E) or biotin-FGF10 (5nM, Kactus, catalog number #FGF-HE010B) in the presence of the anti-FGFR2b mAb of serial dilution (1:5), and the ligand bound was detected with HRP-anti-6X His tags (Abcam, catalog number #ab1187) or streptavidin-protein HRP (Thermo, catalog number #21126) and TMB substrate (Cell Signaling, catalog number #7004P6). Result as shown in Figure 2 and Table 4. Compared to the benchmark, the antibodies of the present disclosure significantly blocked the binding of FGF7 to FGFR2b, but only partially inhibited the binding of FGF10 to FGFR2b, suggesting that the antibodies of the present disclosure may have better safety in terms of corneal toxicity.

[0178] Table 4. Inhibition of FGF7 and FGF10 binding to FGFR2b

[0179]

[0180]

[0181] Example 4. Surface plasmon response (SPR) sensorgram of anti-FGFR2b antibody binding to human FGFR2b antigen

[0182] The binding affinity of anti-FGFR2b mAb to human FGFR2b (FGFR2b-his) was measured using surface plasmon resonance (Octet, Sartorius). Anti-FGFR2b mAb was 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 a blocking reagent. FGFR2b-his protein was diluted in HEPES-buffered saline running buffer containing 0.05% surfactant P20 and then flowed over the immobilized antibody. The results are shown in Figure 3And as shown in Table 5.

[0183] Table 5. Binding affinity of anti-FGFR2b antibodies to human FGFR2b.

[0184]

[0185] Example 5. ADCC of anti-FGFR2b antibodies

[0186] To generate afucosylated (AF) mAbs, customer-supplied expression plasmids were transformed into E. coli and propagated separately at appropriate scale. NucleoBond Xtra Maxi Plus EF Kit was used for large-scale plasmid generation. Constructs containing each antibody heavy and light chain were co-transfected into FUT8-KO CHO cells using PEI. Conditioned media was harvested 9-11 days after transfection. Conditioned media expressing the target Ab was harvested by centrifugation and filtration and then loaded onto a protein A affinity column. The purified Ab was analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0187] Cytotoxic activity was assessed using FACS analysis. Effector cells Human peripheral blood mononuclear cells were obtained from individual human donors ( 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, catalog number #C34564) for 10 minutes at 37°C, washed twice with RPMI 1640 medium (Gibco, catalog number #A10491-01) containing 10% FBS (Gibco, catalog number #10099-141), and then plated in 96-well round-bottom plates at a ratio of effector cells to target cells (20:1). Serial dilutions (1:3) of anti-FGFR2b mAb were added to the designated rows of the detection plate. After incubation at 37°C for 4 hours, 2 μl of propidium iodide staining solution (BD biosciences, catalog number #556547) was added to each well to stain dead cells for 10 minutes at room temperature. Cell analysis was performed directly on Cytoflex (Beckmann Coulter). Figure 4 As shown, the antibodies of the present disclosure (including HC18 and HC18-AF) induced a strong ADCC response to kill cells expressing FGFR2b, and the afucosylated (AF) antibody showed a stronger effect.

[0188] Example 6. Anti-FGFR2b antibody inhibits FGF7- and FGF10-induced FGFR2 phosphorylation in SNU-16 cells

[0189] The effect of anti-FGFR2b antibodies on in vitro tumor cell FGFR2 phosphorylation was measured in SNU-16 cells. Approximately 50,000 SNU-16 cells were plated in RPMI 1640 medium (Gibco, catalog #A10491-01) in a 96-well plate and incubated for 4 hours at 37°C, 5% CO2. Next, SNU-16 cells were treated with serially diluted (1:10) anti-FGFR2b mAbs for 1 hour. SNU-16 cells were then treated with 30ng / mL FGF7 (R&D, catalog #251-KG-01M) or FGF10 (Kactus, catalog #FGF-HE010B) and 20μg / mL heparin (Sigma, catalog #H3149-500KU) and incubated for 5 minutes at 37°C and 5% CO2. To harvest cells for phosphorylated FGFR2 (Tyr653 / 654) detection, spin the 96-well plate at 300 g for 3 minutes. Then slowly aspirate the entire volume in the well without disrupting the cells. Immediately add 50 μL of 1X supplemented lysis buffer (Cisbio, catalog #64FGFR2Y6PEG) and incubate at room temperature with shaking for at least 30 minutes according to the manufacturer's preparation protocol. FGFR2 phosphorylation was read by fluorescence emission at two different wavelengths (665 nm and 620 nm) on a reader (EnVision, Perkin Elmer). The experiment was performed three times. As shown in FIG5 , the antibodies disclosed herein significantly inhibited FGF7- and FGF10-induced FGFR2 phosphorylation in SNU-16 cells, especially FGF7-induced FGFR2 phosphorylation.

[0190] Example 7. Anti-FGFR2b antibody inhibits FGF7 and FGF10-induced ERK1 / 2 phosphorylation in SNU-16 cells

[0191] The effect of anti-FGFR2b antibodies on in vitro tumor cell ERK1 / 2 phosphorylation was measured in SNU-16 cells. Approximately 50,000 SNU-16 cells were plated in RPMI 1640 medium (Gibco, catalog #A10491-01) in a 96-well plate and incubated for 4 hours at 37°C, 5% CO2. Next, SNU-16 cells were treated with serially diluted (1:5) anti-FGFR2b mAbs for 1 hour. SNU-16 cells were then treated with 30ng / mL FGF7 (R&D, catalog #251-KG-01M) or FGF10 (Kactus, catalog #FGF-HE010B) and 20μg / mL heparin (Sigma, catalog #H3149-500KU) and incubated for 15 minutes at 37°C and 5% CO2. To harvest cells for phosphorylated ERK (Thr202 / Tyr204) detection, spin the 96-well plate at 300 g for 3 minutes. Then slowly aspirate the entire volume in the well without disrupting the cells. Immediately add 50 μL of 1X supplemented lysis buffer (Cisbio, catalog #64ERKPEH) and incubate at room temperature with shaking for at least 30 minutes according to the manufacturer's preparation protocol. Reader (EnVision, Perkin Elmer), by two different wavelengths (665nm and 620nm) of fluorescence emission reading ERK1 / 2 phosphorylation. The experiment was performed three times. As shown in Figure 6, the antibody of the present disclosure significantly inhibited FGF7 and FGF10-induced ERK1 / 2 phosphorylation in SNU-16 cells, especially inhibited FGF7-induced ERK1 / 2 phosphorylation.

[0192] Example 8. Anti-FGFR2b antibody inhibits FGF7-induced SNU-16 cell proliferation

[0193] The effect of anti-FGFR2b antibodies on in vitro tumor cell proliferation was measured in SNU-16 cells. Approximately 10,000 SNU-16 cells were plated in RPMI 1640 medium (Gibco, catalog #A10491-01) in a 96-well plate to serum starve the cells and incubated for 16 hours / overnight at 37°C, 5% CO2. Next, SNU-16 cells were treated with serial dilutions (1:10) of anti-FGFR2b mAb for 1 hour. SNU-16 cells were then treated with 50ng / mL FGF7 (R&D, catalog #251-KG-01M) and 5μg / mL heparin (Sigma, catalog #H3149-500KU) and incubated for 4 days at 37°C and 5% CO2. To harvest cells for cell viability assays, the 96-well plate was spun at 300g for 3 minutes. Half of the total volume in the well (about 100 μL) was then slowly aspirated without disrupting the cells. CellTiter-Glo reagent (100 μL) from the CellTiter-Glo Luminescent Cell Viability Assay (Promega, catalog # G7571) was then added according to the manufacturer's protocol. The plate was shaken for 15 minutes at room temperature. During the incubation with the CellTiter-Glo reagent, the plate was protected from light. Cell proliferation was read by luminescence on a SpectraMax M5 (Molecular Devices). This experiment was performed three times. The results are shown in Figure 2. Figure 7 As shown, the inhibitory effect of the antibodies disclosed herein on SNU-16 proliferation is the same as or even better than that of FPA144.

[0194] Example 9. Epitope grouping of anti-FGFR2b mAbs.

[0195] Epitope binning of anti-FGFR2b antibodies was measured by ELISA assay. Each step of each assay was performed by incubating with appropriate reagents for 1 hour at room temperature, except for the initial plate coating step which was performed overnight at 4°C. Between each step, the plates were washed 3 times in PBS containing 0.05% Tween 20.

[0196] To determine the epitope classification of anti-FGFR2b mAb, plates were coated with 1 μg / ml HC18 or BMK FPA144 and then blocked with 2% BSA. Next, the plates were incubated with 0.1 μg / ml biotin-hFGFR2b-Fc (Kactus, catalog #FGR-HM2BB) mixture in the presence of serially diluted (1:5) anti-FGFR2b mAb, and bound antigens were detected with streptavidin-protein HRP (Thermo, catalog #21126) and TMB substrate (Cell Signaling, catalog #7004P6). The results are shown in Table 6, indicating that the antibodies disclosed herein have different epitope groups (epitope bins) compared to FPA144.

[0197] Table 6. Epitope grouping of anti-FGFR2b mAbs.

[0198] Competitive Ab FPA144 HC18 FPA144 98％ 98％ HC18 57％ 94％

[0199] Other Implementations

[0200] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0201] refer to

[0202] 1.Ahn,S.,Lee,J.,Hong,M.,Kim,ST,Park,SH,Choi,MG,Lee,JH,Sohn,TS,Bae,JM,Kim,S.,et al.(2016).FGFR2 in gastric cancer:proteinoverexpression predicts gene amplification and high H-index predicts poorsurvival.Modern pathology:an official journal of the United States andCanadian Academy of Pathology,Inc 29,1095-1103.

[0203] 2.Angal,S.,King,D.J.,Bodmer,M.W.,Turner,A.,Lawson,A.D.,Roberts,G.,Pedley,B.,and Adair,J.R.(1993).A single amino acid substitution abolishes theheterogeneity of chimeric mouse / human(IgG4)antibody.Molecular immunology 30,105-108.

[0204] 3.Beenken,A.,and Mohammadi,M.(2009).The FGF family:biology,pathophysiology and therapy.Nature reviews Drug discovery 8,235-253.

[0205] 4.Catenacci,D.V.T.,Rasco,D.,Lee,J.,Rha,S.Y.,Lee,K.W.,Bang,Y.J.,Bendell,J.,Enzinger,P.,Marina,N.,Xiang,H.,et al.(2020).Phase I Escalation andExpansion Study of Bemarituzumab(FPA144)in Patients With Advanced SolidTumors and FGFR2b-Selected Gastroesophageal Adenocarcinoma.Journal ofclinical oncology:official journal of the American Society of ClinicalOncology 38,2418-2426.

[0206] 5.Grose,R.,and Dickson,C.(2005).Fibroblast growth factor signaling intumorigenesis.Cytokine&growth factor reviews 16,179-186.

[0207] 6.Itoh,N.(2016).FGF10:A multifunctional mesenchymal-epithelialsignaling growth factor in development,health,and disease.Cytokine&growthfactor reviews 28,63-69.

[0208] 7.Katoh,M.,and Katoh,M.(2006).FGF signaling network in thegastrointestinal tract(review).International journal of oncology 29,163-168.

[0209] 8.Korc,M.,and Friesel,R.E.(2009).The role of fibroblast growthfactors in tumor growth.Current cancer drug targets 9,639-651.

[0210] 9.Nagatsuma,A.K.,Aizawa,M.,Kuwata,T.,Doi,T.,Ohtsu,A.,Fujii,H.,andOchiai,A.(2015).Expression profiles of HER2,EGFR,MET and FGFR2 in a largecohort of patients with gastric adenocarcinoma.Gastric cancer:officialjournal of the International Gastric Cancer Association and the JapaneseGastric Cancer Association 18,227-238.

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Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising: a heavy chain complementary 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.

2. The antibody or antigen-binding fragment thereof according to claim 1, which has at least one of the following characteristics: i) The inhibitory effect on the binding of FGF10 to FGFR2b is weak; ii) Undetectable binding affinity to FGFR2c.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 having an amino acid sequence of SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to claim 3, comprising: a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

6.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising 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.

6. The antibody or antigen-binding fragment thereof according to claim 5, comprising: a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

8.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, 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.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a constant region of human IgG, preferably human IgG1.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the constant region comprises one or more modifications that enhance antibody-dependent cellular cytotoxicity (ADCC).

10. The antibody or antigen-binding fragment thereof according to claim 8, which is afucosylated.

11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which 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, a F(Fab')2 fragment, a scFv fragment, a Fv fragment, a Fab' fragment or a domain antibody.

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is capable of inhibiting FGF-induced FGFR2 phosphorylation and cancer cell proliferation.

13. An antibody or antigen-binding fragment thereof that competes for binding to FGFR2b with the antibody or antigen-binding fragment thereof of any one of the preceding claims.

14. The antibody or antigen-binding fragment thereof according to claim 13, which specifically binds to an epitope on FGFR2b, which is the same as the epitope 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 shown in SEQ ID NOs: 7 and 8, respectively.

15. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of the preceding claims. 16 . The nucleic acid according to claim 15 , comprising: 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.

17. An expression vector comprising the nucleic acid according to claim 15 or 16. A host cell comprising the expression vector according to claim 17 .

19. A pharmaceutical composition comprising: (a) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15 or 16, or the expression vector according to claim 17, and (b) a pharmaceutically acceptable carrier.

20. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14 linked to one or more conjugated moieties.

21. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the method comprising culturing the host cell of claim 18 under conditions that allow expression of the antibody or antigen-binding fragment thereof.

22. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20 in the preparation of a medicament for treating a disease or condition associated with abnormal expression of FGFR2b in a subject.

23. A method for inhibiting or reducing FGF-induced tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20.

24. 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 the antibody or antigen-binding fragment thereof of any one of claims 1-14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20.

25. A method for killing tumor cells associated with abnormal expression of FGFR2b and reducing corneal toxicity in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20.

26. A method for treating 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 the antibody or antigen-binding fragment thereof of any one of claims 1-14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20.

27. The use according to claim 22 or the method according to claim 26, wherein the disease or disorder is cancer, and optionally the cancer is characterized by expression or overexpression of FGFR2b.

28. The use of claim 22 or the method of claim 26, wherein the disease or condition is selected from 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 carcinoma and urothelial carcinoma.

29. The use according to claim 22 or the method according to claim 26, wherein the disease or disorder is gastric cancer.

30. The use according to claim 22 or the method according to claim 26, wherein the disease or disorder is FGFR2-positive gastric cancer.

31. The method of any one of claims 23-26, wherein the antibody or antigen-binding fragment thereof of any one of claims 1-14, the nucleic acid of claim 15 or 16, the expression vector of claim 17, the host cell of claim 18, the pharmaceutical composition of claim 19, or the antibody-drug conjugate of claim 20 and at least one additional therapeutic agent are administered sequentially or simultaneously.

32. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

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