Anti-FGFR2b antibody, antibody-drug conjugate thereof, and use thereof

Abstract

Provided are a specific binding antibody targeting a fibroblast growth factor receptor 2b (FGFR2b), an antibody-drug conjugate and a method for using same, and a composition containing the molecule. An anti-FGFR2b antibody, a conjugate or composition thereof may be used as an independent therapy for treatment of cancer / tumor, or used in combination with other therapies / or other anti-cancer agents.

Description

Anti-FGFR2b antibody, antibody-drug conjugate and use thereof Technical Field

[0001] The present invention relates to antibodies that specifically bind to fibroblast growth factor receptor 2b (FGFR2b), antibody-drug conjugates and methods of using the same, as well as compositions containing the same. Background Art

[0002] The fibroblast growth factor (FGF) receptor tyrosine kinase (RTK) family is composed of fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3 and FGFR4, and covers high affinity receptors for up to 18 different FGF ligands. Receptor is a transmembrane tyrosine kinase, and the downstream pathway driven by FGFR signal transduction comprises mitogen-activated protein kinase (MAPK) and AKT pathways that are crucial to cell proliferation, differentiation, survival and migration. The binding of FGF ligands to receptors induces the dimerization of FGF:FGFR complexes. Dimerization causes kinase activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor, as well as the activation of downstream signal transduction of phosphoinositide 3-kinase (PI3K)-AKT and MAPK extracellular signal-regulated kinase (ERK) pathways. The alternative splicing of the IgIII loop in FGFR1-3 produces FGFRIIIb or FGFRIIIc isoforms.

[0003] FGFR2 amplification has been reported in various cancers. FGFR2 amplification affects signaling without altering the receptor's intrinsic kinase activity. FGFR2 protein is overexpressed in approximately 3% of breast cancers, including triple-negative breast cancer, and in approximately 10% of gastric / esophageal cancers. FGFR2 overexpression has also been found in other cancers, including colon, hepatocellular carcinoma, pancreatic, ovarian, uterine, cervical, endometrial, bladder, lung, colon, gliomas, and head and neck cancers. In addition, mutations in the FGFR2 gene have been reported in approximately 12% of endometrial cancers. FGFR2 overexpression has been associated with poor survival in patients with gastric cancer.

[0004] Both preclinical and clinical studies have demonstrated that FGFR-amplified tumors are sensitive to FGFR inhibition and therefore to targeted therapy. Treatment of FGFR2-positive tumors with anti-FGFR2 therapies such as Bemarituzumab (FPA144) has been reported to result in improvements in early survival and advanced disease. “FPA144: A Therapeutic Antibody for Treating Patients with Gastric Cancers Bearing FGFR2 Gene Amplification” (American Association for Cancer Research (AACR) Abstract ID 5446); Powers et al. (2016) “FPA144, A Therapeutic Monoclonal Antibody Targeting the FGFR2b Receptor, Promotes Antibody Dependent Cell-Mediated Cytotoxicity and Stimulates Sensitivity to PD-1 in the 4T1 Syngeneic Tumor Model” (American Association for Cancer Research Abstract ID 1407); Lee et al. (2016) “Antitumor Activity and Safety of FPA144, an ADCC-Enhanced, FGFR2b Isoform-Selective Monoclonal Antibody, in Patients with FGFR2b+ Gastric Cancer and Advanced Solid Tumors” (American Society of Clinical Oncology (ASCO) Abstract ID 2502).

[0005] There remains an unmet medical need for improved anticancer drugs that are effective in FGFR2-expressing cancers. Summary of the Invention

[0006] The present invention provides novel anti-FGFR2b antibodies, antigen-binding fragments thereof, and antibody-drug conjugates, which have advantages such as high affinity and specificity for human FGFR2b. The anti-FGFR2b antibodies, antigen-binding fragments thereof, and anti-FGFR2b antibody-drug conjugates provided herein can be used as standalone therapies or in combination with other therapies or other anticancer agents for the treatment of conditions such as cancer and tumors.

[0007] In a first aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that can specifically bind to FGFR2b.

[0008] In some embodiments, the present invention provides an anti-FGFR2b antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising:

[0009] (1) the three CDRs (HCDR1, HCDR2, HCDR3) contained in VH as shown in SEQ ID NO: 7 or variants thereof; and / or the three CDRs (LCDR1, LCDR2, LCDR3) contained in VL as shown in SEQ ID NO: 8 or variants thereof; or,

[0010] (2) the three CDRs (HCDR1, HCDR2, HCDR3) contained in VH as shown in SEQ ID NO: 12 or its variants; and / or the three CDRs (LCDR1, LCDR2, LCDR3) contained in VL as shown in SEQ ID NO: 13 or its variants.

[0011] In some embodiments, the present invention provides an anti-FGFR2b antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising:

[0012] 1) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1;

[0013] HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2;

[0014] A HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 3;

[0015] LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 4;

[0016] LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 5; and

[0017] LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 6; or

[0018] 2) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1;

[0019] HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2;

[0020] A HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 11;

[0021] LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 4;

[0022] LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 5; and

[0023] A LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 6.

[0024] In some embodiments, the present invention provides anti-FGFR2b antibodies and antigen-binding fragments thereof that specifically bind to FGFR2b, comprising a heavy chain variable region (VH), wherein:

[0025] (1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 7, or consists of SEQ ID NO: 7; or

[0026] (2) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12, or consists of SEQ ID NO: 12.

[0027] In some embodiments, the present invention provides anti-FGFR2B antibodies and antigen-binding fragments thereof that specifically bind to FGFR2B, comprising a light chain variable region (VL), wherein:

[0028] (1) the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 8, or consists of SEQ ID NO: 8; or

[0029] (2) The light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13, or consists of SEQ ID NO: 13.

[0030] In other embodiments, the present invention provides anti-FGFR2b antibodies and antigen-binding fragments thereof that specifically bind to FGFR2b, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0031] (1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:7, or consists of SEQ ID NO:7; the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:8, or consists of SEQ ID NO:8; or

[0032] (2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12, or consists of SEQ ID NO: 12; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13, or consists of SEQ ID NO: 13.

[0033] In some embodiments, the above-mentioned antibodies or antigen-binding fragments thereof further comprise heavy chain and / or light chain constant region sequences derived from the human antibody germline consensus sequence. The light chain constant region is preferably a human kappa or lambda chain constant region. The heavy chain constant region can be a gamma, mu, alpha, delta, or epsilon chain. In some embodiments, the heavy chain constant region is preferably derived from the constant region sequence of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the light chain constant region comprises the sequence shown in SEQ ID NO: 17, or consists of the sequence. In other embodiments, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 16, or consists of the sequence.

[0034] It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein amino acid positions are identified according to the EU index system of Kabat et al. (1991).

[0035] In a specific embodiment, the present invention provides an anti-FGFR2b antibody and an antigen-binding fragment thereof that specifically binds to FGFR2b, which comprises a heavy chain and a light chain, wherein

[0036] 1) the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or consists of SEQ ID NO: 9; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or consists of SEQ ID NO: 10; or

[0037] 2) the heavy chain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14, or consists of SEQ ID NO: 14; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15, or consists of SEQ ID NO: 15.

[0038] In a second aspect, the present invention provides an anti-FGFR2b antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof as described in formula (I):

[0039] Ab-(LD)m[Formula (I)]

[0040] in,

[0041] L is the joint unit;

[0042] D is a small molecule drug with cytotoxicity;

[0043] m is the average number of LD units coupled to Ab, and m is selected from 1 to 10, preferably 2 to 8;

[0044] Ab is an anti-FGFR2b antibody or an antigen-binding fragment thereof, comprising:

[0045] (1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;

[0046] or

[0047] (2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0048] In some embodiments, the small molecule drug D of the present invention is a monomethyl auristatin compound, a camptothecin compound, or a maytansine alkaloid.

[0049] In some embodiments, the monomethyl auristatin compound may be monomethyl auristatin E (MMAE).

[0050] or monomethyl auristatin F (MMAF)

[0051] In some embodiments, the maytansine alkaloid may be DM1, DM3 or DM4.

[0052] In some embodiments, the camptothecin derivative compound may be (Ixitetac),

[0053] In some embodiments, the antibody drug conjugate comprises multiple D components, and the multiple D components can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substance or pharmaceutically active ingredient.

[0054] In some embodiments, the antibody drug conjugate has a drug to antibody ratio (DAR) of 1-15, such as a DAR of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0055] In some embodiments, the DAR is an average DAR.

[0056] In some embodiments, the average DAR is 1-15, such as 1-10.

[0057] As a specific embodiment, the average DAR of the antibody-drug conjugate of the present invention is preferably 2-10, such as 2-8.

[0058] As a specific embodiment, when D is a camptothecin compound, the average DAR of the antibody-drug conjugate of the present invention is preferably 2-8.

[0059] In some embodiments, the cytotoxin is covalently linked to the anti-FGFR2b antibody or antigen-binding fragment thereof in a non-site-specific manner or in a site-specific manner via a linker.

[0060] As used herein, "linker," "linker unit," and "linker" are used interchangeably.

[0061] The "drug-containing linker" used in the present invention refers to a compound obtained by directly or indirectly covalently bonding a drug, such as a small molecule drug with cytotoxicity as described in D, to a linker.

[0062] In some embodiments, the L is a combination of one or more L'.

[0063] In some embodiments, the L' is selected from carbonyl, amino, amide, aminoacyl, -(PEG) n -、-(CH2) n -, containing heteroatoms -(CH2) n-, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimido (mc), maleimidopropionyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl (PAB), dimethylethylenediamine (DMED), N-succinimidyl-4-(2 -pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC).

[0064] In some embodiments, n is independently selected from integers ranging from 1 to 20.

[0065] As a specific embodiment, when L' is selected as (PEG) n When n is independently preferably 2, 4, 6 or 8.

[0066] As a specific embodiment, when L' is selected as (CH2) n When n is independently an integer of 1 to 16,

[0067] As a specific embodiment, when L' is selected as (CH2) containing heteroatoms n When n is independently an integer from 1 to 16, the heteroatom is selected from N, O or S. The number of heteroatoms is independently an integer from 1 to 8.

[0068] In some embodiments, the L may be J-L1-L2-X-L3.

[0069] In some embodiments, the J is selected from

[0070] In some embodiments, L1 is selected from a single bond, -(PEG) n -or-(CH2) n -, the -(CH2) n- may optionally contain 1 to 6 heteroatoms selected from N, O, and S; wherein n is independently selected from an integer from 1 to 20. Preferably, L1 is selected from -(PEG) n -, wherein n is independently 2, 4, 6 or 8.

[0071] In some embodiments, L2 is selected from a single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, or a combination of one or more thereof; wherein the -CH2- and -NH- may be optionally replaced by C 1～6 Alkyl or halogen substituted 1 to 3 times.

[0072] In some embodiments, X is selected from a single bond or a combination of 1 to 4 X', wherein X' is independently selected from amino acids, such as glycine, alanine, phenylalanine, valine, lysine, and citrulline. In some embodiments, X is selected from a single bond or valine-citrulline (val-cit), valine-alanine (val-ala), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), glycine-glycine-phenylalanine-glycine (GGFG).

[0073] In some embodiments, L3 is selected from a single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, -phenyl-, -cyclopropyl-, -cyclobutyl-, -cyclohexyl-, or a combination of one or more thereof; wherein the -CH2-, -NH-, -phenyl-, -cyclopropyl-, -cyclobutyl-, -cyclohexyl- may be optionally replaced by C 1～6 Alkyl, halogen, cyano or hydroxyl groups are substituted 1 to 3 times.

[0074] In another aspect, the present invention provides three linker units of formula (LD) - cytotoxic small molecule drugs, such as formula Cpd3, formula Cpd5 and formula Cpd6:

[0075] In some embodiments, the linker-toxins represented by Formula Cpd3, Formula Cpd5, and Formula Cpd6 provided herein are suitable for coupling to anti-FGFR2b antibodies or antigen-binding fragments thereof.

[0076] Furthermore, the present invention provides an anti-FGFR2b antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the anti-FGFR2b antibody-drug conjugate structure is shown in Formula (I-C3), Formula (I-C5), and Formula (I-C6):

[0077] in,

[0078] m is the average number of connections, and m is independently selected from 1 to 10;

[0079] Ab is an anti-FGFR2b antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;

[0080] or

[0081] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0082] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7, or at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8, or at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0083] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 12, or at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 13, or at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0084] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9, or at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain having an amino acid sequence as shown in SEQ ID NO: 10, or at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0085] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 14, or at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain having an amino acid sequence as shown in SEQ ID NO: 15, or at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0086] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8.

[0087] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 13.

[0088] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9 and a light chain having an amino acid sequence as shown in SEQ ID NO: 10.

[0089] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 14 and a light chain having an amino acid sequence as shown in SEQ ID NO: 15.

[0090] In some embodiments, the antibody or antigen-binding fragment of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0091] As a specific embodiment, the SEQ ID: 12 fragment of the present invention is a humanized antibody.

[0092] As a specific embodiment, the SEQ ID: 13 fragment of the present invention is a humanized antibody.

[0093] As a specific embodiment, the SEQ ID: 14 ​​fragment of the present invention is a humanized antibody.

[0094] As a specific embodiment, the SEQ ID: 15 fragment of the present invention is a humanized antibody.

[0095] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention is Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or sdAb.

[0096] In some embodiments, the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention is of any IgG subtype, such as IgG1, IgG2, IgG3, and IgG4; preferably, the antibody is low or non-fucosylated.

[0097] In some embodiments, the Ab of the present invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0098] In some embodiments, the Ab of the present invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0099] In some embodiments, the Ab of the present invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0100] In some embodiments, the Ab comprises a heavy chain variable region (VH).

[0101] In some embodiments, the heavy chain variable region comprises three complementarity determining regions (HCDRs) from a heavy chain variable region.

[0102] In some embodiments, the Ab comprises a light chain variable region (VL).

[0103] In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs) from a light chain variable region.

[0104] In some embodiments, the Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0105] In some embodiments, the heavy chain variable region of the present invention:

[0106] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 7;

[0107] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7; or

[0108] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 7 or consists of said amino acid sequence, preferably, said amino acid sequence changes do not occur in the CDR regions.

[0109] In some embodiments, the heavy chain variable region of the present invention:

[0110] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 12;

[0111] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0112] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12, or consists of said amino acid sequence. Preferably, said amino acid sequence changes do not occur in the CDR regions.

[0113] In some embodiments, the light chain variable region of the present invention:

[0114] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8;

[0115] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8; or

[0116] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 8 or consists of said amino acid sequence, preferably, said amino acid sequence changes do not occur in the CDR regions.

[0117] In some embodiments, the light chain variable region of the present invention:

[0118] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13;

[0119] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0120] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 13, or consists of said amino acid sequence. Preferably, said amino acid sequence changes do not occur in the CDR regions.

[0121] In some embodiments, the complementarity determining regions HCDR1, HCDR2, and HCDR3 of the three VHs of the present invention are:

[0122] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 7; or

[0123] (ii) relative to the sequence of (i), the single HCDR region comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, more preferably conservative amino acid substitution).

[0124] In some embodiments, the complementarity determining regions HCDR1, HCDR2, and HCDR3 of the three VHs of the present invention are:

[0125] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 12; or

[0126] (ii) relative to the sequence of (i), the single HCDR region comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, more preferably conservative amino acid substitution).

[0127] In some embodiments, the complementarity determining regions LCDR1, LCDR2, and LCDR3 of the three VLs of the present invention are:

[0128] (i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 8; or

[0129] (ii) relative to the sequence of (i), the single LCDR region comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions).

[0130] In some embodiments, the complementarity determining regions LCDR1, LCDR2, and LCDR3 of the three VLs of the present invention are:

[0131] (i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL of SEQ ID NO: 13; or

[0132] (ii) relative to the sequence of (i), the single LCDR region comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions).

[0133] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 1.

[0134] In some embodiments, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence, or HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 2.

[0135] In some embodiments, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, or consists of the amino acid sequence, or HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 3.

[0136] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 4.

[0137] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 5.

[0138] In some embodiments, LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 6.

[0139] In some embodiments, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, or consists of the amino acid sequence, or HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the sequence of SEQ ID NO: 11.

[0140] In some embodiments, the Ab described herein comprises a heavy chain constant region.

[0141] In some embodiments, the Ab described herein comprises a light chain constant region.

[0142] In some embodiments, the Ab described herein comprises a heavy chain constant region and a light chain constant region.

[0143] In some embodiments, the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4.

[0144] In some embodiments, the heavy chain of the present invention

[0145] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9;

[0146] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

[0147] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9, or consists of said amino acid sequence.

[0148] In some embodiments, the heavy chain of the present invention

[0149] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14;

[0150] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0151] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 14, or consists of said amino acid sequence.

[0152] In some embodiments, the light chain of the invention

[0153] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10;

[0154] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10; or

[0155] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10, or consists of said amino acid sequence.

[0156] In some embodiments, the Ab described in the present invention specifically binds to FGFR2b and comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:7, and / or the three complementarity determining regions LCDR1, LCDR2 and LCDR2 contained in the VL as shown in SEQ ID NO:8.

[0157] In some embodiments, the light chain of the invention

[0158] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15;

[0159] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 15; or

[0160] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 15, or consists of said amino acid sequence.

[0161] In some embodiments, the Ab described in the present invention specifically binds to FGFR2b and comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:7, and / or the three complementarity determining regions LCDR1, LCDR2 and LCDR2 contained in the VL as shown in SEQ ID NO:8.

[0162] In some embodiments, the Ab described in the present invention specifically binds to FGFR2b and comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 12, and / or the three complementarity determining regions LCDR1, LCDR2 and LCDR2 contained in the VL as shown in SEQ ID NO: 13.

[0163] In some embodiments, the Ab of the present invention comprises

[0164] VH comprising or consisting of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0165] VL, which comprises or consists of the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0166] In some embodiments, the Ab of the present invention comprises

[0167] VH comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0168] VL, which comprises or consists of the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0169] The present invention provides an antibody-drug conjugate targeting human FGFR2b, its isomers, its pharmaceutically acceptable salts, or mixtures thereof, wherein the antibody-drug conjugate has the following advantages:

[0170] (1) Binds to target cells expressing human FGFR2b with high affinity;

[0171] (2) Entering cells through endocytosis and killing target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;

[0172] (3) treating, preventing, or ameliorating a condition (e.g., cancer) associated with abnormal function or expression of FGFR2b in a subject, or treating, preventing, or ameliorating one or more symptoms of the disease;

[0173] (4) reducing or inhibiting tumor growth or progression in a subject having a tumor that expresses FGFR2b;

[0174] (5) inducing regression (e.g., long-term regression) of tumors expressing FGFR2b;

[0175] (6) exert cytotoxic activity in cells expressing FGFR2b;

[0176] In a third aspect, the present invention provides an isolated polynucleotide molecule encoding any one of the antibodies or antigen-binding fragments thereof according to the first aspect.

[0177] In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect. In some embodiments, the vector is an expression vector.

[0178] In a fifth aspect, the present invention provides a host cell comprising the vector of the fourth aspect or the nucleic acid molecule of the third aspect. In some embodiments, the host cell is prokaryotic, such as Escherichia coli. In other embodiments, the host cell is eukaryotic, such as HEK293 cells, CHO cells, yeast cells, or plant cells.

[0179] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the anti-FGFR2b antibody or antigen-binding fragment thereof according to the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or a mixture thereof according to the second aspect, and a pharmaceutically acceptable excipient.

[0180] In a seventh aspect, the present invention provides use of the anti-FGFR2b antibody or antigen-binding fragment thereof according to the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to the second aspect; or the pharmaceutical composition according to the sixth aspect in the preparation of a medicament for treating and / or preventing FGFR2b-mediated diseases or conditions.

[0181] In one embodiment, the present invention provides the anti-FGFR2b antibody or antigen-binding fragment thereof of the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt, or mixture thereof of the second aspect; or the pharmaceutical composition of the sixth aspect for use in treating and / or preventing. In a specific embodiment, the present invention provides the anti-FGFR2b antibody or antigen-binding fragment thereof of the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt, or mixture thereof of the second aspect; or the pharmaceutical composition of the sixth aspect for use in treating and / or preventing a disease or condition mediated by FGFR2b.

[0182] In an eighth aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by FGFR2b, comprising administering to a subject in need thereof an effective amount of the anti-FGFR2b antibody or antigen-binding fragment thereof according to the first aspect; or the antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or a mixture thereof according to the second aspect; or the pharmaceutical composition according to the sixth aspect.

[0183] In some embodiments, the FGFR2b-mediated disease or condition described in the various aspects above is cancer or tumor.

[0184] In some embodiments, the cancer or tumor is selected from lung cancer, squamous cell lung cancer, lung adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, intrahepatic cholangiocarcinoma, bladder cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, renal cell carcinoma, head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, bile duct cancer, gallbladder cancer, intrahepatic bile duct carcinoma, oral mucosal cancer and urothelial carcinoma.

[0185] In a ninth aspect, the present invention provides a drug combination comprising the anti-FGFR2b antibody or antigen-binding fragment thereof according to the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or a mixture thereof according to the second aspect; or the pharmaceutical composition according to the sixth aspect, and one or more other therapeutic agents.

[0186] In a tenth aspect, the present invention provides a kit comprising the anti-FGFR2b antibody or antigen-binding fragment thereof described in the first aspect; or the anti-FGFR2b antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or a mixture thereof described in the second aspect; or the pharmaceutical composition described in the sixth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0187] Figure 1: Anti-FGFR2b antibody drug conjugate inhibits the proliferation of SNU-5 cells.

[0188] Figure 2: Anti-FGFR2b antibody drug conjugate inhibits the proliferation of SNU-16 cells.

[0189] Figure 3: Effect of HC18 antibody drug conjugate on tumor volume in the SNU-16 mouse subcutaneous xenograft tumor model.

[0190] Figure 4: Effect of HC18 antibody-drug conjugate on mouse body weight in the SNU-16 mouse subcutaneous xenograft tumor model.

[0191] Figure 5: Effects of different HC18-01 antibody drug conjugates on tumor volume in the SNU-16 mouse subcutaneous xenograft tumor model.

[0192] Figure 6: Effects of different HC18-01 antibody drug conjugates on mouse body weight in the SNU-16 mouse subcutaneous xenograft tumor model.

[0193] Figure 7: Antitumor efficacy of different doses of HC18-01-ADC1 on SNU-16 human gastric cancer xenograft model mice.

[0194] Figure 8: Effects of different doses of HC18-01-ADC1 on body weight in SNU-16 human gastric cancer xenograft model mice.

[0195] Figure 9: Antitumor efficacy of different doses of HC18-01-ADC1 on KATO-III human gastric cancer xenograft model mice.

[0196] Figure 10: Effects of different doses of HC18-01-ADC1 on body weight in mice bearing the KATO-III human gastric cancer xenograft model.

[0197] Figure 11: Effect of HC18-01-ADC1 on the average tumor volume in xKATO-III subcutaneous transplanted mice.

[0198] FIG12 : Effect of HC18-01-ADC1 on tumor weight in the xKATO-III subcutaneous xenograft tumor model.

[0199] FIG13 : Effect of HC18-01-ADC1 on mean tumor volume in a mouse human lung cancer tumor xenograft model.

[0200] FIG14 : Effect of HC18-01-ADC1 on tumor weight in a human lung cancer tumor xenograft model.

[0201] Definition of terms

[0202] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0203] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this article, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly provided in the text.

[0204] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0205] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0206] The term "FGFR2b" refers to any recombinant or naturally occurring form of fibroblast growth factor receptor 2b (FGFR2b), a variant or homolog thereof that maintains, for example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of FGFR2b. The variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the naturally occurring FGFR2 protein over the entire sequence or a partial sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion).

[0207] The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain circumstances, e.g., antibodies naturally occurring in camels may comprise only heavy chains.

[0208] The term "anti-FGFR2b antibody" refers to an antibody molecule that specifically binds to FGFR2b and is capable of inhibiting FGFR2b activity. Relative to the absence of the FGFR2b antibody, the anti-FGFR2b antibody is capable of inhibiting FGFR2b activity, for example, by at least partially or completely blocking FGFR2b stimulation, reducing, preventing or delaying FGFR2b activation, or inactivating, desensitizing or downregulating FGFR2b signaling, activity or amount. In some embodiments, the antibody can inhibit FGFR2b activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control.

[0209] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and is capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies (e.g., bispecific antibodies) formed from antibody fragments.

[0210] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.

[0211] "Complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs in the variable domain are typically referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The IMGT database (http: / / imgt.cines.fr / ) and the North CDR definition were based on affinity propagation clustering using a large number of crystal structures.

[0212] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0213] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0214] CDRs can also be identified based on having the same AbM numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the present invention). In some embodiments, the CDRs of the antibodies of the present invention are positioned according to the AbM numbering scheme.

[0215] Unless otherwise indicated, throughout this invention, references to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues) refer to positions numbered according to the AbM numbering system.

[0216] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0217] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0218] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0219] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0220] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0221] The term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0222] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD of the binding protein or cell is 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0223] The term "high affinity" for IgG antibodies refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0224] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0225] The term "median effective concentration (EC50) 50 )" refers to the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

[0226] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0227] The term "antibody drug conjugate" or "ADC" refers to an antibody or antibody fragment covalently coupled to a therapeutically active substance or active pharmaceutical ingredient, such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin that can kill cells (preferably cancer cells) targeted by the ADC. The covalent attachment of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin may be performed in a non-site-specific manner using a linker, or in a site-specific manner.

[0228] The term "site-specific conjugation" refers to a method of specifically linking a therapeutically active substance or active pharmaceutical ingredient to a specific site of an antibody. In some embodiments, the conjugation is accomplished with the aid of a linker.

[0229] The term "cytotoxic agent" is used interchangeably with "cytotoxin" and, as used herein, refers to a substance that inhibits or disrupts cellular function and / or causes cell death or destruction.

[0230] The terms "linker," "linker unit," and "linker" are used interchangeably herein to refer to a chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In some embodiments, the linker may comprise amino acid residues that connect the antigen to the payload. The amino acid residues may form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units. The amino acid residues include naturally occurring ones as well as non-naturally occurring amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.

[0231] According to the property classification, the linkers suitable for the present invention can be tissue protease-degradable linkers, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, ultraviolet-sensitive linkers such as PC4AP.

[0232] The linker of the present invention can also be a combination of one or more linkers. For example, a cathepsin-degradable linker can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention encompasses a single type of linker or a combination of different types of linkers, as long as it is capable of conjugating the antibody of the present invention to the drug.

[0233] The term "load" or "drug load" or "payable load" refers to the average effective load per antibody within the ADC molecule ("payable load" is used interchangeably herein with "therapeutically active substance or active pharmaceutical ingredient"). Drug load can range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody. The term "drug / antibody ratio" or "DAR" refers to the ratio of the therapeutically active substance or active pharmaceutical ingredient (D) conjugated to the antibody to the antibody. The ADCs described herein typically have a DAR of 1-20, and in certain embodiments have a DAR of 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6-10, 3-8, 4-6, 6-10, and 2-4. Representative DAR values ​​are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, typically expressed as a combination of the letter D and a number, wherein the number represents the numerical value of the DAR, for example, D2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is an average DAR, i.e., the average DAR as determined by a detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC). Quantitative DAR values ​​can also be determined. The DAR may be limited by the number of attachment sites on the antibody. For example, where the attachment site is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups or may have only one or a few sufficiently reactive thiol groups through which a linker unit may be attached.

[0234] The term "FGFR2b-mediated disease or condition" refers to a disease state caused or promoted by abnormal signaling (e.g., gene mutation, overexpression, or abnormal activation) of fibroblast growth factor receptor 2b (FGFR2b). When FGFR2b signaling is abnormal, it may lead to a variety of pathological conditions, such as cancer (e.g., gastric cancer, breast cancer, and lung cancer), developmental diseases, fibrosis, and proliferative diseases. In a specific embodiment of the present invention, the term "FGFR2b-mediated disease or condition" refers to a cancer or tumor associated with abnormal expression, mutation, or dysfunction of the FGFR2b gene or protein.

[0235] The term "treat" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

[0236] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0237] The term "effective amount" refers to the amount or dosage of an antibody, conjugate, or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0238] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof can vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or conjugate or composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0239] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0240] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0241] The term "in combination with" refers to the administration of one or more additional therapeutic agents and includes simultaneous (concurrent) and consecutive administration in either order.

[0242] The term "pharmaceutically acceptable salt" refers to a salt of the drug conjugate of the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The antibody drug conjugate of the present application may form a salt with an acid.

[0243] The term "pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical preparation or composition other than the active ingredient that are non-toxic to the subject.

[0244] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. DETAILED DESCRIPTION

[0245] The following examples further illustrate the present invention. However, it should be understood that the examples are described by way of illustration and not limitation, and that various modifications can be made by those skilled in the art. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0246] In some embodiments, the present invention provides a method for preparing an anti-FGFR2b antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-FGFR2b antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-FGFR2 antibody, and optionally isolating the anti-FGFR2b antibody. In certain embodiments, the method further comprises recovering the anti-FGFR2b antibody from the host cell (or host cell culture medium).

[0247] To recombinantly produce an anti-FGFR2b antibody of the invention, nucleic acid encoding the anti-FGFR2b antibody of the invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that specifically bind to nucleic acid encoding an anti-FGFR2b antibody of the invention.

[0248] The anti-FGFR2b antibodies of the present invention, prepared as described herein, can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art. The purity of the anti-FGFR2b antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0249] The prior art describes various methods for conjugating cytotoxic agents or other therapeutic agents to antibodies. For example, conjugation can occur via the amino group of a lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl group at the C-terminus of aspartic acid, glutamic acid, or an activated cysteine ​​sulfhydryl group in the antibody.

[0250] The practice of the present invention will employ, unless explicitly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, which are within the skill of the art.

[0251] The structures of the compounds disclosed herein can be confirmed by conventional methods well known to those skilled in the art. If the disclosure involves the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional technical means in the art.

[0252] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names.

[0253] Example 1: Humanization of murine FGFR2b antibody and preparation of FGFR2b antibody

[0254] The murine antibody of the present invention was prepared with reference to the method disclosed in WO2023185778A1, which can be abbreviated as "HC18". The present invention incorporates the entire text of WO2023185778A1 as a reference.

[0255] The preparation of the control antibody FPA144 antibody of the present invention refers to WO2015017600A1, which is incorporated herein by reference in its entirety.

[0256] The VH and VL sequences of the mouse antibody HC18 were compared with the known human antibody database to find the human germline gene VH and VL sequences with the highest homology to the mouse VH and VL sequences, respectively.

[0257] The humanization design and PTM analysis methods used in the present invention are:

[0258] 1. Analyze the variable region sequence of the selected antibody to distinguish the antigen binding domain (CDR) and framework region (FR).

[0259] 2. Perform homology modeling to obtain the modeled structure of the parent antibody. Calculate the hydrophilicity of the framework regions and identify the amino acids located within the antibody on the framework regions (i.e., hydrophilicity < 15%).

[0260] 3. Select a humanized framework sequence for each of VH and VL based on sequence homology. Graft the CDRs of the parent antibody onto the humanized framework to obtain the sequences of the grafted antibodies, which are defined as VH1 and VL1.

[0261] 4. Compare VH1 and VL1 with the parental VH and VL sequences. Identify all key residues that differ between the grafted and parental antibody framework sequences (i.e., potential backmutation sites).

[0262] 5. Perform one or more back mutations in the VH1 and VL1 sequences step by step according to the following principles:

[0263] a. Critical amino acids for antibody loop interactions.

[0264] b. Amino acids located at the interface between the light and heavy chains.

[0265] c. Key amino acids for antibody core formation.

[0266] 6. Design multiple humanized sequences for each antibody.

[0267] 7. Analyze post-translational modifications and potential degradation sites in the sequence, including deamidation, isomeric oxidation, and glycosylation.

[0268] 8. Identify PTM risk sites on the surface in the structural model, such as N-glycosylation sites, deamidation sites, isomerization sites, oxidation sites, and unpaired cysteine ​​residues, which may affect the binding activity and downstream production of the antibody.

[0269] 9. If there are high-risk PTM sites in the antibody CDR region, the risk sites will be considered for removal.

[0270] Through the above analysis, it was found that there was a high-risk isomerization site DG on the HCDR3 of the mouse antibody HC18. Through PTM removal, DG was replaced with DA. It was found that this replacement had no significant effect on the binding and drugability of the antibody.

[0271] The murine antibody HC18 was humanized to obtain the humanized antibody HC18-01, whose CDR sequences are shown in Table 1. The humanized antibodies were constructed, expressed, purified, and characterized by SDS-PAGE and SEC-HPLC. The results showed that each antibody had a band of approximately 150 kD on a non-reducing SDS-PAGE gel, and bands of approximately 50 kD and 25 kD on a reducing SDS-PAGE gel, consistent with the expected size. The SEC monomer purity of all antibodies was greater than 95%.

[0272] Table 1 Amino acid sequences of CDR regions of humanized antibodies

[0273] Example 2 Preparation of Linker-Toxin

[0274] The preparation of the linkers and toxins used in the present invention, such as deruxtecan, GGFG-Dxd, Dxd, GGFG-Exd, and Val-Ala-PAB, refers to WO2014057687, WO1997046260, and CN101795711, the entire text of which is incorporated herein by reference.

[0275] The linker-toxin Cpd3 used in the present invention and its preparation method are as follows:

[0276] Step 1: (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (3-1, 2.5 g, 3.6 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl -1,2,3,9,12,15-Hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (3-2, 1.6 g, 3.6 mmol) and N,N-diisopropylethylamine (965 mg, 7.5 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), and the reaction solution was stirred at room temperature for 16 hours. The reaction was monitored by LCMS to yield 4-((S)-2-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-3). LCMS (ESI) m / z: 977.3 [M+H] + .

[0277] Step 2: Hexahydropyridine (464 mg, 5.4 mmol) was added directly to the reaction mixture in Step 1, and the reaction mixture was stirred at room temperature for 2 hours. LCMS monitored the reaction completion of the raw material, and the reaction mixture was directly purified by C18 column chromatography to obtain 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-4) (1.8 g). LCMS (ESI) m / z: 755.2 [M+H] + .

[0278] Step 3: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-triacont-31-oic acid (3-5, 1.8 g, 2.38 mmol), 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl ((1S,9S)-9-ethyl 1-H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-4, 1.8 g, 2.38 mmol), N,N-diisopropylethylamine (614 mg, 4.7 The mixture was stirred at room temperature for 3 h. The reaction was monitored by LCMS to obtain 4-((33S,36S)-1-(9H-fluoren-9-yl)-33-isopropyl-36-methyl-3,31,34-trioxo-2,7,10,13,16,19,25,25,28-nonaoxa-4,32- ,35-triaza-heptahistrazino-37-amido)-benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-6). LCMS(ESI)m / z:1400.5[M+H] + .

[0279] Step 4: Piperidine (409 mg, 4.76 mmol) was directly added to the reaction solution of step 3, and the reaction solution was stirred at room temperature for 2 hours. The reaction of the raw materials was completed by LCMS monitoring, and the reaction solution was directly purified by C18 column chromatography to obtain 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-triacontria-33-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-7) (1.2 g). LCMS (ESI) m / z: 1178.4 [M+H] + .

[0280] Step 5: 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-triacontria-33-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4 ':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (3-7, 20 mg, 0.017 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (3-8, 13.7 mg, 0.068 mmol), and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine tetrafluoroborate (22.3 mg, 0.068 mmol) were dissolved in anhydrous N,N-dimethylacetamide (3 mL), and the reaction solution was stirred at room temperature for 16 hours. After separation and purification by C18 column chromatography, 4-((31S,34S)-31-isopropyl-34-methyl-1-(2-(methylsulfonyl)pyrimidin-5-yl)-1,29,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triaza-triazolyl-35-amido-benzyl(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (2.3 mg, yield: 10%), namely Cpd3, was obtained. 1 H NMR (400MHz, DMSO-d6) δ9.95(s,1H),9.37(s,1H),9.14(s,1H),8.46(s,1H),8.21(d,J=6.8Hz,1H),8.06(d,J=7.8H z,1H),7.89(d,J=8.3Hz,1H),7.78(d,J=10.9Hz,1H),7.60(d,J=8.4Hz,2H),7.36(d,J=8.1Hz,2H),7.31(s,1H),6. 53(s,1H),5.45(s,2H),5.28(s,3H),5.08(s,2H),4.42–4.34(m,1H),4.22–4.17(m,1H),3.62–3.53(m,10H),3.52– 3.46(m,28H),2.38(s,3H),2.33(s,1H),2.20(s,3H),2.01–1.81(m,4H),1.30(d,J=7.0Hz,3H),0.90–0.80(m,9H).

[0281] The linker-toxin Cpd5 used in the present invention and its preparation method are as follows:

[0282] Step 1: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (5-1,440 mg, 1.15 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2 -b]Quinoline-10,13-dione (500 mg, 1.15 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL), and N,N'-dicyclohexylcarbodiimide (308 mg, 1.5 mmol), N-hydroxysuccinimide (132 mg, 1.15 mmol) and N,N-diisopropylethylamine (148 mg, 1.15 mmol) were added under ice bath, and then the reaction solution was stirred at room temperature for 16 hours. The reaction was completed by LCMS monitoring. The organic phase was extracted with ethyl acetate and water, concentrated, and purified on a silica gel column using a methanol and dichloromethane system (1:20) to give (9H-fluoren-9-yl)methyl (2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3,650 mg). LCMS (ESI) m / z: 802.3 [M+H] + .

[0283] Step 2: (9H-Fluoren-9-yl)methyl (2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3, 650 mg, 0.81 mmol) was dissolved in N,N-dimethylformamide (10 mL), and then hexahydropyridine (69 mg, 5.4 mmol) was added and the reaction was stirred at room temperature for 2 hours. The reaction of the raw material was completed by LCMS monitoring, and the reaction solution was directly purified by C18 column chromatography to obtain 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4,420 mg). LCMS (ESI) m / z: 580.2 [M+H] + .

[0284] Step 3: 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4, 420 mg, 0.73 mmol), (((9H-fluorene-9 -yl) methoxy) carbonyl) glycylglycyl-L-phenylalanine (5-5, 370 mg, 0.73 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), and N,N'-dicyclohexylcarbodiimide (195 mg, 0.95 mmol), N-hydroxysuccinimide (84 mg, 0.73 mmol) and N,N-diisopropylethylamine (94 mg, 0.73 mmol) were added under ice bath, and the reaction solution was stirred at room temperature for 16 hours. The reaction was monitored by LCMS. The organic phase was extracted with ethyl acetate and water, concentrated, and purified on a silica gel column using a methanol:dichloromethane system (1:15) to give (9H-fluoren-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate (5-6,550 mg). LCMS (ESI) m / z: 1063.5 [M+H] + .

[0285] Step 4: (9H-Fluoren-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate (5-6,550 mg, 0.52 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then hexahydropyridine (44 mg, 0.52 mmol) was added and the reaction solution was stirred at room temperature for 2 hours. The reaction of the raw material was completed as monitored by LCMS, and the reaction solution was directly purified by C18 column chromatography to obtain ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)amine (5-7, 400 mg). LCMS (ESI) m / z: 841.4 [M+H] + .

[0286] Step 5: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-triacont-31-oic acid (347 mg, 0.52 mmol), HATU (274 mg, 0.72 mmol) and N,N-diisopropylethylamine (124 mg, 0.96 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). Then, the mixture was stirred at room temperature for 1 h, and then ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa- The mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction solution was directly purified by C18 column chromatography to obtain (9H-fluoren-9-yl)methyl ((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1 0,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetradecane-44-yl)carbamate (5-9, 350 mg). LCMS (ESI) m / z: 1486.6 [M+H] + .

[0287] Step 6: (9H-fluoren-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,1 5,18-Hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetradec-44-yl)carbamate (5-9, 350 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then hexahydropyridine (20 mg, 0.24 mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. The reaction of the raw material was completed after LCMS monitoring, and the reaction solution was directly purified and separated by C18 column chromatography to give 1-amino-N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (5-10, 200 mg). LCMS (ESI) m / z: 1264.6 [M+H] + .

[0288] Step 7: 1-amino-N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-3, 6,9,12,15,18,21,24-Octoxaheptacosane-27-carboxamide (5-10, 100 mg, 0.079 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (64 mg, 0.3 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine (53 mg, 0.3 mmol), and N-methylmorpholine (182 mg, 1.8 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL), and the reaction solution was stirred at room temperature for 16 hours. The product was separated and purified by C18 column chromatography to give N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetradecane-44-yl)-2-(methylsulfonyl)pyrimidine-5-carboxamide (5 mg), namely Cpd5. LCMS (ESI) m / z: 1448.6 [M+H] + .

[0289] 1H NMR (400MHz, DMSO-d6) δ9.37(d,J=2.1Hz,2H),9.12(s,1H),8.63(s,1H),8.51(d,J=8.6Hz,1H),8.29(s,1H),8.21–8.07(m,2H ),7.99(s,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H),7.21(t,J=15.2Hz,5H),6.52(s,1H),5.60(s,1H),5.42(s,2H),5.20(s,2H ),4.64(d,J=6.3Hz,2H),4.47(s,1H),4.02(s,2H),3.76–3.66(m,4H),3.59–3.53(m,8H),3.51–3.45(m,29H),2.39(s,5H),2.1 8(s,2H),2.00(d,J=7.9Hz,1H),1.84(dd,J=15.4,7.8Hz,2H),1.33–1.20(m,5H),1.12(d,J=6.6Hz,1H),0.87(t,J=6.3Hz,3H).

[0290] The linker-toxin Cpd6 used in the present invention and its preparation method are as follows:

[0291] Step 1: (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanine (6-1, 943 mg, 2.3 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] Quinoline-10,13-dione (1 g, 2.3 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). N,N'-dicyclohexylcarbodiimide (616 mg, 3 mmol), N-hydroxysuccinimide (264 mg, 2.3 mmol) and N,N-diisopropylethylamine (297 mg, 2.3 mmol) were added under ice-cooling, and the reaction solution was stirred at room temperature for 16 hours. The reaction was monitored by LCMS. The organic phase was extracted with ethyl acetate and water, concentrated, and purified on a silica gel column using a methanol:dichloromethane system (1:20) to give (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6-3,730 mg). LCMS (ESI) m / z: 828.3 [M+H] + .

[0292] Step 2: (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6-3, 730 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (10 mL), and then hexahydropyridine (75 mg, 0.88 mmol) was added and the reaction was stirred at room temperature for 2 hours. The reaction of the raw material was completed by LCMS monitoring, and the reaction solution was directly purified by C18 column chromatography to obtain (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (6-4,450 mg). LCMS (ESI) m / z: 606.2 [M+H] + .

[0293] Step 3: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacont-31-oic acid (592 mg, 0.89 mmol), HATU (422 mg, 1.11 mmol) and N,N-diisopropylethylamine (143 mg, 1.11 mmol) were dissolved in anhydrous N,N-dimethylformamide. (5 mL), and then stirred at room temperature for 1 h, and then (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)- The mixture was stirred at room temperature for 4 h. The reaction was monitored by LCMS. The reaction solution was directly purified by C18 column chromatography to give (9H-fluoren-9-yl)methyl ((29S,32S)-33-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1 0,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-triacontanoyl)carbamate (6-5,400 mg). LCMS (ESI) m / z: 1251.6 [M+H] + .

[0294] Step 4: (9H-fluoren-9-yl)methyl((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-29-isopropyl 400 mg, 0.32 mmol) was dissolved in N,N-dimethylformamide (5 mL) and then piperidine (27 mg, 0.32 mmol) was added. The reaction solution was stirred at room temperature for 2 hours. The reaction of the raw material was completed by LCMS monitoring, and the reaction solution was directly purified and separated by C18 column chromatography to obtain 1-amino-N-((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxa-heptacosane-27-amide (6-6, 210 mg). LCMS (ESI) m / z: 1029.6 [M+H] + .

[0295] Step 5: 1-amino-N-((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12 ,15,18,21,24-Octaoxa-heptacosa-27-amide (6-6, 100 mg, 0.097 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (78 mg, 0.39 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine (68 mg, 0.39 mmol) and N-methylmorpholine (59 mg, 0.582 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL), and the reaction solution was stirred at room temperature for 16 hours. The product was purified and separated by C18 column chromatography to obtain N-((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-tricarboxyl)-2-(methylsulfonyl)pyrimidine-5-carboxamide (11.5 mg), namely Cpd6. LCMS (ESI) m / z: 1213.6 [M+H] +

[0296] 1H NMR (400MHz, DMSO-d6) δ9.36(s,2H),9.13(s,1H),8.41(d,J=8.9Hz,1H),8.10(d,J=6.9Hz,1H),7.86(d,J=8.4Hz,1H),7. 79(d,J=11.0Hz,1H),7.30(s,1H),6.53(s,1H),5.54(s,1H),5.42(s,2H),5.24(d,J=18.9Hz,1H),5.11(d,J=19.0Hz,1H), 4.32–4.21(m,1H),4.17–4.06(m,1H),3.58–3.52(m,8H),3.48(s,25H),3.44(s,6H),2.40(s,3H),2.29(dd,J=13.7,7.1H z,1H),2.13(s,2H),1.91(ddd,J=23.2,22.6,7.6Hz,4H),1.27(d,J=7.0Hz,3H),0.87(t,J=7.4Hz,3H),0.83–0.72(m,6H).

[0297] Example 3: FGFR2b-ADC preparation

[0298] The antibody stock solution (HC18-01, 15 mg) was removed from the refrigerator (-80°C), thawed, and added to a centrifuge tube. PB buffer (20 mM, pH 7.2, 751 uL), DTPA-3Na (10 mM, 375 uL), and TCEP (10 mM, 94 uL) were then added and reacted at 25°C for 2 hours. The linker-toxin described in Example 2, such as Cpd3 (5 mM, 272 uL), was then added and the reaction continued at 25°C for another 2 hours. After the reaction, the solution was transferred to an ultrafiltration centrifuge tube (30 kDa) and dialyzed against His / His-HCl (10 mM, pH 5.5) to obtain an ADC stock solution (11.4 mg) with a DAR of 7.5-8 and a SEC purity of ≥95%.

[0299] The linker-toxin used in the above experiments is Cpd3, described in Example 2, as an example. The antibody stock solution can be HC18 or HC18-01; the linker-toxin can be a combination of a linker and a camptothecin compound commonly used in the art, including but not limited to deruxtecan, Cpd3, Cpd5, and Cpd6.

[0300] The above experiments were performed using humanized antibody HC18-01 and other linker-toxins. The ADC samples were validated, and the test results are shown in Table 2 below.

[0301] Table 2-ADC sample test results

[0302] Example 4 Proliferation inhibition experiment

[0303] In this example, using staurosporine as a positive control, the binding ability of HC18-deruxtecan ADC and FPA144 antibody to human FGFR2b on the surface of tumor cells SNU-5 and SNU-16 (cell line sources are shown in Table 3) was tested by FACS. SNU-5 cells do not express FGFR2b on their surface.

[0304] Table 3 Cell line sources

[0305] Experimental method: SNU-5 and SNU-16 were maintained at 37°C, 5% CO2, and each tumor cell line was routinely subcultured. Cells in the logarithmic growth phase were harvested, counted, and plated into 96-well plates (Greiner 96-well plates, #655090). Cells were adjusted to an appropriate cell density and 135 μL of cell suspension was added to each well of a 96-well plate, resulting in cell densities of 5000 / well for SNU-5 and 3000 / well for SNU-16, respectively. 135 μL of culture medium was added to blank wells and incubated overnight at 37°C, 5% CO₂, and 100% RH. 15 μL of serially diluted ADC samples (3000, 300, 30, and 3 nM) was added to each well, and 15 μL of culture medium was added to blank wells. The plates were incubated at 37°C, 5% CO₂ for 6 days. Luminescent cell viability was determined using the Promega CellTiter Glo kit (Promega-G7573) and luminescence was measured using a 2104 EnVision Multilabel Plate Reader (PerkinElmer).

[0306] The results are shown in Figures 1 and 2: HC18-deruxtecan had a higher inhibitory effect on the proliferation of two gastric cancer cells, SNU-5 (Figure 1) and SNU-16 (Figure 2), than FPA144.

[0307] Example 5: Affinity of humanized antibodies for antigens from different species

[0308] This example tests the affinity of the humanized antibody HC18-01 to the antigen protein FGFR2 (IIIb) of humans, cynomolgus monkeys, rats, and mice.

[0309] Experimental methods:

[0310] 1. Antibody Capture

[0311] The experiment used 1×PBS (containing 0.05% Tween 20, pH 7.4) buffer as the test buffer. The antibody was diluted to 1 μg / mL with PBS buffer. The flow rate was set to 10 μL / min and directly captured onto the test surface of the Protein A chip. The capture lasted for 20 seconds, and the capture amount could reach 80-100 RU.

[0312] 2. Testing conditions for FGFR2b samples

[0313] A total of eight assay concentrations were set for the antigen. For human and cynomolgus FGFR2b proteins, the concentration gradient was 0 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 20 nM, 50 nM, and 100 nM, respectively. For mouse and rat FGFR2 proteins, the concentration gradient was 0 nM, 6.25 nM, 12.5 nM, 20 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. Samples were analyzed at a flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 360 s, and a temperature of 25°C.

[0314] 3. Regeneration conditions

[0315] Gly-HCl buffer (pH 1.5, 10 mM) was used as the regeneration buffer at a flow rate of 30 μL / min for 30 s. After regeneration, the chip was stabilized for 60 s before analyzing the next sample.

[0316] 4. Determination of Kinetic Parameters

[0317] The experiment was run using multiple cycles, with analysis time plotted on the horizontal axis and response signal values ​​plotted on the vertical axis. The data were fitted using BIAcore T200 analysis software using a 1:1 Langmuir binding model to determine kinetic constants such as the association rate constant, dissociation rate constant, and association-dissociation constant.

[0318] The results are shown in Table 4. It shows that the HC18-01 antibody has good affinity for FGFR2b of various genera.

[0319] Table 4: Binding activity of humanized antibodies against antigens from different species

[0320] Example 6: Antitumor Activity of ADC in SNU-16 Mouse Subcutaneous Xenograft Tumor Model

[0321] In this example, the tumor inhibition effect of ADC (HC18-deruxtecan) in animals was evaluated, using FPA144 antibody as a control, and the tumor cells used were gastric cancer cell SNU-16.

[0322] Experimental method: 6-8 weeks old female Balb / C nude mice weighing about 20 g (animal source: Weitonglihua) were used, and 10×10 6 SNU-16 cells (SNU-16 cells were cultured adherently in a medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2. The cells were routinely passaged twice a week. When the cells were in the exponential growth phase, they were harvested, counted, and inoculated) with an inoculation volume of 0.2 mL. The cells were inoculated until the average tumor volume reached approximately 150-200 mm 3 At the same time, the mice were randomly divided into groups and cages. There were 8 tumor-bearing nude mice in each group, with a total of 4 groups, including a Vehicle negative control group, a human IgG1-deruxtecan (Dxd) group, an HC18-deruxtecan group, and an FPA144 group (dosage was 10m / kg, administration volume was 1μl / g). The administration method was a single injection of 10mg / kg through the tail vein. The tumor volume was measured twice a week, for a total of 11 times / 5 weeks. The formula for calculating the tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter (mm) and short diameter (mm) of the tumor, respectively. Five weeks after the end of administration, the mice were euthanized, and the tumors were removed and weighed. The changes in tumor volume, tumor weight, and mouse body weight were analyzed, and the tumor inhibition rate was calculated.

[0323] The experimental results, as shown in Table 5 and Figures 3-4, showed no significant differences in body weight between the groups of mice, and no significant changes in body weight were observed during treatment. The specific results are shown in Figure 4, indicating that the mice tolerated the ADC well. HC18-deruxtecan demonstrated significant tumor-suppressing effects, as shown in Figure 3.

[0324] Table 5 Inhibition of tumors by ADC in tumor-bearing mice

[0325] Example 7: Antitumor Activity of ADC in SNU-16 Mouse Subcutaneous Xenograft Tumor Model

[0326] This example evaluates the tumor-suppressing effects of different ADC molecules (HC18-01-deruxtecan, HC18-01-ADC1, HC18-01-ADC2, and HC18-01-ADC3) of the humanized antibody HC18-01 in animals. The tumor cells used are gastric cancer cells SNU-16.

[0327] Experimental method: 6-8 weeks old female Balb / C nude mice weighing about 20 g (animal source: Weitonglihua) were used, and 10×106 SNU-16 cells (SNU-16 cells were cultured adherently in a medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2. The cells were routinely passaged twice a week. When the cells were in the exponential growth phase, they were harvested, counted, and inoculated) with an inoculation volume of 0.2 mL. The cells were inoculated until the average tumor volume reached approximately 150-200 mm 3 At the same time, the mice were randomly divided into groups and cages. There were 8 tumor-bearing nude mice in each group, for a total of 11 groups, including a Vehicle negative control group, an HC18-deruxtecan group, an HC18-01-deruxtecan group, an HC18-01-ADC1 group, an HC18-01-ADC2 group, and an HC18-01-ADC3 group (the experimental groups were administered with a dose of 1 and 3 mg / kg, respectively, and a dosing volume of 1 μl / g). The experimental groups were administered with a single dose of 1 or 3 mg / kg via tail vein injection. The tumor volume was measured twice a week, for a total of 11 times / 5 weeks. The formula for calculating the tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter (mm) and short diameter (mm) of the tumor, respectively. Five weeks after the end of administration, the mice were euthanized, and the tumors were removed and weighed. The changes in tumor volume, tumor weight, and mouse body weight were analyzed, and the tumor inhibition rate was calculated.

[0328] The experimental results are shown in Figures 5 and 6. There were no significant differences in the body weight of mice between the groups, and no significant changes in body weight were observed during treatment. The specific results are shown in Figure 6, indicating that the mice tolerated the ADC well. HC18-01-deruxtecan, HC18-01-ADC1, HC18-01-ADC2, and HC18-01-ADC3 demonstrated significant tumor-suppressing effects, as shown in Figure 5.

[0329] Example 8. Antitumor Effect of Antibody Drug Conjugate HC18-01-ADC1 in SNU-16 Human Gastric Cancer Xenograft Model

[0330] This example further explores the inhibitory effect of different single doses of HC18-01-ADC1 on the growth of human gastric cancer SNU-16 xenografts. The experimental method is the same as that described in Example 8.

[0331] In this example, a vehicle negative control group, a 0.5 mg / kg HC18-01-ADC1 group, a 1 mg / kg HC18-01-ADC1 group, a 3 mg / kg HC18-01-ADC1 group, a 10 mg / kg HC18-01-ADC1 group, and a 10 mg / kg commercially available bemazumab positive control group were set up.

[0332] Table 6 and Figures 7-8 record the efficacy results of the treatment in each experimental group.

[0333] Table 6: Inhibitory effects of different doses of HC18-01-ADC1 on human gastric cancer SNU-16 xenografts

[0334] Figure 7 shows the antitumor efficacy of HC18-01-ADC1 at different doses. A single dose of 0.5-10 mg / kg HC18-01-ADC1 inhibited the growth of SNU-16 xenografts in a dose-dependent manner, and the antitumor effect of 10 mg / kg HC18-01-ADC1 was superior to that of an equivalent dose of bemazumab.

[0335] Figure 8 shows the changes in body weight of mice at different doses. The experimental results showed that there was no significant difference in body weight among the mice in each group.

[0336] Example 9 Antitumor Effect of Antibody Drug Conjugate HC18-01-ADC1 in KATO-III Human Gastric Cancer Xenograft Model

[0337] This example further explores the inhibitory effect of different single doses of HC18-01-ADC1 on the growth of human gastric cancer KATO-III xenografts.

[0338] In this example, a vehicle negative control group, a 0.4 mg / kg HC18-01-ADC1 group, a 2 mg / kg HC18-01-ADC1 group, a 10 mg / kg HC18-01-ADC1 group, a 10 mg / kg commercially available bemazumab positive control group, and a 10 mg / kg HC18-01 naked antibody control group were set up. The experimental methods were the same as those described in Example 11.

[0339] Human gastric cancer KATO-III cells were cultured adherently in IMDM medium (supplemented with 20% fetal bovine serum and 1% antibiotic-antimycotic) at 37°C and 5% CO2. Cells were routinely passaged twice weekly using trypsinization. When cells were in the exponential growth phase and had a viability greater than 95%, they were harvested, counted, and plated.

[0340] Table 7 and Figures 9-10 show the results of the experiments.

[0341] Table 7: Treatment results of HC18-01-ADC1 on KATO-III human gastric cancer xenografts on day 60

[0342] Figure 9 shows the antitumor efficacy of HC18-01-ADC1 at different doses. A single dose of 0.4-10 mg / kg HC18-01-ADC1 potently inhibited the growth of KATO-III xenograft tumors in a dose-dependent manner. The antitumor effects of 0.4 mg / kg HC18-01-ADC1 were comparable to those of 10 mg / kg HC18-01. The antitumor effect of 10 mg / kg HC18-01-ADC1 was superior to that of an equivalent dose of bemazumab.

[0343] Figure 10 shows the changes in body weight of mice at different doses. The experimental results showed that there was no significant difference in body weight among the mice in each group.

[0344] Example 10 Antitumor Effect of Antibody Drug Conjugate HC18-01-ADC1 in Human Gastric Cancer xKATO-III Xenograft Model

[0345] In this example, the in vivo antitumor effects of the test compound HC18-01-ADC1 and its naked antibody HC18-01 against human gastric cancer were evaluated using a human gastric cancer xKATO-III mouse subcutaneous xenograft tumor model. The efficacy of the test compound HC18-01-ADC1 and its naked antibody HC18-01 were compared with those of the test compounds bemazumab and paclitaxel (paclitaxel injection, source: Sichuan Huiyu Pharmaceutical), respectively. The pharmacodynamic effects under different dosing regimens were also explored, providing a reference for the selection of clinical indications and clinical trial design for the test compound HC18-01-ADC1.

[0346] Human gastric cancer xKATO-III cells were cultured adherently in IMDM medium (supplemented with 20% fetal bovine serum and 1% antibiotic-antimycotic) at 37°C and 5% CO2. Cells were routinely passaged twice weekly using trypsinization. When cells were in the exponential growth phase and had a viability greater than 95%, they were harvested, counted, and plated (passage P9).

[0347] Female SCID Beige mice (Shanghai Lingchang Biotechnology Co., Ltd.) aged 6-8 weeks and weighing about 20 g were used and inoculated with 0.2 mL (containing 10*10 6 xKATO-III cells + 50% Matrigel) mixed cell suspension. The average tumor volume reached 202mm 3 The patients were randomly divided into groups and given medication at 37 hours. The medication settings are shown in Table 8.

[0348] Table 8: Dosing regimen of HC18-01-ADC1

[0349] The tumor volume was measured twice a week. The formula for calculating the tumor volume was: V = 0.5a × b 2, a and b represent the long diameter (mm) and short diameter (mm) of the tumor, respectively.

[0350] Efficacy indicators such as TGI, T / C, number of mice with complete tumor remission (CR) and partial tumor remission (PR), and tumor weight inhibition rate (IR) were evaluated. Each indicator was calculated according to the following formula or method:

[0351] 1. TGI (%) = [1-(V Ti / V T0 ) / (V ci / V c0 )]×100%(V Ti : Mean tumor volume of the treatment group on day i of administration, V T0 : Mean tumor volume of the treatment group on day 0 of administration, V ci : Mean tumor volume of the solvent control group on day i of administration, V c0 : mean tumor volume of the solvent control group on day 0 of administration);

[0352] 2. T / C (%) = V T / V c ×100(V T V: average tumor volume of the treatment group; c : average tumor volume of the solvent control group).

[0353] 3. The number of mice with complete remission (CR) or partial remission (PR). CR refers to a mouse tumor volume of 0 mm 3 PR means that the tumor volume of mice decreased by more than 30% compared with the other groups.

[0354] 4. IR (%) = [1-(average tumor weight of the treatment group / average tumor weight of the solvent control group)] x 100%.

[0355] Experimental data are presented as mean ± standard error (SEM). Statistical analysis was performed based on data obtained at the end of the experiment to assess differences between groups. Differences between groups were analyzed using Graphpad Prism 10.0 software and one-way analysis of variance (ANOVA). A p < 0.05 was considered significant.

[0356] The experimental results are shown in Figures 11-12. The test substances HC18-01-ADC1, HC18-01, Bemarituzumab, and Paclitaxel had minimal effects on the body weight of mice bearing xKATO-III subcutaneous tumors. On day 60, there were no significant differences in body weight or rate of change between the drug-treated and vehicle groups. Throughout the experimental period, there were no significant changes in body weight across the groups, and clinical observations showed no abnormalities. The drug dosages used in this example are safe.

[0357] On day 60 (D60), the average tumor volume of the xKATO-III subcutaneous transplant model mice was measured. The effects of the test substances on the tumor weight of the xKATO-III subcutaneous transplant model are shown in Figure 12. Compared with the Vehicle group, except for the 0.4 mg / kg HC18-01-ADC1 group (p>0.05), the average tumor volume of the other treatment groups was significantly reduced (p<0.0001, **** p<0.0001 indicates comparison with the vehicle control group); the average tumor volume of 10 mg / kg HC18-01-ADC1 was significantly smaller than that of 0.4 mg / kg HC18-01-ADC1 (p<0.0001, #### p<0.0001 vs. HC18-01-ADC1 0.4 mg / kg).

[0358] The T / C (%) and TGI (%) of the test substance in the xKATO-III subcutaneous xenograft tumor model were calculated based on the D60 tumor volume, the IR (%) was calculated based on the D60 tumor weight (TW), and the CR rate and PR rate were calculated based on the tumor response. The results are shown in Table 9 below.

[0359] Table 9: D60, results of HC18-01-ADC1 inhibition of xKATO-III subcutaneous transplanted tumors. **** p<0.0001 compared with the solvent control group, #### p<0.0001 vs. HC18-01-ADC1 0.4mg / kg group

[0360] A single dose of HC18-01-ADC1 significantly inhibited the growth of subcutaneous xKATO-III human gastric cancer xenografts in mice. Higher doses within the 2-10 mg / kg range demonstrated a significant dose-dependent inhibitory effect. HC18-01-ADC1 also demonstrated a long-lasting inhibitory effect, with no rebound effect observed 60 days after a single 10 mg / kg dose. At equivalent doses, HC18-01-ADC1 demonstrated superior tumor inhibition to bemarituzumab and paclitaxel.

[0361] Example 11 Antitumor Effect of Antibody Drug Conjugate HC18-01-ADC1 in Human Lung Cancer Xenograft (PDX) Model

[0362] The purpose of this study is to evaluate the anti-tumor effect of the test substance HC18-01-ADC1 in a human lung cancer xenograft (PDX) model and compare its efficacy with the test substances docetaxel and bemarituzumab.

[0363] In this example, a human lung cancer tumor tissue sample (NO.LD1-0025-200783) was selected for drug efficacy experiments, and it was verified that the sample was positive for FGFR2 expression.

[0364] Tumor tissue (NO.LD1-0025-200783, FP2+1 generation) was obtained from the biobank, cleaned and trimmed aseptically, and non-tumor tissues such as blood clots, fat, and internal necrotic foci were removed. The tissue was trimmed into a suitable size of 3×3×3mm. 3 The tissue blocks were then washed and an appropriate amount of Matrigel was added according to the volume of the tumor blocks. The blocks were placed on ice for later use. Nu / Nu mice were treated according to standard operating procedures and 3×3×3mm 3 Tumor tissue was inoculated subcutaneously into the limbs of Nu / Nu mice, with 1-2 sites inoculated per animal. The tumor volume and body weight of the mice were observed twice a week. When the tumor volume reached 100-200 mm 3 After the tumor tissue is successfully revived, the average tumor volume will reach 620mm. 3 Tumor-bearing animals were euthanized and the tumors were removed in a biosafety cabinet. Non-tumor and necrotic tissues were removed and then tumor tissue blocks (FP2+2 generation) were prepared for inoculation. BALB / c nude mice were used for inoculation and the tumor tissue blocks (FP2+2 generation) were divided into several 3×3×3 mm 3 The tissue blocks were inoculated into the subcutaneous tissue of mice (FP2+3 generations). After inoculation, the tumor volume and body weight of the mice were monitored twice a week. When the average tumor volume reached 118 mm 3 (The tumor latency period is 14 days), indicating that the PDX modeling is successful and meets the grouping criteria.

[0365] PDX model mice were dosed according to the schedule disclosed in Table 10.

[0366] Table 10. Dosing regimen of HC18-01-ADC1 for the treatment of human lung cancer xenografts

[0367] Body weight was recorded twice a week at fixed times, and tumor diameter was measured with a vernier caliper twice a week to calculate tumor volume.

[0368] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter (mm) and short diameter (mm) of the tumor, respectively.

[0369] At the end of the experiment, the animals were euthanized, and the tumors were removed by autopsy and weighed.

[0370] 1. Calculate the weight change rate of mice during the experimental period, RCBW (%) = (BW Di -BW D0 ) / BW D0 ×100%(BW Di : Body weight on the i-th day of administration, BW D0 : body weight on day 0 of administration).

[0371] 2. The anti-tumor efficacy of drugs in mice was evaluated by TGI (Tumor Growth Inhibition, %) or relative tumor proliferation rate T / C (%); where TGI (%) = [1-(V Ti / V T0 ) / (V ci / V c0 )]×100%(V Ti : Mean tumor volume of the treatment group on day i of administration, V T0 : Mean tumor volume of the treatment group on day 0 of administration, V ci : Mean tumor volume of the solvent control group on day i of administration, V c0 : mean tumor volume of the solvent control group on day 0 of administration); T / C% = V T / V c ×100%(V T : average tumor volume of the treatment group; V c : average tumor volume of the solvent control group).

[0372] 3. Count the number of mice with complete response (CR) or partial response (PR); CR refers to mice with a tumor volume of 0 mm 3 PR means that the tumor volume decreased by more than 30% compared with the group.

[0373] 4. Calculate tumor regression rate TRR (%), TRR (%) = (1-V Dn / V D0 )×100(V Dn V: tumor volume measured on day n; D0 : tumor volume at day 0 when grouping).

[0374] The test substances HC18-01-ADC1, Bemarituzumab, and Docetaxel had minimal effects on the body weight of human lung cancer PDX mice. Throughout the experimental period, there were no significant changes in the body weight of the animals in each group, and no abnormalities were observed in clinical observations, demonstrating that the drug dosages used in this example are safe.

[0375] The results of this example are shown in Figures 13-14 and Tables 11-12. On day 31, compared with the solvent control group, it was found that the 10 mg / kg Bemarituzumab group had almost no inhibitory effect on tumor volume, so this group of experiments was terminated. Compared with the solvent control group, the average tumor volume of the remaining drug-treated groups was significantly reduced (p<0.001), and the experiment was continued until the end of day 38. At the end of the experiment, compared with the 10 mg / kg Docetaxel group, the average tumor volume of the 10 mg / kg HC18-01-ADC1 group was significantly reduced (p<0.05); the average tumor volume of the 10 mg / kg HC18-01-ADC1 group was significantly smaller than that of the 3 mg / kg HC18-01-ADC1 group (p<0.05). Among them, * p represents the comparison solvent control group, # p represents comparison with the 10 mg / kg HC18-01-ADC1 group. (Note: On day 31, due to the large tumor size of the animals, the experimental process in the vehicle control group and the bemarituzumab group was terminated to meet the ethical standards of experimental animals. The experimental process in the remaining groups continued until the endpoint of day 38.)

[0376] At the end of the experiment, D38, the tumor weights of the remaining groups were measured. The results are shown in Figure 14 and Table 12. Compared with the 10 mg / kg Docetaxel group, the average tumor weight of the 10 mg / kg HC18-01-ADC1 group was significantly reduced (p < 0.01); the average tumor weight of the 10 mg / kg HC18-01-ADC1 group was significantly less than that of the 3 mg / kg HC18-01-ADC1 group (p < 0.01). && p、 & p indicates comparison with the HC18-01-ADC1 10 mg / kg group.

[0377] Table 11: Anti-tumor efficacy and statistical results on day 31

[0378] Table 12: Anti-tumor efficacy and statistical results on day 38 (end point)

[0379] A single dose of HC18-01-ADC1 significantly inhibited tumor growth in a subcutaneous human lung cancer PDX (NO.LD1-0025-200783) mouse model. Higher doses resulted in greater tumor inhibition, demonstrating a clear dose-dependent effect. A single dose of 10 mg / kg HC18-01-ADC1 showed no rebound effect 38 days after administration, demonstrating sustained tumor inhibition. At equivalent doses, HC18-01-ADC1 demonstrated significantly superior tumor inhibition compared to the antibody drug bemarituzumab and the chemotherapy drug docetaxel.

[0380] Example 12: Verification of Plasma Stability of Linker-Toxin Cpd3

[0381] The test ADC molecule HC18-01-ADC1 (concentration 0.2 mg / mL) was incubated at 37°C in plasma from different species (human, monkey, rat, and mouse) for 21 days. Analyte concentrations in plasma were measured at different time points using LC-MS / MS. The control sample in this example was mc-Exd (commercially available). Table 13 below summarizes the experimental parameters for HC18-01-ADC1 (analyte Cpd3) and the benchmark HC18-01-mc-Exd (analyte mc-Exd, i.e., the methanesulfonamide linker of Cpd3 was replaced with a maleic acid linker; the remaining linker components were the same, with the toxin moiety being Exd). Table 13 summarizes the plasma stability of HC18-01-ADC1.

[0382] Release rate (%) refers to the free drug content in plasma measured after a certain period of incubation between ADC and plasma, and the free drug shedding rate in plasma calculated based on the ratio of the free drug content in plasma to the total amount of drug in ADC.

[0383] Experimental results demonstrate that the humanized antibody HC18-01 conjugated to Cpd3 (DAR8) exhibits excellent plasma stability across diverse species. Cpd3 exhibits exceptional stability in humans, monkeys, and rats, and is superior to the control mc-EXD.

[0384] Sequence Listing

Claims

1. An anti-FGFR2b antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b, comprising: (1) the three CDRs (HCDR1, HCDR2, HCDR3) contained in VH as shown in SEQ ID NO: 7 or variants thereof; and / or the three CDRs (LCDR1, LCDR2, LCDR3) contained in VL as shown in SEQ ID NO: 8 or variants thereof; or, (2) the three CDRs (HCDR1, HCDR2, HCDR3) contained in VH as shown in SEQ ID NO: 12 or its variants; and / or the three CDRs (LCDR1, LCDR2, LCDR3) contained in VL as shown in SEQ ID NO: 13 or its variants.

2. The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 1, comprising: 1) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1; HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2; A HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 3; LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 4; LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 5; and LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 6; or 2) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1; HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2; A HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 11; LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 4; LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 5; and A LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:

6.

3. The anti-FGFR2b antibody and antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:7, or consists of SEQ ID NO:7; the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:8, or consists of SEQ ID NO:8; or (2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12, or consists of SEQ ID NO: 12; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13, or consists of SEQ ID NO:

13.

4. The anti-FGFR2b antibody and antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain and a light chain, wherein 1) the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or consists of SEQ ID NO: 9; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or consists of SEQ ID NO: 10; or 2) the heavy chain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14, or consists of SEQ ID NO: 14; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15, or consists of SEQ ID NO:

15.

5. The anti-FGFR2b antibody drug conjugate of formula (I), its isomers, its pharmaceutically acceptable salts or mixtures thereof: Ab-(LD)m[Formula (I)] in, L is the joint unit; D is a small molecule drug with cytotoxicity; m is the average number of LD units coupled to Ab, and m is selected from 1 to 10, preferably 2 to 8; Ab is the anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to claim 5, wherein the anti-FGFR2b antibody or fragment thereof comprises the following CDRs: HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 2, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 11, LCDR1 comprises or consists of the sequence shown in SEQ ID NO: 4, LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 5, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:

6.

7. The antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to claim 5 or 6, wherein the anti-FGFR2b antibody or fragment thereof comprises a light chain variable region and a heavy chain variable region: The light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

12.

8. The antibody drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to any one of claims 5 to 7, wherein: L is a linker unit, which is a combination of one or more L'; the L' is selected from carbonyl, amino, amide, aminoacyl, -(PEG) n -、-(CH2) n -, containing heteroatoms -(CH2) n -, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimido (mc), maleimidopropionyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl (PAB), dimethylethylenediamine (DMED), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC); wherein n is independently selected from an integer ranging from 1 to 20; D is a small molecule drug with cytotoxicity, which is selected from one or more of monomethyl auristatin compounds, camptothecin compounds or maytansine alkaloids; m is the average number of LD units coupled to Ab, wherein m is selected from 1-10.

9. The antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to any one of claims 5 to 8, wherein the conjugate comprises a plurality of D components, and the plurality of D components can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substance or pharmaceutically active ingredient, and the D is preferably a camptothecin compound, more preferably exitecan, Dxd(1), Dxd(2) or SN-38.

10. The antibody drug conjugate, its isomer, its pharmaceutically acceptable salt or mixture thereof according to claim 9, wherein the (LD) moiety is derived from the structures represented by formula Cpd3, formula Cpd5 and formula Cpd6:

11. The antibody-drug conjugate according to any one of claims 5 to 10, its isomer, its pharmaceutically acceptable salt, or a mixture thereof, wherein the antibody-drug conjugate has a structure as shown in Formula (I-C3), Formula (I-C5), and Formula (I-C6): in, m is the average number of connections, and m is independently selected from 1 to 10; Ab is the anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

12. An isolated nucleotide molecule encoding the anti-FGFR2b antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the heavy chain variable region and / or light chain variable region thereof.

13. An expression vector comprising the nucleotide molecule of claim 12.

14. A host cell comprising the nucleotide molecule of claim 12 or the expression vector of claim 13.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the antibody-drug conjugate according to any one of claims 5 to 11, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, and a pharmaceutically acceptable excipient.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the antibody-drug conjugate, its isomer, pharmaceutically acceptable salt or mixture thereof according to any one of claims 5 to 11, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating and / or preventing a disease or condition mediated by FGFR2b.

17. A method for treating and / or preventing a disease or condition mediated by FGFR2b, comprising administering to a subject in need thereof the antibody of any one of claims 1-4; the antibody-drug conjugate, its isomer, its pharmaceutically acceptable salt, or mixture thereof of any one of claims 5-11; or the pharmaceutical composition of claim 15.

18. The use according to claim 16 or the method according to claim 17, wherein the disease or condition is a tumor and / or cancer; the tumor and / or cancer is selected from the group consisting of lung cancer, squamous cell lung carcinoma, lung adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, intrahepatic cholangiocarcinoma, bladder cancer, colon cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, renal cell carcinoma, head and neck cancer, mesothelioma, melanoma, sarcoma, brain tumor, gastroesophageal adenocarcinoma, malignant uterine tumor, gastroesophageal junction adenocarcinoma, bile duct cancer, gallbladder cancer, intrahepatic bile duct cancer, oral mucosal cancer and urothelial carcinoma.