Antibody specifically binding to FGFR2b and drug conjugate thereof

Abstract

The present document provides a monoclonal antibody specifically binding to fibroblast growth factor receptor 2b (FGFR2b) and an antigen-binding fragment thereof, and further comprises an antibody drug conjugate (ADC), and a use of the anti-FGFR2b antibody or ADC in cancer treatment. The antibody drug conjugate comprises an anti-FGFR2 antibody or an antigen-binding fragment thereof linked to a cytotoxic agent or another component.

Description

Antibodies specifically binding to FGFR2b and conjugates thereof to drugs TECHNICAL FIELD

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to fibroblast growth factor receptor 2b (FGFR2b) and methods of their use, further comprising antibody-drug conjugates (ADC) of such antibodies and their use in the manufacture of a medicament. BACKGROUND

[0002] Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinases encoded by four structurally related genes (FGFR1 to FGFR4). The FGFR mRNAs undergo multiple alternative splicing, resulting in multiple isoforms. FGFRs share common structural features, including an extracellular ligand-binding portion composed of different Ig-like domains (alpha isoforms contain all three Ig-like domains D1, D2, and D3; beta isoforms contain only two Ig-like domains D2 and D3 domains but not D1), a transmembrane domain, and an intracellular tyrosine kinase catalytic domain. FGFs bind to the receptors primarily through the D2 and D3 regions of the receptor. In FGFR1-FGFR3, all forms contain the first half of D3, with isoforms containing only the first half of D3 denoted as IIIa forms, while the second half of D3 can use two alternative exons, resulting in IIIb and IIIc forms. There are two variants of FGFR2: FGFR2-IIIb and FGFR2-IIIc (denoted simply as FGFR2b and FGFR2c). FGFR-2 IIIb is primarily expressed in normal epithelial cells, as well as in oral mucosa, esophagus, stomach, colorectal, pancreas, lung, breast, endometrial, cervical, and prostate cancers. The IIIc variant of FGFR is expressed in mesenchymal cells, during epithelial-mesenchymal transition (EMT), in colorectal, pancreatic, bladder, cervical, and prostate cancers. FGFR IIIb and IIIc variants bind different forms of FGF and play autocrine and / or paracrine roles in cancer. Recent reports suggest that switching from IIIb to IIIc variants is associated with the invasive nature of cancer through EMT. (Ishiwata T. Front Biosci (Landmark Ed). 2018; 23(4):626-639.).

[0003] FGFs mediate a variety of responses in various cell types, including proliferation, migration and differentiation, particularly during embryonic development (Ornitz et al, J. Biol. Chem. 271:15292, 1996), and in adults, are involved in tissue homeostasis and repair. The signaling pathways mediated by FGFRs are involved in physiological processes such as neovascularization, cell proliferation and migration, regulation of organ development, wound healing, etc.; when FGFRs are mutated or overexpressed, it can cause overactivation of the FGFR signaling pathway and further induce normal cells to become cancerous. Among them, the overactivation of RAS-RAF-MAPK can stimulate cell proliferation and differentiation; the overactivation of PI3K-AKT can inhibit cell apoptosis; SATA is closely related to promoting tumor invasion and metastasis and enhancing the ability of tumor immune escape; the PLCy signaling pathway is an important pathway for regulating tumor cell metastasis.

[0004] FGFR2b is a splice form of FGFR2, and its expression is usually limited to epithelial cells of the stomach and skin tissues. FGFR2b is a newly emerging biomarker in the clinical study of gastric cancer, and it is a new type of target with the dual functions of biological diagnosis and treatment. The main variation of FGFR gene in gastric cancer is the amplification of FGFR2 gene, and the main manifestation at the protein level is the overexpression of FGFR2b subtype. The overexpressed FGFR2b receptor on tumor cells binds with the ligand to form a homodimer, thereby overactivating the downstream signaling pathway and promoting tumor cell proliferation, migration and angiogenesis (Ahn S, Lee J, Hong M, et al. Mod Pathol. 2016; 29(9): 1095-1103.).

[0005] Among the biological agents targeting FGF / FGFR signals, Bemarituzumab, also known as FPA144, is the only antibody drug that can enter the III phase trial for evaluation. It is a humanized anti-FGFR2b antibody that inhibits tumor progression by inhibiting ligand-induced FGFR2b downstream tumor signaling in tumor cells and enhancing the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Bemarituzumab is undergoing a global phase 3 clinical study for patients with advanced gastric cancer overexpressing FGFR2b as a first-line treatment.

[0006] Bemarituzumab treatment has shown good clinical efficacy in patients with gastric cancer and gastroesophageal junction adenocarcinoma, but a considerable proportion of adverse events in the cornea have been observed. It is speculated that strong inhibition of FGFR10 may be the mechanism of action that leads to corneal toxicity. Therefore, there is still an urgent need in the field of tumor treatment to develop safer and more efficient anti-FGFR2b antibodies and their derived therapeutic agents.

[0007] SUMMARY

[0008] The present application provides an antibody or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 2b (FGFR2b), an antibody-drug conjugate (ADC) comprising the same and use of the anti-FGFR2b antibody or ADC thereof in the manufacture of a medicament for treating cancer.

[0009] In one aspect, the present application provides an antibody that specifically binds to FGFR2b, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises:

[0010] (i) HCDR1 consisting of an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence homology thereto;

[0011] (ii) HCDR2 consisting of an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence homology thereto; and

[0012] (iii) HCDR3 consisting of an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 95% sequence homology thereto;

[0013] and the VL comprises:

[0014] (iv) LCDR1 consisting of an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 95% sequence homology thereto;

[0015] (v) LCDR2 consisting of an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 95% sequence homology thereto; and

[0016] (vi) LCDR3 consisting of an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence having at least 95% sequence homology thereto.

[0017] In some embodiments, the antibody provided by the present application further comprises one or more conservative amino acid residue substitutions or modifications, while still maintaining the specific binding affinity to FGFR2b.

[0018] In some embodiments, at least one of the substitutions or modifications is in one or more of the VH or VL sequences. In some embodiments, at least one of the substitutions or modifications is in one or more of the VH or VL sequences, but outside of any CDR sequence.

[0019] In some embodiments, the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7-11, or a homologous sequence thereof having at least 85% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 7-11.

[0020] In some embodiments, the VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 13-17, or a homologous sequence thereof having at least 85% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 13-17.

[0021] In some embodiments, the antibody further comprises a light chain constant region (CL) and an immunoglobulin heavy chain constant region (CH).

[0022] In some embodiments, the antibody provided herein is a monoclonal antibody.

[0023] In some embodiments, the antibody provided herein is a chimeric antibody.

[0024] In some other embodiments, the antibody provided herein is a humanized antibody.

[0025] In some embodiments, the heavy chain constant region is a constant region of human IgG.

[0026] In some embodiments, the heavy chain constant region is a constant region of human IgG1 and the light chain constant region is a constant region of human kappa light chain.

[0027] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 19-23, or a homologous sequence thereof having at least 85% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 19-23.

[0028] In some embodiments, the LC comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 25-29, or a homologous sequence thereof having at least 85% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 25-29.

[0029] In some embodiments, the chimeric antibody that specifically binds to FGFR2b provided herein cross binds to monkey, mouse FGFR2b protein.

[0030] In some embodiments, the antibodies provided herein further comprise one or more amino acid modifications in the constant region thereof, including modifications that enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0031] In some embodiments, the antibodies provided herein have one or more amino acid modifications that reduce fucose content.

[0032] In some embodiments, the antibodies having one or more amino acid modifications that reduce fucose content exhibit enhanced ADCC activity compared to the unmodified antibodies.

[0033] In one aspect, the present application provides an ADCC-enhanced humanized anti-FGFR2b antibody.

[0034] In some embodiments, the ADCC-enhanced humanized anti-FGFR2b antibody is an afucosylated anti-FGFR2b antibody.

[0035] In some embodiments, the afucosylated humanized anti-FGFR2b antibody is produced by expression using a FUT8 knockout host cell line.

[0036] In another aspect, the present application also provides an isolated antibody or antigen binding fragment thereof that competes for binding to FGFR2b with the antibodies described above.

[0037] In another aspect, the present application also provides an antibody-drug conjugate that specifically binds to FGFR2b, comprising an antibody or antigen binding fragment that specifically binds to human FGFR2b as described in the foregoing aspects.

[0038] In some embodiments, the antibody or antigen binding fragment is linked to a cytotoxic drug moiety, with or without a linker.

[0039] In some embodiments, the antibody or antigen binding fragment is linked to a cytotoxic drug moiety via a linker.

[0040] In some embodiments, the linker is MC-VC-PAB.

[0041] In some embodiments, the cytotoxic drug moiety is Exatecan.

[0042] In one aspect, the present application provides an isolated nucleic acid encoding an antibody or antigen binding fragment thereof that specifically binds to FGFR2b as described in the foregoing aspects.

[0043] In one aspect, the present application provides an expression vector comprising the aforementioned isolated nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present application.

[0044] In another aspect, the present application provides a host cell comprising the aforementioned expression vector of the present application.

[0045] In yet another aspect, the present application provides a method of producing the antibody provided by the present application, wherein the method comprises culturing the host cell of the present application under conditions such that the expression vector of the present application is expressed.

[0046] In some embodiments, the method further comprises purifying the antibody produced by the host cell.

[0047] In yet another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or antibody-drug conjugate, of the present application, and a pharmaceutically acceptable carrier.

[0048] In another aspect, the present application provides a method of treating a FGFR2b- associated disease or condition in a subject, the method comprising administering a therapeutically effective amount of the antibody, antibody-drug conjugate, or pharmaceutical composition of the aforementioned aspect of the present application.

[0049] In another aspect, the present application provides use of the antibody or antigen-binding fragment, antibody-drug conjugate, or pharmaceutical composition of the aforementioned aspect of the present application in the manufacture of a medicament for treating a disease or condition in a subject, wherein the disease or condition would benefit from modulation of FGFR2b abnormal overexpression.

[0050] In some embodiments, the disease or condition is cancer, and optionally, the cancer is characterized by expression or overexpression of FGFR2b.

[0051] In another aspect, the present application provides a pharmaceutical composition characterized by the conjoint administration of the antibody or antigen-binding fragment, antibody-drug conjugate, or pharmaceutical composition of the aforementioned aspect of the present application and a second medicament having a second therapeutic effect.

[0052] In some embodiments, the second medicament having a second therapeutic effect is selected from a second antibody that is not FGFR2b specific.

[0053] In some embodiments, the second medicament having a second therapeutic effect is a cytotoxic chemical drug.

[0054] The anti-FGFR2Ib antibody or antigen-binding fragment thereof of the present application has one or more of the following properties:

[0055] a. binds to FGFR2b with significantly higher affinity than to FGFR2c in vitro, and has an affinity for FGFR-IIIc that is low to undetectable;

[0056] b. the chimeric antibody is capable of blocking the binding of FGF7 to the FGFR2b receptor protein on the surface of human gastric cancer tumor cells, and the humanized antibody is capable of blocking the binding of FGF7, FGF10 to the FGFR2b receptor protein;

[0057] c. binds to FGFR2b-positive tumor cells;

[0058] d. has strong endocytosis in tumor cells expressing FGFR2b;

[0059] e. can mediate the down-regulation of FGFR2 phosphorylated protein, and the significant down-regulation of two key phosphorylated proteins ERK1 / 2 and FRS2a in the downstream RAS-MAPK, PI3K-AKT tumor signaling pathways, and has good inhibition effect on the downstream phosphorylation pathways of FGFR2;

[0060] f. induces ADCC effect;

[0061] g. the humanized FGFR2b antibody conjugate (ADC) of the present application has in vitro killing effect on tumor cells, preferably on FGFR2b-positive tumor cells;

[0062] g. can inhibit tumor growth in vitro;

[0063] h. the combination of the antibody with the PD1 antibody has significantly better inhibitory effect on tumor growth and proliferation than the Anti-mPD-1 group and the FGFR2b monoclonal antibody alone, and has a synergistic effect. Advantages:

[0064] The antibody and antigen-binding fragment or ADC of the present application that specifically binds to FGFR2b are expected to be developed into safe and efficient targeted therapeutic drugs for various solid tumors including gastric cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, etc., and provide more diversified choices for the cancer treatment market. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 shows the results of ELISA method for detecting the binding of FGFR2b chimeric antibody 27A6 to FGFR2 alpha (IIIb) protein and FGFR2 another subtype protein FGFR2 alpha (IIIC) of Example 4 (A. binding to FGFR2 alpha (IIIb), B. binding to FGFR2 alpha (IIIC)).

[0066] Figure 2, the results of detecting the binding activity of FGFR2b chimeric antibody 27A6 to target protein positive tumor cells by FACS method are shown (A. strong binding of chimeric antibody 27A6 to FGFR2b protein on KATO3 cells; B. strong binding of chimeric antibody 27A6 to FGFR2b protein on SNU16 tumor cells).

[0067] Figure 3, the results of detecting the blocking activity of FGFR2b chimeric antibody to the binding of FGFR2b protein on tumor cell surface to its ligand FGF7 are shown.

[0068] Figure 4, the results of detecting the endocytosis effect of FGFR2b chimeric antibody 27A6 in two tumor cells with different FGFR2b expression levels are shown (A. endocytosis effect in KATO3 cells; B. endocytosis effect in MFM-223 cells).

[0069] Figure 5, the results of detecting the inhibition of SNU16 tumor cell proliferation by chimeric antibody 27A6 in vitro.

[0070] Figure 6, the results of detecting the binding of chimeric antibody 27A6 to different species of FGFR2b protein are shown (A. the results of detecting the binding to mouse FGFR2 (IIIb) protein; B. the results of detecting the binding to recombinantly expressed monkey FGFR2 (IIIb) protein).

[0071] Figure 7, the results of detecting the binding of 27A6 humanized antibody to FGFR2b positive tumor by FACS method are shown (A. binding to KATO3 cells; B. binding to SUN16 cells).

[0072] Figure 8, the results of detecting the blocking of FGF7\FGF10 binding to FGFR2b receptor protein by 27A6 humanized antibody by ELISA method are shown (A. blocking FGF7 binding; B. blocking FGF10 binding).

[0073] Figure 9, the results of the internalization of 27A6 humanized antibody in tumor cells are shown (A. internalization activity in KATO3 cells; B. internalization activity in SNU16 cells).

[0074] Figure 10, the inhibitory effect of humanized antibody on FGF7-mediated phosphorylation pathway of FGFR2 positive tumor cells is detected in SNU16 tumor cells.

[0075] Figure 11, the killing effect of 27A6 humanized antibody and its antibody inotuzumab ozogamicin conjugate on FGFR2b positive tumor cells is detected (A. killing effect on SNU16; B. killing effect on KATO3).

[0076] Figure 12, 27A6 humanized antibody ADCC activity test results show (A. ADCC effect on KATO3 tumor cells; B. ADCC effect on SUN16 tumor cells).

[0077] Figure 13, Change curve of SNU16 cell subcutaneous transplanted tumor volume after administration of humanized FGFR2b antibody in BALB / c-Nu mouse SNU16 cell subcutaneous transplanted tumor inhibition test.

[0078] Figure 14, Change curve of BALB / c-Nu mouse body weight after administration of humanized FGFR2b antibody in BALB / c-Nu mouse SNU16 cell subcutaneous transplanted tumor inhibition test.

[0079] Figure 15, Results show the change of 4T1 cell subcutaneous transplanted tumor volume after administration of antibodies in the inhibition test of humanized FGFR2b antibody and Anti-mPD-1 combined use on BALB / c mouse 4T1 cell subcutaneous transplanted tumor.

[0080] Figure 16, Results show the change of BALB / c mouse body weight after administration of antibodies in the inhibition test of humanized FGFR2b antibody and Anti-mPD-1 combined use on BALB / c mouse 4T1 cell subcutaneous transplanted tumor.

[0081] Figure 17, Schematic diagram of MC-VC-PAB linker chemical structure

[0082] Figure 18, MC-VC-PAB-Exatecan chemical structure

[0083] Figure 19, Schematic diagram of mammalian expression vector pHR structure DETAILED DESCRIPTION

[0084] Before the application is described, it is to be understood that this application is not limited to particular methods and experimental conditions described, as such methods and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the application will be limited only by the appended claims.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The term "about," as used herein when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0086] The following description of the application is merely intended to illustrate various embodiments of the application. Thus, specific modifications discussed are not to be interpreted as limiting the scope of the application. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present application, and it is understood that such equivalent embodiments are within the scope of the present application, all references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference in their entirety.

[0087] Target Proteins FGFR and FGFR2 Proteins

[0088] In the present application, "FGFR" encompasses any and all fibroblast growth factor receptor family members (FGFR1-FGFR4), and is intended to encompass any form of FGFR, for example: 1) a native unprocessed FGFR molecule, "full-length" FGFR chain, or naturally occurring variants of FGFR, including, for example, allelic variants; 2) any form of FGFR produced by processing in the cell, for example, different splice forms, for example, FGFR1b, FGFR1c, FGFR2a, FGFR2b, FGFR2c, etc.; or 3) fragments of FGFR subunits (e.g., truncated forms, extracellular / transmembrane domains) or modified forms (e.g., mutant forms, glycosylated / pegylation, His-tag / immunofluorescent fusion forms) produced by recombinant methods.

[0089] In the present application, the expression "FGFR2" and the like refers to a tyrosine protein kinase that functions as a cell surface receptor for fibroblast growth factors.

[0090] Unless specified as from a non-human species, for example, "mouse FGFR2," "monkey FGFR2," and the like, generally "FGFR2" means human FGFR2.

[0091] "Cell surface expressed FGFR2" can include or consist of FGFR2 protein expressed on the surface of a cell that normally expresses FGFR2 protein, or a cell engineered to express FGFR2 on its surface. The expression "cell surface expressed FGFR2" means expressing one or more FGFR2 proteins or extracellular domains thereof on the surface of a cell in vitro or in vivo such that at least a portion of the FGFR2 protein is exposed to the extracellular side of the cell membrane and accessible to an antigen binding portion of an antibody.

[0092] In the present application, "FGFR2b" means the isoform IIIb splice form of FGFR2. An exemplary FGFR2b sequence includes the human FGFR2b protein (e.g., FGFR2b as set forth in the amino acid sequence of SEQ ID NO: 24, encoded by the nucleic acid set forth in SEQ ID NO: 30).

[0093] The technical problem to be solved by the present application is to overcome the defects of unsatisfactory tumor killing effect and safety of antibody drugs in the prior art, and to provide an antibody or antigen-binding fragment thereof specifically binding to FGFR2b, an antibody-drug conjugate (ADC) comprising the same, and application of the antibody-drug conjugate in preparation of a drug for preventing and treating a cancer or tumor disease with abnormal expression of FGFR2b.

[0094] Exemplary antibodies capable of specifically binding to FGFR2b

[0095] In the present application, the antibody or antigen-binding fragment thereof specifically binding to FGFR2b or "FGFR2b antibody" refers to an antibody or antigen-binding fragment or molecular complex thereof comprising at least one complementarity determining region (CDR) specifically binding to or interacting with human FGFR2b, wherein the antibody or antigen-binding fragment thereof has very low binding affinity to FGFR2c.

[0096] The "FGFR2b antibody" of the present application comprises a heavy chain variable region (abbreviated as VH in the present application) and a light chain variable region (abbreviated as VL in the present application), and the VH and VL regions can be further subdivided into complementarity determining regions (CDRs) and more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminal to carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0097] The present application adopts hybridoma technology and performs ELISA and FACS binding screening to obtain a murine antibody 27A6 capable of binding to human FGFR2b while not binding to human FGFR2C; a chimeric antibody (27A6CHI) recombinantly expressing 27A6, the FGFR2b-specific chimeric antibody can strongly bind to FGFR2b target-positive tumors, effectively inhibit the binding of ligand FGF7 to FGFR2 receptor, and inhibit tumor cell proliferation in vitro, and the FGFR2b chimeric antibody also has good endocytosis activity, and can be internalized into cells with high efficiency in tumor cells. Further, the chimeric antibody 27A6 is humanized by CDR grafting and back mutation, and a humanized antibody is obtained. Through in vitro and in vivo antibody activity screening, a humanized anti-FGFR2b antibody with good human FGFR2b binding specificity and high affinity, reduced immunogenicity, and good tumor inhibition effect is finally obtained, which has an affinity to FGFR2b equivalent to that of the parent chimeric antibody 27A6. Further, the present application also develops an anti-FGFR2b antibody-drug conjugate (ADC) having a killing effect on FGFR2b-positive tumors.

[0098] In one aspect, the present application provides an antibody (also referred to as an anti-FGFR2b antibody) or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein the HCDR1, HCDR2 and HCDR3, and the LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, and set forth in SEQ ID NOs: 4, 5 and 6, respectively, as shown in Table 1. In some exemplary embodiments of the present application, the murine antibody parent clone that specifically binds to FGFR2b is obtained by hybridoma screening, comprising the HCDRs and LCDRs shown in Table 1.

[0099] Table 1. CDR amino acid sequences of murine clones

[0100] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b comprises a VH / VL amino acid sequence pair comprising the HCDR / LCDR amino acid sequence pairs in Table 1, wherein the HCDR / LCDR amino acid sequence pairs comprise any of the HCDR amino acid sequences listed in Table 1 paired with any of the LCDR amino acid sequences listed in Table 1.

[0101] In related embodiments, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b comprises a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3).

[0102] The CDR amino acid sequences of the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b are defined according to the Kabat convention.

[0103] In some embodiments, the present application provides an exemplary anti-FGFR2b murine clone designated 27A6 comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, of Table 1.

[0104] In the anti-FGFR2b antibody of the present application, the paired VH and VL together form one FGFR2b antigen binding site.

[0105] In some embodiments, the anti-FGFR2b antibody of the present application is selected from at least one of a murine antibody, a chimeric antibody, a human antibody or a humanized antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single chain antibody, a diabody, a triabody, a tetrabody or a single domain antibody, or a nanobody or a single domain antibody.

[0106] Preferably, the anti-FGFR2b antibody of the present application is a monoclonal antibody.

[0107] In some embodiments, the anti-FGFR2b antibody of the present application can be a monoclonal antibody, for example, of murine origin, chimeric or human origin.

[0108] In some embodiments, the anti-FGFR2b antibody of the present application can be a chimeric antibody.

[0109] In some embodiments, the present application provides a chimeric anti-FGFR2b antibody, which is obtained by simultaneously introducing into a host cell an expression vector fusing a heavy chain variable region sequence of a murine antibody and a human IgG1 constant region sequence and a light chain expression vector fusing a light chain variable region sequence of a murine antibody and a human kappa chain constant region sequence.

[0110] In some embodiments, the chimeric anti-FGFR2b antibody of the present application is designated as 27A6CHI.

[0111] In some embodiments, the anti-FGFR2b antibody of the present application can be a humanized antibody.

[0112] In some exemplary embodiments, the CDR sequences provided in Table 1, for example, are derived from the CDR sequences of the murine monoclonal antibody 27A6, which can be grafted into a human antibody framework sequence with high homology by CDR grafting technology. Key amino acid residues that are different between the murine and human 27A6 antibody sequences are subjected to back mutation design, and finally the humanized heavy chain variable region coding sequence and the humanized light chain variable region coding sequence of the present application are obtained, and then the light and heavy chains are combined for vector construction and transfection expression to obtain the anti-FGFR2b humanized antibody of the present application.

[0113] In some embodiments, the FR region of the antibody (or antigen binding portion thereof) provided by the present application can be identical to the human FR region sequence, or can be naturally or artificially modified.

[0114] In some preferred embodiments, the anti-FGFR2b antibody or antigen-binding portion thereof of the present application is humanized, which is a humanization mutation of the encoding nucleic acid of the variable region of the chimeric anti-FGFR2b antibody by CDR grafting and back mutation according to the present application, and further constructing an expression vector comprising the humanized light chain and heavy chain, constructing an expression vector of light and heavy chain combination, transfecting and screening to obtain a humanized anti-FGFR2b antibody with conservative modification of the variable region amino acid sequence relative to the chimeric antibody and reduced immunogenicity, and affinity to human FGFR2b protein, cross-binding to monkey and mouse FGFR2b, and binding level to human FGFR2b comparable to the chimeric antibody.

[0115] In some preferred embodiments, the anti-FGFR2b antibody or antigen-binding portion thereof of the present application comprises VH and VL as defined in the exemplary anti-FGFR2b antibody sequences listed in Table 2, which form a binding site that specifically binds FGFR2b.

[0116] In some embodiments, the present application obtains 4 humanized heavy chain variable region sequences and 4 humanized light chain variable region sequences by CDR grafting and back mutation, as shown in Table 2; and then combines the light and heavy chains to obtain the humanized body of the present application, as shown in Table 3.

[0117] Table 2. VH amino acid sequences and paired VL amino acid sequences of variable region sequences of exemplary chimeric anti-FGFR2b antibodies and humanized anti-FGFR2b antibodies

[0118] Table 3, combination of heavy chain variable region and light chain variable region of exemplary anti-FGFR2b antibodies

[0119] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds FGFR2b comprises a heavy chain variable region (VH) and a light chain variable region (VL) amino acid, wherein the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7-11, as listed in Table 2.

[0120] In some embodiments, the VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 13-17, as listed in Table 2.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds FGFR2b, the VH contains an amino acid sequence as set forth in SEQ ID NO: 7.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VH contains an amino acid sequence as set forth in SEQ ID NO: 8.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VH contains an amino acid sequence as set forth in SEQ ID NO: 9.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VH contains an amino acid sequence as set forth in SEQ ID NO: 10.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VH contains an amino acid sequence as set forth in SEQ ID NO: 11.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VL contains an amino acid sequence as set forth in SEQ ID NO: 13.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VL contains an amino acid sequence as set forth in SEQ ID NO: 14.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VL contains an amino acid sequence as set forth in SEQ ID NO: 15.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VL contains an amino acid sequence as set forth in SEQ ID NO: 16.

[0130] In some embodiments, the antibody or antigen-binding fragment thereof of the present application that specifically binds to FGFR2b, the VL contains an amino acid sequence as set forth in SEQ ID NO: 17.

[0131] In some exemplary embodiments, the anti-FGFR2b antibody of the present application is a chimeric antibody, such as the heavy chain variable region as set forth in SEQ ID NO: 7 and the light chain variable region as set forth in SEQ ID NO: 13, designated as 27A6CHI.

[0132] In some exemplary embodiments, the anti-FGFR2b antibody of the present application is a humanized antibody.

[0133] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 8 and a light chain variable region as set forth in SEQ ID NO: 14, designated as 27A6H0L0.

[0134] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 15, designated as 27A6H2L2.

[0135] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 16, designated as 27A6H2L3.

[0136] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 17, designated as 27A6H2L4.

[0137] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and a light chain variable region as set forth in SEQ ID NO: 15, designated as 27A6H3L2.

[0138] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and a light chain variable region as set forth in SEQ ID NO: 16, designated as 27A6H3L3.

[0139] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and a light chain variable region as set forth in SEQ ID NO: 17, designated as 27A6H3L4.

[0140] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 15, designated as 27A6H4L2.

[0141] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 16, designated as 27A6H4L3.

[0142] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 17, designated as 27A6H4L4.

[0143] In some embodiments, the anti-FGFR2b antibody of the present application further comprises a heavy chain constant region and a light chain constant region, such as a heavy chain constant region and a heavy chain variable region constitute a full-length heavy chain, and a light chain variable region and a light chain constant region constitute a full-length light chain.

[0144] In some embodiments, the heavy chain constant region of the anti-FGFR2b antibody of the present application is an IgG heavy chain constant region, preferably an isotype of human IgG heavy chain constant region.

[0145] In some embodiments, the human IgG heavy chain constant region is selected from one of human IgGl, IgG2, IgG3 or IgG4 antibody heavy chain constant region.

[0146] In some more preferred embodiments, the heavy chain constant region of the anti-FGFR2b antibody of the present application is an IgGl antibody heavy chain constant region of human.

[0147] In some embodiments, the antibody light chain constant region is selected from any one of human antibody kappa and lambda chain light chain constant region.

[0148] In some embodiments, the antibody light chain constant region is a human antibody kappa type light chain constant region.

[0149] In some embodiments, the human IgGl heavy chain constant region of the anti-FGFR2b antibody of the present application has, for example, an amino acid sequence as set forth in SEQ ID NO: 12.

[0150] In some embodiments, the human kappa type light chain constant region of the anti-FGFR2b antibody of the present application has, for example, an amino acid sequence as set forth in SEQ ID NO: 18.

[0151] In one aspect, the present application provides an antibody or antigen-binding fragment of FGFR2b binding specificity, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence of VH as set forth in any one of the preceding SEQ ID NOs: 7-11 and an IgGl humanized heavy chain constant region (CH) as set forth in SEQ ID NO: 12, and the LC comprises an amino acid sequence of VL as set forth in any one of the preceding SEQ ID NOs: 13-17 and a human kappa type light chain constant region (CL) as set forth in SEQ ID NO: 18; wherein the antibody or antigen-binding fragment comprises an antibody of variable region variant that retains FGFR2b binding specificity while no detectable affinity to FGFR2c.

[0152] In one aspect, the present application provides an antibody or antigen-binding fragment specific for FGFR2b binding, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-23, and the LC comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-29. Exemplary HCs and LCs are shown in Table 4, and exemplary HC and LC combinations are shown in Table 5.

[0153] In some embodiments, the antibody of the present application that specifically binds to FGFR2b is a chimeric antibody.

[0154] In some embodiments, in the chimeric antibody, the HC contains an amino acid sequence set forth in SEQ ID NO: 19.

[0155] In some embodiments, in the chimeric antibody, the LC contains an amino acid sequence set forth in SEQ ID NO: 25.

[0156] In some embodiments, the antibody of the present application that specifically binds to FGFR2b is a humanized antibody.

[0157] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the HC contains an amino acid sequence set forth in SEQ ID NO: 20.

[0158] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the HC contains an amino acid sequence set forth in SEQ ID NO: 21.

[0159] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the HC contains an amino acid sequence set forth in SEQ ID NO: 22.

[0160] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the HC contains an amino acid sequence set forth in SEQ ID NO: 23.

[0161] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the LC contains an amino acid sequence set forth in SEQ ID NO: 26.

[0162] In some embodiments, in the humanized antibody of the present application that specifically binds to FGFR2b, or an antigen-binding fragment thereof, the LC contains an amino acid sequence set forth in SEQ ID NO: 27.

[0163] In some embodiments, the LC of the humanized antibody or antigen-binding fragment thereof of the application that specifically binds to FGFR2b contains an amino acid sequence as set forth in SEQ ID NO: 28.

[0164] In some embodiments, the LC of the humanized antibody or antigen-binding fragment thereof of the application that specifically binds to FGFR2b contains an amino acid sequence as set forth in SEQ ID NO: 29.

[0165] Table 4. Heavy chain (HC) and light chain (LC) sequences of exemplary anti-FGFR2b antibodies of the application

[0166] Table 5. Combinations of heavy chain and light chain sequences of exemplary FGFR2b antibodies of the application

[0167] In some exemplary embodiments, the chimeric anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 25, designated as 27A6CHI.

[0168] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 20 and a light chain as set forth in SEQ ID NO: 26, designated as 27A6H0L0.

[0169] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 27, designated as 27A6H2L2.

[0170] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 28, designated as 27A6H2L3.

[0171] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 29, designated as 27A6H2L4.

[0172] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the application comprises a heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 27, designated as 27A6H3L2.

[0173] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 28, designated as 27A6H3L3.

[0174] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 29, designated as 27A6H3L4.

[0175] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 27, designated as 27A6H4L2.

[0176] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 28, designated as 27A6H4L3.

[0177] In some exemplary embodiments, the humanized anti-FGFR2b antibody of the present application comprises a heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 29, designated as 27A6H4L2.

[0178] Functionally conserved variants of homology

[0179] In the light of the exemplary antibodies of the present application, functionally conserved variants of the aforementioned exemplary anti-FGFR2b antibodies and antigen-binding fragments thereof obtained by using the general antibody humanization technology and genetic recombination technology are also part of the present application, wherein the functionally conserved variants comprise substantially similar homologous sequence variants (VHA) of the aforementioned VH and substantially similar homologous sequence variants (VLA) of the aforementioned VL. "Substantially similar", "homologues" and "homology" are interchangeable, referring to nucleic acid sequences (or their complementary strands) or amino acid sequences that share at least 85% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or 99.5%, or 99.9%) sequence identity when optimally aligned. The "percent (%) sequence identity (homology)" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0180] In certain embodiments, the present application provides function-conserved variants of the anti-FGFR2b antibodies, which include heavy chain variable region variants (VHA) and light chain variable region variants (VLA), wherein the VHA and VLA are generated by introducing conservative or non-conservative amino acid substitutions into the VH and VL sequences of one or more exemplary anti-FGFR2b antibodies provided by the present application, respectively, without substantially altering the binding specificity thereof. Any of the variants of the anti-FGFR2b antibodies and antigen-binding fragments thereof discussed in the present application can be "function-conserved variants", or referred to as "conservatively modified variants".

[0181] In some embodiments, the variants of the anti-FGFR2b antibodies or antigen-binding fragments thereof of the present application are variants in which one or more amino acids of the VH or VL are conservatively modified or conservatively substituted. A "conservatively modified variant" or "conservatively substituted variant" refers to a variant in which an amino acid of the VH and / or VL is substituted with another amino acid having similar chemical properties (e.g., charge, size of side chain, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), but which maintains the specificity for human FGFR2b, affinity close to the chimeric antibody or humanized antibody before mutation, or significantly improved. One skilled in the art knows that a single amino acid substitution in a region of the antibody that is not essential will not substantially alter the biological activity. Furthermore, substitution of structurally or functionally similar amino acids is less likely to significantly disrupt the biological activity.

[0182] Conservatively modified variants of the humanized FGFR2b antibodies can be further obtained using known methods, including but not limited to CDR grafting and back mutation, phage display technology. For example, the binding of the antibody to FGFR2b can be virtually simulated using computer software, and the amino acid residues on the antibody that form the binding interface can be identified, which can serve as key sites for substitution to avoid reduction of binding affinity, or as targets for substitution to achieve stronger binding.

[0183] In certain embodiments, the variants of the anti-FGFR2b antibodies provided by the present application can be further modified or substituted with individual amino acids from one or more CDR regions provided in Table 1, and the resulting variants maintain the specificity and binding affinity of the parent antibodies to FGFR2b.

[0184] The CDRs are the fragments of amino acids that cause antigen binding, and it has been found that one or two of the six CDRs can be changed with individual amino acids while the FGFR2b antibody still maintains the ability to specifically bind to FGFR2b; wherein the HCDR3 region is located at the center of the antigen binding site, and the HCDR3 is the most diversified CDR of the antigen binding site so far in terms of length, amino acid composition and conformation.

[0185] The production of humanized anti-FGFR2b antibody variants of the present application is at the nucleic acid level by modifying one or more residues within one or both variable regions (i.e., VH and / or VL) (e.g., in one or more CDR regions and / or one or more FR regions) of a chimeric FGFR2b antibody of the present application, for example, to conserve the framework regions of the humanized antibody by conservative amino acid modification, to obtain variants of the humanized antibody that have improved binding affinity and / or increased similarity to naturally occurring antibodies of certain species.

[0186] In some embodiments, the conservative amino acid modification mutation can be an amino acid substitution, addition, or deletion, but is preferably a substitution.

[0187] In some embodiments, the amino acid mutations that produce function-conserved variants of the anti-FGFR2b antibodies of the present application can be no more than one, two, three, four, or five amino acid residues within an exemplary CDR region are altered.

[0188] In some embodiments, variants of the antigen binding proteins of the present application that specifically bind FGFR2b include heavy chain variants comprising mutations to HCDR1, HCDR2, and HCDR3 as set forth in Table 1, and / or light chain variants comprising mutations to LCDR1, LCDR2, and LCDR3, wherein one or two of such CDRs have one or two conservative amino acid substitution mutations. Conservative amino acid modifications or substitutions can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which the amino acid residue is substituted by another amino acid residue having a side chain of similar charge, size, and / or shape. Families of amino acid residues having similar side chains are known to those of ordinary skill in the art and can be selected from textbooks of biochemistry.

[0189] In some embodiments, function-conserved variants of the antibodies or antigen binding fragments thereof of the present application that specifically bind FGFR2b comprise one or more variant CDRs (e.g., variants of any one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3) as described herein that have at least 95%, or at least 97%, or at least 98%, or at least 99%, or or at least 99.9% sequence identity or similarity to the amino acid sequence set forth in, for example, SEQ ID NO: 1, 2, 3, 4, 5, and / or 6.

[0190] In certain embodiments, the HCDR3 comprises a substantially similar amino acid sequence having at least 95%, or at least 97%, or at least 98%, or at least 99%, or or at least 99.9% sequence identity to the HCDR3 amino acid sequence set forth in SEQ ID NO: 3 as listed in Table 1.

[0191] In certain embodiments, the LCDR3 comprises a substantially similar amino acid sequence having at least 95%, or at least 97%, or at least 98% or at least 99% or or at least 99.9% sequence identity to the LCDR3 amino acid sequence set forth in SEQ ID NO: 6 as listed in Table 1.

[0192] In some embodiments, the function-conserved variants of the antibodies or antigen-binding fragments thereof of the present application that specifically bind to FGFR2b comprise a heavy chain variable region variant (VHA) and a light chain variable region variant (VLA), wherein the CDRs within the variant antigen-binding protein are not mutated per se, and the conservative amino acid mutations are located in the FR regions. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs), and at least one (or all) of the FR sequences or variable region sequences comprise conservative amino acid substitutions.

[0193] In some embodiments, the function-conserved variants of the antibodies and antigen-binding fragments thereof of the present application that specifically bind to FGFR2b comprise a VHA and / or VLA having, for example, 8 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or 1 amino acid substitutions relative to any of the VH, VL amino acid sequences listed in Table 2 of the present application, wherein the resulting function-conserved variants of the antibodies and antigen-binding fragments thereof maintain specific binding activity to FGFR2b.

[0194] In some embodiments, a variety of function-conserved variants of the antibodies or antigen-binding fragments thereof that specifically bind to FGFR2b comprising a heavy chain variable region variant (VHA) and a light chain variable region variant (VLA) can be readily designed by one of ordinary skill in the art of antibodies based on the exemplary VH and VL amino acid sequences of the present application; wherein the VHA has one or more individual FR region amino acid mutations or combinations thereof as compared to the VH, and the VLA has one or more individual FR region amino acid mutations or combinations thereof as compared to the VL.

[0195] In some embodiments, the VHA and VLA comprise one or more conservative amino acid substitutions, insertions, and / or deletions in the framework regions (VH FR and / or VL FR) relative to the heavy chain variable region (VH) and light chain variable region (VL) prior to mutation.

[0196] Preferably, different amino acid residue positions employ different conservative amino acid substitutions.

[0197] "Conservative substitutions" of an amino acid sequence refer to the replacement of an amino acid residue by a different amino acid residue having similar physical and chemical properties. As is known in the art, conservative substitutions generally do not involve a substantial change in the properties of the protein, and thus can maintain the FGFR2b specific binding function of the antibody.

[0198] In some embodiments, variants of the FGFR2b specific binding antibodies of the application can be obtained by those skilled in the art using known methods including, but not limited to, CDR grafting and back mutation techniques. For example, computer software can be used to virtually model the binding of an antibody to FGFR2b and identify amino acid residues on the antibody that form the binding interface, such residues can be targeted for substitution to avoid a decrease in binding affinity or to achieve stronger binding.

[0199] In some embodiments, variants of the antibodies or antigen binding fragments of the application can contain two or more FR region conservative amino acid mutations in any combination within the FR regions, for example, where certain individual amino acid residues of the humanized FR region are back mutated to the corresponding residues of the murine chimeric antibody FR region sequence, while certain other residues that differ from the original murine chimeric antibody FR region sequence are retained. In certain embodiments, only certain amino acid residues are mutated back to the amino acid residues of the original murine FR sequence. In other embodiments, one or more key amino acid positions of the framework (FR) residues are mutated to the corresponding residues of the murine FR region.

[0200] Generally, function-conservative variants of the antibodies or antigen binding fragments provided by the application that contain certain conservative amino acid modifications retain the ability to specifically bind FGFR2b, while once variants of the antibodies or antigen binding fragments containing one or more FR region mutations are obtained, further desirable properties of the anti-FGFR2b antibody can be achieved while retaining FGFR2b binding specificity, such as improved increased binding affinity, improved or enhanced antagonistic or agonistic biological properties as the case can be, reduced immunogenicity, and the like, in a manner generally known in the art for obtaining antibodies or antigen binding fragments thereof. Antibodies or antigen binding fragments thereof obtained in this manner are encompassed within embodiments of the application.

[0201] Variable region modifications are techniques commonly used in the art to mutate amino acid residues within the VH and / or VL regions to improve one or more antibody activities (e.g., affinity, immunogenicity) of the antibody of interest.

[0202] The VHA and VLA of the function-conservative variants of the humanized anti-FGFR2b antibodies of the present application are generated using the sequences of exemplary anti-FGFR2b antibody VH / VL of the present application as starting material, introducing one or two or more conservative amino acid modifications within the variable region (i.e., VH and / or VL) into one or more framework regions (FRs).

[0203] In some embodiments, the function-conservative variants of the antibodies and antigen-binding proteins of the present application that specifically bind FGFR2b include a VHA having at least 85% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5%) amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7-11; in some embodiments, the function-conservative variants of the antibodies and antigen-binding proteins of the present application that specifically bind FGFR2b include a VLA having at least 85% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5%) amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13-17.

[0204] In some embodiments, the function-conservative variants of the antibodies or antigen-binding proteins of the present application that specifically bind FGFR2b can include a variant VHA or VLA resulting from one or more (e.g., 1, 2, 3, 4, 5) amino acid mutations, such as missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions, to a VH or VL comprising an amino acid sequence set forth in the present application. For example, the present application includes function-conservative variants of antigen-binding proteins that include an immunoglobulin heavy chain variable region variant (VHA) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7-11 but having one or more such mutations, and an immunoglobulin light chain variable region variant (VLA) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13-17 but having one or more such mutations. In certain embodiments, the function-conservative variants of the anti-FGFR2b antibodies provided by the present application include one or more conservative amino acid residue substitutions in the FR sequences within one or more VH set forth in any one of SEQ ID NOs: 7-11 or one or more VL set forth in any one of SEQ ID NOs: 13-17.

[0205] In some embodiments, the function-conservative variants of the antibodies or antigen-binding fragments of the present application that specifically bind FGFR2b retain at least 85%, 90%, 95%, or 100% or more of the binding affinity for FGFR2b compared to the parent antibody.

[0206] In some embodiments, one to ten amino acids in total can be substituted, inserted, or deleted in the variable region sequences listed in Table 2.

[0207] In some embodiments, the VHA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0208] In some embodiments, the VHA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0209] In some embodiments, the VHA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.

[0210] In some embodiments, the VHA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0211] In some embodiments, the VHA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0212] In some embodiments, the VLA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13.

[0213] In some embodiments, the VLA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0214] In some embodiments, the VLA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0215] In some embodiments, the VLA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0216] In some embodiments, the VLA contains a substantially similar amino acid sequence having, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0217] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and a VLA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13.

[0218] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; and a VLA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0219] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and a VLA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0220] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and a VLA having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0221] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0222] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0223] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0224] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0225] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0226] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0227] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a VHA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, and a VLA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0228] Modifications of the constant region

[0229] In certain embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof provided by the present application further comprises an immunoglobulin constant region.

[0230] In certain embodiments, the immunoglobulin is an IgG.

[0231] In certain embodiments, the immunoglobulin constant region comprises a heavy chain constant region and a light chain constant region.

[0232] In certain embodiments, the heavy chain constant region is a heavy chain constant region of human IgG isotype.

[0233] In certain embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1 isotype.

[0234] In certain embodiments, the light chain constant region is a kappa or lambda type light chain constant region.

[0235] In certain embodiments, the light chain constant region is a kappa type light chain constant region.

[0236] In certain embodiments, the variant of the anti-FGFR2b antibody of the present application comprises a constant region mutation that is capable of inducing effector functions such as ADCC or CDC.

[0237] In certain embodiments, the constant region is of IgG1 isotype, which is known to induce ADCC.

[0238] In certain embodiments, the anti-FGFR2b antibodies of the application include one or more modifications in the constant region that enhance ADCC.

[0239] Various methods of mutating to enhance ADCC are described in the art and are well known to those skilled in the art.

[0240] In some embodiments, the application can achieve enhanced ADCC activity by engineering the glycosylation level and pattern of the antibody. Various alterations in the glycosylation pattern can enhance the ADCC activity of an antibody by enhancing its binding to Fc receptors on effector cells.

[0241] In certain embodiments, the anti-FGFR2b antibodies provided by the application are glycoengineered. A "glycoengineered" antibody or antigen binding fragment includes reduced levels of glycosylation, altered glycosylation pattern, or both.

[0242] In some exemplary embodiments, the glycoengineered antibodies provided by the application are expressed in a FUT8 knockout mammalian host cell line that expresses a defucosylated humanized FGFR2b antibody. The defucosylated (i.e., reduced fucose content) antibodies of the application exhibit increased binding to FcyRIII and thus higher ADCC activity.

[0243] In some embodiments, the antibodies provided by the application can further include one or more modifications in the constant region that enhance binding to an activating Fc receptor, and / or enhance ADCC.

[0244] One skilled in the art can also obtain humanized specific binding FGFR2b antibodies with enhanced ADCC effect by engineering the Fc region for binding to FcyR.

[0245] In some embodiments, the glycoengineering of the specific binding FGFR2b antibodies of the application includes any manipulation of the peptide backbone (e.g., modification of the amino acid sequence and / or side chain groups of individual amino acids) and / or manipulation of post-translational modifications by the host cell line (e.g., modification of the glycosylation pattern).

[0246] Heavy chain variants and light chain variants

[0247] In some embodiments, the function-conservative variants of the specific binding FGFR2b antibodies or antigen binding fragments thereof of the application include heavy chain variants (HCA) and light chain variants (LCA).

[0248] In some embodiments, the present application provides a function-conservative variant of an antibody or antigen-binding fragment thereof that specifically binds FGFR2b, comprising a heavy chain variant (HCA) comprising a substantially similar amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 19-23 as set forth in Table 4, and a light chain variant (LCA) comprising a substantially similar amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 25-29 as set forth in Table 4.

[0249] In some embodiments, the HCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

[0250] In some embodiments, the HCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20.

[0251] In some embodiments, the HCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21.

[0252] In some embodiments, the HCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22.

[0253] In some embodiments, the HCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.

[0254] In some embodiments, the LCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0255] In some embodiments, the LCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0256] In some embodiments, the LCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0257] In some embodiments, the LCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0258] In some embodiments, the LCA contains a substantially similar amino acid sequence having at least 85%, at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29.

[0259] In certain embodiments, a variant of an antibody or antigen-binding fragment thereof of the present application that specifically binds FGFR2b comprises a (HCA / LCA) amino acid sequence pair, the HCA and LCA amino acid sequence pair comprising the FGFR2b binding site.

[0260] In some embodiments, a variant of an anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0261] In some embodiments, a variant of an anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

[0262] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0263] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0264] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29.

[0265] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0266] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0267] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29.

[0268] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0269] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0270] In some embodiments, the variant of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present application comprises a HCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a LCA of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29.

[0271] Antigen binding fragment or variant thereof

[0272] In some embodiments, the present application also provides antigen-binding fragments that can specifically bind FGFR2b. Based on the foregoing, antigen-binding fragments of the anti-FGFR2b antibodies, or function-conservative variants thereof, can be further developed in the art to contain various types of antigen-binding fragment variants with modifications or substitutions, including, for example, HCDRs and / or LCDRs as set forth in any of SEQ ID NOs: 1-3 or 4-6 of Table 1, or VHs and / or VLs as set forth in any of SEQ ID NOs: 7-11 or 13-23 of Table 2.

[0273] Other techniques for making antibody fragments will be apparent to those skilled in the art of antibody technology.

[0274] Non-limiting examples of antigen-binding fragments that can be prepared by those skilled in the art in light of the present disclosure that specifically bind FGFR2b include, but are not limited to: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide; and other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies.

[0275] Antigen-binding fragments of antibodies typically comprise at least one variable domain of a VH and a VL.

[0276] In some embodiments, the VH and VL of the application that specifically bind FGFR2b can be positioned relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0277] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain.

[0278] In some embodiments, the variable and constant domains of the present application that specifically bind FGFR2b can comprise any of the configurations listed above, including any of the exemplary configurations listed above, wherein the variable and constant domains can be directly linked to one another or can be linked by an intact or partial hinge or linker region. In addition, the antigen binding fragments can comprise any of the variable and constant domains listed above assembled into a homodimeric or heterodimeric (or other multimeric) configuration non-covalently associated with one another and / or non-covalently associated with one or more monomers, e.g., assembled via disulfide bonds.

[0279] In some embodiments, "antigen binding portions," "antigen binding fragments" of antibodies, and the like, can also include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind FGFR2b to form a complex.

[0280] In some embodiments, the preparation of antigen binding fragments of the anti-FGFR2b antibodies of the present application can be produced using any suitable standard techniques, e.g., constructing DNA comprising a DNA encoding the variable domain and optionally the constant domain of an antibody, from which the aforementioned antigen binding fragments derived from the complete antibody molecule can be expressed by recombinant genetic engineering techniques.

[0281] Encoding nucleic acids

[0282] In a third aspect, the present application also provides an isolated nucleic acid encoding a heavy chain variable region and / or a light chain variable region of the aforementioned antibody or antigen binding fragment thereof that specifically binds FGFR2b, or a functional conservative variant thereof.

[0283] In some embodiments, the present application provides a nucleic acid molecule encoding any of the VH amino acid sequences and / or VL amino acid sequences of Table 2, or a nucleic acid variant molecule thereof that has one to more, preferably one to ten, more preferably one to six, more preferably one to three, most preferably one, silent codon mutations.

[0284] In some embodiments, the isolated nucleic acid comprises a nucleic acid molecule encoding a VH or a variant thereof VHA and / or a VL or a variant thereof VLA, wherein Table 6 comprises the nucleotide sequences of nucleic acid molecules encoding any of the exemplary VH and / or VL amino acid sequences listed in Table 2. In certain embodiments, the isolated nucleic acid molecule comprises a polynucleotide sequence encoding a nucleic acid molecule selected from any of the VHs listed in Table 2, or a substantially similar polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, or a polynucleotide sequence encoding a nucleic acid molecule selected from any of the VLs listed in Table 2, or a substantially similar polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect, the isolated nucleic acid molecule encodes a VH and / or VL of Table 3. Exemplary nucleotide sequences of the encoding nucleic acid molecules are shown in Table 6.

[0285] Table 6. Encoding nucleic acids of variable regions of exemplary anti-FGFR2b antibodies

[0286] In some embodiments, the encoding nucleic acid of a variant of the heavy chain variable region (VHA) of the FGFR2b antibody is a substantially similar variant polynucleotide sequence having at least about 85-99.9% (e.g., at least about 85, at least about 90, at least about 95, at least about 97, at least about 98, at least about 99, at least about 99.5, or at least about 99.9%) sequence identity to the reference nucleotide sequence listed herein, e.g., the encoding nucleotide sequence of the heavy chain variable region VH, such as SEQ ID NO: 32, 33, 34, or 35; or the encoding nucleic acid of a variant of the light chain variable region (VLA) is a substantially similar variant polynucleotide sequence having at least about 85-99.9% (e.g., at least about 85, at least about 90, at least about 95, at least about 97, at least about 98, at least about 99, at least about 99.5, or at least about 99.9%) sequence identity to the reference nucleotide sequence listed herein, e.g., the encoding nucleotide sequence of the light chain variable region VL, such as 37, 38, 39, or 40.

[0287] In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding any of the HC amino acid sequences listed in Table 4, or an amino acid sequence of a substantially similar variant heavy chain (HCA) having at least about 85, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and further comprises a nucleic acid molecule providing an amino acid sequence of any of the LC amino acid sequences listed in Table 4, or an amino acid sequence of a substantially similar variant light chain (LCA) having at least about 85, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0288] "Substantial identity" or "substantially identical" means, when referring to nucleic acids or fragments thereof, having at least about 85%, at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% sequence identity with another nucleic acid (or its complementary strand). In certain instances, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having the same or substantially similar amino acid sequence to the polypeptide encoded by the reference nucleic acid molecule.

[0289] The encoding nucleic acids of the present application can be chemically synthesized, or produced by using molecular biology techniques, e.g., arranging one or more variable and / or constant domains into a suitable fragment arrangement configuration of a peptide chain, encoding nucleotide sequences, or introducing codons, creating cysteine residues, modifying, adding or deleting amino acids, etc.; and / or readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage antibody libraries), or can be synthesized.

[0290] In some embodiments, polynucleotide sequences also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0291] In some embodiments, degenerate codon substitutions can be achieved by generating sequences with, for example, a mixed base or deoxyinosine residue in a position where the third base of one or more selected (or all) codons is variable.

[0292] Vectors, host cells, and methods of making

[0293] In one aspect, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising an encoding nucleic acid expressing an antibody or antigen binding fragment thereof or a variant thereof that specifically binds to FGFR2b, which contains a nucleic acid sequence of the present application encoding the antibody, at least one promoter operably linked to the nucleic acid sequence, and at least one selection marker.

[0294] The isolated polynucleotides of the present application encoding the heavy and light chains of the anti-FGFR2b antibody (e.g., comprising the sequences as shown in Table 6) can be inserted into vectors using recombinant techniques known in the art for further cloning (amplification of the DNA) or for expression of the antibody. Numerous vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence. The vector can also include material to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coat.

[0295] The vector type can include, but is not limited to, plasmid, phagemid, cosmid, and artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), and animal virus.

[0296] In some embodiments, the vector is an expression vector.

[0297] In some exemplary embodiments, the expression vector is a pHr expression vector.

[0298] In some exemplary embodiments, the structure of the pHr expression vector is shown in FIG. 19.

[0299] The expression vector of the present application comprising a polynucleotide sequence encoding the aforementioned antibody or antigen-binding fragment specifically binding to FGFR2b can be introduced into a host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA of the vector provided by the present application is a prokaryote, yeast, or higher eukaryotic cell.

[0300] In some embodiments, the mammalian cell includes, but is not limited to, CHO cell, BHK cell, or NS0 cell.

[0301] In some preferred embodiments, the host cell is a cultured mammalian cell, such as a CHO cell.

[0302] In some most preferred embodiments, the CHO cell is a cultured mammalian cell, such as an Expi CHO-S cell.

[0303] In some embodiments, the host cell is capable of producing glycoengineered antibodies. For example, the host cell line can provide the desired glycosylation machinery during post-translational modification. Examples of such host cell lines include, but are not limited to, cell lines with altered (increased or decreased) activity of glycosylation-related enzymes such as fucosyltransferases (e.g., a-1,6-fucosyltransferase gene (FUT8), (l,3) fucosyltransferase), GDP-fucose transporter (GFT), which can be natural or obtained by genetic engineering.

[0304] In some embodiments, the host cell is a FUT8 gene knockout CHO cell lacking functional FUT8. The FUT8 gene knockout host cell line is fucosylation-deficient and produces afucosylated antibodies.

[0305] The present application transfects a FUT8 gene knockout CHO host cell lacking functional FUT8 with the above-described expression or cloning vector to produce an anti-FGFR2b antibody and is cultured in a modified conventional nutrient medium appropriate for inducing the promoter, selecting transformants, or amplifying the genes encoding the desired sequences.

[0306] The host cells for production of the antibodies provided by the present application can be cultured in a variety of media.

[0307] In some embodiments, hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphate salts), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds that are normally present at final concentrations in the micromolar range), and glucose or equivalent energy source can be supplemented as needed. Any other necessary supplements known to those of ordinary skill in the art can also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0308] In some embodiments, the present application for producing an FGFR2b antibody, includes culturing a FUT8 gene knockout CHO host cell lacking functional FUT8 transfected with the above-described expression or cloning vector in OptiPRO SFM Complex Medium. TM SFM Complex Medium.

[0309] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the first step in purification is to disrupt the cells and remove cellular debris, for example, by centrifugation or ultrafiltration including but not limited to ultrafiltration units. Concentration of the anti-FGFR2b antibody produced by the cells can be purified using, for example, affinity chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, etc., with affinity chromatography being the preferred purification technique.

[0310] In some preferred embodiments, the present application uses Protein A immobilized on a solid phase for immunoaffinity purification of anti-FGFR2b antibodies and antigen-binding fragments thereof. The suitability of Protein A as an affinity ligand depends on the class and isotype of any immunoglobulin Fc domain present in the antibody. The matrix to which the affinity ligand is attached is usually agarose, but other matrices can be used. Following any preliminary purification step(s), the mixture including the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, preferably at low salt concentrations (e.g., about 0 to 0.25 M salt).

[0311] Biological functions of FGFR2b antibodies

[0312] The anti-FGFR2b antibodies or antigen-binding proteins of the present application (or anti-FGFR2b antibodies) or variants thereof can have one or more of the above-mentioned biological properties or any combination thereof. The biological properties of the anti-FGFR2b antibodies described below are not exhaustive. Other biological properties of the antibodies provided by the present application will be apparent to those of ordinary skill in the art in light of the disclosure, including the working examples of the present application.

[0313] In some embodiments, the anti-FGFR2b antibodies of the present application are chimeric antibodies.

[0314] In some embodiments, the chimeric anti-FGFR2b antibodies of the present application have strong binding activity to FGFR2b protein compared to the reference antibody FPA144, and do not bind FGFR2c (e.g., no detectable binding activity).

[0315] In some embodiments, the chimeric anti-FGFR2b antibodies of the present application have an in vitro FGFR2b protein binding activity ELISA binding EC50 of no more than 30 ng / mL, or no more than 20 ng / mL, or no more than 10 ng / mL, or no more than 8 ng / mL, no more than 6 ng / mL, or no more than 5.6 ng / mL, as measured by ELISA.

[0316] In the present application, "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen.

[0317] In some embodiments, the chimeric FGFR2b antibodies of the present application are capable of binding to recombinantly expressed monkey FGFR2b protein, with an EC50 value close to that of the reference antibody FPA144.

[0318] In some embodiments, the chimeric FGFR2b antibody of the present application is capable of binding to recombinantly expressed mouse FGFR2b protein with a stronger binding ability than the reference antibody FPA144, with an EC50 value that is reduced by at least 6-fold compared to the EC50 of the latter.

[0319] In some embodiments, the chimeric FGFR2b antibody of the present application binds to recombinantly expressed mouse FGFR2b protein with an EC50 value that is reduced by at least 6-fold compared to the ELISA binding EC50 of the reference antibody FPA144.

[0320] In some embodiments, the chimeric anti-FGFR2b antibody or antigen binding fragment thereof or function-conservative variant thereof provided by the present application is capable of binding to FGFR2b positive tumor cells.

[0321] In some embodiments, the FGFR2b positive tumor cells to which the chimeric anti-FGFR2b antibody or antigen binding fragment thereof or function-conservative variant thereof provided by the present application binds are gastric cancer cells.

[0322] In some embodiments, the FACS binding EC50 of the FGFR2b chimeric antibody to FGFR2b positive tumor cells is no higher than 1000 ng / mL, or no higher than 800 ng / mL, or no lower than 600 ng / mL, or no higher than 400 ng / mL, or no higher than 350 ng / mL, or no lower than 300 ng / mL, or no higher than 260 ng / mL, or no higher than 220 ng / mL, or no lower than 180 ng / mL, or no higher than 150 ng / mL, or no higher than 120 ng / mL, or no higher than 100 ng / mL, or no higher than 80 ng / mL, or no higher than 50 ng / mL, as determined by FACS.

[0323] In some embodiments, the chimeric FGFR2b antibody of the present application is capable of competing with FGF7 for binding to FGFR2b, effectively blocking the binding of FGF7 to FGFR2b receptor protein on the surface of FGFR2b positive human tumor cells.

[0324] In the present application, "competes for binding" or "inhibits" or "blocks" 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-FGFR) to any detectable extent (e.g., inhibits by at least 85%, or at least 90%, or at least 95%).

[0325] In some embodiments, the FGFR2b chimeric antibody detected by FACS assay has a FACS blocking IC50 of no more than 10000 ng / mL, or no more than 8000 ng / mL, or no more than 7000 ng / mL, or no more than 6000 ng / mL, or no more than 5500 ng / mL, or no more than 5000 ng / mL, or no more than 4800 ng / mL, or no more than 4500 ng / mL, or no more than 4400 ng / mL, or no more than 4300 ng / mL, for blocking the binding of FGF7 to FGFR2b receptor protein on the surface of FGFR2b positive human tumor cells.

[0326] In some embodiments, the FGFR2b chimeric antibody detected by FACS assay has a FACS blocking IC50 of no more than 5000 ng / mL, or no more than 4800 ng / mL, or no more than 4500 ng / mL, or no more than 4400 ng / mL, or no more than 4300 ng / mL, for blocking the binding of FGF7 to FGFR2b receptor protein on the surface of FGFR2b positive human tumor cells.

[0327] In some embodiments, the FGFR2b chimeric antibody of the present application is capable of being effectively internalized into the cell in FGFR2b positive tumor cells, and has a high endocytosis level.

[0328] It is known in the art that the recombinant DT3C protein can inhibit the protein translation mechanism in the cell to induce cell death, and can bind to IgG antibodies of different species.

[0329] In some embodiments, the conjugate of the FGFR2b chimeric antibody of the present application and DT3C is used as a tool for evaluating the internalization efficiency of the chimeric antibody on two tumor cells, and the internalization efficiency is evaluated by detecting the degree of cell death induced thereby.

[0330] In some embodiments, the FGFR2b chimeric antibody of the present application is capable of effectively inhibiting the proliferation of tumor cells driven by the FGG7 / FGFR2b signaling pathway, and has an activity of inhibiting tumor growth in vitro comparable to that of the reference antibody.

[0331] In some embodiments, the FGFR2b chimeric antibody of the present application can cause a decrease in the live cell fluorescence signal of tumor cells at a concentration level of 3-100 μg / ml in the presence of the FGF7 ligand.

[0332] In some embodiments, the FGFR2b chimeric antibody of the present application can cause a decrease in the live cell fluorescence signal of tumor cells at a concentration level of 3-80 μg / ml in the presence of the FGF7 ligand.

[0333] In some embodiments, the chimeric FGFR2b antibodies of the application can cause a decrease in live cell fluorescent signal of tumor cells at a concentration level of 3-50 μg / ml in the presence of FGF7 ligand.

[0334] In some embodiments, the chimeric FGFR2b antibodies of the application can cause a decrease in live cell fluorescent signal of tumor cells at a concentration level of 8 μg / ml in the presence of FGF7 ligand.

[0335] In some embodiments, the chimeric FGFR2b antibodies of the application can cause a decrease in live cell fluorescent signal of tumor cells at a concentration level of 20 μg / ml in the presence of FGF7 ligand.

[0336] In some embodiments, the chimeric FGFR2b antibodies of the application can cause a decrease in live cell fluorescent signal of tumor cells at a concentration level of 50 μg / ml in the presence of FGF7 ligand.

[0337] In some embodiments, the antibodies of the application that specifically bind FGFR2b are humanized antibodies.

[0338] In some embodiments, the humanized antibodies of the application that specifically bind FGFR2b have high affinity for the human FGFR2b antigen.

[0339] The binding affinity of the antibodies and antigen-binding fragments provided herein can be expressed by the KD(M) value, which represents the ratio of the dissociation rate to the association rate (koff / kon) when the binding between the antigen and the antigen-binding molecule (e.g., antibody and antigen-binding fragment) reaches equilibrium. The antigen-binding affinity (e.g., KD) can be determined using suitable methods known in the art, including, for example, employing Bio layer interferometry (BLI) to determine the affinity of FGFR2 humanized antibodies to bind to human FGFR2 IIIb-mFc protein using the method described in Example 11. After obtaining the sensorgram data, the association constant (ka) and dissociation constant (kd) are analyzed using Octet BLI Analysis software to fit the ideal binding dissociation curve, and the equilibrium dissociation constant KD (Kd / Ka) between the antibody and antigen is calculated.

[0340] In some embodiments, the chimeric anti-FGFR2b antibodies of the application have an affinity KD(M) for FGFR2 IIIb-mFc no higher than 2.82E-10.

[0341] In some embodiments, the humanized anti-FGFR2b antibody of the present application has an affinity KD(M) for FGFR2 IIIb-mFc (amino acid sequence as set forth in SEQ ID NO: 41, encoding nucleic acid sequence as set forth in SEQ ID NO: 42) of no more than 4.20E-9, or no more than 3.60E-9, or no more than 3.32E-9, or no more than 2.92E-9, or no more than 2.60E-9, or no more than 2.40E-9, or no more than 2.22E-9, or no more than 810E-10, or no more than 6.10E-10, or no more than 4.85E-10, or no more than 4.20E-10, or no more than 3.92E-10, or no more than 3.85E-10, or no more than 3.60E-10, or no more than 3.32E-10, or no more than 2.98E-10, or no more than 2.72E-10.

[0342] In some embodiments, the chimeric anti-FGFR2b antibody of the present application has an affinity KD(M) for FGFR2 IIIb-mFc (amino acid sequence as set forth in SEQ ID NO: 41, encoding nucleic acid sequence as set forth in SEQ ID NO: 42) comparable to the aforementioned chimeric FGFR2b antibody.

[0343] In some embodiments, the humanized FGFR2b antibody of the present application has an activity of binding to FGFR2b positive tumor cells.

[0344] In some embodiments, the humanized FGFR2b antibody of the present application has an activity of binding to FGFR2b positive gastric cancer tumor cells.

[0345] In some embodiments, the humanized FGFR2b antibody of the present application has a FACS binding EC50 of no more than 6000 ng / mL, or no more than 5000 ng / mL, or no more than 2000 ng / mL, or no more than 800 ng / mL, or no more than 550 ng / mL, or no more than 470 ng / mL, or no more than 350 ng / mL, or no more than 260 ng / mL, or no more than 220 ng / mL, or no more than 150 ng / mL, in FACS assay for detecting the binding of the humanized FGFR2b antibody to FGFR2b positive tumor cells.

[0346] In some embodiments, the humanized FGFR2b antibody of the present application has a FACS binding EC50 of no more than 3000 ng / mL, or no more than 2000 ng / mL, or no more than 1000 ng / mL, or no more than 500 ng / mL, or no more than 300 ng / mL, or no more than 200 ng / mL, in FACS assay for detecting the binding of the humanized FGFR2b antibody to FGFR2b positive tumor cells.

[0347] In some embodiments, the humanized FGFR2b antibody of the present application is capable of effectively inhibiting the binding of FGF7 and FGF10 to FGFR2b receptor protein, and blocking the tumor cell proliferation driven by FGF10 / FGFR2b signaling pathway.

[0348] In some embodiments, the humanized FGFR2b antibody of the present application has an in vitro tumor growth inhibition activity comparable to that of the parent chimeric antibody.

[0349] In some embodiments, the humanized FGFR2b antibody of the present application has a blocking ability of FGF7 binding close to that of the parent chimeric antibody.

[0350] In some embodiments, the humanized FGFR2b antibody of the present application has a blocking ability of FGF10 binding close to that of the parent chimeric antibody.

[0351] In some embodiments, the humanized FGFR2b antibody of the present application has endocytosis activity in FGFR2b positive tumor cells.

[0352] In some embodiments, the endocytosis efficiency of the humanized FGFR2b antibody of the present application is positively correlated with the tumor expression abundance of FGFR2b.

[0353] In some embodiments, the endocytosis efficiency of the humanized FGFR2b antibody of the present application is stronger than that of the reference antibody FPA14.

[0354] In some embodiments, the endocytosis efficiency of the humanized FGFR2b antibody of the present application is positively correlated with the tumor expression abundance of FGFR2b.

[0355] In some embodiments, the chimeric antibody and the humanized antibody of the present application can both mediate the down-regulation of FGFR2 phosphorylated protein, and significantly down-regulate two key phosphorylated proteins ERK1 / 2 and FRS2a of the downstream RAS-MAPK and PI3K-AKT tumor signaling pathways.

[0356] In some embodiments, the chimeric antibody and the humanized antibody of the present application have good inhibitory effect on the downstream phosphorylation pathway of FGFR2, and the inhibitory ability is comparable to that of the control antibody FPA144.

[0357] In some embodiments, the humanized FGFR2b antibody of the present application can specifically bind to the target cell surface antigen FGFR2b, induce ADCC effect, and the ADCC activity is comparable to that of the reference antibody FPA144-AF.

[0358] In some embodiments, the humanized FGFR2b antibody of the present application has inhibitory effect on the growth and proliferation of FGFR2b positive tumors.

[0359] In some embodiments, the humanized FGFR2b antibody of the present application has a significantly better inhibitory effect on the growth and proliferation of FGFR2b-positive tumors than the control FPA144-AF.

[0360] In some embodiments, the humanized FGFR2b antibody of the present application has an inhibitory effect on gastric cancer tumors at a concentration of 6-18 mg / mL, preferably 8-15 mg / mL, and more preferably 10-12 mg / mL.

[0361] In some embodiments, the humanized FGFR2b antibody of the present application has an inhibitory effect on tumors, which is evaluated by tumor volume (Tv = a * b 2 / 2, a represents the long diameter, and b represents the short diameter) and tumor growth inhibition rate (TGI, TGI = (1-RTV 实验组 / RTV 对 照组 ).

[0362] In some embodiments, the humanized FGFR2b antibody of the present application has an inhibitory effect on subcutaneous transplanted tumors of mouse 4T1 cells in combination with Anti-mPD-1.

[0363] In some embodiments, the inhibitory effect of the combination of the humanized FGFR2b antibody and Anti-mPD-1 on tumors is better than that of Anti-mPD-1 alone and the humanized FGFR2b antibody alone, which is evaluated by TGI as the pharmacodynamic evaluation index and the Chou-Talalay formula (q = E combination / (EA drug + EB drug - EA drug * EB drug), q > 1.15 indicates synergistic effect).

[0364] In some embodiments, the humanized FGFR2b antibody of the present application has an inhibitory TGI of 3%-40% on tumors.

[0365] Antibody drug conjugate (ADC)

[0366] In a fourth aspect, the present application provides a humanized antibody-drug conjugate (humanized anti-FGFR2b-ADC) that specifically binds to FGFR2b, wherein the humanized antibody-drug conjugate comprises the humanized antibody or antigen-binding fragment that specifically binds to human FGFR2b according to the preceding aspect.

[0367] In some embodiments, the humanized anti-FGFR2b antibody or antigen-binding fragment is connected to the therapeutic drug through a linker or without a linker.

[0368] In some embodiments, the humanized anti-FGFR2b antibody or antigen-binding fragment is connected to the therapeutic drug through a linker.

[0369] In some embodiments, the therapeutic drug comprises a cytotoxic agent (i.e., a cytotoxin), a chemotherapeutic drug, or a radioisotope.

[0370] In some embodiments, the cytotoxic agent comprises any agent that is detrimental to the growth, viability, or proliferation of a cell.

[0371] In some embodiments, the humanized anti-FGFR2b-ADCs of the present application can be used to deliver cytotoxic agents locally, e.g., for the treatment of cancer, and the humanized anti-FGFR2b-ADCs of the present application can target the delivery of cytotoxic agents to tumors and intracellular accumulation therein, and thus are particularly useful in situations where systemic administration of these unconjugated cytotoxic agents can cause unacceptable levels of toxicity to normal cells as well as to the tumor cells one wishes to eliminate.

[0372] In some preferred embodiments, the cytotoxic agent is a chemotherapeutic agent, such as a growth inhibitory agent, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anticancer drug, a toxin, or a highly reactive radioisotope.

[0373] In some preferred embodiments, the cytotoxic agent can also be a small molecule toxin and a chemotherapeutic drug.

[0374] In some embodiments, the small molecule toxin in the antibody-drug conjugate (ADC) of the present application includes, but is not limited to, a tubulin inhibitor, a DNA damaging agent, a maytansinoid, or a topoisomerase I inhibitor, an auristatin, a hemi-streptol, vinblastine, vincristine, pyrrolobenzodiazepine, paclitaxel, docetaxel, a cryptophycin.

[0375] In some embodiments, the cytotoxic agent includes, but is not limited to, a tubulysin, a camptothecin analog, a maytansinoid, an auristatin.

[0376] In some embodiments, the cytotoxic payload is a topoisomerase I inhibitor.

[0377] In some embodiments, the cytotoxic agent is a camptothecin or a camptothecin analog.

[0378] In some embodiments, the camptothecin analog includes, but is not limited to, irinotecan, exatecan, topotecan, or Dxd or a derivative thereof, 9-aminocamptothecin.

[0379] In some embodiments, the cytotoxic agent is Dxd or a derivative thereof.

[0380] In some embodiments, the cytotoxic agent is Exatecan.

[0381] According to certain embodiments, the cytotoxic agent conjugated to the anti-FGFR2b antibody of the present application is a maytansinoid, such as DM1 or DM4, a distamycin derivative.

[0382] According to certain embodiments, the cytotoxic agent conjugated to the anti-FGFR2b antibody of the present application is an auristatin, such as MMAE, MMAF or a derivative thereof.

[0383] In some embodiments, the cytotoxic agent conjugated to the anti-FGFR2b antibody of the present application includes, but is not limited to, macromolecular bacterial and plant toxins, such as diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, abrin, modeccin, alpha-sarcin, crotin, pseudomonas exotoxin, botulinum toxin, bryodin, saporin, pokeweed antiviral protein. Such macromolecular toxins can be conjugated to the anti-FGFR2b antibody provided herein using methods known in the art.

[0384] In certain embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof in the ADC provided herein is conjugated to a cytotoxic agent (e.g., any of the cytotoxic agents disclosed above) via a linker (also referred to as a linking group). The linker is any group or moiety that links or bonds the antibody or antigen-binding protein described herein to a therapeutic moiety, such as a cytotoxic agent. Suitable linkers can be found, for example, in antibody-drug conjugates and immunotoxins.

[0385] Suitable binding agents or linkers (i.e., linking groups) for the antibody conjugates described herein are sufficiently stable to exploit the circulating half-life of the antibody and at the same time capable of releasing its payload upon antigen binding and / or antigen-mediated internalization of the conjugate. Any linker molecule or linker technology known in the art can be used to generate or construct the ADCs of the present application.

[0386] In certain embodiments, the linker is one of a cleavable linker and a non-cleavable linker.

[0387] In some embodiments, the linker is a cleavable linker.

[0388] In certain embodiments, the linker is stable under physiological conditions.

[0389] In certain embodiments, the linker is cleavable under a particular physiological environment, thereby facilitating the release of the cytotoxic agent in the cell. For example, the linker can be an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker, or a disulfide-containing linker.

[0390] In some embodiments, the linker comprises an enzyme cleavable moiety. Exemplary enzyme cleavable moieties include, but are not limited to, a peptide bond, an ester bond, a hydrazone, and a disulfide bond.

[0391] In some embodiments, the linker that can conjugate the antibody of the application to a therapeutic drug moiety includes, but is not limited to, a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide-pentapeptide linker, a glucuronide linker, or a thioether linker.

[0392] In some embodiments, the bifunctional linker includes, but is not limited to, for example, PAB (p-aminobenzyloxy carbonyl) or its derivatives p-aminobenzyloxy carbonyl (PABC), MC (6-maleimidocaproyl), MP (maleimidopropanoyl), SPP (N-succinimidyl 4-(2-pyridinylthio) pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate), SIAB (N-succinimidyl (4-iodoacetyl) aminobenzoate), bis-azido compounds (such as bis(p-azidobenzamido) hexanediamine), bis-diazo derivatives (such as bis-(p-diazo benzoyl)-ethylenediamine), diisocyanates (such as 2,6-diisocyanatotoluene), and bis-active fluorides (such as 1,5-difluoro-2,4-dinitrobenzene), and variants and combinations thereof.

[0393] In certain embodiments, the linker is a peptide linker in which one or more side chains of the amino acids are linked to a side chain group, as described below. In some embodiments, the peptide linker comprises lysine, valine, and citrulline.

[0394] In some embodiments, the peptide linker comprises lysine, valine, and alanine.

[0395] In some embodiments, the linker comprises valine and alanine.

[0396] In some embodiments, the peptide linker comprises a dipeptide, tripeptide, or tetrapeptide, for example, wherein the peptide is valine-citrulline (val-cit or VC), glutamic acid-valine-citrulline (EVC), glycine-glycine-phenylalanine (GGF), or glycine-glycine-phenylalanine-glycine (GGFG).

[0397] In some embodiments, the peptide linker is valine-citrulline (val-cit or VC).

[0398] In some exemplary embodiments, in an exemplary ADC of the application, the linker comprises a moiety having the structure: MC-VC-PAB, as in Formula I:

[0399] wherein, Disulfide bonds are bonds to the antibody or antigen binding protein (e.g., through lysine residues) and are bonds to a cytotoxic agent.

[0400] In some exemplary embodiments, the cytotoxic agent in the exemplary ADCs of the application is Exatecan.

[0401] In one embodiment, the exemplary ADCs of the application are prepared by first linking MMAE and the linker MC-VC-PAB to form the linker-payload intermediate MC-VC-PAB-Exatecan (Formula II):

[0402] Preparation of FGFR2b-specific-ADCs

[0403] The ADCs provided herein can be prepared by any suitable method known in the art.

[0404] In certain embodiments, the nucleophilic group of the antibody is reacted with the bifunctional linker reagent first, followed by attachment to the cytotoxic agent, or vice versa, i.e., the nucleophilic group of the cytotoxic agent is reacted with the bifunctional linker first, followed by attachment to the antibody.

[0405] In certain embodiments, the cytotoxic agent can contain (or be modified to contain) a thiol-reactive functional group that can react with the cysteine thiol of a free cysteine in the antibody provided herein. Exemplary thiol-reactive functional groups include, for example, maleimides, iodoacetamides, pyridyl disulfides, haloacetyl groups, succinimidyl esters (e.g., NHS, N-hydroxysuccinimide), isothiocyanates, sulfonyl chlorides, 2,6-dichlorotriazinyl groups, pentafluorophenyl esters, or phosphoramidates (Hermanson, G., Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0406] In some embodiments, the cytotoxic agent and the antibody can be linked in one step by in situ activation and reaction to form the ADC.

[0407] In some preferred embodiments, the preparation of the FGFR2b-specific-ADCs of the application comprises the steps of:

[0408] The antibody interchain disulfide bonds are first opened using the TCEP reducing agent; followed by post-purification preparation through thiol coupling reactions, further purified using gel chromatography.

[0409] In some embodiments, the molar feed ratio of TCEP: antibody is between 2 and 10.

[0410] In some embodiments, the molar charge ratio of TCEP: antibody is 4-8.

[0411] In some embodiments, the molar charge ratio of drug-linker: antibody is 3-30.

[0412] In some embodiments, the molar charge ratio of drug-linker: antibody is 6-25.

[0413] In some embodiments, the molar charge ratio of drug-linker: antibody is 8-20.

[0414] In some embodiments, the molar charge ratio of drug-linker: antibody is 10-18.

[0415] In some embodiments, the molar charge ratio of drug-linker: antibody is 12-16.

[0416] In some embodiments, the FGFR2b antibody of the present application is conjugated to the anti-FGFR2b antibody at A 280 The highest peak of absorption is of the anti-FGFR2b-ADC (anti-FGFR2b-MC-VC-PAB-Exatecan) is at A 370 .

[0417] In certain embodiments, conjugate moieties are attached to specific types of amino acid residues that are surface-exposed in the antibody, such as cysteine residues or lysine residues, at random.

[0418] In certain embodiments, conjugate moieties are attached to specifically defined sites to provide an ADC population with high uniformity and batch-to-batch consistency in drug / antibody ratio (DAR) and site of attachment.

[0419] In some embodiments, the number of polymers of MV-VC-PAB-Exatecan attached to the anti-FGFR2b antibody can vary, and if multiple polymers are attached, they can be the same or different molecules.

[0420] In certain embodiments, in the preparation of the ADC provided herein, the antibody (or antigen-binding fragment) that specifically binds FGFR2b is conjugated to one or more cytotoxic agents at an antibody: agent ratio (DAR) of about 1 :20, or about 1 : 15, or about 1 : 12, or about 1 : 10, or about 1 : 8, about 1 : 6, or about 1 : 5.

[0421] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the anti-FGFR2b-ADC of the present application is 2-10.

[0422] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the anti-FGFR2b-ADC of the present application is 3-8.

[0423] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the anti-FGFR2b-ADC of the present application is 5-7.

[0424] In some exemplary embodiments, the average DAR value is calculated in advance according to the known concentrations of naked antibody and drug-linker respectively, the absorbance of antibody and drug-linker at A 280 , A 370 , and then the antibody concentration and drug concentration are calculated according to the following formula, the ratio of drug to antibody concentration:

[0425] A 280 = ε D,280 C D + ε A,280 C A

[0426] A 370 = ε D,370 C D + ε A,370 C A .

[0427] Wherein, A 280 and A 370 represent the absorbance of antibody drug conjugate aqueous solution at 280 nm and 370 nm, ε A,280 and ε A,370 represent the molar absorbance coefficient of antibody at 280 nm and 370 nm, ε D,280 and ε D,370 represent the molar absorbance coefficient of drug-linker at 280 nm and 370 nm, C A represents the antibody concentration, C D represents the drug-linker concentration.

[0428] It is calculated that the absorbance coefficient is, ε A,280 = 210600, ε A,370 = 0, ε D,280 = 5280, ε D,370 = 19560.

[0429] Biological activity of exemplary ADC

[0430] In some embodiments, the anti-FGFR2b antibody-ADC of the present application is anti-FGFR2b-MC-VC-PAB-Exatecan.

[0431] In some embodiments, the anti-FGFR2b-MC-VC-PAB-Exatecan of the present application has the following biological activities:

[0432] (1) specifically binds FGFR2b in vitro;

[0433] (2) can specifically bind FGFR2b positive cells;

[0434] (3) has cytotoxicity to FGFR2b positive cells and weak cytotoxicity to cells with low FGFR2b expression but high FGFR2c expression;

[0435] (4) has a strong killing effect on FGFR2b positive tumor cells.

[0436] In some embodiments, the anti-FGFR2b-MC-VC-PAB-Exatecan of the present application has a strong killing effect on FGFR2b positive tumor cells.

[0437] In some embodiments, the FGFR2b positive tumor cells are gastric cancer cells.

[0438] In some embodiments, the killing activity of the anti-FGFR2b antibody ADC of the present application on FGFR2b positive tumor cells is stronger than that of the FGFR2b monoclonal antibody without conjugated Exatecan.

[0439] In some embodiments, the killing rate of the anti-FGFR2b-MC-VC-PAB-Exatecan of the present application on FGFR2b positive tumor cells is higher than 80%, higher than that of the FGFR2b monoclonal antibody without conjugated Exatecan.

[0440] Pharmaceutical compositions

[0441] In one aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human monoclonal antibodies or antigen-binding fragments thereof that specifically bind FGFR2b, or an ADC of an anti-FGFR2b antibody as described in the preceding aspects of the present application, and one or more pharmaceutically acceptable carriers, excipients and other agents formulated together to provide improved transfer, delivery, tolerability, etc.

[0442] In some embodiments, the pharmaceutical composition comprises a composition comprising an antibody, antigen-binding fragment thereof, or conjugate as provided herein, further comprising one or more antioxidants, such as methionine, that will reduce oxidation of the antibody or antigen-binding fragment. Such reduction in oxidation will prevent or reduce loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Accordingly, in certain embodiments, the present application provides a composition comprising one or more antibodies disclosed herein and one or more antioxidants, such as methionine. In certain embodiments, the present application also provides a method of preventing oxidation, extending shelf life, and / or improving efficacy of an antibody or antigen-binding fragment as provided herein by admixing the antibody or antigen-binding fragment with one or more antioxidants, such as methionine.

[0443] In some embodiments, the pharmaceutical composition can be formulated as an injectable composition.

[0444] In some embodiments, the pharmaceutically acceptable carrier can comprise, for example, an aqueous vehicle, such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water for Injection, or Dextrose and Lactated Ringer's Injection.

[0445] Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, or solid forms suitable for producing liquid solutions or suspensions or emulsions upon dissolution. Formulations for injection can include: (1) sterile and / or pyrogen-free solutions that can be used immediately for injection; (2) sterile dry soluble products that are combined with a solvent only immediately before use, such as lyophilized powder; (3) sterile suspensions that can be used immediately for injection; (4) sterile dry insoluble products that are combined with a vehicle only immediately before use; (5) and sterile and / or pyrogen-free emulsions. Solutions can be aqueous or non-aqueous.

[0446] In certain embodiments, a unit dose of a parenteral formulation can be packaged in an ampule, vial, or a syringe with a needle. All formulations for parenteral administration should be sterile and pyrogen-free, as is known and practiced in the art. In certain embodiments, sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.

[0447] In some embodiments, the solvent can contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art. In some embodiments, the buffer is at approximately neutral pH. The sterile filtered solution is then lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In some embodiments, the resulting solution will be dispensed into vials for lyophilization. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. In some embodiments, to reconstitute, sterile and / or non-pyrogenic water for injection or other liquid suitable carrier is added to the lyophilized powder. The precise amount depends on the selected therapy to be administered and can be determined empirically.

[0448] Combination therapy

[0449] In some embodiments, the disclosed antibodies, including anti-FGFR2b-ADCs, can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the disclosed antibodies can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anti-cancer drug.

[0450] In some embodiments, the present application provides combination therapy methods of administering to a subject in need thereof a therapeutic combination of any of the foregoing anti-FGFR2b antibodies or antigen-binding fragments thereof, including anti-FGFR2b-ADCs, with one or more additional therapeutically active ingredients.

[0451] In certain embodiments, the disclosed anti-FGFR2b antibodies or antigen-binding fragments, including anti-FGFR2b-ADCs, can be administered simultaneously with the additional therapeutic agent(s), and the antibody or antigen-binding fragment, including anti-FGFR2b-ADC, and the additional therapeutic agent(s) can be administered as part of the same pharmaceutical composition. In certain embodiments, administration of the disclosed antibodies or antigen-binding fragments prior to or following administration of another agent is also considered administration "in combination" with that agent, even if the anti-FGFR2b antibodies or antigen-binding fragments of the present application and the other agent are administered by different routes. Where possible, the additional therapeutic agent(s) administered in combination with the disclosed antibodies or ADCs are administered according to the time course set forth in the product information sheet for the additional therapeutic agent(s) or according to protocols well known in the art.

[0452] The present application includes pharmaceutical compositions in which the foregoing anti-FGFR2b antibodies or antigen-binding fragments thereof and anti-FGFR2b-ADCs are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0453] In some embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof, and anti-FGFR2b-ADCs of the present application can be co-formulated and / or administered in combination with one or more additional specifically therapeutic antibody components selected from the group consisting of: another FGFR2 antagonist, another EGFR family member such as ErbB3 or ErbB4 antagonist, an anti-ErbB2, such as ErbB3 or ErbB4 antagonist, a MET antagonist, an EGFR antagonist, an IGF1 R antagonist, an IGF1 R antagonist, a DGFR-a inhibitor, a PDGFR-beta inhibitor, or a small molecule kinase inhibitor, a VEGF-inhibitor, a DLL4 antagonist.

[0454] In some embodiments, the additional therapeutically active component is a second specifically antibody.

[0455] In some embodiments, the second specifically antibody includes, but is not limited to, a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin antibody), a LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), a CD20xCD3 bispecific antibody, a PD-1 blocker (e.g., an anti-PD-1 antibody such as pembrolizumab or nivolumab), and the like.

[0456] In some embodiments, other therapeutic agents that can be advantageously administered in combination with the antibodies provided herein also include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines, such as, but not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, or their respective receptors.

[0457] In some embodiments, the target patient population for the therapeutic compositions of the present application specifically includes patients having tumors that overexpress FGFR2 or express a mutated FGFR2, such as patients having FGFR2-expressing gastric cancer, pancreatic cancer, breast cancer.

[0458] In some embodiments, the additional therapeutic agents that can be combined with any of the anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, described herein include one or more chemotherapeutic agents.

[0459] In some embodiments, examples of chemotherapeutic agents that can be combined with any of the anti-FGFR2 antibodies or antigen-binding fragments thereof described herein, including anti-FGFR2 antibody ADCs, include alkylating agents, aziridines, nitrosoureas, antibiotics, antimetabolites, purine analogs, pyrimidine analogs, alkyl sulfonates, folic acid supplements, aldehyde phosphonamide glycosides, taxanes, platinum analogs, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0460] In some embodiments, the additional therapeutically active ingredients also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens (including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, onapristone, and toremifene (Fareston); and anti-androgens (including, for example, flutamide, nilutamide, bicalutamide, leuprolide, and goserelin); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0461] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, of the present application can also be administered and / or co-formulated in combination with an antiviral agent, an antibiotic, an analgesic, a corticosteroid, a steroid, oxygen, an antioxidant, a COX inhibitor, a cardioprotectant, a metal chelator, IFN-γ, and / or an NSAID.

[0462] Use in the manufacture of a medicament

[0463] In another aspect, the present application provides the use of the anti-FGFR2b antibodies or antigen-binding fragments thereof, ADCs thereof, or pharmaceutical compositions comprising the same of the foregoing aspects for the manufacture of a medicament for the treatment, prevention, and / or amelioration of any disease or disorder associated with FGFR2b expression, signaling, or activity, or mediated by FGFR2b expression, signaling, or activity, or any disease or disorder that can be treated by blocking FGFR2b signaling or otherwise inhibiting FGFR2b activity and / or signaling.

[0464] In some embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof, ADCs thereof, or pharmaceutical compositions comprising the same are used in the manufacture of a medicament for the treatment of a cancer associated with abnormal FGFR2 expression.

[0465] In some embodiments, the FGFR2b expression abnormality-associated cancer comprises one or more of the following cancers: gastric cancer, glioblastoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, leukemia, liver cancer, leiomyosarcoma, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngeal carcinoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, primary and / or recurrent cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, squamous cell carcinoma, synovial sarcoma, thyroid cancer, triple negative breast cancer, uterine cancer, and Wilms' tumor, astrocytoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, clear cell renal cell carcinoma, colorectal cancer, microsatellite- intermediate colorectal cancer, cutaneous squamous cell carcinoma, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fibrosarcoma,

[0466] In some embodiments, the FGFR2b expression abnormality-associated cancer comprises one or more of the following solid tumors or cancers: including but not limited to gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell lung cancer), colon cancer, prostate cancer, cervical cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor (e.g., glioma such as glioblastoma), and hematological malignancy.

[0467] In certain embodiments, the cancer is selected from the group consisting of gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colon adenocarcinoma, and breast invasive ductal carcinoma.

[0468] Therapeutic uses

[0469] In one aspect, the present application provides a method of treating cancer, comprising administering to a patient in need thereof an anti-FGFR2b antibody or antigen-binding fragment thereof of the present application, a pharmaceutical composition comprising an anti-FGFR2b antibody of the present application, an ADC of the anti-FGFR2b antibody, or a pharmaceutical composition having the anti-FGFR2b antibody or antigen-binding fragment thereof of the anti-FGFR2b antibody or an ADC of the anti-FGFR2b antibody as an active ingredient, or a pharmaceutical composition comprising the anti-FGFR2b antibody or antigen-binding fragment thereof of the anti-FGFR2b antibody or an ADC of the anti-FGFR2b antibody in combination with any additional therapeutically active ingredient mentioned in the present application.

[0470] In another aspect, the present application provides a method of treatment, comprising administering to a subject in need thereof a therapeutic composition comprising a therapeutically effective amount of an anti-FGFR2b antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, comprising the amino acid sequence of any anti-FGFR2b antibody or variant thereof described in the present application.

[0471] In some embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof or anti-FGFR2b antibody ADCs of the present application are particularly useful for treating, preventing and / or ameliorating any disease or disorder associated with FGFR2b expression, signaling or activity, or mediated by FGFR2b expression, signaling or activity, or any disease or disorder that can be treated by blocking FGFR2 signaling or otherwise inhibiting FGFR2b activity and / or signaling, e.g., particularly a tumor that is FGFR2b positive.

[0472] In certain embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof and anti-FGFR2b antibody ADCs of the present application are used to treat one or more of the following cancers: gastric cancer, pancreatic cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, leukemia, liver cancer, leiomyosarcoma, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngeal carcinoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, primary and / or recurrent cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, squamous cell carcinoma, synovial sarcoma, thyroid cancer, triple negative breast cancer, uterine cancer, and Wilms' tumor, astrocytoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, clear cell renal cell carcinoma, microsatellite- intermediate colorectal cancer, skin squamous cell carcinoma, diffuse large B-cell lymphoma, fibrosarcoma, glioblastoma, glioblastoma multiforme.

[0473] In some embodiments, the FGFR2b expression abnormality-associated cancer comprises one or more of the following solid tumors or cancers: gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell lung cancer), colon cancer, prostate cancer, cervical cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor (e.g., a glioma such as glioblastoma), and a hematologic malignancy.

[0474] In certain embodiments, the cancer is selected from any one or more of the group consisting of gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colon adenocarcinoma, and breast invasive ductal carcinoma.

[0475] In some embodiments, the cancer is a primary cancer.

[0476] In some embodiments, the cancer is a metastatic and / or recurrent cancer.

[0477] In some aspects, the anti-FGFR2b antibodies or antigen-binding fragments thereof, and ADCs of anti-FGFR2b antibodies of the present application, are useful for treating FGFR2bb-positive primary and / or metastatic tumors, i.e., tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and adnexa, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver, and urinary tract, as well as specialized sensory organs, e.g., tumors arising in the eye.

[0478] In the context of the therapeutic methods described herein, the anti-FGFR2b antibodies or antigen-binding fragments thereof can be administered as a monotherapy (i.e., as the only therapeutic agent), or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein), or as an ADC (examples of which are also described elsewhere herein).

[0479] Accordingly, the present application provides methods of treating cancer in a subject having a tumor that overexpresses FGFR2b, the method comprising administering to the subject an anti-FGFR2b antibody or antigen-binding fragment thereof, or an ADC thereof, provided herein.

[0480] In some embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof, in the methods of treating a tumor or cancer that overexpresses FGFR2b of the present application, comprise: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 1; a HCDR2 having the amino acid sequence of SEQ ID NO: 2; a HCDR3 having the amino acid sequence of SEQ ID NO: 3; a LCDR1 having the amino acid sequence of SEQ ID NO: 4; a LCDR2 having the amino acid sequence of SEQ ID NO: 5; and a LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0481] Therapeutic methods

[0482] In certain embodiments, the present application also provides methods of treating cancer, reducing tumor growth, and / or causing tumor regression in a subject, the method comprising administering to a subject in need thereof an antibody-drug conjugate (ADC) comprising an anti-FGFR2b antibody or antigen-binding fragment thereof and a cytotoxin, wherein the anti-FGFR2b antibody or antigen-binding fragment thereof comprises: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 1; a HCDR2 having the amino acid sequence of SEQ ID NO: 2; a HCDR3 having the amino acid sequence of SEQ ID NO: 3; a LCDR1 having the amino acid sequence of SEQ ID NO: 4; a LCDR2 having the amino acid sequence of SEQ ID NO: 5; and a LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0483] In some aspects, the cytotoxin is a maytansinoid.

[0484] In some aspects, the cytotoxin is a maytansinoid.

[0485] In some aspects, the cytotoxin is a maytansinoid.

[0486] In some aspects, the cytotoxin is a maytansinoid.

[0487] A therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as the subject's body weight, age, medical history, current medications, state of health, and the likelihood of cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. A person of ordinary skill in the art (e.g., a physician or veterinarian) can reduce or increase the dosage as appropriate, as indicated by these and other conditions or requirements.

[0488] In certain embodiments, an antibody or antigen-binding fragment provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the antibody or antigen-binding fragment is administered at a dose of about 50 mg / kg or less, and in certain embodiments of these examples, the dose is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less.

[0489] In certain embodiments, the dose administered can be varied during the course of therapy. For example, in certain embodiments, the initial dose administered can be higher than the subsequent doses administered. In certain embodiments, the dose administered can be varied during the course of therapy depending on the subject's response.

[0490] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered, or several divided doses can be administered over time.

[0491] The antibodies disclosed herein can be administered by any route known in the art, such as parenterally (e.g., subcutaneously, intraperitoneally, intravenously (including intravenous infusion), intramuscularly, or intradermally).

[0492] In some embodiments, the method of administration employs a multiple-dose regimen, wherein the anti-FGFR2b antibody or antigen-binding fragment thereof, including anti-FGFR2b antibody ADC, or comprising anti-FGFR2b antibody or anti-FGFR2b antibody ADC, is administered to the subject over a defined time period.

[0493] In some embodiments, an additional therapeutically active ingredient, e.g., any of the agents listed above or a derivative thereof, can be administered prior to, concurrently with, or soon after administration of an anti-FGFR2b antibody or antigen-binding fragment thereof, including an anti-FGFR2b antibody ADC; (for purposes of the present application, such administration schemes are considered administration of the antibody "in combination" with the additional therapeutically active ingredient).

[0494] In some embodiments, the multiple-dose regimen comprises sequential administration of multiple doses of an anti-FGFR2b antibody or antigen-binding fragment thereof provided herein, including an anti-FGFR2b antibody ADC, to the subject, wherein "sequential administration" means that each dose of the antibody or ADC is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months).

[0495] In some exemplary embodiments, the "sequential administration" comprises sequential administration of a single initial dose of an anti-FGFR2b antibody or antigen-binding fragment thereof, including an anti-FGFR2b antibody ADC, to the patient, followed by administration of one or more secondary doses of the anti-FGFR2b antibody or antigen-binding fragment thereof or anti-FGFR2b antibody ADC, and optionally followed by administration of one or a third dose of the anti-FGFR2b antibody or antigen-binding fragment thereof or ADC. In some exemplary embodiments, "initial dose", "secondary dose", and "third dose" refer to the chronological order of administration of the anti-FGFR2b antibody or antigen-binding fragment thereof or anti-FGFR2b antibody ADC. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as "baseline dose"); the "secondary dose" is the dose administered after the initial dose; the "third dose" is the dose administered after the second dose. The initial, secondary, and third doses can all contain the same amount of the anti-FGFR2b antibody or antigen-binding fragment thereof or anti-FGFR2b antibody ADC of the present application, but generally can differ from each other in terms of frequency of administration. In certain embodiments, the amount of the anti-FGFR2b antibody or antigen-binding fragment thereof or anti-FGFR2b antibody ADC of the present application contained in the initial dose, secondary dose, and / or third dose differs from each other (e.g., adjusted upward or downward as appropriate) over the course of the treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as a "loading dose", followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0496] Examples

[0497] The following examples are provided to better illustrate the claimed invention and are not intended to limit the scope of the invention. All specific compositions, materials and methods described herein (including all whole or part combinations thereof) are included within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the invention, but merely to illustrate particular embodiments within the scope of the invention. Those skilled in the art will readily develop other equivalent compositions, materials and methods without departing from the scope of the invention. It is to be understood that many variations can be made to the procedures described herein and such variations are to be considered within the bounds of the invention. The inventors intend to cover all such variations within the scope of the invention.

[0498] Example 1. Preparation of human FGFR2 IIIb-hFc protein

[0499] 1.1 Construction of eukaryotic expression vector of FGFR2 IIIb-hFc fusion protein

[0500] The coding sequence of human FGFR2 IIIb-hFc extracellular domain (amino acid sequence SEQ ID NO: 24, nucleic acid as shown in SEQ ID NO: 30) and the coding sequence of human Fc protein were connected together through the restriction enzyme sites at both ends, and cloned into the mammalian expression vector pHR, thereby obtaining the eukaryotic expression plasmid pHR-GFR2 IIIb-hFc of FGFR2 IIIb-hFc fusion protein.

[0501] Human FGFR2 beta (IIIb) hFc

[0502] 1.2 Expression and purification of FGFR2 IIIb-hFc fusion protein

[0503] FGFR2 IIIb-hFc fusion protein expression: The pHR FGFR2 IIIb-hFc plasmid was transiently transfected into expiCHO S cells (Thermo Fisher) using ExpiFecta minute e CHO transfection kit (Gibco) according to the manufacturer's instructions. After the transfected ExpiCHO-S cells were cultured in ExpiCHO-S expression medium for 10 days, the cell supernatant was collected.

[0504] FGFR2IIIb-hFc fusion protein purification: The resulting cell supernatant (CHO cell, Chinese hamster ovary cell, ATCC, CRL-2092) was centrifuged (6000 x g, 10 min s) and filtered (0.45 μm filter was used) and then purified. The purification conditions were as follows: instrument: AKTA; flow rate: 1 mL / min; pre-packed column: Hitrap MabSelect SuRe Protein A (GE, #11-0034-93-GEC), equilibrated with 10 volumes of buffer 1 (PBS, Solaibio, P1010, pH 7.4); eluent A: buffer 1 (PBS, pH 7.4); eluent B: buffer 3 (100 mM glycine, 150 mM NaCl, pH 3.0); elution sequence: eluted with eluent A (about 30 mL) until the A280 value was stable, and then eluted with eluent B. The eluate corresponding to the peak with an A280 value of 30 or more was collected, and 15 μL / mL of neutralization buffer (1 M Tris-HCl, pH 9.0) was added to the collection tube in advance to maintain the pH of the resulting antibody solution at 7.0-8.0. The resulting antibody solution was subjected to buffer exchange (the buffer was replaced with PBS) and concentration using a 10 KD ultrafiltration tube.

[0505] Example 2. Production of anti-human FGFR2b monoclonal antibody

[0506] The mouse anti-human FGFR2b monoclonal antibody was obtained by conventional hybridoma technology as follows: The fusion protein of human FGFR2IIIb-hFc prepared in Example 1 was mixed with fast immune adjuvant (Bio-oil, KX0210041) at a volume ratio of 1:1, and then the adjuvant and antigen mixture was used to immunize Balb / c mice (purchased from Sibeifu Biotechnology Co., Ltd.) by intramuscular injection. After serum titer detection, the mice that met the fusion criteria were euthanized, the spleen was collected and a single cell suspension was prepared, and hybridoma was produced by electrofusion with SP20 myeloma cells (Nanjing Kebai, CBP60881). The fused cells were plated in a 96-well cell culture plate and grown for 10 days. After 10 days, the supernatant was taken for ELISA and FACS binding activity screening, and the parent clone 27A6 capable of binding human FGFR2b was obtained. The single clone with FGFR2b binding activity was isolated from the parent clone mixed cell library by limited dilution, and the sequence of the variable region of the antibody of the single clone was retrieved and sequenced. The heavy chain variable region sequence and the light chain variable region sequence of murine clone 27A6 were finally obtained by sequencing.

[0507] Heavy chain variable region sequence and light chain variable region sequence of murine clone 27A6

[0508] 27A6 VH

[0509] 27A6 VL

[0510] Example 3. Expression and purification of 27A6 chimeric antibody

[0511] The C-terminus of the heavy chain variable region VH1 (amino acid sequence as SEQ ID No: 01) of 27A6 murine antibody was linked to human IgG1 constant region (amino acid sequence of heavy chain constant region as SEQ ID No: 02), and the coding sequence of the heavy chain of the chimeric antibody was inserted into pHR expression vector. Similarly, the C-terminus of the light chain variable region LH1 (amino acid sequence as SEQ ID No: 06) of 27A6 murine antibody was linked to human kappa chain constant region (amino acid sequence of light chain constant region as SEQ ID No: 03), and the coding sequence of the light chain of the chimeric antibody was inserted into pHR expression vector. The obtained light chain and heavy chain expression vectors were simultaneously transfected into Expi CHO-S cells (Gibco, A29133) using transfection reagent (Gibco, A29129). The specific operation is as follows: the cell density was adjusted one day before transfection so that the cell density on the transfection day could reach 6x106 / mL. 4% of OptiPRO TM SFM complex medium (purchased from Gibco) was used as transfection buffer, and the plasmid DNA was diluted to 0.8 μg / mL. The Expi Fecta transfection reagent (Gibco, A29129) was mixed with the DNA for 5 minutes at room temperature. TM After mixing the CHO supplement (purchased from Gibco), the obtained cationic transfection reagent / DNA mixture was gently poured into the Expi CHO-S cell suspension, gently mixed and incubated at 37°C with 5% CO2 overnight. After 18-22 hours of overnight incubation, 24% of the volume of post-transfection culture was added to the culture flask, and the cells were incubated at 37°C with 5% CO2 for 10 days. TM The CHO supplement (purchased from Gibco) and 0.6% of the volume of post-transfection culture were added to the culture flask, and the cells were incubated at 37°C with 5% CO2 for 10 days. TM The CHO supplement (purchased from Gibco) and 0.6% of the volume of post-transfection culture were added to the culture flask, and the cells were incubated at 37°C with 5% CO2 for 10 days. The collected cell supernatant was centrifuged at 10,000 rpm for 10 minutes before purification, and the supernatant was filtered using a 0.22 μm filter. The Protein A column was equilibrated using 5 column volumes of equilibration buffer (10 mM PB pH 7.0). After adding the filtered supernatant to the purification column, 10 column volumes of equilibration buffer were used for equilibration. 5 mL of elution buffer (100 mM Gly-NaCl, pH 3.8) was added, and the eluate was collected. The concentration of the antibody was detected by A280 method, and the purity of the antibody was determined by SEC-HPLC method, and finally the purity of the recombinant chimeric antibody 27A6 was greater than 95%.

[0512] Example 4. FGFR2b chimeric antibody binding to FGFR2 protein

[0513] The binding activity of FGFR2b chimeric antibody to FGFR2 alpha (IIIb) protein and FGFR2 another subtype protein FGFR2 alpha (IIIC) was detected by Elisa experiment method. Specifically, the recombinant expressed human FGFR2 alpha (IIIb) protein (Sino Biological, 16485-H08H) and human FGFR2 alpha (IIIC) protein (Sino Biological, 10824-H08H) were diluted to 1 μg / mL with coating solution (0.2 mol / L Na2CO3 8 mL, 0.2 mol / L NaHCO3 17 mL mixed, and 75 mL distilled water was added, and the pH was adjusted to 9.6), and 50 μL / well was added to the enzyme-labeled plate (Corning, 3590) hole, and incubated at 4°C overnight. After discarding the liquid in the hole, 200 μL of washing solution (PBS + 0.5‰ Tween-20) was added to each hole and washed 3 times. Then 200 μL of blocking solution (PBS + 0.5‰ Tween-20 + 5% skim milk powder) was added to each hole, and incubated at 37°C for 2 hours. Discard the liquid in the hole, add 200 μL of washing solution to each hole, and wash 3 times. The 27A6 chimeric antibody, the reference antibody FPA144 (the sequence is derived from the FDA Global Material Registration Database) and the isotype control antibody hIgG1 were diluted by 4 times gradient from 10 μg / mL as the starting concentration, a total of 12 concentration points, and 50 μL of each concentration of antibody was added to the corresponding Elisa plate hole, and incubated at 37°C for 1 hour. Discard the liquid in the hole, wash the plate 3 times with 200 μL of washing solution. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-170) to each hole, incubate at 37°C for 1 hour, discard the liquid in the hole, and then add 200 μL of washing solution to each hole and wash 3 times. Add 50 μL of color developing solution (Sigma, T4444) to each hole, incubate at room temperature for 3 minutes, then add 50 μL of 2 mol / L H2SO4 to stop the reaction. Read the OD value at 450 nm on the enzyme-labeled instrument (Thermo Scientific Multiskan FC, 8D-030A), and plot and analyze the data with the antibody concentration as the abscissa and the OD450nm value as the ordinate. The results are shown in Figure 1.

[0514] As shown in Figure 1, the 27A6 chimeric antibody has strong binding activity to FGFR2 IIIb compared with the reference antibody FPA144, and does not bind to FGFR2 IIIC.

[0515] Example 5. FGFR2b chimeric antibody binding to FGFR2b positive tumor cells

[0516] The binding activity of FGFR2b chimeric antibody to target protein positive tumor cells was detected by flow cytometry method, and the specific method was as follows: KATO3 (purchased from Shengene) and SNU16 (purchased from Nanjing Kebai) cells were inoculated in U-shaped 96-well plates (BIOFIL, 002096) at a density of 1x10 5 The chimeric antibody 27A6, the reference antibody FPA144 and the isotype control antibody hIgG1 were prepared at a starting concentration of 20 μg / mL, and then diluted by 3 times in turn, a total of 12 concentration points. 50 μL of each concentration of antibody was added to the cell well, incubated at 4°C for 30 minutes, and the cells were washed twice with FACS buffer (1% BSA in PBS solution). 50 μL of PE-labeled goat anti-human Fc secondary antibody (Jackson, 109-116-098) was added to each well, incubated at 4°C for 30 minutes, and the cells were washed twice with FACS buffer. 100 μL of FACS buffer was added to each well to resuspend the cells, and the fluorescence intensity of the cells was detected by flow cytometry (Beckman Coulter Bioscience, A00-1-1102). The antibody concentration was plotted as the abscissa, and the PE MFI was plotted as the ordinate. The results are shown in Figure 2.

[0517] As shown in Figure 2, compared with the negative control hIgG1 and the reference antibody FPA144, the chimeric antibody 27A6 had strong binding to FGFR2b protein on both KATO3 and SNU16 tumor cells.

[0518] Example 6. Identification of the antagonistic activity of FGFR2b chimeric antibody

[0519] The blocking activity of FGFR2b chimeric antibody on the binding of FGFR2b protein on the surface of tumor cells to its ligand FGF7 was detected by flow cytometry method, and the specific method was as follows: KATO3 cells were inoculated in U-shaped 96-well plates (BIOFIL, 002096) at a density of 1x10 5The KATO3 cells (purchased from Sino Biological) and MFM-223 cells (purchased from Nanjing Keygen) were seeded at a density of 4x104cells / well in a U-shaped 96-well plate (BIOFIL, 002096) and centrifuged at 500g for 3 minutes, and the cells were washed twice with FACS buffer (PBS solution containing 1% BSA). The chimeric antibody 27A6, the reference antibody FPA144 and the isotype control antibody hlgG1 were configured at 50μg / mL, and then sequentially diluted by 3 times to obtain 12 concentration points. 25μL of each concentration of antibody was added to the cell well, and 25μL of FGF7 (Sino Biological, 10210-H07E) ligand solution with a final concentration of 3μg / mL was also added to each well, and the two were mixed uniformly and incubated at 4°C for 30 minutes. The cells were centrifuged at 500g for 3 minutes, washed twice with FACS buffer, and 50μL of anti-His-tag protein FITC-labeled secondary antibody (Invitrogen, MA1-81891) was added to each well, and incubated at 4°C for 30 minutes. The cells were washed twice with FACS buffer, resuspended with 100μL of FACS buffer, and the fluorescence intensity of the cells was detected using a flow cytometer (Beckman Coulter, A00-1-1102). The antibody concentration was plotted as the abscissa, and the FITC MFI was plotted as the ordinate. The results are shown in Figure 3.

[0520] As shown in Figure 3, compared with the negative control hlgG1 and the reference antibody FPA144, the chimeric antibody 27A6 can effectively block the binding of FGF7 to the FGFR2b receptor protein on the surface of KATO3 human gastric cancer tumor cells.

[0521] Example 7. Endocytosis effect of FGFR2b chimeric antibody

[0522] The recombinant DT3C protein can inhibit protein translation mechanism in cells to induce cell death, and can bind to IgG antibodies of different species. In this embodiment, the antibody forms a conjugate with DT3C and is used as a tool for evaluating the internalization efficiency of the chimeric antibody on two tumor cells, and the internalization efficiency is evaluated by detecting the degree of cell death induced. The specific method is as follows: KATO3 cells (purchased from Sino Biological) and MFM-223 cells (purchased from Nanjing Keygen) were seeded at a density of 4x104 4The density of 2x10 cells / well was seeded in 96-well plates. Then the chimeric antibody 27A6 was configured with an initial concentration of 20 μg / ml, 2-fold gradient dilution of 10 concentration points, 40 μL of the corresponding concentration of antibody and 40 μL of 4 μg / ml DT3C protein solution (Cusabio, CSB-EP360556CQR1) were added to each well, then mixed, pre-incubated at room temperature for 30 minutes, 50 μL of DT3C-antibody conjugate was added to the cell well with cells plated, and incubated in a 37°C incubator for 72 hours. The endocytosis efficiency of the antibody was determined by detecting the fluorescence signal of the living cells by Cell Counting-Lite 3D kit (Novus, DD1102).

[0523] According to the results of Figure 4, the chimeric antibody 27A6 can effectively internalize into the cell in two tumor cells with different FGFR2b expression levels.

[0524] Example 8. Inhibition of tumor growth in vitro by FGFR2b chimeric antibody

[0525] To identify FGFR2b antagonistic antibodies, the chimeric antibody was incubated with tumor cells expressing FGFR2b, and then the proliferation of tumor cells was detected. The specific method is as follows: the density of SNU16 cells was adjusted to 2x10 5 The initial concentration of the chimeric antibody to be tested was configured to 100 μg / ml, 2.5-fold gradient dilution of 3 concentration points, 50 μL of the corresponding dilution concentration of the antibody was sequentially added to the cell well, incubated at 37°C, 5% CO2 culture condition for 30 minutes, then 10 μL of mixed solution with a final concentration of 100 ng / ml FGF7 (Sino Biological, 10210-H07E) and 20 μg / mL heparin (MCE, HY-17567A / CS-3922) was added to each well, and continued to be cultured at 37°C, and the fluorescence signal of the living cells was detected by Cell Counting-Lite 3D kit (Novus, DD1102) after 72 hours.

[0526] As shown in Figure 5, in the presence of FGF7 ligand, the fluorescence signal of living cells of SNU16 tumor cells after the action of 27A6 chimeric antibody group and FPA144 reference antibody group at three concentrations of antibodies was reduced, showing that 27A6 antibody and FPA144 antibody can effectively inhibit the proliferation of tumor cells driven by FGG7 / FGFR2b signaling pathway.

[0527] Example 9. FGFR2b chimeric antibody binds to monkey and mouse FGFR2b protein

[0528] The cross-binding activity of 27A6 antibody to FGFR2b proteins of different species was detected by ELISA method. The specific experimental method is as follows: mouse FGFR2 (IIIb) (Acro, FGB-M52H5) protein and monkey FGFR2 (IIIb) (Acro, FGB-C52H6) protein were diluted to 1 μg / mL with coating solution, 50 μL / well was added to the enzyme-labeled plate hole, and incubated at 4°C overnight. Discard the liquid in the hole, add 200 μL of washing solution to each hole, and wash the plate 3 times. Add 200 μL of blocking solution to each hole, and incubate at 37°C for 2 hours. Discard the liquid in the hole, add 200 μL of washing solution to each hole, and wash the plate 3 times. Chimeric antibody 27A6, reference antibody FPA144 was diluted by 4 times gradient from 10 μg / mL, a total of 8 concentration points, 50 μL of each concentration of antibody was added to the corresponding Elisa plate hole, and incubated at 37°C for 1 hour. Discard the liquid in the hole, wash the plate 3 times with 200 μL of washing solution, add 50 μL of HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-170) to each hole, and incubate at 37°C for 1 hour. After discarding the liquid in the hole, add 200 μL of washing solution to each hole and wash 3 times. Add 50 μL of color developing solution (Sigma, T4444) to each hole, incubate at room temperature for 3 minutes, then add 50 μL of 2 mol / L H2SO4 to stop the reaction. Read the OD value at 450 nm on the enzyme-labeled instrument (Thermo Scientific Multiskan FC, 8D-030A), and plot the antibody concentration as the horizontal coordinate and the OD450nm value as the vertical coordinate for data analysis. The results are shown in Figure 6.

[0529] As shown in Figure 6, compared with reference antibody FPA144, chimeric antibody 27A6 can bind to recombinantly expressed monkey FGFR2 (IIIb) protein, and the EC50 values of the two antibodies are close. The detection results of binding to recombinantly expressed mouse FGFR2 (IIIb) protein show that chimeric antibody 27A6 has stronger binding capacity than antibody FPA144, and its EC50 value is 5.8 times lower than that of the latter.

[0530] Example 10. Humanization of FGFR2b antibody

[0531] This example uses CDR grafting to humanize the variable region sequences of the 27A6 murine antibody. Bioinformatics analysis is used to determine the human sequences with the highest homology to the murine 27A6 light and heavy chain variable region sequences. The CDR sequences of the murine 27A6 light and heavy chain variable regions are grafted into human germline genes. Structure analysis is used to determine the key amino acid residues that differ between the murine and human 27A6 antibody sequences, and a back mutation design is performed. Four humanized heavy chain variable region sequences and four humanized light chain variable region sequences are obtained, as shown in Table 2, which are expressed according to the light and heavy chain combinations in Table 3, resulting in nine humanized antibodies.

[0532] Table 2. VH amino acid sequences and paired VL amino acid sequences of the variable regions of exemplary chimeric anti-FGFR2b antibodies and humanized anti-FGFR2b antibodies

[0533] Table 3. Heavy chain variable region and light chain variable region combinations of exemplary anti-FGFR2b antibodies

[0534] Table 6. Nucleic acids encoding the variable regions of exemplary anti-FGFR2b antibodies

[0535] Example 11. Detection of the binding affinity of humanized FGFR2b antibodies to human FGFR2b antigen

[0536] FGFR2 humanized antibody affinity detection based on Bio layer interferometry (BLI) technology, FGFR2IIIb-mFc was gradient diluted with PBST, the initial concentration was 100 nM, and then 7 concentration points were obtained by 2-fold gradient dilution, and zero concentration control wells were set. FGFR2 humanized antibody was diluted to 5 μg / mL with Loading Buffer (1xPBS, pH 7.4, with 0.02% Tween-20, 0.1% BSA). The running conditions of the molecular interaction instrument ForteBio's Octet System (Sartorius, model Octet R8) were set as follows: temperature 30℃, Shake speed 1000 rpm. The AHC2 sensor was used to solidify the antibody to be tested, the threshold was 1.5 nm, and the solidification time was 180 s; the gradient diluted FGFR2IIIb-mFc protein sample was combined, the binding time was 120 s; the dissociation time was 300 s; and the regeneration buffer (10 mM glycine, PH 1.7) was used for regeneration for 30 s. After obtaining the sensorgram data, the Octet BLI Analysis software was used to analyze the binding constant (ka) and the dissociation constant (kd), and the ideal binding and dissociation curve was fitted to calculate the equilibrium dissociation constant KD (Kd / Ka) between the antibody and the antigen. As shown in Table 7, the affinities of 3 humanized antibodies are comparable to that of the parent chimeric antibody 27A6CHI.

[0537] Table 7. FGFR2b antibody binding affinity to FGFR2IIIb-hFc fusion protein

[0538] Example 12. Detection of the binding activity of humanized FGFR2b antibody to FGFR2b positive tumor cells

[0539] The binding activity of humanized antibodies to two tumor cells with different FGFR2b expression levels was detected by flow cytometry, and the specific method is described in Example 5. According to the results shown in Figure 7, 27A6H0L0 has no binding activity to the two tumor cells, and the other humanized antibodies can bind to the two tumor cells, and the binding activity is different. Among them, the binding activity of 27A6H4L2, 27A6H4L3 and 27A6H4L4 to the two tumor cells is close to that of the parent chimeric antibody 27A6CHI.

[0540] Example 13. Blocking of FGF7, FGF10 and FGFR2b receptor protein binding by humanized antibody

[0541] The blocking effect of the humanized FGFR2b antibody on the binding of FGF7, FGF10 and FGFR2b receptor protein was detected by Elisa method, and the blocking activity of the humanized antibody on FGF7 and FGF10 mediated FGFR2 signal pathway was evaluated. The specific method is as follows: FGF7 (Sino Biological, 10210-H07E) and FGF10 (Acro, FG0-H5145) were diluted to 5 μg / mL with coating solution (0.2 mol / L Na2CO3 8 mL, 0.2 mol / L NaHCO3 17 mL mixed, and 75 mL distilled water was added, and the pH was adjusted to 9.6) and incubated at 4°C overnight. After discarding the liquid in the wells, 200 μL of washing solution (PBS + 0.5‰ Tween-20) was added to each well, and the plate was washed 3 times. Then 200 μL of blocking solution (PBS + 0.5‰ Tween-20 + 3% BSA) was added to each well, and incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing solution was added to each well, and the plate was washed 3 times. The starting concentration of each antibody was 100 μg / mL, which was diluted by 3 times in turn, a total of 8 concentration points, and 40 μL of corresponding concentration of antibody was taken, mixed with 40 μL of 10 μg / mL FGFR2b-mFC protein, and incubated at 37°C for 30 minutes. Then 50 μl of pre-incubated antibody and FGFR2b-mFC mixture was transferred to the FGF7 or FGF10 coated plate well, and incubated at 37°C for 1 hour, and the liquid in the well was discarded, and the plate was washed with washing solution for 3 times. 50 μL of HRP labeled goat anti-mouse Fc secondary antibody (Jackson, 115-005-008) was added to each well, and incubated at 37°C for 1 hour, and the liquid in the well was discarded, and the plate was washed with washing solution for 3 times. 50 μL of color developing solution (Sigma, T4444) was added to each well, and incubated at room temperature for 3 minutes, and then 50 μL of 2 mol / L H2SO4 was added to terminate the reaction. The OD value at 450 nm was read on the enzyme marker (Thermo Scientific Multiskan FC, 8D-030A), and the antibody concentration was taken as the abscissa, and the OD450nm value was taken as the ordinate to plot and analyze the data, and the results are shown in Figure 8.

[0542] According to the results shown in Figure 8, the six humanized antibodies in the embodiment can all block the binding of FGF7 to FGFR2b protein, wherein the blocking ability of the three humanized antibodies 27A6H4L2, 27A6H4L3 and 27A6H4L4 to block the binding of FGF7 is close to that of the parent chimeric antibody 27A6CHI, and the IC50 value of the FPA144 antibody differs by 2.8 times. Similarly, the six humanized antibodies in the example can also all block the binding of FGF10 to FGFR2b protein, wherein the blocking ability of the three humanized antibodies 27A6H4L2, 27A6H4L3 and 27A6H4L4 to block the binding of FGF10 is close to that of the parent chimeric antibody 27A6CHI, and the blocking activity of the FPA144 antibody to the binding of FGF10 to FGFR2b protein is weakened, indicating that our humanized antibodies have the property of reducing the blocking of the FGF10 / FGFR2b signaling pathway. The current eye toxicity inducement of the FGFR2 antagonist under clinical research is caused by long-term blocking of the FGF10 / FGFR2b signaling pathway by the antibody.

[0543] Example 14. Endocytosis effect of humanized FGFR2b antibodies

[0544] In this embodiment, the endocytosis efficiency of humanized FGFR2b antibodies in two tumor cells was detected by endpoint method. The specific experimental method is as follows: KATO3 and SNU16 cells were inoculated in 96-well plates at a density of 5x10 4 cells / well, and incubated in a 37°C, 5% CO2 incubator overnight. The humanized antibodies were diluted with complete culture medium to 16 μg / mL, and then 30 μL of the corresponding concentration of antibodies and 30 μL of goat anti-human IgG1 Fc secondary antibody (Sino Biological, SSA015) pre-labeled with pHAb Amine and Thiol Reactive Dyes (Promega, G9841) at a final concentration of 20 μg / mL were mixed well, and incubated at 37°C in the dark for 30 minutes. The cell plate was taken out of the incubator, and 50 μL of labeled antibody was added to the designated well, and the cells were resuspended after incubation at 37°C for 24 hours, and washed twice with FACS buffer. The cells were resuspended with 100 μL of FACS buffer, and the PE fluorescence signal of the cells was detected by flow cytometry and the data was analyzed by plotting.

[0545] As shown in the results of Figure 9, except for 27A6H0L0, other 9 humanized antibodies have endocytosis activity in KATO3 and SNU16 cells, the endocytosis efficiency of the antibodies is positively correlated with the expression abundance of FGFR2b in tumors, and the endocytosis effect of each humanized antibody is stronger in KATO3 cells overexpressing FGFR2b, among which the endocytosis amount of three humanized antibodies 27A6H3L2, 27A6H3L3 and 27A6H3L4 is the highest, and is higher than that of the reference antibody FPA144. In SNU16 cells with moderate expression of FGFR2b, the endocytosis amount of each humanized antibody is related to the affinity of the antibody, and the endocytosis amount of three humanized antibodies 27A6H4L2, 27A6H4L3 and 27A6H4L4 with higher affinity is also higher, and the endocytosis activity is comparable to that of the reference antibody FPA144.

[0546] Example 15. Inhibition of phosphorylation pathway of tumor cells by humanized FGFR2b antibodies

[0547] The effect of humanized antibodies on FGF7-mediated FGFR2 tumor cell phosphorylation pathway was detected in SNU16 tumor cells. The specific method is as follows: SUN-16 cells were resuspended with 1640 medium containing 1% FBS, and the cell density was adjusted to 5x10 5The cells were seeded at 1 x 104 / well into a 6-well plate and incubated overnight. The test antibody was added at a concentration of 10 pg / mL, and the cells were incubated at 37°C for 24 hours. Then, a mixture of FGF7 (Sino Biological, 10210-H07E) and heparin (MCE, HY-17567A / CS-3922) was added to each well at a final concentration of 100 ng / mL and 20 pg / mL, respectively, and the cells were incubated at 37°C for 15 minutes. The culture medium was discarded, and the cells were lysed by adding 130 pL of RIPA lysis buffer containing phosphatase inhibitors to each well. The lysate was centrifuged at 13000 rpm at 4°C for 10 minutes, and 120 pL of cell supernatant was transferred to a new EP tube. Then, 30 pL of 5x Loading Buffer was added to each tube, and the mixture was denatured at 100°C for 5 minutes. After denaturation, the samples were subjected to protein electrophoresis, and then the proteins were transferred to a PVDF membrane by blotting. The membrane was placed in TBST containing 5% skim milk powder and incubated at room temperature for 1 hour. Then, antibodies against GAPDH (Solarbio, K200057M), P-FGFR (Cell Signaling, #3476), P-FRS2-a (Cell signaling, #3861), FGFR2 (Cell signaling, #11835), and P-Erk1 / 2 (Cell signaling, #4370) were added to the membrane, respectively, and incubated at 4°C overnight. After washing the membrane with TBST, the membrane was incubated with corresponding horseradish peroxidase-conjugated anti-rabbit (Sinobiological, SSA004) and horseradish peroxidase-conjugated anti-mouse (Jackson, 115-005-008) secondary antibodies at room temperature for 1 hour. ECL Western Blot Substrate (Thermo Scientific TM , #32209) was used for development, and the ECL luminescence was directly imaged using a Tanon 5200 chemiluminescence imaging system to obtain the experimental results.

[0548] According to the results shown in Figure 10, 27A6CHI (chimeric antibody) and its exemplary humanized antibodies 27A6H4L2 and 27A6H4L4 can all mediate the down-regulation of FGFR2 phosphorylated protein and the significant down-regulation of two key phosphorylated proteins ERK1 / 2 and FRS2a in the downstream RAS-MAPK and PI3K-AKT tumor signaling pathways, indicating that the two humanized antibodies have good inhibitory effect on the downstream phosphorylation pathway of FGFR2, and the inhibitory ability is comparable to that of the control antibody FPA144.

[0549] Example 16. In vitro killing effect of humanized FGFR2b antibody conjugate (ADC) on tumor cells

[0550] 16.1 Humanized FGFR2b antibody conjugated with MC-VC-PAB-Exatecan (Exatecan)

[0551] In this embodiment, the anti-FGFR2 antibody is conjugated with the cytotoxin Exatecan through the MC-VC-PAB linker (as shown in Figure 17), which has the following structure:

[0552] The antibody is conjugated with the MC-VC-PAB-Exatecan linker moiety (as shown in Figure 18) by first opening the inter-chain disulfide bond of the antibody using the TCEP reducing agent (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Catalog No. 51805-45-9, purity 98%), with a molar feed ratio of TCEP: antibody of 6.5, and adding 50 mmol / L TCEP aqueous solution in a 5 mg / mL antibody solution at room temperature with continuous stirring, and placing it in a 37°C incubator for 2 hours; then the MC-VC-PAB-Exatecan is coupled with the antibody through a thiol coupling reaction, with a molar feed ratio of drug-linker: antibody of 15, and first adding a DMSO solution with a volume ratio of 5% in the antibody solution after the reaction is complete, then adding 10 mmol / L drug-linker in batches with continuous stirring, continuously stirring for 1 minute to obtain a uniform solution, and placing it in a 25°C water bath for 3 hours.

[0553] The antibody drug conjugate is purified by gel chromatography, as follows: first, flush the pipeline with 1M NAOH solution at a flow rate of 20 ml / min, then place the inlet tube in the phosphate buffer (chromatography eluent) to balance until the PH changes to the PH of the eluent (7), then set the flow rate to 1 ml / min, load the chromatography column, set the flow rate to 5 ml / min, and until the PH is 7, load at a flow rate of 2.55 ml / min, when the UV rises to 30, start collecting, and until the UV falls back to 30, end the collection. The collected antibody drug conjugate sample is sterile filtered using a 0.22 μm filter as needed, and concentrated to the desired concentration using a 30Kd or 50Kd concentration tube.

[0554] The concentration and DAR (drug antibody coupling ratio) value of the antibody drug conjugate are determined. The concentration and average DAR value of the antibody drug conjugate are detected by ultraviolet spectrophotometry using ultraviolet absorbance, and it is determined that the antibody has the highest absorption peak at A 280 The highest absorption peak of the MC-VC-PAB-Exatecan drug-linker is at A 370, the antibody and drug-linker concentrations need to be calculated in advance according to the known concentrations of naked antibody and drug-linker, respectively, and the absorbance of A 280 , A 370 , and then the antibody concentration and drug concentration are calculated according to the following formula, and the ratio of drug to antibody concentration is the average DAR value:

[0555] A 280 = ε D,280 C D + ε A,280 C A

[0556] A 370 = ε D,370 C D + ε A,370 C A

[0557] A 280 and A 370 represent the absorbance of the antibody-drug conjugate aqueous solution at 280 nm and 370 nm, ε A,280 and ε A,370 represent the molar absorbance coefficient of the antibody at 280 nm and 370 nm, ε D,280 and ε D,370 represent the molar absorbance coefficient of the drug-linker at 280 nm and 370 nm, C A represents the antibody concentration, and C D represents the drug-linker concentration.

[0558] The calculated absorbance coefficients are, ε A,280 = 210600, ε A,370 = 0, ε D,280 = 5280, and ε D,370 = 19560. The average DAR value of the antibody-drug conjugate is 6.39784.

[0559] 16.2 Detection of tumor cell killing effect of humanized FGFR2b antibody conjugate (ADC)

[0560] The density of SNU-16 cells was adjusted to 1 × 10 5Cells were added at a concentration of 50 μL / mL to each well of a 96-well plate and cultured overnight. Humanized FGFR2b antibody and antibody conjugate (ADC) were initially prepared at 250 μg / mL and serially diluted 3-fold to 10 concentration points. 50 μL of the corresponding antibody concentration was added to each well and cultured at 37°C. After 120 hours, the fluorescence signal intensity of live cells was detected using a Cell Counting-Lite 3D kit (Novizan, DD1102). Tumor cell killing rate (%) = (1 - average signal value of experimental group / average signal value of negative control group) × 100%.

[0561] As shown in Figure 11, the icinotecan conjugate of the example antibody 27A6H3L3 showed strong killing effects on both FGFR2b-positive tumor cells SNU16 and KATO3. In vitro comparison of the killing curves of the two tumor cells showed that the icinotecan conjugate of antibody 27A6H3L3 was more sensitive to the cytotoxicity of SNU16 tumor cells.

[0562] Example 17. Design and preparation of ADCC-enhanced FGFR2b antibody

[0563] Using the CHO-K1 (FUT8 knockout) cell line, a defucosylated humanized antibody against JH032 was expressed. The obtained light and heavy chain expression vectors were simultaneously transfected into the company's self-developed CHO-K1 (FUT8 knockout) cells using a transfection reagent (Gibco, A29129). The cell density was adjusted to 4 × 10⁶ cells one day before transfection. 6 Cells / ml. Continue culturing CHO-K1 cells (FUT8 knockout) to achieve a cell density of 6 × 10⁶ cells / ml on the day of transfection. 6 Cells / ml. Take 4% OptiPRO. TM SFM complexing medium (purchased from Gibco) was used as the transfection buffer. 0.8 μg of plasmid DNA was added to each milliliter of transfection buffer, mixed well, and then ExpiFecta was added. TM Mix the CHO reagent (purchased from Gibco) thoroughly, and gently pour the cationic transfection reagent / DNA mixture into the Expi CHO-s cell suspension. Gently mix and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM Excipients (purchased from Gibco) and 0.6% ExpiCHO by volume of the transfected culture. TMThe enhancer (purchased from Gibco) was mixed gently and the culture was continuously maintained until the 10th day or when the cell viability was less than or equal to 70%, and then the culture was collected. The ADCC-enhanced FGFR2b antibody was purified by the antibody purification method in Example 3, and finally FGFR2b antibody with a purity of greater than 95% was obtained.

[0564] Example 18. ADCC activity of humanized FGFR2b antibody

[0565] The antibody-dependent cellular cytotoxicity effect (ADCC) induced by the humanized FGFR2b antibody on KATO3 and SNU16 cells was detected by ADCC Bioassay effector cells (Rhinogen, Cat#: RA-CK01). KATO3 and SNU16 cells were centrifuged at 1200 rpm for 5 minutes, and after the cells were washed with PBS, ADCC detection medium (phenol red-free 1640, Gibco, Cat#: 11835030; 1% FBS, Gibco, Cat#: 10099-141C, inactivated at 56°C for 30 minutes) was added to resuspend the cells, and the cell viability was about 95%. The KATO3 and SNU16 cell density was adjusted to 2.5 x 106 cells / ml, and 30 μl of 7.5 x 104 target cells per well was added to a 96-well plate (Corning, Cat#: 3917). The antibody to be tested was diluted to have a working starting concentration of 15 μg / ml, and was serially diluted at a 5-fold gradient, a total of 10 concentrations, and 30 μl per well was added to the corresponding well, and was incubated at 37°C in a 5% CO2 incubator for 45 minutes. The ADCC Bioassay effector cells were collected by centrifugation at 1200 rpm for 5 minutes and were washed with PBS, and the effector cells were resuspended in ADCC detection medium to a density of about 8.33 x 105 cells / ml, and 30 μl of 2.5 x 104 effector cells per well was added to a 96-well plate, and the effector-to-target ratio was 1:3. The sample was incubated at 37°C in a 5% CO2 incubator for 6 hours, and then was taken out to equilibrate to room temperature, and then an equal volume of 90 μL of Bright-Lite Luciferase Assay System (Vazyme, Cat#: DD1204-03) was added to each well, and the tip was gently blown 5 times (without air bubbles), and was placed in the dark for 5 minutes for lysis, and the EnVision 2105 detected the 96-well plate Luciferase. hIgG1 (Yikui Shenzhou, Cat#: MA14MA3102) was used as a homotypic antibody control, and FPA144-AF was used as a reference antibody.

[0566] As shown in Figure 12, the negative homotypic control hIgG1 did not induce ADCC effect, and the FGFR2b humanized antibodies could specifically bind to the target cell surface antigen FGFR2b and induce ADCC effect, and the ADCC activity was comparable to the reference antibody FPA144-AF.

[0567] Example 19. Inhibition of SNU16 cell subcutaneous transplanted tumor in BALB / c-Nu mice by humanized FGFR2b antibody

[0568] The experimental material was selected from BALB / c-Nu mice (Sibeifu (Beijing) Biotechnology Co., Ltd.) female, 6-8 weeks old. After the mice were quarantined, the SNU16 cells in the logarithmic growth phase were collected and adjusted to a concentration of 1E8 / mL. Before inoculation, the cells were mixed with Matrigel at a ratio of 1:1. Each mouse was inoculated with 0.1 mL of the mixed solution subcutaneously in the right anterior axillary region, and each mouse was inoculated with 5E6 cells. When the tumor cell volume grew to about 100 mm 3 , the mice were randomly divided into 5 groups according to the tumor volume: model control group (hIgG1, 10 mg / kg), positive control group (FPA144-AF, 10 mg / kg), test product group (27A6H4L2-AF, 27A6H4L3-AF, 27A6H4L4-AF, 10 mg / kg), 5 mice in each group. After grouping, the administration was started. hIgG1, FPA144-AF and 27A6H4L2-AF, 27A6H4L3-AF, 27A6H4L4-AF were dissolved in PBS to 2 mg / mL, respectively, and were injected intraperitoneally, with a dose of 5 mL / kg, twice a week, for 5 consecutive weeks. The body weight and tumor diameter were measured twice a week, and the tumor volume (Tv = a*b 2 / 2, a represents the long diameter, and b represents the short diameter) and tumor growth inhibition rate (TGI, TGI = (1-RTV 实验组 / RTV 对照组 ) x 100%; RTV = V t / V0, V0 is the tumor volume at the beginning of the experiment, and V t is the tumor volume at the end of the experiment).

[0569] The results are shown in Figure 13. After administration, the inhibition of SNU16 tumor by 27A6H4L4-AF was significantly better than that by FPA144-AF, and the TGI at the end of the experiment was 33.5% and 3.92%, respectively. The test products 27A6H4L2-AF, 27A6H4L3-AF, 27A6H4L4-AF (10 mg / kg) and the positive control FPA144-AF (10 mg / kg) had little effect on the change of animal body weight (shown in Figure 14).

[0570] Example 20. Inhibition of 4T1 cell subcutaneous transplanted tumor in BALB / c mice by humanized FGFR2b antibody Anti-mPD-1 combination

[0571] The experimental material was selected from BALB / c mice (Sibeifu (Beijing) Biotechnology Co., Ltd.) female, 7-9 weeks old. After the mice were quarantined, the logarithmic growth phase of 4T1 cells was collected and adjusted to a concentration of 1E6 / mL. Each mouse was subcutaneously inoculated with 0.1 mL of the mixed solution in the right anterior axillary, and each mouse was inoculated with 1E5 cells. When the tumor cell volume grew to about 100 mm 3 When the tumor cell volume grew to about 100 mm

[0572] The results are shown in Figure 15. The inhibitory effect of 27A6H4L3 and 27A6H4L4 on 4T1 tumor was significantly better than that of Anti-mPD-1; the inhibitory effect of 27A6H4L4 combined with Anti-mPD-1 was significantly better than that of 27A6H4L4 or Anti-mPD-1 alone; the TGI of Anti-mPD-1, 27A6H4L3, 27A6H4L4, 27A6H4L3 combined with Anti-mPD-1, and 27A6H4L4 combined with Anti-mPD-1 at the end of the experiment was 5.66%, 33.87%, 47.51%, 7.51%, and 80.02%, respectively; the calculated q of 27A6H4L4 combined with Anti-mPD-1 was 1.59, indicating that the combination of the two drugs was synergistic; 27A6H4L3 combined with Anti-mPD-1 had no synergistic effect; Anti-mPD-1, 27A6H4L3, 27A6H4L4 alone, and Anti-mPD-1 combination had no significant effect on the change of animal weight (as shown in Figure 16).

Claims

1. An antibody or an antigen-binding fragment thereof specifically binding to FGFR2b, comprising a heavy chain variable region (VH) and a light chain variable region (VL), characterized in that, the VH comprises: (i) a HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence homology thereto; (ii) a HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence homology thereto; and (iii) a HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence homology thereto; and the VL comprises: (iv) a LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence homology thereto; (v) a LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence homology thereto; and (vi) a LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence homology thereto. 2.The antibody or the antigen-binding fragment thereof of claim 1, characterized in that, the anti-VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-11 or a homologous sequence thereof, wherein the homologous sequence has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7-11; the VH comprises: a) an amino acid sequence set forth in SEQ ID NO: 7, or b) an amino acid sequence set forth in SEQ ID NO: 8, or c) an amino acid sequence set forth in SEQ ID NO: 9, or d) an amino acid sequence set forth in SEQ ID NO: 10, or e) an amino acid sequence set forth in SEQ ID NO:

11. 4.The antibody or the antigen-binding fragment thereof of any one of claims 1-3, characterized in that, the anti-VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-17 or a homologous sequence thereof, wherein the homologous sequence has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13-17; the VL comprises: a) a light chain variable region set forth in SEQ ID NO: 13; or b) a light chain variable region set forth in SEQ ID NO: 14; or c) a light chain variable region set forth in SEQ ID NO: 15; or d) a light chain variable region set forth in SEQ ID NO: 16; or e) a light chain variable region set forth in SEQ ID NO: 17; and the HC comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-23 or a homologous sequence thereof, wherein the homologous sequence has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 19-23. ​ ​ ​ ​ ​ ​ ​ ​ ​ 3. The antibody or antigen-binding fragment of claim 1, wherein ​ ​ ​ ​ ​ ​ ​ ​ 5. The antibody or antigen binding fragment of any one of claims 1-3, characterized in that, ​ ​ ​ ​ ​ ​ 6. An antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising a heavy chain (HC) and a light chain (LC), characterized in that, ​ 7. The antibody or antigen-binding fragment thereof of claim 6, wherein the antibody or antigen-binding fragment thereof is, The LC comprises an amino acid sequence selected from any one of SEQ ID NOs: 25-29, or a homologous sequence thereof, wherein the homologous sequence has at least 85% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 25-29.

8. An antibody-drug conjugate that specifically binds to FGFR2b, characterized in that, The antibody or antigen-binding fragment that specifically binds to human FGFR2b of any one of claims 1-7, linked to a cytotoxic drug moiety, with or without a linker.

9. The antibody-drug conjugate according to claim 7, characterized in that, The linker is MC-VC-PAB, and the cytotoxic drug moiety is exatecan.

10. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody or antigen-binding fragment that specifically binds to FGFR2b of any one of claims 1-9.

11. A pharmaceutical composition, characterized by, The antibody or antigen-binding fragment of any one of claims 1-7, or the antibody-drug conjugate of claim 8 or 9, and a pharmaceutically acceptable carrier.

12. Use of the antibody or antigen-binding fragment of any one of claims 1-7, the antibody-drug conjugate of claim 8 or 9, or the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating a disease or condition in a subject.

13. Use according to claim 12, characterized in that, The disease or condition is cancer, wherein the cancer is a FGFR2b expression abnormality associated disease or condition.

14. Use according to claim 12 or 13, characterized in that The cancer is a solid tumor, wherein the solid tumor is selected from any one of ovarian cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, renal cell cancer, head and neck cancer, mesothelioma, melanoma, sarcoma, and brain tumor.

15. Use according to claim 12 or 13, characterized in that, The cancer is a solid tumor, wherein the solid tumor is selected from any one of breast invasive ductal carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, pancreatic adenocarcinoma, colon adenocarcinoma, and gastroesophageal junction adenocarcinoma.

16. The use according to claim 12, characterized in that The treatment further comprises administering a second therapeutic agent, wherein the second therapeutic agent is selected from any one of a second antibody against FGFR2b and a cytotoxic chemotherapeutic agent.

17. Use according to claim 16, characterised in that The second antibody against FGFR2b is an anti-PD-1 antibody; and the cytotoxic chemotherapeutic agent is selected from any one of a platinum agent, paclitaxel, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C, and doxorubicin hydrochloride.

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