Anti-FGFR2b antibody-drug conjugate and use thereof

Abstract

An antibody-drug conjugate (ADC) targeting human FGFR2 and use thereof, particularly an ADC specifically binding to a human FGFR2b isoform, having relatively strong killing activity against tumor cells endogenously expressing FGFR2b, such as KATO III and SNU-16, and especially exhibiting a relatively high tumor inhibition rate in an in-vivo efficacy model.

Description

Anti-FGFR2b antibody-drug conjugates and their applications

[0001] The present disclosure claims priority to the Chinese patent application filed with the Patent Office of China on December 18, 2023, with application number 202311755620.1, entitled “Anti-FGFR2b Antibody-Drug Conjugates and Their Applications,” and the Chinese patent application filed with the Patent Office of China on December 11, 2024, with application number 202411822694.7, entitled “Anti-FGFR2b Antibody-Drug Conjugates and Their Applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of antibodies, and in particular to anti-FGFR2b antibody-drug conjugates and applications thereof. Background Art

[0003] The fibroblast growth factor (FGF) family consists of 22 known FGFs, which are divided into seven subfamilies based on activity and sequence similarity. Currently, only four fibroblast growth factor receptors (FGFRs) 1-4 and their subtypes are known to bind to FGF. When FGFRs bind to ligands and heparin, they induce FGFR dimerization, autophosphorylation of the intracellular tyrosine kinase domain, and activation of multiple intracellular signal transduction pathways such as proliferation (STATs, RAS / p38 / JNKs, and RAS / MAPK / ERK), survival (STATs and PI3K / AKT), and cytoskeleton regulation (PLC / Ca 2+ ), promoting cell proliferation, survival, and differentiation, and participating in the regulation of physiological processes in the body, such as embryonic development, organ development, wound healing, and angiogenesis. Different subtypes have different ligands. FGFR2b is a high-affinity receptor of the FGF7 subfamily. Different subtypes have different tissue expression. FGFR2b is primarily expressed in epithelial tissues (such as the epithelial cells on the surface of the colorectal lumen, the epithelial cells from the base to the lower third of the esophagus, the epithelial parietal cells on the surface of the gastric cavity, the squamous epithelium on the surface of the uterus, and vascular smooth muscle cells), while FGFR2c is primarily expressed in interstitial tissues.

[0004] When FGFR is overexpressed or mutated, it causes abnormalities in the FGFR signaling pathway, promoting tumor development and progression by promoting cell proliferation, survival, migration, and angiogenesis. FGFR2 is overexpressed and mutated in a variety of solid tumors, with the FGFR2b isoform expressed in gastric cancer, squamous cell carcinoma of the non-small cell lung, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer. Gastric cancer is a common and highly prevalent malignant tumor worldwide, with an annual incidence of over one million patients and a mortality rate second only to lung and breast cancer. However, current treatment options for gastric cancer are very limited, especially for the high proportion of HER2-negative patients, who lack effective treatment options. Statistics show that approximately 30% of patients with HER2-negative advanced gastric and gastroesophageal junction cancer are FGFR2b-positive. Immunohistochemistry and sequencing have confirmed that FGFR2b isoforms, but not FGFR2c, are specifically overexpressed in gastric cancer tissues, while FGFR2b expression is undetectable in adjacent tissues and normal organs (except skin). Therefore, FGFR2b may serve as a therapeutic target for these gastric cancer patients. Furthermore, FGFR2b is highly expressed (2+ / 3+ IHC) in 31% of squamous cell non-small cell lung cancers, 13% of triple-negative breast cancers, 40% of ovarian cancers, 4% of pancreatic cancers, and 22% of intrahepatic bile duct cancers.

[0005] Antibody-drug conjugates (ADCs) are a new type of tumor treatment method. They are composed of antibodies (antibodies) that selectively recognize antigens on the surface of cancer cells, a drug payload (payload), and a linker. By combining antibodies targeting specific antigens with cytotoxic drugs, they combine the tumor targeting of antibody drugs with the powerful killing effects of traditional small molecule chemotherapy, accurately attacking tumor cells while reducing damage to normal cells. Summary of the Invention

[0006] In one aspect, the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the general structural formula is Pc-(LD) n ,in,

[0007] D is a cytotoxic drug;

[0008] L is a linker unit;

[0009] Pc is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2b; and

[0010] n is a real number from 1 to 16.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof has one or more of the following properties:

[0012] (1) Not more than 5×10 -8 M's affinity binds to FGFR2;

[0013] (2) binds to human, mouse or monkey FGFR2; and

[0014] (3) Binds to FGFR2b but not to FGFR2c.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1-3 and / or the LCDR1-3 are selected from the following combinations:

[0016] (1) the HCDR1-3 comprise the sequences shown in SEQ ID NOs. 9-11; or / and the LCDR1-3 comprise the sequences shown in SEQ ID NOs. 12-14;

[0017] (2) the HCDR1-3 comprise the sequences shown in SEQ ID NOs. 15-17; or / and the LCDR1-3 comprise the sequences shown in SEQ ID NOs. 18-20;

[0018] (3) the HCDR1-3 comprise the sequences shown in SEQ ID NOs. 21-23; or / and the LCDR1-3 comprise the sequences shown in SEQ ID NOs. 24-26; or,

[0019] (4) The HCDR1-3 and / or the LCDR1-3 comprise a sequence having at least 80% identity, or a sequence having at most 3 insertion, deletion or substitution mutations, compared with any CDR in any of the HCDR1-3 and LCDR1-3 in groups (1) to (3); preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0020] In some embodiments, the HCDRs and LCDRs of the antibody or antigen-binding fragment thereof are divided according to the Kabat numbering system.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following:

[0022] (1) the heavy chain variable region comprises the sequence shown in SEQ ID NO. 1, or / and the light chain variable region comprises the sequence shown in SEQ ID NO. 2;

[0023] (2) the heavy chain variable region comprises the sequence shown in SEQ ID NO. 3, or / and the light chain variable region comprises the sequence shown in SEQ ID NO. 4;

[0024] (3) the heavy chain variable region comprises the sequence shown in SEQ ID NO. 5, or / and the light chain variable region comprises the sequence shown in SEQ ID NO. 6; or,

[0025] (4) The heavy chain variable region and / or the light chain variable region comprise a sequence having at least 80% identity, or a sequence having at most three insertion, deletion or substitution mutations, compared with the heavy chain variable region and / or the light chain variable region in any one of groups (1) to (3); preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from the constant region of human or mouse IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is selected from the human or mouse κ constant region or λ constant region; optionally, the antibody or its antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO.7, and the light chain consists of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO.8.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; and / or, (3) a fully human antibody or fragment thereof;

[0028] Preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0029] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv fragment, scFv, nanobody or affibody.

[0030] In some embodiments, the cytotoxic drug is selected from a microtubule inhibitor, a DNA damaging agent or a topoisomerase inhibitor, the microtubule inhibitor includes but is not limited to dolastatin, auristatin, maytansine, tubulysins and cryptomycins, the DNA damaging agent includes but is not limited to PBD drugs, and the topoisomerase inhibitor includes but is not limited to camptothecin drugs.

[0031] In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor or an auristatin.

[0032] In some embodiments, the cytotoxic drug is selected from the compound represented by formula (DI),

[0033] in,

[0034] R 1 、R 2 The atoms to which it is attached together form a 5-6 membered heterocyclic ring, said 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, said 5-6 membered heterocyclic ring being optionally substituted by one or more deuterium atoms;

[0035] R 4 Selected from H or C1-C3 alkyl;

[0036] R 5 is selected from H, halogen, CN, =O, OH, NH2 or C1-C3 alkyl;

[0037] R 6 Selected from H or C1-C3 alkyl;

[0038] R 7 is selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by deuterium, halogen, CN, ═O, OH, NH2 or C1-C3 alkyl.

[0039] In some embodiments, the R 1 、R 2 Together with the atoms they are connected to form

[0040] In some embodiments, R 4Selected from H.

[0041] In some embodiments, R 5 Selected from H.

[0042] In some embodiments, R 6 Selected from H.

[0043] In some embodiments, R 7 Selected from cyclopropyl.

[0044] In some embodiments, the compound represented by formula (DI) is selected from

[0045] In some embodiments, the cytotoxic drug is selected from

[0046] In some embodiments, the linker unit is selected from Wherein, m1 is selected from integers 2 to 8, L 1 is selected from a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted with one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl, L 2 Selected from The a-end of the linker unit is covalently linked to the antibody or its antigen-binding fragment, and the b-end is covalently linked to the cytotoxic drug.

[0047] In some embodiments, the L 1 is selected from the peptide residue represented by Val-Cit or Gly-Gly-Phe-Gly.

[0048] In some embodiments, m1 is 5.

[0049] In some embodiments, the linker unit is Its a-end is covalently linked to the antibody or its antigen-binding fragment, and its b-end is covalently linked to the cytotoxic drug.

[0050] In some embodiments, n is selected from real numbers of 1 to 16, for example, n is selected from real numbers of 2 to 12, for example, n is selected from real numbers of 4 to 10, for example, n is selected from real numbers of 3 to 9, for example, n is selected from real numbers of 4 to 8, for example, n is selected from real numbers of 6 to 8, for example, n is selected from real numbers of 3 to 5.

[0051] In some embodiments, n is selected from a real number of 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.9 7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0052] In some embodiments, n is a real number selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0053] In some embodiments, n is a real number selected from 3 to 5, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0054] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0055] wherein Pc and n are as defined above.

[0056] In another aspect, the present disclosure provides one or more isolated nucleic acid molecules, which can be nucleotides, deoxynucleotides and / or ribonucleotides of any length in isolated form, encoding the aforementioned antibodies or antigen-binding fragments thereof.

[0057] In some embodiments, the present disclosure provides an expression vector comprising the nucleic acid molecule described above.

[0058] In some embodiments, the present disclosure provides an isolated host cell comprising the nucleic acid molecule described above, or the expression vector described above; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0059] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned general formula Pc-(LD) n The invention relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0060] In some embodiments, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutic drugs.

[0061] On the other hand, the present disclosure provides a method for treating mammalian tumors, comprising administering a therapeutically effective amount of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above to a mammal, preferably a human, in need of such treatment.

[0062] In some embodiments, the methods further comprise the use of additional therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or, for example, chemotherapeutic drugs.

[0063] In some embodiments, the tumor is a tumor that expresses FGFR2b.

[0064] In some embodiments, the tumor is selected from the group consisting of gastric cancer, non-small cell lung cancer, squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

[0065] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above in the preparation of a drug for treating tumors.

[0066] In some embodiments, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutic drugs.

[0067] In some embodiments, the tumor is a tumor that expresses FGFR2b.

[0068] In some embodiments, the tumor is selected from the group consisting of gastric cancer, non-small cell lung cancer, squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

[0069] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above in treating tumors.

[0070] In some embodiments, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors; or such as chemotherapeutic drugs.

[0071] In some embodiments, the tumor is a tumor that expresses FGFR2b.

[0072] In some embodiments, the tumor is selected from the group consisting of gastric cancer, non-small cell lung cancer, squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

[0073] On the other hand, the present disclosure provides a method for treating tumors having the general formula Pc-(LD) n The invention relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. Beneficial effects:

[0074] The antibody-drug conjugates disclosed herein have the following advantages:

[0075] 1. It can specifically recognize the FGFR2b target and has higher selectivity for FGFR2b than FGFR2c;

[0076] 2. Compared with existing monoclonal antibody therapy, it has better tumor inhibition effect and has broad clinical application prospects in the treatment of tumors and other diseases.

[0077] 3. As of the date of completion of this invention, no ADC drug targeting FGFR2b has entered the clinical stage. Through research, the inventors have obtained an FGFR2b ADC that has better efficacy than bemarituzumab at the same or lower dose. In addition, it has been verified that the FGFR2b-ADC disclosed herein can significantly inhibit tumor growth in a bemarituzumab-resistant gastric cancer patient tumor transplant mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1. Binding activity of antibodies and ADCs to tumor cells;

[0079] Figure 2. Inhibitory effect of ADC on tumor cell proliferation in vitro;

[0080] Figure 3. ADC regulation of SNU-16-#232 tumor growth and weight changes in tumor-bearing mice;

[0081] Figure 4. ADC regulation of KATOIII-#729 tumor growth and weight change curve of tumor-bearing mice;

[0082] Figure 5. Comparison of binding activity of control antibodies and ADCs to tumor cells;

[0083] Figure 6. Comparison of the inhibitory effect of ADC on tumor cell proliferation in vitro with that of control ADC. DETAILED DESCRIPTION

[0084] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0085] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0086] Definitions and Explanations of Terms

[0087] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0088] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0089] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0090] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0091] The term FGFR (Fibroblast Growth Factor Receptors) is used herein to refer to fibroblast growth factor receptors, which generally include four types: FGFR1-FGFR4 (also known as CD331-334). FGFRs are members of the tyrosinase receptor family and are type I transmembrane proteins. They consist of an extracellular domain containing a ligand-binding site comprised of two or three immunoglobulin-like domains (IgI to IgIII); a single transmembrane domain; and an intracellular domain containing the tyrosine kinase domain. FGFRs typically function as dimers. The extracellular domain comprises three Ig-like domains, D1 (IgI), D2 (IgII), and D3 (IgIII). D1 and the acidic box form the autoinhibitory region; D2 and D3 are responsible for ligand binding (D2 binds to heparan sulfate on the cell surface, while D3 has two forms, IIIb and IIIc, due to alternative splicing; only the IIIc isoform has been identified in FGFR4). Based on the number of Ig-like domains, FGFRs can be divided into two types: the α type, which contains the IgI, IgII, and IgIII regions; and the β type, which contains only IgII and IgIII. For example, in this disclosure, FGFR2b includes "FGFR2(α)b" and "FGFR2(β)b."

[0092] Human FGF is classified into 22 types of FGF (FGF1 to FGF14 and FGF16 to FGF23). Except for FGF19, FGF21, and FGF23, which are endocrine, the rest are produced paracrinely. Among them, FGF7 can only bind to type IIIb FGFR2. FGFR binding to FGF mediates the activation and transmission of signaling pathways such as RAS-RAF-MAPK, PI3K-AKT, JAK-STAT, and PLCγ. FGFR gene mutations are commonly found in solid tumors such as lung cancer, liver cancer, intrahepatic bile duct cancer, breast cancer, gastric cancer, uterine cancer, and bladder cancer. The types and frequencies of FGFR mutations vary among different cancer types.

[0093] FGFR has the following activities: (1) binds to FGF; (2) this binding causes FGFR dimerization; (3) this dimerization causes FGFR phosphorylation at specific tyrosine residues in FGFR; (4) this phosphorylation promotes the recruitment of adaptor proteins such as FGFR substrate 2α (FRS2α); and (5) this transduces signals generated by FGF stimulation to cells or tissues expressing FGFR or activates signal transduction.

[0094] The terms "FGFR2b" and "FGFR2IIIb" are used interchangeably to refer to the fibroblast growth factor receptor 2IIIb splice form. An exemplary human FGFR2IIIb is shown in GenBank accession number NP_075259.4 (date: July 7, 2013). The FGFR2 IIIb protein typically binds to one or two or more FGFs selected from FGF1, FGF3, FGF7 (KGF), FGF10, FGF22, and FGF23. The FGFR2 IIIb protein may bind to other FGFs and may not bind to mutant forms of the FGFs included in the above group.

[0095] The terms "FGFR2c" and "FGFR2IIIc" are used interchangeably to refer to the fibroblast growth factor receptor 2IIIc splice form. An exemplary human FGFR2IIIc is shown in GenBank accession number NP_000132.3 (date: July 7, 2013). The FGFR2 IIIc protein typically binds to one or two or more FGFs selected from FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21, and FGF23. The FGFR2 IIIc protein may bind to other FGFs and may not bind to mutant forms of the FGFs included in the above group.

[0096] In some embodiments, the antibodies of the present disclosure bind to FGFR2b with high affinity and bind to FGFR2c with low affinity or do not bind to FGFR2c. Typically, the antibodies or antigen-binding fragments thereof of the present disclosure bind to FGFR2b with an affinity or binding activity that is 10-fold, 100-fold, 1000-fold, or 10,000-fold greater than that of FGFR2c.

[0097] The term "antibody-drug conjugate" (ADC) refers to an antibody or antigen-binding fragment thereof linked to a biologically active drug via a stable linker unit. The linker unit can be a covalent bond or a non-covalent interaction such as electrostatic forces. To form an immunoconjugate, various linkers known in the art can be used.

[0098] The term "DAR" or "drug-to-antibody ratio" refers to the average number of small molecule cytotoxic drugs attached to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0099] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0100] The term "antibody" is used in the broadest sense herein and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain, thereby being able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used for transplanting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0101] The term "antibody" herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains. The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0102] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0103] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0104] The term "antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically being present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus and other methods known in the art.

[0105] The term "antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and the like.

[0106] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope on the same antigen.

[0107] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0108] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0109]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0110] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0111] The term "single-domain antibody" (sdAb) herein refers to a single-domain antibody (sdAb) constructed by cloning the variable region of a heavy chain antibody. This sdAb is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a sdAb consisting of only one heavy chain variable region.

[0112] Papain digestion of intact antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains carry free thiol groups. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0113] "Fv fragment" is the smallest fragment produced by IgG and IgM that contains a complete antigen binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as Fab. The VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0114] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO.37) may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).

[0115] The term "diabody" refers to a protein whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0116] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0117] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0118] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0119] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, naked antibodies are not glycosylated when expressed by host cells that do not have their own glycosylation machinery (e.g., glycosylase). In certain embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0120] The term "variable region" refers to the region of an antibody heavy or light chain involved in antigen binding. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity-determining regions because their spatial structure precisely complements the antigen epitope. The CDRs of the heavy chain variable region can be abbreviated as HCDRs, and the CDRs of the light chain variable region can be abbreviated as LCDRs. The terms "framework region" and "FR region" are used interchangeably herein to refer to the amino acid residues in the heavy or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is generally composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0121] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0122] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence relative to the variable domains of antibodies. The "heavy chain constant region" is selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of the full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" is selected from the Fc or CH3 domain.

[0123] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region is selected from a constant kappa domain or a constant lambda domain.

[0124] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can be, for example, removed during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and therefore, the Fc region may or may not include Lys447.

[0125] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0126] 1) Alanine (A), serine (S), threonine (T);

[0127] 2) Aspartic acid (D), glutamic acid (E);

[0128] 3) Asparagine (N), glutamine (Q);

[0129] 4) Arginine (R), Lysine (K), Histidine (H);

[0130] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0131] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0132] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0133] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone bonding or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for direct expression of antibodies of the present invention in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, for example, EP 2101 823B1, which is incorporated herein by reference).

[0134] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the present invention contain polynucleotide sequences and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES) and polyadenylation signal sites to direct the efficient transcription of the genes carried on the expression vector. The expression vectors of the present invention may also contain the following polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0135] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0136] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0137] Herein, “n is a real number from 1 to 16” means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0138] In this article Indicates the junction site.

[0139] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120 (which is incorporated herein by reference). Unless otherwise indicated, wedge and dashed wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0140] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0141] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0142] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0143] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0144] In this article, C m -C n , means having an integer number of carbon atoms in the range of mn.

[0145] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C6 alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0146] The "C1-C6 alkyl group" described herein may further include a "C1-C3 alkyl group".

[0147] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0148] The term "heterocyclyl" refers to a fully saturated or partially saturated monocyclic, fused, spiro or bridged ring group, which contains 1-5 heteroatoms or heteroatom groups (i.e., heteroatom-containing atomic groups) in its ring atoms, wherein the "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6 or 7 ring atoms, and containing 1-3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. The term "5-6 membered heterocyclyl" refers to a heterocyclyl having 5 or 6 ring atoms, wherein the ring atoms contain 1-3 heteroatoms or heteroatom groups independently selected from the above. Examples of 4-membered heterocyclyls include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclyls include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclyls include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclyls include, but are not limited to, diazepanyl. "4-7 membered heterocyclyl" may include, but are not limited to, "4-7 membered heterocyclylalkyl," "5-6 membered heterocyclyl," "5-6 membered heterocyclylalkyl," and the like.

[0149] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0150] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0151] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0152] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0153] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0154] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0155] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0156] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0157] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0158] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0159] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0160] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0161] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0162] The pharmaceutical compositions disclosed herein may also include therapeutic antibodies or chemotherapeutic agents, such as PD-1 / PD-L1 inhibitors. The pharmaceutical compositions may be packaged as a combination within a single drug packaging box, or the anti-FGFR2b antibody-drug conjugates disclosed herein may be packaged separately from other therapeutic agents in separate drug boxes.

[0163] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0164] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0165] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety.

[0166] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0167] Non-limiting example cancers include gastric cancer, non-small cell lung cancer, squamous cell carcinoma, triple-negative breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, intrahepatic bile duct cancer, colorectal cancer and esophageal cancer. In some embodiments, cancer comprises FGFR2 gene amplification. In some embodiments, FGFR2 amplification comprises> 3 FGFR2: CEN10 (chromosome 10 centromere) ratio. In some embodiments, the cancer comprising FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, the degree of overexpression of FGFR2IIIb comprising FGFR2 overexpression is higher than FGFR2IIIc. In some embodiments, the normalized level of FGFR2IIIb expressed by the cancer comprising FGFR2 amplification exceeds 2 times, 3 times, 5 times or 10 times the normalized level of FGFR2IIIc expression. In some embodiments, the expression level is normalized to GUSB. In some embodiments, cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification. In some embodiments, gastric cancer comprises FGFR2 gene amplification. In some embodiments, gastric cancer comprising FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, gastric cancer comprising FGFR2 gene amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the normalized level of gastric cancer expressing FGFR2IIIb comprising FGFR2 gene amplification is more than 2 times, 3 times, 5 times or 10 times the normalized level of FGFR2IIIc expression. In some embodiments, the expression level is normalized to GUSB. In some embodiments, gastric cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification. In some embodiments, overexpression is mRNA overexpression. In some embodiments, overexpression is protein overexpression.

[0168] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

[0169] Example

[0170] Example 1: Preparation of anti-human FGFR2b antibodies and control antibodies

[0171] 1.1 Preparation of anti-human FGFR2b monoclonal antibody

[0172] Our company developed three anti-human FGFR2b monoclonal antibodies, Ab-1, Ab-2, and Ab-3, in-house. The nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of these antibodies were recombined into the expression vector pTT5, which carries a signal peptide and the heavy chain constant region (CH) and light chain constant region (CL) sequences of human IgG1 antibodies, to generate recombinant plasmids expressing VH-CH and VL-CL. Table 1 shows the VH and VL sequences of the antibodies, the signal peptide sequence, and the CH and CL sequences of human IgG1 antibodies. Table 2 shows the Kabat analysis results of the CDR sequences.

[0173] Table 1 Variable region, signal peptide, and constant region sequences of anti-FGFR2b monoclonal antibodies

[0174] Table 2 Kabat analysis results of anti-FGFR2b monoclonal antibody CDR sequences

[0175] The expression plasmid and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma-Aldrich, Catalog No. G8270-5KG) were added. On the sixth day of transfection, the cell culture supernatant was collected.

[0176] Antibodies from cell culture supernatants were purified using a Protein A column (purchased from Cytiva, Catalog No. 17549802). The Protein A column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4) (purchased from Sangon, Catalog No. B548117-0500). The clarified culture supernatant was then loaded onto the Protein A column at a flow rate of 10 mL / min. After loading, the Protein A column was washed with equilibration buffer at a volume 3-5 times the bed volume of the Protein A column. Proteins bound to the Protein A column were eluted with elution buffer (50 mM NaAc-HAc, pH 3.5). The eluted protein was collected and adjusted to a neutral pH by adding Tris buffer (purchased from Sinopharm, Catalog No. 30188336). After the sample was appropriately concentrated, it was further purified using PBS-equilibrated gel chromatography Superdex200 (purchased from Cytiva, product number 28990946) to remove aggregates, collect the monomer peak, and then sterile filter it using a 0.22 μm filter (purchased from Millipore, product number SLGVR13SL). The concentration was determined using Nanodrop (purchased from Thermofisher), the antibody purity was determined using HPLC-SEC, and the antibody endotoxin content was detected using an endotoxin detection kit (purchased from Andus). After passing the test, the samples were aliquoted for use and stored at -80°C.

[0177] 1.2 Preparation of control antibodies

[0178] The antibody sequence of Amgen's anti-FGFR2b positive control antibody Bemarituzumab (FPA144) is the HuGAL-FR21 sequence in patent US8603987B2. The sequence obtained based on this patent is shown in Table 3 and is named FPA144-hIgG1. The antibody sequences of Regeneron's anti-FGFR2b positive control antibodies mAb1 and mAb2 are from patent WO2022087243A1. The sequences obtained based on this patent are shown in Table 3. The isotype control antibody is an antibody against fluorescein isothiocyanate (FITC) that does not bind to FGFR2. The sequence is shown in Table 3 and is named Isotype. Reference 1.1 for the expression and purification of antibodies to obtain control antibodies.

[0179] Table 3 Heavy and light chain sequences of control antibodies Note: Antibody variable region + constant region (italic part)

[0180] Surface plasmon resonance (SPR) analysis was used to determine the affinity of antibodies for human FGFR2(β)b protein. Antibodies were captured using a Protein A chip (Cytiva, Catalog No. 29-127-558). Sample and running buffer used HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cytiva, Catalog No. BR-1006-69). The flow-through cell was set to 25°C and the sample block to 16°C. Both were preconditioned with running buffer. In each cycle, the test antibody was first captured using the Protein A chip. A single concentration of human FGFR2(β)b-His antigen was then injected, and the binding and dissociation processes between the antibody and antigen were recorded. Finally, the chip was regenerated using Glycine pH 1.5 (Cytiva; BR-1003-54). Binding was measured by injecting various concentrations of human FGFR2(β)b-His protein in solution for 240 seconds at a flow rate of 30 μL / min, starting at 200 nM and diluted 1:1 for a total of five concentrations. The dissociation phase was monitored for 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with 10 mM glycine solution (pH 1.5) for 30 seconds at a flow rate of 30 μL / min. Bulk refractive index differences were corrected by subtracting the response obtained from the reference channel. A blank injection (double referencing) was also subtracted. To calculate the apparent KD and other kinetic parameters, a Langmuir 1:1 model was used. The association rates (ka), dissociation rates (kd), and binding affinities (KD) of the antibodies to human FGFR2(β)b-His protein are shown in Table 4. All three candidate humanized antibodies have good affinity for the target antigen, human FGFR2(β)b.

[0181] Table 4 Affinity of antibodies to human FGFR2b protein

[0182] Example 2: Preparation of Antibody-Drug Conjugates

[0183] 2.1 Preparation / Source of Drug-Linker Compounds

[0184] The structure of drug-linker 1 is shown below. Its preparation refers to patent document WO2023217227A1 (Example 39).

[0185] The structure of drug-linker 2 is shown below, and it was purchased from MedChemExpress (MCE) with the product number HY-15575.

[0186] 2.2 Conjugation of Antibodies and Drug-Linkers

[0187] A 10-fold molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to a 2-10 mg / ml antibody solution (1×PBS, pH 7.4). The mixed solution was mixed and incubated at 37°C on a thermostatic metal shaker for 2 hours. A 15-fold molar equivalent of the aforementioned drug-linker 1 compound was then added, and the reaction mixture was mixed and reacted at 25°C for 4 hours. After the reaction, the solution was ultrafiltered three times with 10 mM NaAC-HAc pH 5.5 buffer to remove residual unreacted free small molecules, thereby obtaining an antibody-drug conjugate.

[0188] A 2.0-2.5-fold molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride was added to a 2-10 mg / ml antibody solution (1×PBS, pH 7.4). The mixed solution was mixed and incubated at 37°C on a thermostatic metal shaker for 3 hours. A 15-fold molar equivalent of the aforementioned drug-linker 2 compound was then added, and the reaction mixture was mixed and reacted at 25°C for 16 hours. After the reaction, the solution was ultrafiltered three times with 1×PBS buffer to remove residual unreacted free small molecules to obtain an antibody-drug conjugate.

[0189] 2.3 ADC Purity and DAR Value Testing

[0190] SEC Purity Analysis: SEC-HPLC was used to analyze the protein samples to characterize the size uniformity of the recombinant protein and determine its purity. The HPLC used in this method was an Agilent 1260, with a TSKgel G3000SWXL column (purchased from Tosoh Bioscience). The mobile phase consisted of 200 mM phosphate buffer, pH 7.0, and isopropanol (Merck, 1.01040.4008) (v / v 9:1). The detection temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0191] DAR value determination: The DAR value of the ADC molecule was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-glycan modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37°C for 1 hour to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected onto a Waters ACQUITY Protein BEH molecular exclusion chromatography column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were as follows: spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, parent ion scan range 800-3000, etc. Finally, the mass spectrometry data analysis software Biopharma Finder was used. 4.1. The molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component were calculated by deconvolution processing using the Respect algorithm, and the DAR value of the ADC sample to be tested was calculated based on this information, as shown in Table 5.

[0192] Table 5 Preparation, DAR value and purity of antibody drug conjugates

[0193] Example 3: Binding activity of anti-FGFR2b antibodies and FGFR2b-ADCs to tumor cells

[0194] The human FGFR2b high-expressing KATOIII (purchased from Nanjing Kebai Biotechnology), medium-high-expressing SNU-16 (purchased from Nanjing Kebai Biotechnology), medium-low-expressing OVCAR3 (purchased from ATCC), and low-expressing NUGC4 (purchased from JCRB) tumor cell lines were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was aspirated, washed once with PBS buffer, and then treated and collected with Versene (purchased from Gibco, catalog number: 15040066). After cell counting, the cells were washed twice with PBS phosphate buffer (purchased from Hyclone, catalog number: SH30256.01) and diluted to 2×10 650 μL of cells per milliliter were added to each well of a 96-well FACS reaction plate. 1% (v / v) fetal bovine serum (Excell Bio, Cat. No. FSP500) in PBS phosphate buffer was added as flow cytometry (FACS) buffer and washed twice by centrifugation at 1500 rpm at 4°C. 100 μL of diluted antibody was added to each well and incubated at 4°C for 1 hour. Washed three times by centrifugation with FACS buffer, 50 μL of Alexa Fluor 647 fluorescently labeled goat anti-human IgG (H+L) secondary antibody (Jackson Immuno, Cat. No. 109-605-088) was added to each well and incubated at 4°C for 1 hour. Washed three times by centrifugation with FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze using a FACS system (BD, FACS Canto II).

[0195] The test results are shown in Table 6 and Figure 1. The binding activity of ADC-1 to tumor cell lines was weaker than that of Ab-1. The binding activity of ADC-2 and ADC-3 to tumor cells did not change significantly. The binding activity of ADC-5, ADC-6, and ADC-7 to tumor cells did not change significantly.

[0196] Table 6 Binding activity of anti-FGFR2b antibodies and FGFR2b-ADC to tumor cells

[0197] Example 4: Endocytic effect of anti-FGFR2b antibodies

[0198] The KATOIII cells used in this experiment (purchased from Nanjing Kebai Biotechnology, catalog number: CBP60483) were cultured in RPMI 1640 + 10% FBS (purchased from Gibco, catalog number: 10491; Gibco, catalog number: 10091-148, respectively). KATOIII cells were resuspended in flow cytometry buffer (purchased from Biolegend, catalog number: 420201) and the cell density was adjusted to 2×10 6 50 μL of cell suspension was added to each well of a round-bottom 96-well plate (purchased from Corning, catalog number: 3799) so that the number of cells in each well was 1×10 5. 50 μL of serially diluted antibody (starting concentration 100 nM, 3-fold dilution) was then added, and the cells were incubated at 4°C for 1 hour. After 1 hour, unbound antibody was washed away, and the cells were resuspended in flow cytometry buffer. The control group was continued to be incubated at 4°C, while the endocytosis group was incubated at 37°C for 4 hours. After 4 hours, the cells were washed twice with PBS, and Alexa Fluor 647 fluorescently labeled goat anti-human IgG Fcγ secondary antibody (purchased from Jackson Immuno, cat. no. 109-605-098) was added. The cells were incubated at 4°C for 1 hour, washed twice with PBS, and the Alexa Fluor 647 fluorescence signal (MFI) was analyzed by FACS. GraphPad Prism 9.0 software was used for calculation and graphing. The wells containing the isotype negative control antibody were defined as negative controls. Endocytosis efficiency = (control group MFI - endocytosis group MFI) / control group MFI × 100%.

[0199] The results are shown in Table 7. In this experiment, the negative isotype control Isotype had no obvious endocytic activity in KATO III cells at a saturated concentration of 100 nM. The three anti-FGFR2b antibodies Ab-1, Ab-2, and Ab-3 all had a certain degree of endocytic activity in KATO III cells (endocytic rate 40%-43.5%). The endocytic activity of the anti-FGFR2b monoclonal antibodies of the present invention was better than that of the control antibody FPA144-hIgG1 (endocytic rate 30.5%).

[0200] Table 7 Endocytic activity of anti-FGFR2b antibodies in KATOIII cells

[0201] Example 5: Inhibitory effect of ADC on tumor cell proliferation in vitro

[0202] The culture medium used in this experiment for KATOIII cells (purchased from Nanjing Kebai Biotechnology, catalog number: CBP60483) was RPMI 1640 + 10% FBS, the culture medium for SNU16 cells (purchased from Nanjing Kebai Biotechnology, catalog number: CBP60502) was RPMI 1640 + 10% FBS, and the culture medium for OVCAR3 cells (purchased from ATCC, catalog number: HTB-161) was 1640 + 20% FBS + 10 μg / mL insulin (purchased from Solarbio, catalog number 40112ES25).

[0203] In this experiment, KATOIII was a cell line with high FGFR2b expression, SNU16 was a cell line with medium to high FGFR2b expression, and OVCAR3 was a cell line with medium FGFR2b expression. KATOIII, SNU16, and OVCAR3 cells were digested with Versene (purchased from Gibco, Cat. No. 15040066) and resuspended in their respective culture media to adjust the cell density. KATOIII, SNU16, and OVCAR3 cells were cultured at 5×10 3 Cells / 90 μL were seeded in a 96-well plate (purchased from Corning, catalog number: 3610) and cultured overnight in a 37°C 5% CO2 incubator. The next day, the antibody-drug conjugate was serially diluted with complete culture medium according to the experimental design, and 10 μL of the diluted ADC was transferred to the corresponding well plate. The cell plate was cultured in a 37°C 5% CO2 incubator for 5 days. After 5 days, 100 μL of CellTiter-Glo detection reagent (purchased from Promega, catalog number: G9243, refer to the product manual for usage) was added to each well, and the fluorescence value was read on the Envision instrument (purchased from PerkinElmer, model: Envision 2105) to detect cell viability.

[0204] Positive and negative control groups were established as controls for 0% and 100% cell killing, respectively. The positive control group did not receive the test drug, with all other procedures identical to those of the experimental group. The negative control group did not receive cells, but received the same volume of culture medium, with all other procedures identical to those of the experimental group. The cell killing rate was calculated as follows: Cell killing rate = ((positive control - sample well reading) / (positive control - negative well reading)) × 100%. GraphPad Prism 9.0 software was used for calculation and graphing.

[0205] The results are shown in Table 8 and Figure 2. ADC drugs loaded with different toxins all showed good proliferation inhibitory activity against FGFR2b-expressing tumor cells in vitro, and the IC50 of the killing activity was positively correlated with FGFR2b expression.

[0206] Table 8 Inhibitory effect of ADC on tumor cell proliferation

[0207] Example 6: In vivo efficacy of ADC in gastric cancer-bearing mice

[0208] FGFR2b-positive human gastric cancer cells SNU-16-#232 and KATO-III-#729 were selected to establish an in vivo model in mice (CB17-SCID: female, 6-8 weeks, Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.), and this model was used to evaluate the anti-tumor efficacy of candidate molecules in vivo.

[0209] 6.1 ADC inhibits tumor growth in human gastric cancer SNU-16-#232

[0210] The inoculation time of human gastric cancer cells SNU-16-#232 (self-extracted primary cells, purchased from Nanjing Kebai Biotechnology) was designated as day 0 of the experiment. On day 0 of the experiment, human gastric cancer cells SNU-16-#232 were harvested and inoculated to the desired number and in the logarithmic growth phase (confluence was approximately 80%, and fresh culture medium was replaced with cells one day before inoculation). First, the cell suspension was collected into a 50mL centrifuge tube and centrifuged at 300g for 7 minutes. The cells were resuspended in an appropriate amount of serum-free RPMI1640 culture medium (purchased from Gibco, catalog number: 61870-036). 500μL of the cell suspension was counted on a cell counter (Beckman, SIC-TP-573). Finally, based on the cell count results, the cell density was adjusted to 200×10 using serum-free culture medium. 6 cells / mL, placed on ice, and transferred to the SPF animal room through a transfer window for inoculation modeling. Before inoculation, the above cell suspension was mixed with Matrigel matrix glue (purchased from Corning Bio, product number: 356237) in equal proportions, and 100 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

[0211] After inoculation of gastric cancer cells SNU-16-#232, tumor growth was monitored. When the mean tumor volume was between 100-150 mm 3 About, select the mice that tumor volume is suitable to be randomly divided into groups, 6 mice in each group, each group carries out corresponding drug tail vein administration according to scheme and measures tumor volume and mouse weight change, specific dosage regimen is as shown in Table 9, wherein PBS is negative control group, ADC-4 and ADC-8 are isotype controls, ADC-2, ADC-3, ADC-5, ADC-6 and ADC-7 are ADC candidate molecules, and ADC candidate molecules select the unit weight administration mass identical with isotype control. When the molecule with maytansine structure prepares antibody-drug conjugate, it is usually more toxic than the molecule with camptothecin structure, and the dosage of maytansine ADC is lower than camptothecin ADC when medication is used clinically. Therefore, when designing the experiment, the molecular dosage with drug-connector 2 structure (see Example 2) is lower than the ADC dosage with drug-connector 1 structure.

[0212] The tumor inhibition rate was calculated as follows: tumor inhibition rate (%) = [1-(Vt(treatment group)-V0(treatment group)) / (Vt(isotype control group)-V0(isotype control group))] × 100%, where V0 is the average tumor volume at the time of grouping, and Vt is the average tumor volume at a certain measurement after treatment.

[0213] The results are shown in Table 10 and Figure 3. On day 44 after administration, all groups showed good efficacy, and there was no obvious abnormal change in the weight of the mice during the administration period. ADC-8 showed stronger nonspecific killing than ADC-4, so the molecule with the drug-linker 2 structure had a relatively smaller window of efficacy.

[0214] Table 9 ADC efficacy and dosing regimen in human gastric cancer SNU-16-#232 tumor-bearing mice

[0215] Table 10 ADC efficacy results in mice bearing human gastric cancer SNU-16-#232 tumors

[0216] 6.2 ADC inhibits tumor growth in human gastric cancer KATOIII-#729

[0217] The inoculation time of human gastric cancer cells KATOIII-#729 (self-extracted primary cells, purchased from Nanjing Kebai Biological) was set as day 0 of the experiment. On day 0 of the experiment, human gastric cancer cells KATOIII-#729 that had been cultured to the required number and were in the logarithmic growth phase (confluence was about 80%, and fresh culture medium was replaced for the cells one day before inoculation) were collected and inoculated. First, the culture medium in the cell culture flask was removed and washed twice with phosphate buffered saline (PBS, purchased from Hyclone, catalog number: SH30256.01), followed by addition of an appropriate amount of 0.25% trypsin digestion solution (purchased from Gibco, catalog number: 25200-072), and the bottom of the flask was gently shaken to ensure that the trypsin digestion solution was evenly covered on the cell surface. The flask was then placed in a 37°C environment for digestion for 5 minutes, and then 10% fetal bovine serum (Fetal Bovine Serum) was added. The digestion reaction was terminated with complete medium containing serum, FBS (purchased from Gibco, catalog number: 10091-148 / 2418958P), and the cells adhering to the bottom of the flask were gently blown off the culture flask. The digested cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 350 g for 5 minutes. An appropriate amount of serum-free RPMI1640 medium was aspirated to resuspend the cells and filtered through a 70 μm mesh. 500 μL of the cell suspension was counted on a cell counter. Finally, based on the cell counting results, the cell density was adjusted to 100 × 10 6 cells / mL, placed on ice, and transferred to the SPF animal room through a transfer window for inoculation modeling. Before inoculation, the above cell suspension was mixed with Matrigel matrix gel in equal proportions, and 200 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

[0218] After inoculation of gastric cancer cells KATOIII-#729, tumor growth was monitored. When the mean tumor volume was 200 mm 3Mice with appropriate tumor volumes were randomly divided into groups of 6 mice each. Each group received tail vein administration of the corresponding drugs according to the protocol, and the tumor volume and mouse body weight changes were measured. The specific dosing regimen is shown in Table 11, where PBS was the negative control group, and ADC-4 and ADC-8 were different isotype controls.

[0219] The results are shown in Table 12 and Figure 4. All candidate molecules showed good efficacy, with no significant differences between the molecules. ADC-8 showed stronger nonspecific killing than ADC-4, so the molecule with the drug-linker 2 structure had a relatively smaller efficacy window.

[0220] Table 11 ADC efficacy and dosing regimen in human gastric cancer KATOIII-#729 tumor-bearing mice

[0221] Table 12 ADC efficacy results in mice bearing human gastric cancer KATOIII-#729

[0222] Example 7: FGFR2b-ADC efficacy verification

[0223] 7.1 Preparation of Control FGFR2b-ADC

[0224] The preparation method of the positive control FGFR2b ADC refers to patents WO2022087243A1 and WO2022015656A1.

[0225] The IgG of mAb1 and mAb2 were mutated at N297Q to achieve site-directed conjugation. The heavy chain sequences of the mutated mAb1 and mAb2 are shown in Table 13. Referring to the antibody expression and purification methods in Section 1.1 of Example 1, antibodies mAb1 (N297Q) and mAb2 (N297Q) were obtained.

[0226] Table 13 Comparison of sequences of anti-FGFR2b antibody IgG after N297Q mutation Note: Antibody variable region + constant region (italic part)

[0227] The structure of amino-azide linker AL1 (CAS: 134179-38-7) is shown below. It was purchased from MCE with the product number HY-W021401.

[0228] The structure of branch connector BL7 (CAS: 2253947-15-6) is shown below. Its preparation refers to patent document WO2018218004A1.

[0229] The drug-linker 3 structure (CAS No.: 2699066-62-9) is shown below and was purchased from MCE with the product number HY-157407.

[0230] Transglutaminase (purchased from MCE, Catalog No. HY-P2962) was dissolved in PBS to 10 mg / mL. The purified antibody sample was diluted to 2 mg / mL in PBS. The dissolved enzyme and antibody were mixed at a 10:1 ratio. A 150-fold molar equivalent of linker AL1 or BL7 was added and the mixture was incubated at 37°C overnight. Excess enzyme and linker were then removed using a Protein A column. MS analysis revealed increases of 804 Da for DAR4 and 1232 Da for DAR8, respectively.

[0231] Drug-linker 3 was conjugated to the linker of the antibody using click chemistry. AL1- or BL7-linked antibodies (1× PBS, pH 7.4) were mixed with ≥10 molar equivalents of 10 mg / mL drug-linker 3 in DMSO containing 5%-15% (v / v) organic solvent. The reaction was allowed to proceed overnight at room temperature. Excess free small molecules were removed using cation exchange, and the sample was then dialyzed into storage buffer using a dialysis cassette.

[0232] Referring to the ADC purity and DAR value detection method in Example 2.3, the SEC purity and DAR value of the ADC sample were obtained, as shown in Table 14.

[0233] Table 14 Preparation, DAR value and purity of antibody drug conjugates

[0234] 7.2 Binding activity of anti-FGFR2b antibodies and FGFR2b-ADCs to tumor cells

[0235] The binding activity of anti-FGFR2b antibodies and FGFR2b-ADCs to tumor cells was tested using the method described in Example 3. The results are shown in Table 15 and Figure 5 . The binding activity of the anti-FGFR2b antibodies and FGFR2b-ADCs disclosed herein to tumor cells was stronger than that of the control anti-FGFR2b antibodies and FGFR2b-ADCs.

[0236] Table 15 Binding activity of anti-FGFR2b antibodies and FGFR2b-ADC to tumor cells

[0237] 7.3 Inhibitory Effect of FGFR2b-ADC on Tumor Cell Proliferation in Vitro

[0238] The in vitro proliferation inhibitory activity of FGFR2b-ADC on tumor cells was tested using the method of Example 5. The results are shown in Table 16 and Figure 6 . The in vitro proliferation inhibitory activity of the FGFR2b-ADC of the present disclosure on tumor cells was stronger than that of the control FGFR2b-ADC.

[0239] Table 16 Inhibitory effect of ADC on tumor cell proliferation

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD) n ,in, D is a cytotoxic drug; L is a linker unit; Pc is an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b; and n is a real number from 1 to 16; Wherein, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1-3 or / and the LCDR1-3 are selected from the following combinations: (1) the HCDR1-3 are sequences shown in SEQ ID NOs. 9-11; or / and the LCDR1-3 are sequences shown in SEQ ID NOs. 12-14; (2) the HCDR1-3 are sequences shown in SEQ ID NOs. 15-17; or / and the LCDR1-3 are sequences shown in SEQ ID NOs. 18-20; (3) the HCDR1-3 are sequences shown in SEQ ID NOs. 21-23; or / and the LCDR1-3 are sequences shown in SEQ ID NOs. 24-26; or, (4) The HCDR1-3 and / or the LCDR1-3 are sequences having at least 80% identity with any CDR in the HCDR1-3 and LCDR1-3 in any of groups (1) to (3), or sequences having at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following: (1) the heavy chain variable region is the sequence shown in SEQ ID NO.1, or / and the light chain variable region is the sequence shown in SEQ ID NO.2; (2) the heavy chain variable region is the sequence shown in SEQ ID NO.3, or / and the light chain variable region is the sequence shown in SEQ ID NO.4; (3) the heavy chain variable region is the sequence shown in SEQ ID NO.5, and / or the light chain variable region is the sequence shown in SEQ ID NO.6; or, (4) The heavy chain variable region and / or the light chain variable region are sequences having at least 80% identity to the heavy chain variable region and / or the light chain variable region in any one of groups (1) to (3), or sequences having at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, wherein: The antibody or its antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from the constant region of human or mouse IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is selected from the human or mouse κ constant region or λ constant region; optionally, the antibody or its antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO.7, and the light chain consists of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO.

8.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The antibodies or antigen-binding fragments thereof are: (1) chimeric antibodies or fragments thereof; (2) a humanized antibody or a fragment thereof; and / or, (3) a fully human antibody or a fragment thereof; preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 4, wherein: The antigen binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv fragment, scFv, nanobody or affibody.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents or topoisomerase inhibitors; the tubulin inhibitors include but are not limited to dolastatins, auristatins, maytansines, tubulysins and cryptomycins, the DNA damaging agents include but are not limited to PBD drugs, and the topoisomerase inhibitors include but are not limited to camptothecin drugs.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, wherein: The cytotoxic drug is selected from topoisomerase I inhibitors or auristatins.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein: The cytotoxic drug is selected from the compound represented by formula (DI), in, R 1 , R 2 The atoms to which it is attached together form a 5-6 membered heterocyclic group, the 5-6 membered heterocyclic group contains 1 or 2 oxygen atoms as ring atoms, the 5-6 membered heterocyclic group is optionally substituted by one or more D atoms; R 4 Selected from H or C1-C3 alkyl; R 5 is selected from H, halogen, CN, =O, OH, NH2 or C1-C3 alkyl; R 6 Selected from H or C1-C3 alkyl; R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: The R 1 , R 2 Together with the atoms they are connected to form 10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: R 4 Selected from H.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: R 5 Selected from H.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: R 6 Selected from H.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, wherein: R 7 Selected from cyclopropyl.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 13, wherein: The compound represented by formula (DI) is selected from 15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 14, wherein: The cytotoxic drug is selected from 16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the linker unit is selected from in, m1 is selected from integers 2 to 8, L 1 A peptide residue selected from 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl, L 2 Selected from The a-end of the linker unit is covalently linked to the antibody or its antigen-binding fragment, and the b-end is covalently linked to the cytotoxic drug.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 16, wherein: The L 1 A peptide residue selected from the group consisting of Val-Cit and Gly-Gly-Phe-Gly.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 16, wherein: m1 is 5.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 16 to 18, wherein: The linker unit is Its a-end is covalently linked to the antibody or its antigen-binding fragment, and its b-end is covalently linked to the cytotoxic drug.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: n is selected from real numbers of 1 to 16, for example, n is selected from real numbers of 2 to 12, for example, n is selected from real numbers of 4 to 10, for example, n is selected from real numbers of 3 to 9, for example, n is selected from real numbers of 4 to 8, for example, n is selected from real numbers of 6 to 8, for example, n is selected from real numbers of 3 to 5.

21. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: n is a real number selected from 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 , 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.

0.

22. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: n is a real number selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: n is a real number selected from 3 to 5, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.

0.

24. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof: wherein Pc comprises the antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, and n comprises a real number described in any one of claims 20 to 23.

25. One or more isolated nucleic acid molecules, which may be nucleotides, deoxynucleotides and / or ribonucleotides of any length in isolated form, encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 25.

26. An expression vector comprising the nucleic acid molecule of claim 25.

27. An isolated host cell, wherein Comprising the nucleic acid molecule of claim 25, or the expression vector of claim 26; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

28. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24 and a pharmaceutically acceptable excipient; preferably, the pharmaceutical composition further comprises other therapeutic agents or chemotherapeutic drugs; preferably, the other therapeutic agents comprise PD-1 / PD-L1 inhibitors.

29. A method for treating a tumor in a mammal, wherein: The method comprises administering a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24 or a drug combination according to claim 28 to a mammal, preferably a human, in need of such treatment; preferably, the method further comprises the use of an additional therapeutic agent, such as other antibodies, typically comprising a PD-1 / PD-L1 inhibitor, or, for example, a chemotherapeutic drug; preferably, the tumor is a tumor expressing FGFR2b; more preferably, the tumor is selected from the group consisting of: gastric cancer, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

30. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or the pharmaceutical composition according to claim 28, in the preparation of a medicament for treating a tumor; preferably, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors, or such as chemotherapeutic drugs; preferably, the tumor is a tumor expressing FGFR2b; more preferably, the tumor is selected from the group consisting of: gastric cancer, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, colorectal cancer.

31. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or the pharmaceutical composition according to claim 28 in treating tumors; preferably, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors, or such as chemotherapeutic drugs; preferably, the tumor is a tumor expressing FGFR2b; more preferably, the tumor is selected from the group consisting of: gastric cancer, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, colorectal cancer.

32. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or a pharmaceutical composition according to claim 28 for treating a tumor; preferably, the pharmaceutical composition further comprises other therapeutic agents, such as other antibodies, typically comprising PD-1 / PD-L1 inhibitors, or such as chemotherapeutic drugs; preferably, the tumor is a tumor expressing FGFR2b; more preferably, the tumor is selected from the group consisting of: gastric cancer, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

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