Anti-FGFR2b antibody, conjugate, and method of use

Abstract

Anti-FGFR2b antibodies and their antigen-binding fragments are provided herein, as are immunoconjugates containing them, and compositions containing antibodies and immunoconjugates. Methods for producing and using antibodies and immunoconjugates for treating FGFR2b-related diseases or disorders are also provided. The antibodies of this disclosure can bind to human FGFR2b and block the interaction between KGF ligands and FGFR2b. These antibodies bind to human glycosylated and aglycosylated FGFR2b with optimized binding rates, interfering with human KGF:FGFR2b interactions and finding applications in various therapeutic and prophylactic methods.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to PCT application PCT / CN2024 / 070825, titled "Anti-FGFR2b Antibodies, Conjugates and Methods of Use," filed on 5 January 2024, which is incorporated herein by reference in its entirety.

[0002] Reference to electronic sequence listings This application includes a sequence listing submitted electronically in .XML format, which is incorporated herein by reference in its entirety. The .XML copy, created on 17 December 2024, is named "01368-0001-00PCT.xml" and has a size of 116,698 bytes. The sequence listing contained in this .XML file is part of this specification and is incorporated herein by reference in its entirety.

[0003] field This disclosure relates to anti-FGFR2b antibodies, antigen-binding fragments thereof, immunoconjugates comprising the antibody or its antigen-binding fragment, compositions comprising the antibody, its antigen-binding fragment, or immunoconjugate, and methods for treating disorders responsive to FGFR2b antagonism. [Background technology]

[0004] Fibroblast growth factor 7 (FGF-7), also known as keratinocyte growth factor (KGF), is an epithelial cell mitogen that acts only through a subset of the FGF transmembrane tyrosine kinase receptor isoform (FGFR2b isoform). The KGF receptor, FGFR2b, is primarily expressed on the surface of epithelial cells. KGF binds to FGFR2b with high selectivity and specificity.

[0005] FGFR2b signaling drives downstream pathways, including the mitogen-activated protein kinase (MAPK) and AKT pathways, which are essential for cell proliferation, differentiation, survival, and migration. Overexpression of FGFR2b RNA has been detected in a wide range of tumor tissues, including the colon, prostate, and stomach. (J.Nat.Canc.Inst., Vol.98, Issue1-12, Part1 (2006)). Therefore, FGFR2b is a clinically validated target with high prevalence in multiple tumor types. Preclinical and clinical studies have demonstrated that FGFR-amplifying tumors are sensitive to FGFR inhibition and thus susceptible to therapeutic targets. Treatment of FGFR2-positive tumors with anti-FGFR2 therapies such as bemarituzumab (also known as HGS1036, FP-1039, FPA144, GSK3052230) results in improved early survival and progressive disease. Gemo et al., Cancer Res (2014) 74(19_Supplement): 5446. However, bemarituzumab benefits only a portion of patients with relatively high FGFR2b overexpression. In addition, a 26% discontinuation rate due to corneal toxicity has been observed with the use of bemarituzumab. Wainberg, Zev A., et al. NCT03694522 (2021): 160-160.

[0006] Various FGFR2b antibodies are conjugated into cytotoxic payloads containing thorium-227 (aplutumab, also known as BAY1179470, BAY2304058) and auristatin (aplutumab xadotin, also known as BAY1187982, BAY1179470ADC). Aplutumab-TTC (anti-FGFR2 antibody, a chelating moiety covalently conjugated to the antibody, and the alpha-emitting radionuclide thorium-227) has been reported to inhibit tumor growth in several xenograft models. Wickstroem et al. (2019) Int J Radiat Oncol Biol Phys 105(2):410-422;Wittemer-Rump et al. (2014) “Pharmacokinetic and Pharmacodynamic (PK / PD) Modeling of Preclinical Data of a Novel Anti-Fibroblast Growth Factor Receptor 2 (FGFR2) Antibody (BAY1179470) to Guide Dosing in Phase 1,” AACR Abstract ID 672.

[0007] Furthermore, clinical trials revealed that appletumabu xadotin was poorly tolerated, with the minimum tolerated dose falling below the pre-clinical estimated therapeutic threshold. Therefore, the trials were terminated prematurely. (Sommer et al. (2016) Cancer Res 76(21):6331-6339; Sommer et al. (2014) “FGFR2-ADC Potently and Selectively Inhibits Growth of Gastric and Breast Cancer Xenograft Models,” AACR Abstract ID 4491.) There remains a critical unmet medical need for improving effective anticancer drugs in FGFR2b-expressing cancers. This disclosure provides anti-FGFR2b antibodies and immunoconjugates exhibiting various desirable characteristics for the treatment of target diseases, such as cancer in humans.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 7

Summary of the Invention

[0009] This disclosure relates to anti-FGFR2b antibodies and their antigen-binding fragments that specifically bind to FGFR2b, as well as methods for producing and using them. Humanized, mouse, or chimeric anti-FGFR2b antibodies are provided individually and as immunoconjugates. These antibodies bind to human glycosylated and aglycosylated FGFR2b with optimized binding rates, interfere with human KGF:FGFR2b interactions, and find use in various therapeutic and prophylactic methods. This disclosure includes isolated antibodies, their derivatives, fragments, and conjugates, pharmaceutical formulations comprising one or more anti-FGFR2b antibodies and immunoconjugates, and cell lines that produce antibodies. The amino acid and nucleotide sequences of the antibodies are also provided.

[0010] In the embodiment, the antibody or its antigen-binding fragment specifically binds to human FGFR2b and includes heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3, as well as light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3. HCDR1 contains the sequence IYWX1N (sequence number 77), HCDR2 contains the sequence IYPENX2DTNYX3GKFKG (SEQ ID NO: 78), HCDR3 contains the sequence GGFDY (sequence number 5), LCDR1 contains the sequence RASENIYSNLA (sequence number 6), LCDR2 contains the sequence TATNLAX4 (sequence number 79), LCDR3 contains the sequence QHFYGILYT (sequence number 8), X1 is M or L, X2 is G or A, X3 is S or N, and X4 is D or E.

[0011] In this embodiment, the antibody or its antigen-binding fragment specifically binds to human FGFR2b and includes HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR3. HCDR1 contains the sequence DTYIH (sequence number 15), HCDR2 contains the sequence RIDPAX1GNTMFASEFQG (SEQ ID NO: 80), HCDR3 contains the sequence SKIHYDYDEGFAY (sequence number 17), LCDR1 contains the sequence RASESVDDYGYSFLH (sequence number 18), LCDR2 contains the sequence RASNLES (sequence number 19), LCDR3 contains the sequence QQSNQNPRT (sequence number 20), X1 is either E or N.

[0012] In this embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b is (i) Heavy chain variable region, (a) HCDR1 having sequence IYWLN (sequence number 3), (b) HCDR2 having sequence QIYPENADTNYSGKFKG (sequence number 4), (c) HCDR3 having sequence GGFDY (sequence number 5), A heavy chain variable region including, (ii) Light chain variable region, (d) LCDR1 having sequence RASENIYSNLA (sequence number 6), (e) LCDR2 having sequence TATNLAE (sequence number 7), (f) LCDR3 having sequence QHFYGILYT (sequence number 8), Including a light chain variable region, Includes.

[0013] In this embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b is (i) Heavy chain variable region, (a) HCDR1 having sequence DTYIH (sequence number 15), (b) HCDR2 having the sequence RIDPAEGNTMFASEFQG (sequence number 16), (c) HCDR3 having the sequence SKIHYDYDEGFAY (sequence number 17), A heavy chain variable region including, (ii) Light chain variable region, (d) LCDR1 having sequence RASESVDDYGYSFLH (sequence number 18), (e) LCDR2 having sequence RASNLES (sequence number 19), (f) LCDR3 having sequence QQSNQNPRT (sequence number 20), Including a light chain variable region, Includes.

[0014] In this embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b is (i) A heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to that of SEQ ID NO: 9, and a light chain variable region (VL) containing an amino acid sequence at least 95% identical to that of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 21, and a light chain variable region (VL) containing an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 22.

[0015] In the embodiment, in an antibody or its antigen-binding fragment that specifically binds to human FGFR2b, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NOs. 9 and / or 10, and / or SEQ ID NOs. 21 and / or 22, are inserted, deleted, or substituted.

[0016] In this embodiment, the antibody or antigen-binding fragment that specifically binds to human FGFR2b is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0017] In the embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b is either reduced-glycosylation, non-glycosylation, or low-fucosylation.

[0018] In the embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b includes an increase in the bisecting GlcNac structure.

[0019] In this embodiment, the antibody or its antigen-binding fragment that specifically binds to human FGFR2b has the Fc domain of IgG1.

[0020] In the embodiment, the pharmaceutical composition comprises an antibody or its antigen-binding fragment that specifically binds to human FGFR2b, and a pharmaceutically acceptable carrier.

[0021] In the embodiment, the isolated nucleic acid encodes an antibody or its antigen-binding fragment that specifically binds to human FGFR2b.

[0022] In the embodiment, the vector comprises a nucleic acid encoding an antibody or its antigen-binding fragment that specifically binds to human FGFR2b.

[0023] In the embodiment, the host cell comprises a nucleic acid encoding an antibody or its antigen-binding fragment that specifically binds to human FGFR2b, or a vector containing such nucleic acid. In the embodiment, the process for producing an antibody or its antigen-binding fragment that specifically binds to human FGFR2b comprises culturing such host cells in a culture medium and recovering the antibody or its antigen-binding fragment from the culture medium. In the embodiment, the purified composition comprises an anti-human FGFR2b antibody or its antigen-binding fragment produced by such a process.

[0024] In embodiments, the immunoconjugate, or a pharmaceutically acceptable salt thereof, solvate, or hydrate thereof, comprises an antibody or its antigen-binding fragment that specifically binds to human FGFR2b, and a cytotoxic agent.

[0025] In this embodiment, such an immunoconjugate is given by the formula: Ab-(CL-(D) m ) n , or comprising a pharmaceutically acceptable salt, solvate, or hydrate thereof, in the formula, Ab is an antibody or its antigen-binding fragment that specifically binds to human FGFR2b. C is a conjugator, L is a linker, D is a cytotoxic agent, m is an integer between 1 and 8. n is between 1 and 10.

[0026] In this embodiment, m is 1.

[0027] In this embodiment, n is 3, 4, 5, 6, 7, 8, 9, or 10.

[0028] In this embodiment, C is a given expression (CI), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), (C-IIIb), or (C-IV): [ka] [ka] It has, * indicates a bond in which the conjugator connects to Ab.

[0029] In the embodiment, L is given by formula (LI), (L-II), or (L-IIII): [ka] It has, Su is a hydrophilic residue, * indicates a linker that connects to a conjugator.

[0030] In this embodiment, Su is [ka] That is the case.

[0031] In this embodiment, Su is [ka] That is the case.

[0032] In this embodiment, the cytotoxic agent (D) is [ka] It is selected from the group consisting of the following.

[0033] In this embodiment, the cytotoxic agent (D) is [ka] It is selected from the group consisting of the following.

[0034] In this embodiment, the immunoconjugate is given by the following formula: [ka] [ka] It is represented by one of the following: or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof.

[0035] In this embodiment, n is 3, 4, 5, 6, 7, 8, 9, or 10.

[0036] In this embodiment, n is approximately 8.

[0037] In this embodiment, the immunoconjugate is given by the following formula: [ka] [ka] It is represented by one of the following: or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof.

[0038] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0039] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0040] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0041] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0042] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0043] In this embodiment, the immunoconjugate is given by the following formula: [ka] It is represented by or its pharmaceutically acceptable salts, solvates, and / or stereoisomers, where n is approximately 8 and Ab is, (i) A heavy chain variable region (VH) containing the amino acids of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0044] In the embodiment, the pharmaceutical composition comprises an immunoconjugate and a pharmaceutically acceptable carrier.

[0045] In the embodiment, the kit includes an antibody or its antigen-binding fragment that specifically binds to FGFR2b, a pharmaceutical composition, or an immunoconjugate, and instructions for their use.

[0046] In the kit embodiment, the antibody or its antigen-binding fragment forms a complex with FGFR2b, which is detected by assays including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and / or Western blotting.

[0047] In one embodiment, a method for treating small cell lung cancer or gastric cancer comprises administering an effective amount of a pharmaceutical composition, a purified composition, or an immunoconjugate to a patient in need of treatment.

[0048] In one embodiment, a method for treating tubulin inhibitor-resistant tumors comprises administering an effective amount of a pharmaceutical composition, purified composition, or immunoconjugate to a subject in which FGFR2b is accumulated or overexpressed in a biological sample.

[0049] In the embodiment, the pharmaceutical composition, purified composition, or immunoconjugate is used in the manufacture of a pharmaceutical product for treating small cell lung cancer or gastric cancer in a patient.

[0050] In the embodiment, the pharmaceutical composition, purified composition, or immunoconjugate is used to treat a patient's small cell lung cancer or gastric cancer.

[0051] In the embodiment, the pharmaceutical composition, purified composition, or immunoconjugate is administered in combination with at least one other therapeutic agent.

[0052] In this embodiment, the other therapeutic agent is a chemotherapeutic agent.

[0053] In embodiments, the other therapeutic agent includes at least one immune checkpoint inhibitor.

[0054] In this embodiment, the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody.

[0055] In this embodiment, the anti-PD1 antibody is tislerizumab.

[0056] In this embodiment, the method for producing an immunoconjugate is: (i) Culturing host cells transformed with an isolated nucleic acid comprising an anti-human FGFR2b antibody or a sequence encoding its antigen-binding fragment, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acids of SEQ ID NO: 10, or a heavy chain containing the amino acid sequence of SEQ ID NO: 21 and a light chain containing the amino acids of SEQ ID NO: 22. (ii) Expressing the antibody or its antigen-binding fragment, (iii) Recovering the expressed antibody or its antigen-binding fragment, (iv) Conjugate at least one cytotoxic agent (D) to an antibody or its antigen-binding fragment using a linker (L) and a conjugator (C) so that an immunoconjugate is formed, Includes.

[0057] This summary of the present invention is not intended, nor should it be construed, to represent the entirety or scope of the present disclosure. Furthermore, any reference made herein to “the present disclosure” or its embodiments should be understood to mean certain embodiments of the present disclosure, and not to be construed as limiting all embodiments to a particular description. The present disclosure is described in the summary of the present invention, as well as in the detailed description and accompanying drawings, at varying levels of detail, and is not intended to be limited in scope by any inclusion or exclusion of elements, components, etc., in the summary of the present invention. Features from any of the disclosed embodiments can be used in combination with each other without limitation. Furthermore, other features and advantages of the present disclosure will become apparent to those skilled in the art by examining the following detailed description and accompanying drawings. [Brief explanation of the drawing]

[0058] [Figure 1] This is a modified version of Tiong, KHet al. (2013) Dec;18:1447-68, showing the basic structure of the fibroblast growth factor receptor (FGFR). FGFR consists of three extracellular immunoglobulin-like (Ig-like) domains (D1, D2, and D3), a single transmembrane helix, intracellular split tyrosine kinase domains (TK1 and TK2), and an acidic box. Ig-like domains II (D2) and III (D3) form ligand-binding pockets and have distinct domains. FGFR splice variants are shown, which are formed from alternative splicing of D3. IgIIIb and IgIIIc splice variants are commonly found in the FGFR2 gene. Epithelial tissues mainly express the IgIIIb isoform, while mesenchymal tissues express IgIIIc. Hatched boxes represent shortened carboxyl terminals. [Figure 2]The amino acid sequence (SEQ ID NO: 1) of human FGFR2, provided under NCBI accession number P21802, is shown. The bolded part is the beta-IIIb(D2+D3) domain, which is approximately Pro253-Glu378, and this was used for immunization. The amino acid sequence (SEQ ID NO: 2), accession number AAB21431.1, of human keratinocyte growth factor is also provided. [Figure 3A] This report compares the native protein-binding activity of different humanized anti-FGFR2b antibodies using FGFR2b-overexpressing Expi293 cells. It shows a comparable comparison of human FGFR2b binding among BGAm1823, BGAh9239, BGAm1371, and BGAh9179. [Figure 3B] This shows a comparison of the innate protein-binding activity of different humanized anti-FGFR2b antibodies using FGFR2b-overexpressing Expi293 cells. Comparable cynomolgus monkey FGFR2b binding is demonstrated among BGAm1823, BGAh9239, BGAm1371, and BGAh9179. [Figure 3C] This report compares the intrinsic protein-binding activity of different humanized anti-FGFR2b antibodies using FGFR2b-overexpressing Expi293 cells. Comparable mouse FGFR2b binding is shown among BGAm1823, BGOH9239, BGAm1371, and BGOH9179. [Figure 3D] This shows a comparison of the native protein-binding activity of different humanized anti-FGFR2b antibodies using FGFR2b-overexpressing Expi293 cells. Comparable human FGFR2c binding is demonstrated among BGAm1823, BGOh9239, BGAm1371, and BGOH9179. [Figure 4A] This section compares the recombinant protein binding activity of different humanized anti-FGFR2b antibodies using ELISA. The ELISA results for BGAm1823, BGAh9239, BGAm1371, and BGAh9179 against human FGFR2b protein are shown. [Figure 4B]This section compares the recombinant protein binding activity of different humanized anti-FGFR2b antibodies using ELISA. The results of ELISA for BGAm1823, BGAh9239, BGAm1371, and BGAh9179 against cynomolgus monkey FGFR2b protein are shown. [Figure 4C] This section compares the recombinant protein binding activity of different humanized anti-FGFR2b antibodies using ELISA. The ELISA results for BGAm1823, BGAh9239, BGAm1371, and BGAh9179 against the human FGFR2c protein are shown. [Figure 5A] The SPR binding reaction rates of the selected chimeric FGFR2b binder are shown. [Figure 5B] The SPR binding reaction rates of the selected chimeric FGFR2b binder are shown. [Figure 5C] The SPR binding reaction rates of the selected chimeric FGFR2b binder are shown. [Figure 5D] The SPR binding reaction rates of the selected chimeric FGFR2b binder are shown. [Figure 6] The results of the Ba / F3 proliferation assay are shown. Net proliferation of Ba / F3 cells overexpressing FGFR2 has been reported after treatment with the anti-FGFR2b antibody of this disclosure. [Figure 7-1] The results of the Ba / F3 proliferation assay are shown. Net proliferation of Ba / F3 cells overexpressing FGFR2 has been reported after treatment with the anti-FGFR2b antibody of this disclosure. [Figure 7-2] Same as above. [Figure 8] This shows the primary structural alignment of amino acid sequences for major anti-FGFR2b antibody candidates. Typical sequence diversity is observed. [Figure 9] This study demonstrates the antitumor effects of different concentrations of BGA3457 and BGA9823 compared to the control BGA8723 in a xenotransplantation (CDX) model derived from a cell line using SNU-16 (FGFR2b high expression) cells. [Figure 10-1]This study demonstrates the antitumor effects of different concentrations of BGA3457 and BGA9823 compared to the control group BGA8723 in a xenotransplantation (CDX) model derived from a cell line using HSC-39 (gastric signet ring cell adenocarcinoma; expressed in FGFR2b) cells. [Figure 10-2] This study demonstrates the antitumor effects of different concentrations of BGA3457 and BGA9823 compared to the control group BGA8723 in a xenotransplantation (CDX) model derived from a cell line using HSC-39 (gastric signet ring cell adenocarcinoma; expressed in FGFR2b) cells. [Figure 11] Figures A and B show the antitumor activity of BGA3457 in xenograft models of HSC-39 (gastric signet ring cell adenocarcinoma, a cell line with moderate to low overexpression of human FGFR2b) + SNU-5 (gastric cancer) co-inoculated in a ratio of 2:1 or 5:1, respectively, to the control group BGA1421. [Figure 12] Figures A and B show the antitumor activity of BGA9823 in xenograft models of HSC-39 (gastric signet ring cell adenocarcinoma, a cell line with moderate to low overexpression of human FGFR2b) + SNU-5 (gastric cancer) co-inoculated in a ratio of 2:1 or 5:1, respectively, to the control group BGA1421. [Figure 13A] This study demonstrates cell toxicity by immunoconjugates BGA9823, BGA3457, and the control BGA8723 in multiple cell lines with different expression levels. The following cell line was evaluated: SNU16 (gastric cancer, high overexpression of FGFR2b). [Figure 13B] This study demonstrates cell toxicity by immunoconjugates BGA9823, BGA3457, and the control BGA8723 in multiple cell lines with different expression levels. The following cell line was evaluated: SNU601-h2b (gastric cancer, moderate FGFR2b overexpression). [Figure 13C] This study demonstrates cell killing by immunoconjugates BGA9823, BGA3457, and the control BGA8723 in multiple cell lines with different expression levels. The following cell line was evaluated: H1048-h2b (small cell lung cancer, moderate to low overexpression of FGFR 2b). [Figure 13D]This study demonstrates cell killing by immunoconjugates BGA9823, BGA3457, and the control BGA8723 in multiple cell lines with different expression levels. The following cell line was evaluated: HSC-39 (gastric signet ring cell adenocarcinoma, moderate to low overexpression of FGFR2b). [Figure 14A] This study demonstrates that BGA3457 exhibits nanomolar-range toxicity in cell lines with diverse FGFR2b expression. [Figure 14B] This study demonstrates that BGA3457 exhibits nanomolar-range toxicity in cell lines with diverse FGFR2b expression. [Figure 14C] This study demonstrates that BGA3457 exhibits nanomolar-range toxicity in cell lines with diverse FGFR2b expression. [Figure 14D] This study demonstrates that BGA3457 exhibits nanomolar-range toxicity in cell lines with diverse FGFR2b expression. [Figure 15] Figures A and B show that BGA3457 demonstrated a potent antitumor effect compared to the comparator BGA8723 in the FGFR2bhighSNU-16(15A) and FGFR2bmediumHSC-39(15B)CDX models. No weight loss was observed in any of the experiments. [Figure 16] This shows that BGA3457 demonstrated a statistically significant bystander killing effect in vitro. [Figure 17A] The results show that BGA3457 and BGA9823 exhibited good plasma stability in a human model, with minimal release of free payload (<1%) and stable DAR values ​​at 8. [Figure 17B] This study demonstrates that BGA3457 and BGA9823 exhibited good plasma stability in a mouse model, with minimal release of free payload (<1%) and stable DAR values ​​at 8. [Figure 18A] This study demonstrates that BGA3457 has an acceptable PK profile in tumor-carrying mice. BGA3457 exhibits nonlinear PK at 1–10 mg / kg in mice, suggesting target-mediated clearance (TMDD). [Figure 18B] This study demonstrates that BGA3457 has an acceptable PK profile in hFcRn mice. BGA3457 exhibits nonlinear PK at 1–10 mg / kg in mice, suggesting target-mediated clearance (TMDD). [Figure 19] This study shows that BGA3457 exhibited a generally favorable PK profile at 1, 3, and 10 mg / kg (mpk) in a cynomolgus monkey model. [Figure 20A] A and B are Western blots showing the effects of anti-FGFR2b antibodies on FGF7-induced or FGF10-induced FGFR phosphorylation. The anti-FGFR2b antibody slightly inhibited FGF7-induced signaling and had little to no effect on FGF10-induced signaling. [Figure 20B] Same as above. [Figure 21] The antitumor effect of BGA3457 in a patient-derived xenotransplant (PDX) model is shown in comparison to the vehicle and the control group BGA8723. [Figure 22] This study shows the effect of treatment with BGA3457 or the control BGA1421 on corneal thickness in mice. [Modes for carrying out the invention]

[0059] Abbreviation As used herein and in the appended claims, the following abbreviations apply: API (Active Pharmaceutical Ingredient) Complementarity-determining regions in CDR immunoglobulins CHO Chinese hamster ovaries DS API Concentration that yields 50% efficacy or binding in EC50 ELISA enzyme-linked immunosorbent assay FFPE formalin-fixed, paraffin-embedded. FGF (Fibroblast Growth Factor) FR Framework Domain HC heavy chain Concentration that results in 50% inhibition of IC50 IgG (Immunoglobulin G) IV Intravenous injection IHC immunohistochemistry or immunohistochemistry KGF (Keratinocyte Growth Factor) mAb monoclonal antibody NCBI (National Center for Biotechnology Information) PD-1 Program Death 1 PD-L1 programmed death ligand 1 VH (Variable Region of Immunoglobulin Heavy Chain) VK Immunoglobulin Kappa Light Chain Variable Region VL (Variable Region of Immunoglobulin Light Chain) v / v volume percentage w / v mass-volume percentage

[0060] definition The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0061] Unless otherwise defined below or elsewhere in this document, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0062] "FGFR2b" refers to a cell receptor. The amino acid sequence of human FGFR2b (SEQ ID NO: 1) can also be found under accession number P21802 (FGFR2b_HUMAN) or UniProtKB / Swiss-Prot:P21802.1. The bolded region in the following sequence is the beta-IIIb (D2+D3) domain, which is approximately Pro253-Glu378, and was used for immunization to create the antibodies disclosed herein. [ka]

[0063] The ligand for FGFR2b is known as keratinocyte growth factor ("KGF"), and the following amino acid sequence can also be found in accession number AAB21431.1 (SEQ ID NO: 2). [ka]

[0064] The terms "anti-FGFR2b antibody" and "antibody that binds to FGFR2b" refer to an antibody that can bind to FGFR2b with sufficient affinity to be useful as a diagnostic and / or therapeutic agent for targeting FGFR2b.

[0065] "FGFR2b expression positive" refers to elevated levels of FGFR2b expression (protein and / or mRNA) by malignant cells in a tumor compared to a suitable control. The level of FGFR2b mRNA expression can be compared to the mRNA expression levels of one or more reference genes frequently used in quantitative RT-PCR. In some embodiments, the level of FGFR2b expression (protein and / or mRNA) by malignant cells and / or infiltrating immune cells in a tumor is determined to be "overexpressed" or "elevated" based on a comparison with the level of FGFR2b expression (protein and / or mRNA) by a suitable control. For example, the expression level of the control FGFR2b protein or mRNA may be a level quantified in non-malignant cells of the same type or in sections from corresponding normal tissue. In some preferred embodiments, FGFR2b expression in a tumor sample is determined to be elevated if the FGFR2b protein (and / or FGFR2b mRNA) in the sample is at least 10%, 20%, or 30% higher than that of the control. "KGF expression positive" is defined similarly.

[0066] The units, prefixes, and symbols used herein are provided in the form recognized by the International System of Units (SI).

[0067] As used herein, including in the attached claims, singular words such as "a," "an," and "the" include their corresponding plural forms unless the context clearly indicates otherwise.

[0068] The term "or" is used to mean "and / or" unless the context clearly indicates otherwise, and is interchangeable with it.

[0069] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the specified features or components, with or without other(s). Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] The terms “for example” and “that is” as used herein are used merely as examples without any intention of limitation and should not be construed as referring only to items explicitly listed herein.

[0071] Unless otherwise specified or evident from the context, the terms “about” and “approximately” as used herein refer to a value or composition that falls within the tolerance range of a particular value or composition determined by those skilled in the art, which in part depends on how the value or composition is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean within or greater than one standard deviation, according to the convention of the art. “About” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% more or less than the stated value. For example, about 5 mg may contain any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms may mean a value of up to one order of magnitude or up to five times the stated value. Where specific values ​​or compositions are provided in this disclosure, unless otherwise specified, the meaning of “approximately” should be assumed to be within the tolerance range of those specific values ​​or compositions.

[0072] Terms such as "at least," "more than," and "greater than or equal to," for example, "at least one," are not limiting but are understood to include values ​​greater than the stated value, such as at least one, two, three, four, etc. Any larger number or fractional part in between is also included. Conversely, the term "less than or equal to" includes each value less than the stated value. For example, "less than or equal to 10 nucleotides" includes 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fractional part in between is also included. Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limiting but are understood to include values ​​greater than the stated value, such as at least two, three, four, five, etc. Any larger number or fractional part in between is also included.

[0073] In this specification, a range can be expressed as a range from one specific value to another specific value that is "about". When such a range is expressed, it may include one specific value and / or another specific value. Similarly, when a value is expressed as an approximation using the antecedent "about", it is understood that a specific value forms a separate case. Furthermore, it should be understood that each endpoint of a range can have significant meaning not only in relation to other endpoints but also independently of other endpoints. As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the listed range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0074] The term "reference" refers to the subject of comparison used as a standard or counter on which the comparison is made. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control, which is a drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those of the one being evaluated.

[0075] The terms “improve,” “increase,” “inhibit,” and “reduce” indicate values ​​relative to a baseline or other reference measure. In some embodiments, a suitable reference measure may include a measurement in a particular system (e.g., a single individual) under otherwise equivalent conditions, in the absence of a drug or treatment (e.g., before and / or after) or in the presence of a suitable equivalent reference drug. In some embodiments, a suitable reference measure may include a measurement in an equivalent system that is known to or expected to respond in an equivalent manner when the relevant drug or treatment is present.

[0076] The term “isolated” means (1) a substance separated from at least some components that were previously associated with it or would normally be associated with it, and / or (2) a substance present in a composition comprising one or more known or unknown contaminants in limited or specified amounts or concentrations. In some embodiments, the isolated substance may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85–90%, 85–95%, 85–100%, 90–95%, 90–100%, or 95–100%) of the other non-material components that the substance was previously associated with, e.g., other components or contaminants that the substance was previously or would normally be associated with. In certain cases, a substance is isolated when it is present in a composition containing molecules of the same or similar type in limited or reduced amounts or concentrations. For example, in certain cases, nucleic acid, DNA, or RNA substances are isolated when they are present in a composition containing non-substance nucleic acid, DNA, or RNA molecules in limited or reduced amounts or concentrations. Another example is polypeptide substances, isolated when they are present in a composition containing non-substance polypeptide molecules in limited or reduced amounts or concentrations. In certain embodiments, the amount may be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, the limited amount may be an amount that is 100% or less of the amount of the substance in the composition, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or less of the amount of the substance in the composition (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain cases, the composition is pure or substantially pure with respect to the selected substance. In some embodiments, the isolated substance has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0077] The term “drug” may refer to any class of molecules or entities, or more molecules or entities, which may be, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, conjugates (e.g., antibody drug conjugates), cells, or organisms (e.g., fractions or extracts thereof), or components thereof. In some embodiments, the drug may be available in isolated or pure form. In some embodiments, the drug may be available in crude or impure form. In some embodiments, the drug may be provided as a population, collection, or library that can be screened to identify or characterize the members contained therein.

[0078] "To bring into contact" is used in its simple and straightforward sense and refers to the process of bringing at least two distinct species (e.g., a chemical compound including a biomolecule or cell) close enough to react, interact, or physically touch. The two species could be, for example, the anti-FGFR2b antibody and FGFR2b antigen described herein. In certain embodiments, contact includes, for example, enabling the anti-FGFR2b antibody described herein to interact with the FGFR2b antigen.

[0079] As used herein, the terms “administer” and “administer” mean, when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Cell therapy includes contact of a reagent with cells, as well as contact of a reagent with a fluid where the fluid is in contact with cells.

[0080] The terms “subject” or “patient” as used herein include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient having or at risk of having one of the disorders described herein).

[0081] In one aspect, “treating” any disease or disorder means improving the disease or disorder (i.e., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another aspect, “treating,” “treating,” or “treatment” means alleviating or improving at least one physical parameter, including those that may not be identifiable by the patient. In yet another aspect, “treating,” “treating,” or “treatment” means modulating the disease or disorder either physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both. The disease or disorder may be related to the binding of FGFR2b to its ligand KGF ("FGFR2b-related disease").

[0082] The terms “effective dose” or “therapeutic dose” in relation to a compound mean an amount that can alleviate symptoms in whole or in part, or that can slow or halt further progression or worsening of those symptoms. As will be apparent to those skilled in the art, it should be expected that the effective dose of the compounds disclosed herein may vary depending on the severity of the indication being treated.

[0083] As used herein, “antitumor effect” or “antitumor efficacy” refers to a biological effect that may manifest as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an extension of overall survival or progression-free survival, an extension of life expectancy, or an improvement in various physiological symptoms associated with tumors.

[0084] The term “sample” generally refers to an aliquot of material obtained from or resulting from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may include cells or organisms such as a population of cells, tissue, or animal (e.g., human). In some embodiments, the source of interest may include biological tissue or biological fluid. In some embodiments, biological tissue or biological fluid may include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomit, and / or combinations or components thereof. In some embodiments, biological fluids may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cell permeable fluid. In some embodiments, biological tissue or specimens may be obtained, for example, by aspiration, biopsy (e.g., fine-needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or perfusion (e.g., bronchoalveolar, tubal, nasal cavity, eye, oral cavity, uterus, vagina, or other lavage or perfusion). In some embodiments, biological specimens include cells obtained from an individual. In some embodiments, specimens are “primary specimens” obtained directly from the source of interest by any suitable means. In some embodiments, as is evident from the context, the term “specimen” refers to preparations obtained by processing a primary specimen (e.g., by removing one or more components of the primary specimen and / or by adding one or more agents to the primary specimen). Such “processed samples” may include, for example, nucleic acids or proteins extracted from a sample, or nucleic acids or proteins obtained by subjecting a primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, isolation of specific components and / or purification.

[0085] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may include antibody production, activation of specific immune-qualified cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or expressed by recombination. Antigens may be specific to certain tissues, such as cancer cells, or they may be broadly expressed. Furthermore, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. In one particular embodiment, the antigen is all or a fragment of FGFR2b.

[0086] As used herein, the term “antibody” refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a more reversible and specific manner than covalent bonding. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH or HCVR) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL, Vκ, or LCVR) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), and interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0087] The location of the CDR and framework areas can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT, which are described in more detail below.

[0088] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, modified human antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies. Antibodies may belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0089] In this specification, the terms “monoclonal antibody” or “mAb” or “Mab” refer to a substantially homogeneous group of antibodies, i.e., the antibody molecules within that group have identical amino acid sequences, except for the possibility of naturally occurring mutations that may exist in small amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies having different amino acid sequences within their variable domains, particularly within their CDRs, which are often specific to different epitopes. The modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of the antibody by any particular method. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497, U.S. Patent No. 4,376,110, Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988, and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein may be any immunoglobulin class such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas that produce monoclonal antibodies can be cultured in vitro or in vivo. High-titer monoclonal antibodies can be obtained through in vivo generation. In this case, cells from individual hybridomas are intraperitoneally injected into mice, such as Balb / c mice, stimulated with pristine, to generate ascites containing high concentrations of the desired antibody. From such ascites, or from the culture supernatant, isotype IgM or IgG monoclonal antibodies can be purified using column chromatography methods well known to those skilled in the art.

[0090] Unless otherwise specified, “antigen-binding fragment” means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which it is bound by a full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, bispecific antibodies, linear antibodies, single-chain antibody molecules, e.g., single-chain Fv(ScFv), VHH (e.g., from species such as camelids or cartilaginous fish), antibodies formed from nanobodies and antibody fragments, and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0091] Antigen-binding fragments can be produced by any means. For example, in some embodiments, antigen-binding fragments can be produced enzymatically or chemically by fragmentation of intact antibodies. In some embodiments, antigen-binding fragments can be produced by recombination (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, antigen-binding fragments can be produced entirely or partially synthetically. In some embodiments, antigen-binding fragments may have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 amino acids or more (e.g., 50-100, 50-150, 50-200, or 100-200 amino acids). In some embodiments, antigen-binding fragments may have a length of at least about 200 amino acids.

[0092] The “target” is a binding motif, antigen-binding system, or any molecule to which a binding agent, such as an antibody, is bound. In some embodiments, the target is the antigen or epitope of the Disclosure. The “target cell” may express the antigen or epitope.

[0093] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. The variable regions of an antibody interact with an antigen via non-covalent forces at multiple sites. Generally, the more interactions there are, the stronger the affinity.

[0094] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more hypervariable regions (e.g., one or more CDRs) compared to the parental antibody without such changes, and such changes result in an improvement in the affinity of the antibody for the antigen. Herein, this process was used to humanize the parental mouse clones.

[0095] The term "binding" generally refers to non-covalent bonds between two or more entities. "Direct" binding involves physical contact between the entities or parts. "Indirect" binding involves physical interactions through physical contact with one or more intermediate entities. The binding between two or more entities can be evaluated in any of a variety of contexts, for example, when the interacting entities or parts are studied alone, or in the context of a more complex system (e.g., while covalently or otherwise associated with a carrier entity and / or within a biological system such as a cell).

[0096] "Binding affinity" generally refers to the total strength of the non-covalent binding interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Binding affinity can be measured and / or expressed in several ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). Generally, the affinity of molecule X for its partner Y can be represented by the equilibrium dissociation constant (K D ).

[0097] The term "equilibrium dissociation constant (K D )" is the dissociation rate constant (k off (kd), time -1 ) divided by the association rate constant (k on (ka), time -1 , M -lIt refers to the result of dividing by k. on and k off This can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA. Therefore, the dissociation constant (K D K) is the concentration of a given antibody, such as an anti-FGFR2b antibody, or its antigen-binding fragment, which, in equilibrium, occupies half of the available binding sites of the FGFR2b target epitopes in the system. D The smaller the value, the higher the affinity the antibody shows for its target antigen. The equilibrium dissociation constant can be measured using any known method in the art.

[0098] As used herein, an antibody or antigen-binding antibody fragment “specifically” or “selectively” binding to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target compared to other proteins, but this specificity does not require absolute binding specificity. “Specific” or “selective” binding reactions determine the presence of an antigen in a heterogeneous population of proteins and other biologics in, for example, blood, serum, plasma, or tissue samples. Therefore, under specific, designated immunoassay conditions, the antibody or its antigen-binding fragment specifically binds to a particular antigen at least twice as strongly as the background level, and does not specifically bind to other antigens present in the sample in significant amounts. In one embodiment, under designated immunoassay conditions, the antibody or its antigen-binding fragment specifically binds to a particular antigen at least ten times as strongly as the background level of binding, and does not specifically bind to other antigens present in the sample in significant amounts.

[0099] In certain embodiments, a molecule that specifically binds to an antigen is a molecule that binds to another antigen. A Compared to K, at least 2log, 2.5log, 3log, 4log, or greater than K A It then binds to the antigen.

[0100] In further embodiments, the molecule selectively binds to the target if the binding affinity between the molecule and the target is more than 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times greater than the binding affinity between the molecule and the non-target. In some embodiments, the binding affinity is about 10 -5 Less than M, approximately 10 -6 Less than M, approximately 10 -7 Less than M, approximately 10 -7 Less than M, or about 10 -9 When it is M, the molecule selectively binds to the target.

[0101] In another embodiment, the molecule that specifically binds to the target is approximately 1 × 10⁶ -7 The dissociation constant of M (K D ) binds. In some embodiments, the antigen-binding molecule is K D is approximately 1 x 10 -9 M ~ approx. 5×10 -9 When M, it specifically binds to the antigen with "high affinity". In some embodiments, the antigen-binding molecule is K D is 1 x 10 -10 M ~ approx. 5×10 -10 In this case, it specifically binds to the antigen with "very high affinity". In one embodiment, the antigen-binding molecule is 10 -9 M's K D It has. In one embodiment, the off speed is approximately 1 × 10 -5 It is less than.

[0102] In this specification, the term "human antibody" means an antibody containing only human immunoglobulin protein sequences. Human antibodies may contain mouse glycans if they are produced in mice, mouse cells, or mouse cell-derived hybridomas. Similarly, "mouse antibody" or "rat antibody" means an antibody containing only mouse immunoglobulin protein sequences or an antibody containing only rat immunoglobulin protein sequences, respectively.

[0103] The term "humanized" or "humanized antibody" means a form of antibody that includes sequences derived from non-human (e.g., murine) antibodies and human antibodies. Such antibodies contain the minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody includes substantially all of at least one, typically two, variable domains, and all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FR regions are the FR regions of human immunoglobulin sequences. A humanized antibody also optionally includes at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Fc). When it is necessary to distinguish a humanized antibody from a rodent parental antibody, the prefix "hum", "hu", "Hu", or "h" is added to the name of the antibody clone. The humanized form of a rodent antibody generally includes the same CDR sequences as the rodent parental antibody, but may include certain amino acid substitutions to enhance affinity, to enhance the stability of the humanized antibody, to remove post-translational modifications, or for other reasons.

[0104] As used herein, the term "binding agent" or "binder" refers to any molecule (e.g., an antibody) that can bind specifically to a given binding partner (e.g., an antigen).

[0105] The term "identity" refers to the overall relatedness between macromolecules, e.g., between nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The term "identical" or "percent identity" is a numerical score determined for a pair of aligned amino acid sequences or nucleic acid sequences. The percent identity measures the number of identical residues ("identity") between two sequences in relation to the length of the alignment over a "comparison window". This number represents the % of amino acid residues or nucleotides that are the same between the two sequences and indicates the degree of similarity of the primary structure.

[0106] Examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0107] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput.Appl.Biosci.4:11-17,(1988). This algorithm is incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using either a BLOSUM62 matrix or a PAM250 matrix, as well as the algorithm of Needleman and Wunsch, J.Mol.Biol.48:444-453,(1970), which is incorporated into the GAP program of the GCG software package, using a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0108] As used herein, the term “amino acid” refers to an organic compound containing an amine (-NH2) and carboxyl (-COOH) functional group, as well as a side chain (R group) specific to each amino acid. The term “amino acid” includes natural or unnatural, proteinogenic or non-proteinogenic, synthetic, D or L optical isomers, as well as amino acid analogs and peptide mimes. Natural amino acids are those encoded by the genetic code and those that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. “Proteogenic” means that an amino acid is one of the 20 naturally occurring amino acids found in proteins. Examples of proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteinogenic" means that an amino acid is either not found in nature in proteins or is not directly produced by cellular mechanisms (for example, it is a product of post-translational modification). Non-exclusive examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, beta-alanine, ornithine, and citrulline. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid mimetic refers to chemical compounds that have a different structure from the general chemical structure of amino acids but function in a similar way to natural amino acids. The terms "non-naturally occurring amino acids" and "unnatural amino acids" refer to amino acid analogs not found in nature, synthetic amino acids, and amino acid mimetic compounds.

[0109] Family of amino acid residues having side chains is defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR(or CDR) or within a framework region(or CDR) of an antibody or its antigen-binding molecule may be replaced with amino acid residues having similar side chains.

[0110] Exemplary amino acid classifications are summarized in Tables 1 and 2 below. [Table 1] [Table 2]

[0111] With respect to amino acid sequences, it will be understood by those skilled in the art that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences, which alter, add, or delete a single amino acid or a small number of amino acids within the encoded sequence, constitute a “conservatively modified variant” in which the alteration replaces an amino acid with a chemically similar amino acid. Such conservatively modified variants are not excluded from polymorphic variants, interspecific homologs, and alleles.

[0112] The most commonly occurring substitutions are isoleucine / valine, tyrosine / phenylalanine, aspartic acid / glutamic acid, lysine / arginine, methionine / leucine, aspartic acid / asparagine, glutamic acid / glutamine, leucine / isoleucine, methionine / isoleucine, threonine / serine, tryptophan / phenylalanine, tyrosine / histidine, tyrosine / tryptophan, glutamine / arginine, histidine / asparagine, histidine / glutamine, lysine / asparagine, lysine / glutamine, lysine / glutamic acid, phenylalanine / leucine, phenylalanine / methionine, serine / alanine, serine / asparagine, valine / leucine, and valine / methionine. The following eight groups each contain exemplary amino acids that can be considered conserved substitutions with one another (see, for example, Creighton, Proteins (1984)). 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) [Table 3]

[0113] "Conservative amino acid substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, skeletal structure and rigidity), allowing for frequent modifications without altering the protein's biological activity or other desired properties, such as antigen affinity and / or specificity. Generally, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th Ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to impair biological activity. Exemplary conservative substitutions are listed in Table 3 above.

[0114] As used herein, “peptide” is defined in its broadest sense in its various grammatical forms as a compound of two or more subunit amino acids, amino acid analogs, or other peptide mimetic compounds. The subunits may be linked by peptide bonds or by other bonds (e.g., esters, ethers, etc.). When a peptide chain is short, for example, of two, three or more amino acids, it is generally called an oligopeptide. When a peptide chain is longer, the peptide is usually called a polypeptide or protein. Full-length proteins, analogs, variants, and fragments thereof are included by definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, because ionizable amino and carboxyl groups are present intramolecularly, certain peptides may be obtained as acidic or basic salts, or in a neutral form. Peptides may be obtained directly from source organisms or produced recombinantly or synthetically.

[0115] The amino acid sequences of the antibodies are from Kabat et al., 1979, Dep't Health, Educ., Welfare, Pub. Health Serv., Nat'l Inst. Health ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Numbering can be done using any known numbering scheme, including the one described by Pluckthun, J.Mol.Biol., 2001, 309:657-70 ("AHo" numbering scheme).

[0116] Table 4 below shows examples of numbering schemes for CDR amino acid residues in the heavy chain variable domain and light chain variable domain.

[0117] When an antibody is said to contain a CDR by a specific definition (e.g., Kabat), that definition specifies the minimum number of CDR residues present in the antibody (i.e., KabatCDR). This does not rule out the possibility of other residues being present that fall within other conventional CDR definitions but outside the specified definition. For example, an antibody containing a Kabat-defined CDR may include, among other possibilities, an antibody in which the CDR contains a KabatCDR residue but no other CDR residues, and an antibody in which HCDR1 is a composite Chothia-KabatHCDR1, other CDRs contain a KabatCDR residue, but no additional CDR residues based on other definitions.

[0118] Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply differences in sequences where they do not exist, and one of ordinary skill in the art can readily confirm sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise noted, the "EU numbering scheme" is generally used when referring to residues within the constant region of an antibody heavy chain (e.g., as reported in Kabat et al. (supra)). [Table 4]

[0119] The terms "cancer" and "cancerous" refer to or describe a physiological state in a mammal characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More detailed examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, e.g., small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, e.g., gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, rectal cancer, colon cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal carcinoma, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0120] "Effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, effector cells can include, but are not limited to, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, and B lymphocytes. Effector cells can be cells of any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0121] "Effector function" refers to the biological consequences of the interaction between the Fc region of an antibody and an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector functions can be antigen-binding dependent, antigen-binding independent, or both. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. While we do not wish to be bound by any theory, ADCC is generally understood to involve effector cells possessing Fc receptors (FcRs) recognizing antibody-coated target cells (e.g., cells expressing the antigen to which the antibody binds on their surface) and subsequently killing them. Effector cells mediating ADCC may include, but are not limited to, one or more immune cells, including natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0122] As used herein, the term "cytotoxic activity" refers to activity that reduces or diminishes the cell viability of the cell line being tested.

[0123] As used herein, the term “bystander killing” refers to a situation in which a drug from an ADC is released from a target cell after internalization and degradation of the ADC, or the drug is released into the extracellular space. In either case, the drug is subsequently taken up by surrounding cells or bystander cells, which may or may not express the ADC target antigen, and kills these cells.

[0124] As used herein, the term “pharmaceutically acceptable excipients” includes all physiologically compatible solvents, dispersions, isotonic agents, and absorption retarders, etc. Excipients may be suitable for administration intravenously, intramuscularly, subcutaneously, parenterally, rectally, spinally, or epidermally (e.g., by injection or infusion).

[0125] The term "buffer solution" includes agents that maintain the pH of a solution of the compositions of this disclosure within an acceptable range.

[0126] The term “vector” refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be integrated into the host cell's genome and replicate in conjunction with the host genome. Furthermore, certain vectors consist of sequences that direct the expression of a functionally linked inserted gene. Such vectors may be referred to herein as “expression vectors.” Standard techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), are used for vector manipulation.

[0127] As used herein, “host cell” means any cell of any organism used for the purpose of producing a recombinant protein encoded by an expression vector, or growing an expression vector introduced into a host cell. “Mammalian recombinant host cell” means a mammalian host cell containing a heterologous expression vector, which may or may not be integrated into the host cell chromosome. “Bacterial recombinant host cell” means a bacterial host cell containing a heterologous expression vector, which may or may not be integrated into the host cell chromosome.

[0128] The terms “toxin,” “payload,” or “cytotoxic agent” are used herein to refer to molecules that inhibit or reduce the expression of molecules within a cell, inhibit or reduce cellular function, induce apoptosis, and / or cause cell death. This term includes toxins (including their fragments and / or variants), such as radioisotopes, chemotherapeutic agents, and small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin. Examples of cytotoxic agents include auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoids, pyrrolobenzodiazepines (PBD), lysine, lysine A chain, combrastatin, duocalmycin, dorastatin, doxorubicin, daunorubicin, taxol, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycete Examples of radioactive isotopes include, but are not limited to, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modesin A chain, alpha-sarcin, geronin, mitogenin, retostrictosin, phenomycin, enomycin, curisin, crotin, and calicheamicin, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0129] As used herein, the term “residue” refers to a chemical portion of a compound remaining after a chemical reaction. For example, the terms “amino acid residue” or “N-alkyl amino acid residue” refer to the product of an amide coupling or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling partner, for example, a water molecule being expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in the incorporation of an amino acid residue or N-alkyl amino acid residue into the product.

[0130] As used herein, “sugar,” “sugar group,” or “sugar residue” refers to a carbohydrate portion that may include a 3-carbon (triose) unit, a 4-carbon (tetrose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, “sugar,” “sugar group,” or “sugar residue” may include furanoses (e.g., ribofuranose, fructofuranose) or pyranoses (e.g., glucopyranose, galactopyranose), or a combination thereof. In some cases, “sugar,” “sugar group,” or “sugar residue” may include aldoses or ketoses, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-exclusive examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other “sugars” or “sugar groups” or “sugar residues” include polysaccharides and / or oligosaccharides (including, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose). In some cases, the “sugar” or “sugar group” or “sugar residue” is an amino sugar. In some cases, the “sugar” or “sugar group” or “sugar residue” is a glucamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the rest of the molecule via its amino group to form an amide bond with the rest of the molecule (i.e., glucamide).

[0131] The "alkyl" group is a saturated linear or branched acyclic hydrocarbon having 1 to 10 carbon atoms, usually 1 to 8 carbon atoms, or in some embodiments, 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Typical alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-hexyl, while saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted. In certain embodiments, where the alkyl groups described herein are said to be “substituted,” they may be substituted with any substituent or a number of substituents (as seen in the exemplary compounds and embodiments disclosed herein), as well as halogens (chloro, iodine, bromo, or fluoro), hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfonyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, B(OH)2, or O(alkyl)aminocarbonyl.

[0132] The "alkenyl" group is a linear or branched acyclic hydrocarbon having 2 to 10 carbon atoms, usually 2 to 8, and containing at least one carbon-carbon double bond. Representative linear and branched (C2C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutyrenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, etc. The double bond of the alkenyl group can be unconjugated or conjugated to other unsaturated groups. The alkenyl group may be unsubstituted or substituted.

[0133] As used herein, “alkynyl” refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynnyls can be optionally substituted and may be linear, branched, or cyclic. Alkynnyls are radicals having 2 to 20 carbon atoms, i.e., C 2-20 Alkynyl radicals are radicals having 2 to 12 carbon atoms, i.e., C 2-12 Alkynyl radicals are radicals having 2 to 8 carbon atoms, i.e., C 2-8 Alkynyl radicals are radicals having 2 to 6 carbon atoms, i.e., C 2-6 Alkynyl radicals, and radicals having 2 to 4 carbon atoms, i.e., C 2-4 This includes, but is not limited to, alkynyl radicals. Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl, and butynyl.

[0134] A "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group having 3 to 10 carbon atoms, having a monocyclic ring or multiple fused or crosslinked rings, which may be optionally substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, and in other embodiments, the number of ring carbon atoms is in the range of 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or multiple ring or crosslinked ring structures such as adamantyl. Examples of unsaturated cycloalkyl groups include, in particular, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl. Cycloalkyl groups may be substituted or unsubstituted. Examples of such substituted cycloalkyl groups include cyclohexanone.

[0135] An "aryl" group is an aromatic carbocyclic group having 6 to 14 carbon atoms, having a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthryl). In some embodiments, the aryl group has 6 to 14 carbon atoms in the ring portion of the group, and in other embodiments, it has 6 to 12 or even 6 to 10 carbon atoms. Specific examples of aryls include phenyl, biphenyl, naphthyl, etc. The aryl group may be substituted or unsubstituted. The phrase "aryl group" also includes groups containing fused rings, for example, fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0136] As used herein, "aryloxy" refers to the monovalent radical portion of an aromatic compound, where the ring atom is a carbon atom and the ring is substituted with an oxygen radical; that is, the aromatic compound contains a single bond to the oxygen atom and the radical is localized to the oxygen atom (e.g., in the case of phenoxy, C6H5-O-). The aryloxy substituent bonds to the compound it substitutes through this oxygen atom. The aryloxy is optionally substituted. The aryloxy is a radical having 6 to 20 ring carbon atoms, i.e., C 6-20 Aryloxy radicals are radicals having 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy radicals, and radicals having 6-10 ring carbon atoms, i.e., C 6-10 This includes, but is not limited to, aryloxy radicals. Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthroxy.

[0137] A "heteroaryl" group is an aryl ring system having 1 to 4 heteroatoms as ring atoms in a heteroaromatic ring system, with the remaining atoms being carbon atoms. In some embodiments, the heteroaryl group contains 5 to 6 ring atoms in the ring portion of the group, and in other embodiments, it contains 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-restrictive examples include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), im This includes, but is not limited to, groups such as dazopyridyl (e.g., azabenzimidazolyl, 3Himidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl.

[0138] A "heterocyclyl" is a non-aromatic cycloalkyl group in which 1 to 4 of the ring carbon atoms are independently replaced by heteroatoms independently selected from the group consisting of O, S, and N. In some embodiments, the heterocyclyl group has 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. Heterocyclyls can also be bonded to other groups at any ring atom (i.e., any carbon or heteroatom of the heterocyclic ring). Heterocyclyl groups may be substituted or unsubstituted. Heterocyclyl groups include unsaturated, partially saturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl groups. The term "heterocyclyl" also includes fused ring species (e.g., those containing fused aromatic and non-aromatic groups such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxynyl, and benzo[1,3]dioxolyl). This phrase also includes, but is not limited to, bridged polycyclic ring systems containing heteroatoms, such as quinuclidyl.Representative examples of heterocyclyl groups include azilidinyl, azetidinyl, pyrrolidyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranil, dioxolyl, furanil, thiophenyl, pyrrolyl, pyrrolidyl, imidazolyl, imidazolinil, pyrazolyl, pyrazolinil, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinil, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinil, thiomorpholinil, and tetrahydrothiophenyl. Lanyl (e.g., tetrahydro-2H-pyranil), tetrahydrothiopyranil, oxatian, dioxyl, dithianil, pyranyl, pyridyl, pyrimidinil, pyridazinil, pyrazinil, triazinil, dihydropyridyl, dihydrodithinyl, dihydrodithionyl, homopiperazinil, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolidinil, benzotriazolyl, benzimidazolyl, benzofuranil, benzothiophenyl, benzthiazolyl, benzooxadi Azolyl, benzoxadinyl, benzodithinyl, benzoxathinyl, benzothiadinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl, e.g., 1H-imidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridinyl-2(3H)-onyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolidinyl, quinoxal This includes, but is not limited to, the groups yl, quinazolinyl, cinolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiadinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxynyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Typical substituted heterocyclyl groups may be monosubstituted or multiple substituted, such as pyridyl or morpholinyl groups that are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents as listed below.

[0139] The "alkoxy" or "alkoxyl" group is -O(alkyl), where alkyl is defined above.

[0140] The "alkoxyalkyl" group is -(alkyl)O(alkyl), where each alkyl group is independently defined as above.

[0141] The "amine" group is a radical with the formula -NH2.

[0142] The "hydroxylamine" group is represented by formula: N(R # )OH or NHOH radical, where R # This refers to a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0143] The "alkoxyamine" group is represented by the formula: -N(R # ) an O-alkyl or -NHO-alkyl radical, where R # This is as defined above.

[0144] The "aralcoxyamine" group is represented by formula: N(R # ) an O-aryl or NHOaryl radical, where R # This is as defined above.

[0145] The "aminocarbonyl" group is represented by the formula: -C(=O)N(R # )2, -C(=O)NH(R # ), or a radical of C(=O)NH2, where each R # This is as defined above.

[0146] The "acylamino" group is represented by the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ) is a radical, in which each alkyl and R # They are independent, as defined above.

[0147] The "O(alkyl)aminocarbonyl" group has the formula: -O(alkyl)C(=O)N(R # )2,-O(alkyl)C(=O)NH(R # ), or -O(alkyl)C(=O)NH2 radical, where each R # They are independent, as defined above.

[0148] The "N-oxide" group is represented by the formula: -N + -O - It is radical.

[0149] The "carboxyl" group is a radical with the formula: C(=O)OH.

[0150] The "ketone" group is represented by the formula: C(=O)(R # ) is a radical, in the formula R # This is as defined above.

[0151] The "aldehyde" group is a radical with the formula -CH(=O).

[0152] The "ester" group is represented by the formula: C(=O)O(R # ) or OC (=O) (R # ) is a radical, in the formula R # This is as defined above.

[0153] The "urea" group has the formula: -N(alkyl)C(=O)N(R # )2,-N(alkyl)C(=O)NH(R # ), -N(alkyl)C(=O)NH2, -NHC(=O)N(R # )2, -NHC(=O)NH(R #) or NHC(=O)NH2 # is a radical, wherein each alkyl and R # is as defined above.

[0154] The "imine" group is a radical of the formula: -N=C(R # )2 or -C(R # )=N(R # ) wherein each R # is as defined above.

[0155] The "imide" group is a radical of the formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2, wherein each R # is as defined above.

[0156] The "urethane" group is a radical of the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # ), -N(R # )C(=O)O(R # ), or -NHC(=O)O(R # ), wherein each R # is as defined above.

[0157] The "amidine" group is a radical of the formula: -C(=N(R # ))N(R # )2, -C(=N(R # ))NH(R # ), -C(=N(R # ))NH2, -C(=NH)N(R # )2, -C(=NH)NH(R # ), -C(=NH)NH2, -N=C(R # )N(R # )2, -N=C(R # )NH(R # ), -N=C(R # )NH2, -N(R # )C(R # )=N(R # ), -NHC(R #)=N(R # ), -N(R # )C(R # )=NH, or -NHC(R # )=NH radical, and each R # They are independent, as defined above.

[0158] The "guanidine" group is represented by the formula: -N(R # )C(=N(R # ))N(R # )2, -NHC(=N(R # ))N(R # )2, -N(R # )C(=NH)N(R # )2, -N(R # )C(=N(R # ))NH(R # ), -N(R # )C(=N(R # ))NH2, -NHC(=NH)N(R # )2, -NHC(=N(R # ))NH(R # ), -NHC(=N(R # ))NH2, -NHC(=NH)NH(R # ), -NHC(=NH)NH2, -N=C(N(R # )2)2, -N=C(NH(R # ))2, or -N=C(NH2)2 radical, where each R # They are independent, as defined above.

[0159] The "enamine" group is represented by the formula: -N(R # )C(R # )=C(R # )2, -NHC(R # )=C(R # )2, -C(N(R # )2)=C(R # )2, -C(NH(R # ))=C(R # )2, -C(NH2)=C(R # )2, -C(R # )=C(R # )(N(R # )2), C(R #)=C(R # )(NH(R # )) or -C(R # )=C(R # )(NH2) is a radical, and in the formula, each R # They are independent, as defined above.

[0160] The "oxime" group is represented by the formula: -C(=NO(R # ))(R # ), -C(=NOH)(R # ), -CH(=NO(R # )), or -CH(=NOH) radical, where each R # They are independent, as defined above.

[0161] The "hydrazide" group is represented by the formula: -C(=O)N(R # )N(R # )2, -C(=O)NHN(R # )2, -C(=O)N(R # )NH(R # ), -C(=O)N(R # )NH2, -C(=O)NHNH(R # )2, or the radical of -C(=O)NHNH2, where each R # They are independent, as defined above.

[0162] The "hydrazine" group is represented by the formula: -N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ) 、 -N(R # )NH2, -NHNH(R # )2, or -NHNH2 radical, where each R # They are independent, as defined above.

[0163] The "hydrazone" group is represented by the formula: -C(=NN(R # )2)(R # )2, -C(=NNH(R # ))(R #)2, -C(=N-NH2)(R # )2, -N(R # )(N=C(R # )2), or -NH(N=C(R # )2) is a radical, in the formula, each R # They are independent, as defined above.

[0164] The "azide" group is a radical with the formula -N3.

[0165] The "isocyanate" group is a radical with the formula: N=C=O.

[0166] The "isothiocyanate" group is a radical with the formula: N=C=S.

[0167] The "cyanate" group is the radical of formula OCN.

[0168] The "thiocyanate" group is a radical of formula SCN.

[0169] The "thioether" group is represented by the formula -S(R # ) is a radical, in the formula R # This is as defined above.

[0170] The "thiocarbonyl" group is represented by the formula: -C(=S)(R # ) is a radical, in the formula R # This is as defined above.

[0171] The "sulfinyl" group is represented by the formula: -S(=O)(R # ) is a radical, in the formula R # This is defined above.

[0172] The "sulfone" group is represented by the formula: -S(=O)2(R # ) is a radical, in the formula R # This is as defined above.

[0173] The "sulfonamide" group is represented by the formula: -S(=O)2N(R #)2, or -S(=O)2NH(R # ), or -S(=O)2NH2 radical, where each R # They are independent, as defined above.

[0174] The "phosphonate" group is represented by the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ), or -OP(=O)(OH)(R # ) is a radical, in the formula, each R # They are independent, as defined above.

[0175] The "phosphine" group is represented by the formula: -P(R # )2 radicals, in the formula, each R # They are independent, as defined above.

[0176] With the exception of alkyl groups, where a group described herein is said to be "substituted," it may be substituted with any suitable substituent(s). Exemplary examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo, or fluoro), alkyl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfinyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, oxygen (=O), B(OH)2, O(alkyl)aminocarbonyl, cycloalkyl (this is simple These are aryloxys, aralkyloxys, heterocyclyloxys, and heterocyclylalkoxys (which may be cyclic or condensed or uncondensed polycyclic) (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocyclyls (which may be monocyclic or condensed or uncondensed polycyclic) (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiadinyl), monocyclic or condensed or uncondensed polycyclic aryl or heteroaryls (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridadinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl).

[0177] As used herein, the term “pharmaceutically acceptable salt” means a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic and organic acids and bases).

[0178] As used herein, unless otherwise indicated, the term “solvate” means a compound or a salt thereof that further contains a solvent, bonded by non-covalent intermolecular forces, in stoichiometric or non-stoichiometric amounts. In one embodiment, the solvate is a hydrate.

[0179] As used herein, unless otherwise indicated, the term “hydrate” means a compound or a salt thereof that further contains water, either stoichiometric or nonstoichiometric, bound by non-covalent intermolecular forces.

[0180] All pharmaceutically acceptable salts, solvates, and / or hydrates of the compounds illustrated herein are within the scope of this disclosure.

[0181] As used herein, unless otherwise indicated, the terms “stereoisomer” or “stereoisomerically pure” mean a stereoisomer of a compound that substantially contains no other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center substantially contains no opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers substantially contains no other diastereomers of that compound. Typical stereoisomerically pure compounds contain more than 80% by weight of one stereoisomer and less than 20% by weight of other stereoisomers of that compound, more than 90% by weight of one stereoisomer and less than 10% by weight of other stereoisomers of that compound, more than 95% by weight of one stereoisomer and less than 5% by weight of other stereoisomers of that compound, or more than 97% by weight of one stereoisomer and less than 3% by weight of other stereoisomers of that compound. Compounds may have chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomers, including mixtures thereof, are included in the embodiments disclosed herein. The use of stereoisomerically pure forms of such compounds, and the use of mixtures of those forms, are encompassed by the embodiments disclosed herein. For example, mixtures containing equimolar or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be synthesized asymmetrically or resolved using standard techniques such as chiral columns or chiral resolving agents.See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen, SH, et al., Tetrahedron 33:2725 (1977), Eliel, EL, Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962), and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0182] It should also be noted that the compound may include E isomers and Z isomers, or mixtures thereof, as well as cis isomers and trans isomers, or mixtures thereof. In certain embodiments, the compound is isolated as either a cis or trans isomer. In other embodiments, the compound is a mixture of cis and trans isomers.

[0183] A "tautomer" refers to an isomer of a compound that is in equilibrium with itself. The concentrations of these isomers vary depending on the environment in which the compound is found; for example, they may differ depending on whether the compound is in a solid state or in an organic solution or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomers, which are referred to as tautomers of each other: [ka]

[0184] As will be readily apparent to those skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of a compound are within the scope of this disclosure.

[0185] It should also be noted that compounds may contain unnatural proportions of atomic isotopes in one or more atoms. For example, a compound may contain tritium ( 3 H), Iodine-125( 125 I), Sulfur-35( 35 S), or carbon-14 ( 14 It may be radioactively labeled with radioactive isotopes such as C, or deuterium ( 2 H), carbon-13 ( 13 C), or nitrogen-15( 15 The compounds may be isotope-enriched, such as N). As used herein, “isotope-substituted compound” refers to an isotope-enriched compound. The term “isotope-enriched” refers to an atom having an isotope composition other than the atom’s natural isotope composition. “Isotope-enriched” may also refer to a compound containing at least one atom having an isotope composition other than the atom’s natural isotope composition. The term “isotope composition” refers to the amount of each isotope present in a given atom. Radiolabeled and isotope-enriched compounds are useful as therapeutic agents, e.g., cancer and inflammation treatments; research reagents, e.g., binding assay reagents; and diagnostic agents, e.g., in vivo contrast agents. All isotope variations of the compounds described herein, whether radioactive or not, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isotope-substituted compounds of the compounds are provided, for example, the isotope-substituted compounds are deuterium, carbon-13, or nitrogen-15 enriched compounds.

[0186] Certain groups, subgroups, substituents, and atoms are represented by a wavy line that intersects with a bond or multiple bonds, indicating the atom to which the group, subgroup, substituent, or atom is bonded. For example: [ka] A phenyl group substituted with a propyl group, as illustrated, has the following structure: [ka] It has.

[0187] Where used herein, diagrams showing substituents bonded to cyclic groups (e.g., aromatic, heteroaromatic, fused rings, and saturated or unsaturated cycloalkyl or heterocycloalkyl groups) via inter-ring atom bonds mean, unless otherwise specified, that the substituent can substitute for the cyclic group at any ring position on the cyclic group or on any ring of a fused ring group, according to the techniques described herein or techniques known in the art relating to this disclosure.

[0188] Figures showing substituents attached to an acyclic group via a bond between two atoms are, unless otherwise specified, meant to indicate that the substituent can be attached to any atom of the bond through which the substituent bond passes, according to the techniques described herein or techniques known in the art relating to this disclosure. Therefore, for example, [ka] It includes.

[0189] Please note that if there is a discrepancy between the structure shown and the name given to that structure, the shown structure should take precedence.

[0190] In the attached claims and the preceding description, unless the context requires otherwise by express wording or implicit meaning, the term “comprise,” or variations such as “comprises” or “comprising,” is used in a comprehensive sense, that is, to clearly indicate the presence of the features shown, but not to exclude the presence or addition of further features in the various embodiments of this disclosure. Detailed explanation

[0191] The present invention provides antibodies that specifically bind to human FGFR2b, their antigen-binding fragments, and antibody-pharmaceutical conjugates. Furthermore, the present disclosure provides antibodies, their antigen-binding fragments, and antibody-pharmaceutical conjugates that have desirable pharmacokinetic properties and other desirable attributes and can therefore be used to reduce the likelihood of cancer or to treat cancer. The present disclosure further provides pharmaceutical compositions comprising antibodies, their antigen-binding fragments, and antibody-pharmaceutical conjugates, as well as methods for producing and using such pharmaceutical compositions for the prevention and treatment of FGFR2b-related diseases.

[0192] Anti-FGFR2b antibody This disclosure provides antibodies and antigen-binding fragments that specifically bind to FGFR2b. The antibodies and antigen-binding fragments may be, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human-modified antibodies, single-chain antibodies (scFv), Fab fragments, Fab' fragments, or F(ab')2 fragments. The antibodies and antigen-binding fragments of this disclosure include, but are not limited to, each of the antibodies or antigen-binding fragments described below and produced as described below.

[0193] In embodiments, the antibody or antigen-binding fragment provided herein binds to a target human antigen, e.g., human FGFR2b, with a higher affinity than to another species of the target antigen, e.g., non-human FGFR2b. In embodiments, the antibody or antigen-binding fragment binds to human FGFR2b with an affinity 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher than to another species of FGFR2b, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In embodiments, the antibody or antigen-binding fragment described herein that binds to a target human antigen binds to another species of target antigen with an affinity less than 10%, less than 15%, or less than 20% of the binding of the antibody or antigen-binding fragment to the human antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

[0194] In one embodiment, the antigen-binding molecule is approximately 1 × 10⁻⁶ -7 M ~ approx. 1×10 -13 M's K D It binds to human FGFR2b. In another embodiment, the antigen-binding molecule is approximately 1 × 10⁻⁶ -10 M ~ approx. 5×10 -10 K D It binds to human FGFR2b. In some embodiments, the antigen-binding molecule is approximately 1 × 10⁻⁶ -7 M ~ approx. 1×10 -13 M's K D It binds to human FGFR2b. In yet another embodiment, the antigen-binding molecule is approximately 1 × 10⁻⁶ -10 M ~ approx. 5×10 -10 M's K D It then binds to human FGFR2b.

[0195] In one embodiment, the degree of binding of an anti-FGFR2b antibody to unrelated non-FGFR2b proteins is less than approximately 10% of the binding of this antibody to FGFR2b, as measured, for example, by radioimmunoassay (RIA). In a particular embodiment, the dissociation constant (K) of the antibody that binds to FGFR2b is D) are ≤1M, ≤100nM, ≤10nM, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (for example, 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13 M) is the answer.

[0196] In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a heavy chain variable region including one, two, or three of the following: a heavy chain complementarity-determining region (HCDR) 1 containing the amino acid sequence IYWX1N (where X1 is M or L), an HCDR2 containing the amino acid sequence IYPENX2DTNYX3GKFKG (where X2 is G or A, and X3 is S or N), and an HCDR3 containing the amino acid sequence GGFDY. In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a heavy chain variable region including one, two, or three of the following: an HCDR1 containing the amino acid sequence IYWLN (SEQ ID NO: 3), an HCDR2 containing the amino acid sequence QIYPENADTNYSGKFKG (SEQ ID NO: 4), and an HCDR3 containing the amino acid sequence GGFDY (SEQ ID NO: 5).

[0197] In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a light chain variable region including one, two, or three of the following: a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence RASENIYSNLA, an LCDR2 containing the amino acid sequence TATNLAE (where X4 is D or E), and an LCDR3 containing the amino acid sequence QHFYGILYT. In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a light chain variable region including one, two, or three of the following: an LCDR1 containing the amino acid sequence RASENIYSNLA (SEQ ID NO: 6), an LCDR2 containing the amino acid sequence TATNLAE (SEQ ID NO: 7), and an LCDR3 containing the amino acid sequence QHFYGILYT (SEQ ID NO: 8).

[0198] In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, comprises (a) a heavy chain variable region comprising HCDR1 comprising the amino acid sequence IYWLN (SEQ ID NO: 3), HCDR2 comprising the amino acid sequence QIYPENADTNYSGKFKG (SEQ ID NO: 4), and HCDR3 comprising the amino acid sequence GGFDY (SEQ ID NO: 5), and (b) a light chain variable region comprising LCDR1 comprising the amino acid sequence RASENIYSNLA (SEQ ID NO: 6), LCDR2 comprising the amino acid sequence TATNLAE (SEQ ID NO: 7), and LCDR3 comprising the amino acid sequence QHFYGILYT (SEQ ID NO: 8).

[0199] In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a heavy chain variable region comprising one, two, or three of the following: HCDR1 comprising the amino acid sequence DTYIH, HCDR2 comprising the amino acid sequence RIDPAX1GNTMFASEFQG (X1 is E or N), and HCDR3 comprising the amino acid sequence SKIHYDYDEGFAY. In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a heavy chain variable region comprising one, two, or three of the following: HCDR1 having the amino acid sequence DTYIH (SEQ ID NO: 15), HCDR2 having the amino acid sequence RIDPAEGNTMFASEFQG (SEQ ID NO: 16), and HCDR3 having the amino acid sequence SKIHYDYDEGFAY (SEQ ID NO: 17).

[0200] In the embodiment, the disclosed antibody and its antigen-binding fragment, which specifically bind to FGFR2b, include a light chain variable region comprising one, two, or three of the following: LCDR1 comprising the amino acid sequence RASESVDDYGYSFLH (SEQ ID NO: 18), LCDR2 comprising the amino acid sequence RASNLES (SEQ ID NO: 19), and LCDR3 comprising the amino acid sequence QQSNQNPRT (SEQ ID NO: 20).

[0201] In some embodiments, the Disclosure provides antibodies and antigen-binding fragments that specifically bind to FGFR2b, wherein the antibodies and antibody fragments (e.g., antigen-binding fragments) include a VH domain containing an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO: 21 (Table 8), and the antibodies or antigen-binding fragments include six complementarity-determining regions (CDRs) in SEQ ID NO: 9 or SEQ ID NO: 21 shown in Table 8. The Disclosure also provides an antibody or antigen-binding fragment that specifically binds to FGFR2b, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR (HCDR) containing the amino acid sequence of any one of the HCDRs listed in Table 8. In one embodiment, the Disclosure provides an antibody or antigen-binding fragment that specifically binds to FGFR2b, wherein the antibody comprises (or alternatively comprises) one, two, three, or more HCDRs containing the amino acid sequence of any of the HCDRs listed in Table 8.

[0202] This disclosure provides antibodies and antigen-binding fragments that specifically bind to FGFR2b, wherein the antibodies and antibody fragments include a VH domain containing an amino acid sequence identical to at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21 (Table 8), or the sequence described in SEQ ID NO: 9 or SEQ ID NO: 21, and a VL domain containing an amino acid sequence identical to at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 22, or the sequence described in SEQ ID NO: 10 or SEQ ID NO: 22 (Table 8). This disclosure also provides antibodies and antigen-binding fragments that specifically bind to FGFR2b, wherein the antibodies or antigen-binding fragments include light chain CDRs (LCDRs) and HCDRs of single antibodies listed in Table 8. In particular, this disclosure provides an antibody or antigen-binding fragment that specifically binds to FGFR2b, wherein the antibody or antigen-binding fragment comprises (or alternatively comprises) one, two, three or more LCDRs and one, two, three or more HCDRs of any one of the antibodies listed in Table 8.

[0203] Specific binding was achieved even when the heavy chain variable region and the light chain variable region had approximately 78% sequence identity with each other. Therefore, amino acids that are at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO: 9 or SEQ ID NO: 21. The claim relating to a genus of antibodies having a heavy chain variable region (HCVR) containing an acid sequence and a light chain variable region (LCVR) containing an amino acid sequence that is at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 10 or sequence number 22 has structural support.

[0204] In certain embodiments, an HCVR sequence that is identical to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21 by at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence may include substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2b antibody or its antigen-binding fragment containing that sequence retains the ability to specifically bind to FGFR2b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 9 or SEQ ID NO: 21. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 9 or SEQ ID NO: 21. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR).

[0205] In certain embodiments, an LCVR sequence that is identical to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 22 by at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence may include substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FGFR2b antibody or its antigen-binding fragment containing that sequence retains the ability to specifically bind to FGFR2b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10 or SEQ ID NO: 22. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10 or SEQ ID NO: 22. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR).

[0206] The structural consensus sequences disclosed herein are intended to guide those skilled in the art with regard to methods for predicting other species that specifically bind to FGFR2b and fall within the scope of the claimed structural / functional genera. This disclosure provides common structural features of antibody genera or their antigen-binding fragments that correlate with the described functions. The common structural features disclosed as species represent the full diversity of the claimed genera.

[0207] Modification of the FC domain framework In some embodiments, the Fc region is modified by altering the effector function of the antibody by substituting at least one amino acid residue with a different amino acid residue. For example, one or more amino acids can be substituted with different amino acid residues, resulting in an antibody that has a modified affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand with modified affinity may be, for example, the Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patents 5,624,821 and 5,648,260 (both by Winter et al.).

[0208] In another embodiment, one or more amino acid residues may be substituted with one or more different amino acid residues so that the antibody has modified C1q binding and / or reduced or absent complement-dependent cell-mediated cytotoxicity (CDC). This technique is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0209] In another embodiment, one or more amino acid residues are modified to alter the antibody's ability to immobilize complement. This technique is described, for example, in Publication WO94 / 29351 by Bodmer et al. In a particular embodiment, one or more amino acids of the antibody or its antigen-binding fragment of the Disclosure are replaced with one or more allotype amino acid residues for the IgG1 subclass and kappa isotype. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of kappa isotypes, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0210] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the antibody's ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or to increase the antibody's affinity for the Fcγ receptor. This approach is described, for example, in Presta's publication WO00 / 42072. Furthermore, binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0211] In another embodiment, the glycosylation of an antibody is modified. For example, a non-glycosylated antibody (i.e., an antibody with no or reduced glycosylation) can be produced. By modifying glycosylation, for example, the affinity of the antibody to an "antigen" can be increased. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, by making one or more amino acid substitutions, one or more variable region framework glycosylation sites are removed, thereby removing glycosylation at that site. Such nonglycosylation can increase the affinity of the antibody to an antigen. Such approaches are described, for example, in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0212] Additionally or alternatively, antibodies with altered glycosylation types can be produced (e.g., low-fucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNAc structures). Such modified glycosylation patterns have been shown to enhance the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with modified glycosylation pathways. Cells with modified glycosylation pathways have been described in the Art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with modified glycosylation. For example, Hang et al., EP1,176,195, describe a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, and therefore antibodies expressed in such cell lines exhibit low fucosylation. Presta's publication WO03 / 035835 describes the Lecl3 cell line, a variant CHO cell line with reduced ability to bind fucose to Asn(297)-linked carbohydrates, which also results in reduced fucosylation of antibodies expressed in its host cells (see also Shieldsetal., (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s WO99 / 54342 describes a cell line manipulated to express glycoprotein-modified glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) so that antibodies expressed in the manipulated cell line show increased bisecting GlcNac structure, resulting in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0213] In another aspect, when reduction of ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to possess only moderate ADCC and little CDC effector function (Moore G.L., et al., 2010 MAbs, 2:181-189). However, natural IgG4 has been found to be less stable in acidic buffers or under stress conditions such as elevated temperature (Angal, S. 1993 Mol Immunol, 30:105-108, Dall'Acqua, W. et al, 1998 Biochemistry, 37:9266-9273, Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably binding the antibody to IgG4 Fc manipulated with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the less desirable intrinsic properties of IgG4 is its ability to dynamically separate its two heavy chains in solution to form half an antibody, which in vivo generates a bispecific antibody via a process called "Fab arm exchange" (Van der Neut Kolfschoten M., et al., 2007 Science, 317:1554-157). A serine-to-proline mutation at position 228 (EU numbering system) appeared to inhibit the separation of the IgG4 heavy chain (Angal, S. 1993 Mol Immunol, 30:105-108, Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and γFc region have been reported to affect antibody interactions with the Fcγ receptor (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040, Mukherjee, J. et al., 1995 FASEB J, 9:115-119, Armour, K. et al., 1999 Eur J Immunol, 29:2613-2624, Clynes, RA et al, 2000 Nature Medicine, 6:443-446, Arnold JN, 2007 Annu Rev immunol, 25:21-50). Furthermore, some IgG4 isoforms that occur rarely in the human population can also produce different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To produce antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc region of human IgG4 and introduce several modifications. These modified IgG4 Fc molecules can be found in Sequence IDs 83-88 of U.S. Patent No. 8,735,553 by Li et al. Production of FGFR2b antibodies

[0214] FGFR2b antibodies and their antigen-binding fragments (including disclosed ADCs) can be produced by any means known in the art, including but not limited to recombinant expression, chemosynthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression may be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0215] This disclosure further provides polynucleotides encoding antibodies as described herein, for example, polynucleotides encoding a variable region or segment of the heavy chain or light chain containing a complementarity-determining region as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region is at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in nucleic acid sequence to the polynucleotide represented by SEQ ID NO: 13 or SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the light chain variable region is at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in nucleic acid sequence to SEQ ID NO: 14 or SEQ ID NO: 26. In some embodiments, the antibodies described herein are encoded by a polynucleotide encoding a heavy chain variable region that is identical to the nucleic acid sequence of at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the polynucleotide represented by SEQ ID NO: 13 or SEQ ID NO: 25, and a polynucleotide encoding a light chain variable region that is identical to the nucleic acid sequence of at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the polynucleotide represented by SEQ ID NO: 14 or SEQ ID NO: 26. SEQ ID NOs: 13 and 14 may be paired as heavy chain and light chain, respectively, and SEQ ID NOs: 25 and 26 may be paired as heavy chain and light chain, respectively.

[0216] The polynucleotides of this disclosure can encode the variable region sequences of anti-FGFR2b antibodies. They can also encode both the variable and constant regions of the antibody. Some of the polynucleotide sequences encode polypeptides containing the variable regions of both the heavy and light chains of the exemplified anti-FGFR2b antibodies.

[0217] This disclosure also provides expression vectors and host cells for the production of anti-FGFR2b antibodies. In embodiments, the expression vector comprises a polynucleotide encoding the antibody described herein. In embodiments, the host cell comprises a polynucleotide encoding the antibody described herein. In embodiments, the host cell comprises a vector comprising a polynucleotide encoding the antibody described herein.

[0218] The choice of expression vector varies depending on the host cell in which the vector is intended to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably ligated to a polynucleotide encoding an anti-FGFR2b antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent the expression of the inserted sequence, except under controlled induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducible conditions without biasing the population for coding sequences in which the expression product is better tolerated by the host cell. In addition to promoters, other regulatory elements may also be included for the efficient expression of the anti-FGFR2b antibody or its antigen-binding fragment. These elements may include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by incorporating the appropriate enhancer into the cell line being used (see, for example, Scharfetal., ResultsProbl.CellDiffer.20:125,1994, and Bittneretal., Meth.Enzymol.,153:516,1987). For example, SV40 enhancers or CMV enhancers can be used to increase expression in mammalian host cells.

[0219] The host cells for harboring and expressing the anti-FGFR2b antibody vector may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of this disclosure. Other suitable microbial hosts include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, it is also possible to create expression vectors containing expression regulatory sequences (e.g., origins of replication) that are adapted to the host cell. Furthermore, various well-known promoters may exist, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or phage-lambda-derived promoter systems. Promoters typically have, optionally, operator sequences to control expression and ribosome-binding site sequences for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express anti-FGFR2b antibodies. Insect cells can also be used in combination with baculovirus vectors.

[0220] In other embodiments, mammalian host cells are used to express and generate the anti-FGFR2b antibody of this disclosure. Examples include hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines having exogenous expression vectors. These include any normally lethal, or normal or abnormally immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for polypeptide expression is commonly described, for example, in Winnacker, From GenestoClones, VCHPublishers, NY, NY, 1987. Mammalian host cell expression vectors may contain expression regulatory sequences such as origins of replication, promoters, and enhancers (see, e.g., Queenetal., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Appropriate promoters may be constitutive, cell type-specific, stage-specific, and / or tunable or modifiable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0221] Any of the aforementioned host cells can be modified using any of the aforementioned expression vectors by transient or stable transfection. Suitable transfection methods include the use of cationic lipids, polyethyleneimines, Lipofectamine®, or ExpiFectamine®, or electroporation, or other means. Those skilled in the art are aware of numerous suitable means for transfection to achieve recombinant antibody expression.

[0222] When culturing transfected or transformed host cells, the anti-FGFR2b antibody of this disclosure, or its antigen-binding fragment, can be secreted directly into the cell culture medium (for example, by using an appropriate secretion-inducing signal peptide) and recovered therefrom.

[0223] In the embodiment, the recovered anti-FGFR2b antibody, or its antigen-binding fragment, may be used in the composition. In the embodiment, the anti-FGFR2b antibody, or its antigen-binding fragment, is first conjugated or linked to at least one therapeutic agent via a linker that forms a conjugate to form a conjugate. This will be described in more detail below. Conjugated anti-FGFR2b antibody and its antigen-binding fragment

[0224] The present invention provides, in particular, anti-FGFR2b antibodies and their antigen-binding fragments that conjugate to one or more therapeutic agents via a linker to produce conjugates (also referred herein as “immunoconjugates” or “antibody-pharmaceutical conjugates”). In some embodiments, the therapeutic agents include, or are, cytotoxic agents, drugs, and / or radioisotopes. The therapeutic agents are also referred herein as “growth inhibitors,” “payloads,” or “toxins,” and may be abbreviated as “D.”

[0225] In this embodiment, the immunoconjugate is given by the following formula A: Ab-(CL-(D) m )n (A), or having a pharmaceutically acceptable salt, solvate, or hydrate thereof, in the formula, Ab is an antibody or its antigen-binding fragment disclosed herein, C is a conjugator, L is a linker, D is a cytotoxic agent, m is an integer between 1 and 8. n is between 1 and 10.

[0226] In a specific embodiment, m is 1.

[0227] In this embodiment, the immunoconjugate is given by the following formula A-1: Ab-(CLD) n (A-1) or having a pharmaceutically acceptable salt, solvate, or hydrate thereof, in the formula, Ab is an antibody or its antigen-binding fragment disclosed herein, C is a conjugator, L is a linker, D is a cytotoxic agent, n is between 1 and 10.

[0228] In some embodiments, n is 1 to 10 or 3 to 10. In some embodiments, n is about 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 7 to 9. In some embodiments, n is about 8.

[0229] In this embodiment, C is a given expression (CI), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), (C-IIIb), or (C-IV): [ka] [ka] It has, In the formula, * indicates a bond in which the conjugator connects to Ab.

[0230] In the embodiment, L is given by formula (LI), (L-II), or (L-IIII): [ka] It has, In the formula, Su is a hydrophilic residue, and * indicates a bond that connects the linker to the conjugator.

[0231] In this embodiment, Su is [ka] That is the case.

[0232] In this embodiment, Su is [ka] That is the case.

[0233] Cytotoxic agent D may be any drug that is harmful to cells (e.g., can kill cells). Examples of cytotoxic agents include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and any of the aforementioned analogues or homologues.

[0234] Other therapeutic or cytotoxic agents to which the anti-FGFR2b antibody or its antigen-binding fragment can be conjugated as disclosed herein include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and / or 5-fluorouracil decarbazine), topoisomerase I inhibitors such as exatecan derivatives (e.g., maleimidomaleimidocaproyl), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU)). This includes, but is not limited to, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and / or cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and / or doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mitramycin, and / or anthramycin (AMC)), and antimitotic agents (e.g., vincristine and / or vinblastine).

[0235] The anti-FGFR2b antibodies or their antigen-binding fragments described herein can also be conjugated with one or more radioisotopes (e.g., radioactive iodine) to produce cytotoxic radiopharmaceuticals for treating diseases, disorders, or conditions described herein, such as cancers described herein.

[0236] The anti-FGFR2b antibodies or their antigen-binding fragments provided herein can be conjugated with any one of the compounds described in WO2023 / 125530, which is incorporated herein by reference as a whole, as well as with the compounds exemplified herein, such as Tables 5A and 5B, and the prior art exatecan derivatives.

[0237] In some embodiments, D is a topoisomerase inhibitor. In some embodiments, D is a residue of an exatecan analog. In some embodiments, D is represented by one of the formulas in Table 5A. In some embodiments, D is represented by one of the formulas in Table 5B.

[0238] [Table 5-1] [Table 5-2] [Table 5-3]

[0239] [Table 6]

[0240] In some embodiments, referring to Tables 5A and 5B, the immunoconjugate includes P2. In some embodiments, the immunoconjugate includes P3. In some embodiments, the immunoconjugate includes P4.

[0241] In some embodiments, CLD is represented by one of the formulas in Table 6. [Table 7-1] [Table 7-2]

[0242] Referring to Table 6, LD2-1 and LD2-2 are commercially available and were purchased from MedChemExpressCO.LTD (Shanghai). The antibodies are bound via a conjugator, which is generally shown on the left side of the molecule, except for LD2-1 (see Table 6).

[0243] As used herein, BGA3457 is an immunoconjugate containing the anti-FGFR2b humanized antibody BGAh9179 and LD2-3.

[0244] As used herein, BGA9823 is an immunoconjugate comprising the anti-FGFR2b humanized antibody BGAh9239 and LD2-3.

[0245] As used herein, BGA8723 is an immunoconjugate comprising the anti-FGFR2b humanized antibody bemarituzumab (Amgen) and LD2-3.

[0246] To generate an anti-FGFR2b immunoconjugate, the antibody or fragment thereof of this disclosure may be conjugated to any of the conjugates, linkers, and / or payloads provided herein and / or shown above. For clarity, even if a conjugator, linker, or payload is shown as part of a combined construct above Table 6 or below Tables 7A-7B, individual conjugator, linker, or payload portions may be mixed and combined with any other conjugator, linker, payload, antibody, or its antigen-binding fragment to form an immunoconjugate.

[0247] In some embodiments, the immunoconjugate is represented by one of the formulas in Table 7A, or by a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof. [Table 8-1] [Table 8-2]

[0248] Referring to Table 7A, Ab is any anti-FGFR2b antibody or its antigen-binding fragment disclosed herein, and n is 1 to 10 or 3 to 10. In embodiments, n is about 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 7 to 9. In embodiments, n is about 8.

[0249] In some embodiments, the immunoconjugate is represented by one of the formulas in Table 7B, or by a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof.

[0250] [Table 9-1] [Table 9-2]

[0251] Referring to Table 7B, Ab is any anti-FGFR2b antibody or its antigen-binding fragment disclosed herein.

[0252] Treatment method The antibodies, antigen-binding fragments, and conjugates of this disclosure are useful for a variety of applications, including, but not limited to, methods for treating FGFR2b-related disorders or diseases. In certain embodiments, the FGFR2b-related disorder or disease is a tubulin inhibitor-resistant tumor or cancer. In some embodiments, subjects having such a disorder or disease have FGFR2b accumulation or overexpression in a biological sample.

[0253] Accordingly, this specification provides a method for treating cancer, which comprises administering to a patient in need of treatment an effective amount of an antibody, antigen-binding fragment and conjugate provided herein, or a pharmaceutical composition provided herein. In some embodiments, the cancer is FGFR2b positive. In some embodiments, the cancer is small cell lung cancer or gastric cancer.

[0254] The antibodies, antigen-binding fragments, and conjugates disclosed herein may be administered by any suitable means, for example, parenteral, intrapulmonary, and intranasal administration, and, if local treatment is desired, intra-lesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any suitable route, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules, including but not limited to single doses, multiple doses at various points in time, bolus doses, and pulse infusions, are contemplated herein.

[0255] Combination therapy In one embodiment, the FGFR2b antibody and its antigen-binding fragment, as well as the immunoconjugate, of this disclosure can be used in combination therapy. Combination therapy refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic portions). In some embodiments, two or more regimens may be administered simultaneously. In some embodiments, such regimens may be administered sequentially (e.g., all “dose” of the first regimen are administered, followed by any dose of the second regimen). In some embodiments, such drugs are administered in overlapping drug regimens. For clarity, combination therapy does not require (or even to be administered simultaneously) the individual drugs to be administered together in a single composition, but in some embodiments, two or more drugs, or their active portions, may be administered together in a combination composition, or further combined in a combination compound (e.g., as part of a single chemical complex or covalent conjugate). Such administration also includes simultaneous administration of each active component in multiple containers or in separate containers (e.g., capsules, powders, and liquids). The powder and / or liquid may be reconstituted or diluted to the desired dose before administration. Therefore, the phrase “in combination with ~” means that the anti-FGFR2b antibody, antibody-conjugated fragment, or antibody-pharmaceutical conjugate is administered to the subject concurrently with, immediately before, or immediately after the administration of the additional therapeutic agent. In certain embodiments, the anti-FGFR2b antibody, antibody-conjugated fragment, or antibody-pharmaceutical conjugate is administered as a co-formulation with the additional therapeutic agent. The co-formulation can be prepared at any point in time, including immediately before administration to the patient.

[0256] The FGFR2b antibody and its antigen-binding fragment, as well as the immunoconjugate, are used in combination therapy with the FGFR2b antibody and its antigen-binding fragments of this disclosure. Examples of therapeutic agents for this combination therapy include chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents (e.g., Abraxane®)), docetaxel, carboplatin, topotecan, deruxtecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacitidine, ifosfamide, oxaliplatin, pemetrexed disodium, and cyanoacrylate. Clophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multi-kinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeted drugs (e.g., rituximab, ofatumumab, RO5) 072759, LFB-R603), CD52-targeted drugs (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19-targeted drugs (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors Examples include, but are not limited to, agents (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), HGF / SF inhibitors (e.g., AMG102), EGEN-001, or polo-like kinase 1 inhibitors (e.g., BI672).

[0257] Other therapeutic agents for use in combination therapy include, for example, immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody. Anti-PD-1 antibodies include, but are not limited to, tislerizumab, pembrolizumab, and nivolumab. Tislerizumab is disclosed in US 8,735,553. Pembrolizumab (formerly MK-3475), disclosed by Merck in US Patents 8,354,509 and 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab is approved for the treatment of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is in clinical study for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, renal cancer, and Hodgkin lymphoma. In some embodiments, the anti-PD-1 antibody is tislerizumab.

[0258] Other immune checkpoint antibodies used in combination therapy may include anti-TIGIT antibodies. Such anti-TIGIT antibodies include, but are not limited to, those disclosed in WO2019 / 129261, which is incorporated herein by reference as a whole.

[0259] Pharmaceutical compositions and preparations Also provided are compositions comprising a pharmaceutical composition containing an anti-FGFR2b antibody or its antigen-binding fragment, as disclosed in Table 8; polynucleotides comprising a sequence encoding an anti-FGFR2b antibody or antigen-binding fragment; and immunoconjugates comprising any one of the disclosed anti-FGFR2b antibody or antigen-binding fragment. These compositions may further comprise suitable carriers or diluents, such as pharmaceutically acceptable excipients containing buffers well known in the art.

[0260] kit This disclosure provides, in particular, a kit comprising an anti-FGFR2b antibody or its antigen-binding fragment or conjugate, for example, one of the disclosed immunoconjugates, and instructions for use and / or instructions for administration. In some embodiments, the kit comprises at least one anti-FGFR2b antibody, its antigen-binding fragment or its conjugate, and a pharmaceutically acceptable carrier, and instructions for use and / or instructions for administration.

[0261] Kits for use in the various methods disclosed herein are also provided. Instructions may include instructions for administering one or more pharmaceutical compositions described herein to a subject in order to achieve the intended activity in the subject. The kits may further include instructions for selecting a person suitable for treatment based on identifying whether the person is in need of treatment. In some embodiments, instructions include instructions for administering at least one anti-FGFR2b antibody or its antigen-binding fragment or conjugate to a subject in need of treatment.

[0262] Instructions for administering one or more doses of at least one anti-FGFR2b antibody or its antigen-binding fragment or conjugate generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers in the kit may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions accompanying the kits of this disclosure are typically those found on the label or in the accompanying leaflet. The label or accompanying leaflet may indicate that one or more pharmaceutical compositions described herein are used for the treatment, delay of onset, and / or mitigation of a disease, disorder, or condition in a subject.

[0263] In some embodiments, the kits provided herein are housed in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, flasks, and flexible packaging. Packaging for use in combination with specific devices, such as injection devices, is also intended. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper puncturable by a subcutaneous needle). The container may also have a sterile access port.

[0264] The kit may include additional components such as buffers and interpretation information. The kit may include a container and label, or one or more accompanying documents on or associated with the container. In some embodiments, this disclosure provides a product comprising the contents of the kit described above.

[0265] In the embodiment, the antibody or antigen-binding fragment provided in the kit forms a complex with FGFR2b, which is detected by the assay. The assay may be enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and / or Western blotting. [Examples]

[0266] Example 1. Generation of mouse anti-FGFR2b antibody Mouse immunization To generate antibodies against FGFR2b, BALB / c mice were immunized with 200 μl of an antigen mixture containing human FGFR2 beta(IIIb) domain protein (FGF-HM2BD, KACTUS) and adjuvants (full Freund's adjuvant (F5881, Sigma) for prime immunization and Sigma adjuvant system (S6322, Sigma) for subsequent immunization). Figure 2 shows the amino acid sequence of human FGFR2 (SEQ ID NO: 1), provided by NCBI accession number P21802. The bolded domain is the beta-IIIb(D2+D3) domain, which is approximately Pro253-Glu378, and this was used for immunization. Animals were injected intraperitoneally and subcutaneously every two weeks. Serum titers against FGFR2b protein were determined by ELISA to monitor the humoral immune response. Animals with sufficient FGFR2b-specific antibody titers received a final boost of FGFR2b domain protein for antibody screening. Plasma cell screening using the Beacon Optofluidic System Three to five days after the final boost, the spleen was harvested, ground up, and prepared as a single-cell suspension. Plasma cells were isolated using a mouse CD138-positive selection kit (STEMCELL) according to the manufacturer's instructions. The density was 6.25 × 10⁶. 6Enriched plasma cells were imported into the channel at a concentration of 1 / ml and incorporated into the NanoPen chamber of the OptoSelect 14K chip (Berkeley Lights) according to the manufacturer's instructions. To screen for huFGFR2b-specific plasma cells, human FGFR2 alpha(IIIb) protein (FGF-HM4ABB, KACTUS) conjugated beads (520-00053, BerkeleyLights) and AlexaFluor488 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) were imported into the channel at a concentration of 5 μg / ml. After import, the freeze valve was turned on and the exposure time of the Alexa Fluor 488 phosphor in the FITC channel was set to 1000 milliseconds. Bloom-like positive signals were captured by time-lapse imaging with 3-minute intervals and 10 cycles. As a counterscreen to roll out unwanted signals, a human FGFR2 alpha(IIIc) protein (FGR-HM4CDB, KACTUS) conjugated bead assay was performed. Plasma cells showing a FGFR2b-specific positive signal were individually exported to 96-well plates filled with lysis buffer.

[0267] Example 2. Cloning, sequencing, and expression of antibody VH and VL genes. First-chain cDNA was synthesized, and the entire cDNA was amplified using the Opto Plasma B Discovery cDNA Synthesis Kit (Berkeley Lights) according to the manufacturer's instructions. The VH and VL genes of the antibodies were amplified using the Opto Plasma B Discovery Sanger Prep Kit (Berkeley Lights) according to the manufacturer's instructions. The amplified VH and VL genes were cloned into mammalian expression vectors containing the human IgG1 gene and the kappa chain constant region gene, respectively, and sequenced. The amino acid sequences of three HCDRs, three LCDRs, VH, VL, HC, and LC of representative humanized antibodies BGAh9179 and BGAh9239, as well as the DNA sequences of HC and LC, are listed in Table 8 as SEQ ID NOs: 3-26. The antibodies were expressed in Expi293 (trademark) cells and purified by affinity chromatography. The sequences of the parental mouse antibodies BGAm1823 and BGAm1371, as well as the reference antibodies BGA1421, BGA2354, and BGA2311, are also shown in Table 8. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8]

[0268] Example 3. Humanization of a chimeric anti-FGFR2b antibody. Antibodies BGOH9239 and BGOH9179 are humanized antibodies developed from the parental mouse antibodies BGAm1823 and BGAm1371, respectively. A multifaceted approach was applied to construct antibodies superior to the parental mouse antibodies BGAm1371 and BGAm1823. The heavy and light chains were optimized for humanization.

[0269] To humanize candidate BGAm1371 and BGAm1823, we searched for human germ cell IgG genes for sequences sharing high homology with the cDNA sequences of BGAm1371 and BGAm1823 using BLAST analysis of the human immunoglobulin gene database IMGT (imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). The human IGVH and IGVL genes, which are frequently present in the human antibody repertoire (Glanville 2009 PNAS106:20216-20221) and show high homology to BGAm1371 and BGAm1823, respectively, were selected as templates for humanization.

[0270] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol248: Antibody Engineering, Methods and Protocols, Humana Press), and humanized antibody variants (BGAh9239 and BGAh9179) were processed into human IgG1 format using an expression vector developed in-house. In the initial round of humanization, mutations from mouse amino acid residues to human amino acid residues in the framework region were induced by simulated 3D structure. Structurally important mouse framework residues were retained in the humanized variants to maintain the standard structure of the CDR, while the remaining mouse framework residues were reverse-mutated one by one to their corresponding human framework residues, generating a large number of humanized variants. These were then purified and characterized to determine which mouse framework residues could be replaced with human framework residues while maintaining the desired biophysical and biochemical properties. In the second round of humanization, key mouse framework residues identified during the first round of humanization were combined to generate the desired humanized versions of mouse BGAm1823 and BGAm1371. Specifically, the CDR of BGAm1823VL was transplanted into the framework of the human germline variable gene IGVK1-39 without retaining mouse residues to obtain the humanized VL sequence of BGOH9239 (SEQ ID NO:10). The CDR of BGAm1823VH was transplanted into the framework of the human germline variable gene IGVH1-69 while retaining two mouse residues (Y27, F91) to obtain the humanized VH sequence of BGAh9239 (SEQIDNO:9). The CDR of BGAm1371VL was transplanted into the framework of the human germline variable gene IGVK4-1 while retaining one mouse residue (L4) to obtain the humanized VL sequence of BGAh9179 (SEQIDNO:22). The CDR of BGAm1371VH was transplanted into the framework of the human germline variable gene IGVH1-46 while retaining two mouse residues (I29, A71) to obtain the humanized VH sequence of BGAh9179 (SEQIDNO:21).

[0271] BGAh9239 and BGAh9179 were constructed in humanized full-length antibody form using in-house developed expression vectors containing subcloning sites that can be easily adapted to the constant regions of human IgG1 and kappa chains, respectively. Expression and purification of BGAh9239 and BGAh9179 were achieved by co-transfection of the two constructs into Expi293® cells and purification using a protein A column (Cat: 17543801, GE Life Sciences). The purified antibodies were concentrated to 0.5–5 mg / mL in PBS, divided into aliquots, and stored in a freezer at -80°C.

[0272] The BGAh9239 and BGAh9179 humanized antibody variants were further processed to remove post-translational modification (PTM) sites within the CDR to improve their molecular and biophysical properties for use in human therapy. Considerations included amino acid composition, thermal stability (Tm), and surface hydrophobicity while maintaining antibody function. Normal sequence diversity was observed among the top candidates (Figure 8).

[0273] In summary, engineered versions of the humanized monoclonal antibodies BGAh9239 and BGAh9179 were generated and characterized in detail using the process described above. The results showed that all humanized variants were very similar in terms of binding affinity and functional activity, such as inhibition of downstream signaling via FGFR2b.

[0274] Example 4. Determination of binding affinity and specificity of anti-FGFR2b antibody The binding affinity and specificity of purified anti-FGFR2b antibodies were determined by ELISA and FACS. Briefly, 2 mg / ml of human FGFR2b (FGR-HM1BD, KACTUS) or FGFR2c (FGR-HM2CD, KACTUS) protein was coated onto 96-well ELISA plates. 50 mL of serially diluted antibody was incubated for 30–60 minutes, washed, and then incubated with HRP-conjugated goat anti-hIgG secondary antibody (Abcam, ab98624). After incubation and washing, the plates were stained with HRP substrate and absorbance was measured. Selected candidates were serially diluted and incubated with human FGFR2b, huFGFR2c, cynoFGFR2b, or mouse FGFR2b cells at 4°C for 30 minutes. After washing twice with FACS buffer, diluted Alexa Fluor 647 goat anti-human IgG secondary antibody was added and incubated with the cells in the dark at 4°C for 30 minutes. After washing twice with FACS buffer, the cells were resuspended in FACS buffer and read using a Becton Dickinson LSR Fortessa® cell analyzer. Nonspecific binding of these antibodies was evaluated by FACS binding to parental 293T blank cells. Titration curves were generated using the sigmoid dose-response nonlinear approximation in Dotmatics GraphPad® software.

[0275] Table 8 shows the sequences of the antibodies tested. BGA1421 is the comparative antibody bemarituzumab, and BGA2354 is the comparative antibody M047 (Bayer; BAY1895344). The isotype IgG control is CB-6 (also known as etesevimab or JS016), which is a recombinant neutralizing human IgG1 anti-SARS-CoV-2 monoclonal antibody.

[0276] As shown in Figures 3A-3D and Tables 9 and 10, all selected candidates specifically bind to FGFR2b with high affinity and cross-bind to FGFR2b in cynomolgus monkeys and mice. [Table 11] [Table 12]

[0277] Example 5. Binding rate of anti-FGFR2b antibody The binding affinity and kinetics of purified anti-FGFR2b antibodies were determined at room temperature by surface plasmon resonance (Biacore 8K, GE Life Sciences). Briefly, mouse anti-human IgG Fc antibodies were immobilized on amine-coupled activated CM5 biosensor chips (catalog number BR100530, GE Life Sciences). Purified monoclonal antibody candidates were flowed onto the chip surface and captured by anti-human IgG antibodies. Next, serial dilutions of soluble human FGFR2b protein (his-tag, in-house developed) were injected onto the antibody capture surface, and changes in the surface plasmon resonance signal were analyzed to determine the binding rate (k) using a 1:1 Langmuir binding model (BIA evaluation software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K) was calculated. D ) to ratio k off / k on The calculations were performed as follows. The binding rate profiles of the selected monoclonal anti-FGFR2b antibodies are shown in Table 11 below. Detailed binding curves for the selected candidate antibodies and comparison antibodies are shown in Figures 5A to 5D. [Table 13]

[0278] Example 6. In vitro proliferation inhibition assay in BaF3_hFGFR2b OE cells The inhibitory activity of antibodies against ligand-induced cell proliferation was evaluated in the FGFR2b-modified BaF3 cell line (BaF3_hFGFR2b OE). BaF3 cell growth is dependent on interleukin-3 (IL-3). The BaF3 system allows for the testing of kinases and kinase inhibitors. While some protein kinases can isolate BaF3 cells from IL-3, kinase inhibitors antagonize this effect. Cells were seeded at 30,000 cells / well in RPMI-1640 medium containing 10% fetal bovine serum in 96-well plates. Anti-FGFR2b antibody at a concentration of 10 μg / ml was added to the assay plate. Recombinant human FGF7 or FGF10 protein (40 ng / ml) was added to the assay plate in the presence of heparin (5 μg / ml). The plates were incubated in an incubator at 37°C, 5% CO2, and 95% humidity for 72 hours. After incubation, 50 μl of Cell Titer Glo reagent was added to each well, the plate was mixed for 1 minute, and then incubated at room temperature for 10 minutes. Luminescence signals were detected and recorded. The control antibodies BGA1421 and BGAm1823 (mouse parent antibodies) were shown to potently inhibit both FGF7-induced and FGF10-induced BaF3_hFGFR2b cell proliferation (Figures 6 and 7, respectively). BGAm1371 was shown to inhibit FGF7-induced BaF3_hFGFR2b cell proliferation, but its potency was one-tenth that of BGA1421 and BGAm1823. BGAm4461 did not strongly inhibit FGF7-induced or FGF10-induced BaF3_hFGFR2b cell proliferation (Figure 6 and data not shown).

[0279] The Ba / F3 system and Figures 6 and 7 will be further explained in Example 12 below.

[0280] Example 7. In vitro inhibition of FGFR2 signaling in SNU-16 cells The inhibition of the FGFR2 signaling pathway by humanized antibodies was investigated. SNU-16 cells were cultured in RPMI-1640 medium containing 10% FBS, collected, washed twice with free medium, and seeded in serum-free RPMI-1640 at 1.5E6 cells / well in 12-well plates. The plates were incubated overnight in an incubator at 37°C, 5% CO2, and 95% humidity. Anti-FGFR2b antibody at a concentration of 10 μg / ml was added to the assay plate. Recombinant human FGF7 or FGF10 protein (10 ng / ml) was added to the assay plate in the presence of heparin (5 μg / ml). The plates were incubated in an incubator at 37°C, 5% CO2, and 95% humidity for 2 hours. After 2 hours of incubation, cells were harvested and washed twice with cold PBS. NP40 lysis buffer containing 1X PMSF and 1X protease plus phosphatase inhibitor was added to the cell pellet. The pellet was suspended by pipetting the mixture up and down. The mixture was gently inverted and each was placed on ice for 10 minutes, for a total of 30 minutes. The mixture was centrifuged at 12000 × rpm for 10 minutes to pelletize the cell debris. The supernatant was transferred to a new tube and the total protein concentration was quantified using a BCA kit. The protein samples were then divided and stored at -80°C for further Western blotting to detect pFGFR and total FGFR2 expression.

[0281] Western blotting procedure: The fully thawed protein sample was mixed with 4XSDS loading buffer and heated at 37°C for 10 minutes. The mixture was then centrifuged at approximately 3500 × rpm for 5 minutes. Equal volumes of protein were loaded onto an SDS-PAGE gel, and then the protein was transferred to a PVDF membrane using iBlot. The membrane was then subjected to Western blotting analysis, focusing on phosphorylated FGFR and total FGFR2.

[0282] The results are shown in Figures 20A-20B. In Figure 20B, n=2 for BGAh9179 and n=1 for BGA1421. Treatment with the comparative BGA1421 and BGAm1823 resulted in downregulation of phosphorylated FGFR in both FGF7-induced and FGF10-induced SNU-16 cells (Figure 20A). BGAm1371 weakly inhibited FGF7-induced FGFR phosphorylation in SNU-16 cells (Figure 20A). BGAh9179 showed little to no downregulation of FGF10-induced FGFR phosphorylation, and the downregulation was less than that of the comparative BGA1421 (Figure 20B). The comparative BGA2311 did not induce downregulation of phosphorylated FGFR in either FGF7-induced or FGF10-induced SNU-16 cells (Figure 20A). Therefore, the anti-FGFR2b antibody of this disclosure reduced the interaction between FGFR2b and its ligand, KGF.

[0283] Example 8: BGA3457 does not cause corneal dystrophy in mice. The effects of the immunoconjugate BGA3457 on corneal dystrophy were observed in Balb / c nude mice (6-8 weeks old). HSC-39 cells were 5.0 × 10⁶ cells along with Matrigel (1:1 v / v ratio). 6 The cells were injected subcutaneously into the right flank. The tumor volume was calculated using the formula: V = 0.5 × (a × b 2 The calculation was performed using the formula (where a and b are the major and minor diameters of the tumor, respectively). The tumor volume was approximately 200 mm³. 3At the required time, mice were randomly assigned to each group. Animals were intravenously administered either a vehicle, BGA3457 (10 mg / kg, Q2W x 2), or BGA1421 (10 mg / kg, BIW x 8). At the endpoint, mice were euthanized using carbon dioxide, their eyes were collected, fixed in Davidson's solution (95% ethanol / formaldehyde / acetic acid / distilled water: 30 mL / 20 mL / 10 mL / 20 mL), and transferred to 75% ethanol after 24 hours. Corneal abnormalities were analyzed by H&E staining. Corneal thickness of mice was measured using image scope software (Leica Biosystems). The difference in mean data between the comparison groups was analyzed for significance using appropriate analysis with GraphPad Prism (GraphPad Software, Inc.). p<0.05 was considered statistically significant.

[0284] The results are shown in Figure 22. When mice were administered the comparator drug BGA1421 (10 mg / kg, BIW x 8), their corneas became significantly thinner compared to the vehicle. However, administering BGA3457 (10 mg / kg, Q2W x 2) to mice did not affect corneal thickness. The corneal thickness of four mice in each group was quantified. Therefore, anti-FGFR2b ADCs with low blocking function (BGA3457) reduce corneal dystrophy in mice.

[0285] Example 9. Determination of affinity of humanized anti-FGFR2b antibody To determine affinity, antibodies were captured with anti-human Fc antibodies and used in affinity assays based on surface plasmon resonance (SPR) technology. The SPR signals were analyzed, and the association rate (ka) and dissociation rate (kd) were calculated using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. Rmax represents the maximum possible SPR signal generated by the interaction between the ligand-analyte pair and is expressed in response units (RU). The results of the SPR measurement of the binding profiles of anti-FGFR2b antibodies are summarized in Tables 12 and 13. BGAh9239 and BGA9179 showed similar binding profiles to human FGFR2b and cynomolgus monkey FGFR2b, respectively, and were similar to the parent antibodies BGAm1823 and BGAm1371. [Table 14] [Table 15]

[0286] Example 10. Binding activity of humanized anti-FGFR2b antibody to native FGFR2b To evaluate the binding activity of anti-FGFR2b antibodies to native FGFR2b on living cells, Expi293 cells were modified to overexpress human FGFR2b, cynomolgus monkey FGFR2b, mouse FGFR2b, or human FGFR2c. Expi293 cells expressing the antigens were seeded in 96-well plates and incubated with serially diluted anti-FGFR2b antibodies. Goat anti-human IgG [Alexa Fluor® 647] (Jackson ImmunoResearch, catalog number 109-605-003) was used as a secondary antibody to detect antibodies binding to the cell surface. Changes in mean fluorescence intensity (MFI) of humanization candidates BGAh9239 and BGAh9179 were measured by flow cytometry. The EC50 values ​​for dose-dependent binding to native human FGFR2b, cynomolgus monkey FGFR2b, mouse FGFR2b, and human FGFR2c were determined using a nonlinear approximation sigmoid dose-response curve from GraphPad Prism™. As shown in Figures 3A-3D and Table 14, both the humanized anti-FGFR2b antibodies BGAh9239 and BGAh9179 showed high binding affinity to native human FGFR2b, cynomolgus monkey FGFR2b, and mouse FGFR2b on living cells, but no detectable binding was observed to native human FGFR2c. [Table 16]

[0287] Example 11. Binding activity of humanized anti-FGFR2b antibody to recombinant FGFR2b To determine affinity, the antibody was captured with an anti-human Fc antibody and used in an affinity assay based on surface plasmon resonance (SPR) technology.

[0288] To investigate whether humanized anti-FGFR2b antibodies bind to recombinant FGFR2b protein, ELISA plates were coated with human FGFR2b, cynomolgus monkey FGFR2b, or human FGFR2c and left overnight at 4°C. After washing and blocking, serially diluted anti-FGFR2b antibodies were added to the plates and incubated at room temperature for 1 hour (recombinant neutralizing human IgG1 anti-SARS-CoV-2 monoclonal antibody isotype human IgGCB-6 (also known as etesevimab or JS016) was used as a negative control). After washing, diluted goat anti-human IgG antibody [HRP] (Abcam, catalog no. Ab98624) was added to the plates and incubated at room temperature for 30 minutes. After washing, TMB substrate was added to the plates and developed, then stopped with TMB substrate stop solution. Absorbance was read at 450 nm using a plate reader. Titration curves were constructed using a nonlinearly fitted S-shaped dose-response from GraphPad, and the EC50s are shown in Table 15. As shown in Table 15 and Figures 4A-4C, BGAh9239, BGAm1823, BGAh9179, and BGAm1371 showed high binding affinity to human and cynomolgus monkey FGFR2b proteins, but no binding to human FGFR2c was detected. [Table 17]

[0289] Example 12. Blockade test using anti-FGFR2b antibody in Ba / F3 proliferation assay The Ba / F3 system is used to investigate the sensitivity of driver variants to downstream oncogenic signaling pathways and therapeutic agents. Driver-dependent Ba / F3 cells die when driver-involved signaling pathways are inhibited, but this effect can be reversed by reintroduction of FGF7 or FGF10.

[0290] Figures 6 and 7 show the results of a Ba / F3 proliferation assay. Net proliferation of Ba / F3 cells overexpressing FGFR2b is reported after treatment with the anti-FGFR2b antibody of this disclosure. Normal Ba / F3 cells die immediately after exogenous FGF7 or FGF10 withdrawal. In Figure 6, BGAm1371 showed weaker blockade than the control BGA1421 (a potent ligand blocker). In Figure 7, BGAm1371 escaped FGF10 blockade and, when induced by FGF10, did not inhibit net growth of Ba / F3 cells overexpressing FGFR2b. The control BGA2311 binds at the N-terminus and is not considered a ligand blocker. Therefore, BGAm1371 may partially block FGFR2b signaling pathways, such as those mediated by FGF7.

[0291] The Ba / F3 system and Figures 6 and 7 are further explained in Example 6 described above.

[0292] Example 13. FGFR2b immunoconjugates BGA3457 and BGA9823 showed equivalent internalization. The internalization activity of the FGFR2b immunoconjugates BGA3457 and BGA9823 (linker / payload structures are shown in row 3 of Table 6 (i.e., LD2-3), and antibody sequences are shown in BGAh9179 and BGAh9239, respectively, in SNU16 (high FGFR2b-expressing gastric cancer cell line), SNU601-h2b (medium FGFR2b-expressing gastric cancer cell line), H1048-h2b (medium to low FGFR2b-expressing lung cancer cell line), and HSC-39 (low FGFR2b-expressing gastric cancer cell line) was evaluated by the pHrodo® assay. The pHrodo® assay is a method for determining phagocytosis of apoptotic cells by macrophages using pH-sensitive fluorescently labeled succinimidyl ester pHrodo® (Miksa, M. et al., 2009. Journal of Immunological Methods, 342(1-2), pp.71-77). FACS analysis can be used to assess the level of phagocytosis using the fluorescent label. After phagocytosis, phagosomes and lysosomes fuse. This fusion lowers the pH within the engulfed compartment due to the acidity of the lysosome. Low pH is detected by a pH-sensitive fluorescent dye. The signal becomes stronger as cells are engulfed.

[0293] Serially diluted FGFR2b immunoconjugates were incubated with pHrodo® labeling reagents at room temperature for 5 minutes to form labeling complexes. Subsequently, 1E5 cells / well were incubated with the labeling complexes at 37°C, 5% CO2 for 0, 6, 16, and 24 hours. At the endpoint, cells were subjected to FACS assays to determine the level of pHrodo®-positive cell population as an immunoconjugate-internalized cell population. BGA3457 and BGA9823 showed better internalization compared to isotypes in high and moderate FGFR2b-expressing cell lines SNU16, SNU601-h2b, and H1048-h2b (Figures 13A, 13B, and 13C, respectively). BGA3457 and BGA9823 showed lower internalization in the low-FGFR2b-expressing cell line HSC-39 (Figure 13D).

[0294] Example 14. In vitro cell-killing activity of FGFR2b immunoconjugate The cytotoxic activity of the FGFR2b immunoconjugate BGA3457 against SNU16 (high FGFR2b-expressing cancer cell line), SNU601-h2b (medium FGFR2b-expressing cancer cell line), H1048-h2b (medium to low FGFR2b-expressing cancer cell line), and HSC39 (low FGFR2b-expressing cancer cell line) was evaluated using the CellTiter-Glo assay. SNU16, SNU601-h2b, and H1048-h2b were seeded at 2000 cells / well, and HSC39 at 1000 cells / well in 96-well plates and incubated overnight at 37°C. Serially diluted FGFR2b BGA3457 was added, and the cells were cultured at 37°C and 5% CO2 for 6 days. Target cell killing was measured using the CellTiter-Glo detection kit (Promega). The results are shown in Figure 14. BGA3457 showed good cytotoxicity in cell lines with high to moderate FGFR2b expression at low concentrations of FGFR2b immunoconjugate (Figures 14A-14C). In cell lines with low FGFR2b expression, high concentrations of FGFR2b immunoconjugate were required to kill cells (Figure 14D).

[0295] In vivo efficacy trial of ADC Example 15. Efficacy of BGA3457 and BGA9823 in cell line-derived xenograft models. Xenotransplantation (CDX) models derived from gastric adenocarcinoma cell lines were generated in mice using SNU-16 (high FGFR2b expression) or HSC-39 (moderate FGFR2b expression) cells (Figures 9, 10, and 15).

[0296] SNU-16 cells and HSC-39 cells were cultured in RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were collected on the day of transplantation and resuspended in cold (4°C) serum-free RPMI-1640 medium. Cell density was set to 5 × 10⁶. 7 The solution was adjusted to the desired cell / mL concentration, and the cells were placed on ice before inoculation.

[0297] Female Balb / c nude mice aged 6-8 weeks were purchased, housed in ventilated cages, and given free access to food and water. They were acclimatized for approximately one week before cell inoculation. Gastric adenocarcinoma SNU-16 cells and HSC-39 cells were inoculated with Matrigel (1:1 v / v ratio) in 5.0 × 10⁶ cells. 6 The cells were injected subcutaneously into the right flank of the mice.

[0298] The tumor volume is calculated using the formula: V = 0.5 × (a × b 2 The calculation was performed using the formula (where a and b are the major and minor diameters of the tumor, respectively). The tumor volume was approximately 200 mm³. 3 Upon reaching a certain stage, the mice were randomly divided into study groups of 7-8 mice each. The animals were intravenously administered either vehicle, iso-control (linker + payload only) (10 mg / kg or 3 mg / kg, single dose), BGA9823 (10 mg / kg, 3 mg / kg, or 1 mg / kg, single dose), BGA3457 (10 mg / kg, 3 mg / kg, or 1 mg / kg, single dose), or BGA8723 (10 mg / kg, BIW). Animal body weight and tumor volume were measured twice weekly. Data are presented as mean tumor volume ± standard error of mean (SEM). Tumor growth (TGI) was calculated using the following formula: %TGI=[1-(treated Tt-treated T0) / (vehicle Tt-vehicle T0)]×100% Mean tumor volume of the drug-treated group on day Tt=t Mean tumor volume of the drug-treated group on T0=0 day Vehicle Tt = t day, mean tumor volume of the vehicle group Mean tumor volume of the vehicle group on day T0 = 0

[0299] Data were analyzed using ANOVA in the CDX model. ***p<0.001, ****p<0.0001. Results are shown in Figures 9, 10, 15A, and 15B. BGA9823 and BGA3457 showed significant antitumor activity in the SNU-16 (Figures 9 and 15A) and HSC-39 (Figures 10 and 15B) xenograft models. In the SNU-16 xenograft model, both BGA9823 and BGA3457 showed dose-dependent efficacy. At dose levels of 3 mg / kg and 10 mg / kg, both BGA9823 and BGA3457 showed superior antitumor effects compared to the control group BGA8723 (Figures 9 and 15A). In the HSC-39 xenograft model, both BGA9823 and BGA3457 at 3 mg / kg showed comparable efficacy to the control BGA8723 (Figures 10 and 15B). Furthermore, both BGA9823 and BGA3457 significantly inhibited tumor growth at 10 mg / kg, demonstrating superior antitumor effects compared to the control BGA8723 (Figures 10 and 15B). All animals tolerated the treatment well without significant weight loss or abnormal clinical observations.

[0300] Example 16. Efficacy of BGA3457 in a human gastric cancer PDX model. A human gastric cancer PDX model (LD1-0017-200652, moderate FGFR2b expression, Shanghi LIDE Biotech Co.,Ltd) was generated. Briefly, approximately 50-90 mg of tumor fragments were transplanted subcutaneously into the right flank of female Balb / c nude mice (6-8 weeks old). The tumor volume was calculated using the formula: V = 0.5 × (a × b 2 The calculation was performed using the formula (where a and b are the major and minor diameters of the tumor, respectively). The tumor volume was approximately 150 (i.e., 100-200) mm². 3At this point, the mice were randomly divided into four groups of three. The animals were administered intravenously with either a vehicle, BGA3457 (3 or 10 mg / kg, single dose), or BGA8723 (10 mg / kg, BIW). Animal body weight and tumor volume were measured twice weekly. Data are presented in Figure 21 as mean tumor volume ± standard error of mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI=[1-(treated Tt-treated T0) / (vehicle Tt-vehicle T0)]×100% Mean tumor volume of the drug-treated group on day Tt=t Mean tumor volume of the drug-treated group on T0=0 day Vehicle Tt = t day, mean tumor volume of the vehicle group Mean tumor volume of the vehicle group on day T0 = 0

[0301] Data were analyzed using tobit in the PDX model. ****p<0.0001. Results are shown in Figure 21. In human patient-derived gastric cancer models, BGA3457 showed superior antitumor efficacy compared to the control BGA8723 at dose levels of 3 mg / kg and 10 mg / kg. All animals were well-tolerated to treatment without significant weight loss or abnormal clinical observations.

[0302] Example 17. In vivo efficacy of co-inoculated HSC-39+SNU-5 gastric cancer xenografts with FGFR2b immunoconjugates BGA3457 and BGA9823 on proliferation of xenografts. The tumor growth inhibitory abilities of the anti-FGFR2b immunoconjugates BGA9823 and BGA3457 of this disclosure were tested in co-incubation models with HSC-39 (gastric signet ring cell adenocarcinoma, a human FGFR2b overexpressing cell line) and SNU-5 (gastric cancer cell line) in a 2:1 or 5:1 ratio (Figures 11A-12B).

[0303] HSC-39 and SNU-5 were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were incubated at 37°C in a humidified incubator with 5% CO2. Cells were collected on the day of transplantation and washed with serum-free medium. Female BALB / c nude mice aged 6-8 weeks were purchased, housed in ventilated cages, and given free access to food and water, and were acclimated for approximately one week before inoculation. Total 5 × 10 6 HSC-39 cancer cells and SNU-5 cancer cells were mixed with Matrigel in a 2:1 or 5:1 ratio and subcutaneously transplanted into the flanks of mice. Tumor volume was measured using calipers, and the tumor volume was calculated using the formula V = 0.5(a × b). 2 ) was calculated. The tumor volume was approximately 150 mm 3 At this stage, mice were randomly divided into three groups of five. On day 0 of treatment, a single dose of either the vehicle, a comparative anti-FGFR2b monoclonal antibody (BGA1421), or an FGFR2b immunoconjugate (BGA3457, Figure 11, or BGA9823, Figure 12) was administered intravenously at 10 mpk. Tumor volume was measured twice weekly. Data are presented as mean tumor volume ± standard error of mean (SEM). In the vehicle group with an HSC-39:SNU-5 ratio of 2:1, approximately 50% of the tumors were FGFR2b-positive at the baseline and approximately 40% at the endpoint. In the vehicle group with an HSC-39:SNU-5 ratio of 5:1, approximately 60% of the tumors were FGFR2b-positive at the baseline and approximately 20% at the endpoint.

[0304] As shown in Figures 11A, 11B, 12A, and 12B, treatment with BGA3457 and BGA9823 induced complete tumor regression in ratios of 2:1 and 5:1, respectively. While the control BGA1421 inhibited tumor growth, it was not more potent than the anti-FGFR2b immunoconjugates of this disclosure, which may be due to the limited exclusion of the FGFR2b-positive population by the control BGA1421. These results demonstrate that the FGFR2b-immunoconjugates BGA3457 and BGA9823 exhibited significant killing effects against both FGFR2b-positive and FGFR2b-negative tumors under co-inoculation conditions. Example 18: Bystander lethality of FGFR2b immunoconjugate BGA3457

[0305] To investigate the bystander effect of BGA3457, a co-culture killing assay was established using a mixture of FGFR2b-positive and FGFR2b-negative cells. Using luciferase as a readout, BGA3457 effectively killed FGFR2b-negative HuTu-80-NanoLuc cells (manipulated with NanoLuc luciferase) co-cultured with FGFR2b-positive SNU-16 cells (Figure 16). The maximum killing efficacy reached 90.74%, and the IC50 value was 34.75 ng / mL. An isotype ADC (CB-6 anti-SARS-CoV-2 monoclonal antibody conjugated to LD2-3) was used as a negative control, but it did not show a bystander killing effect.

[0306] Example 19. Preparation of toxins P3 and P4 UPLC analysis method Method A: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B maintained for 0.2 minutes, 10%~95% B for 5.8 minutes, 95% B maintained for 0.5 minutes, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC(registered trademark) BEHC18 1.7 μm

[0307] Method B: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B maintained for 0.5 minutes, 10%~90% B for 2.5 minutes, 90% B maintained for 0.2 minutes, Flow rate: 0.6 mL / min, Column: ACQUITYUPLC(registered trademark) BEHC18 1.7 μm

[0308] Method C: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B maintained for 0.2 minutes, 10%~90% B for 1.3 minutes, 90% B maintained for 0.3 minutes, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC(registered trademark) BEHC18 1.7 μm

[0309] P1 and P2 are commercially available and were purchased from MedChemExpressCO.LTD (Shanghai).

[0310] P3 synthesis procedure This specification provides synthetic procedures for preparing P3 and P4, toxins shown in Tables 5A-5B, which are useful in the immunoconjugates of this disclosure. [ka]

[0311] N-((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]indolidino[1,2-b]quinoline-1-yl)-3-hydroxy-2,2-dimethylpropanamide (P3).

[0312] To a mixture of P3a (5.0 mg, 0.042 mmol) and HATU (16 mg, 0.042 mmol) in DMF (1 mL), DIEA (21 μL, 16 mg, 0.13 mmol) and exatecan mesylate (23 mg, 0.043 mmol, purchased from MedChemExpress CO. LTD) were added. The resulting brown mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by preparative HPLC (TFA) (Method: Column: XBridgePrepC18OBD5um19*150 mm, Mobile phase: A-Water (0.1% TFA): B-Acetonitrile, Flow rate: 20 mL / min). The fraction was lyophilized to obtain P3 (15 mg, yield 65.5%) as a white powder. MS(ESI)m / z:536.4[M+H] +

[0313] 1H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.59-5.54 (m, 1H), 5.42 (s, 2H), 5.18 (q, J = 19.2 Hz, 2H), 4.87 (t, J = 5.2 Hz, 1H), 3.45 (dd, J = 10.2, 4.8 Hz, 1H), 3.41-3.28 (m, 1H), 3.15 (t, J = 5.6 Hz, 2H), 2.40 (s, 3H), 2.24-2.07 (m, 2H), 1.92-1.80 (m, 2H), 1.11 (d, J = 7.6 Hz, 6H), 0.87 (t, J = 7.2 Hz, 3H). P4 synthesis procedure [ka]

[0314] Step 1 Diethyl 2-fluoro-2-methylmalonate (P4b) A solution of compound P4a (10.00 g, 57.40 mmol) in THF (200 mL) was cooled to 0°C. 60% NaH in oil (3.21 g, 80.37 mmol) was gradually added to the mixture and stirred at 0°C for 30 minutes. Then, N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (NSFI, 19.91 g, 63.20 mmol) was gradually added to the mixture at 0°C and the mixture was heated to room temperature and stirred for 16 hours. After the reaction was complete, the suspension was filtered and the filtrate was concentrated. PE (100 mL) was added to the residue, the precipitate was filtered, and the filtrate was concentrated to obtain compound P4b (12.50 g, crude) as a pale yellow oil. 1 HNMR(400MHz, CDCl3) δ4.30(q,J=7.2Hz,4H),1.79(d,J=22.0Hz,3H),1.31(t,J=7.2Hz,6H). 19 FNMR (376 MHz, CDCl3) δ-157.50.

[0315] Step 2 3-Ethoxy-2-fluoro-2-methyl-3-oxopropanoic acid (P4c) To a solution of compound P4b (1.00 g, 5.20 mmol) in EtOH (5 mL), a solution of KOH (321 mg) in H2O (50 μL) and EtOH (2 mL) were added dropwise at 0°C. The mixture was stirred at room temperature for 2 hours. The mixture was diluted with water (20 mL) and washed with DCM (20 mL x 3). The aqueous solution was adjusted to pH=3 with 1 N HCl and then extracted with ELISA (50 mL x 3). The organic layer was dried, bonded, dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound P4c (470 mg, 55.0% yield) as a colorless oil. 1 HNMR(400MHz, CDCl3) δ8.31(brs,1H),4.32(q,J=7.2Hz,2H),1.83(d,J=22.0Hz,3H),1.33(t,J=7.2Hz,3H). 19 FNMR (376MHz, CDCl3) δ-157.59.

[0316] Step 3 2-Fluoro-3-hydroxy-2-methylpropanoic acid (P4d) To a solution of compound P4c (200 mg, 1.22 mmol) in isopropanol (4 mL), 2 M LiBH4 (1.22 mL, 2.44 mmol) was added at 0°C. The mixture was stirred at room temperature for 2 hours. The mixture was quenched dropwise with 2 N HCl (1.22 mL) at 0°C, diluted with H2O (10 mL), and extracted with RINKAN (50 mL x 3). The organic layer was bonded, dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound P4d (92 mg, yield 61.7%) as a colorless oil. 1 HNMR(400MHz, CDCl3)δ4.01-3.81(m,2H),1.58(d,J=21.2Hz,3H). 19 FNMR (376 MHz, CDCl3) δ-163.98.

[0317] Step 4 N-((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]indolidino[1,2-b]quinoline-1-yl)-2-fluoro-3-hydroxy-2-methylpropanamide(P4)

[0318] To a solution of compound P4d (23 mg, 0.19 mmol) in DMF (2 mL), exatecan mesylate (50 mg, 0.094 mmol), HATU (54 mg, 141 mmol), and DIEA (36 mg, 0.28 mmol) were added. The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5um 19*150 mm; Mobile phase: A-Water (0.1% formic acid): B-Acetonitrile; Flow rate: 20 mL / min). The fractions were lyophilized to obtain the following two isomers:

[0319] Isomer 1:P4: White solid (11 mg, yield 21.9%). UPLC-MS, RT=3.52 min. 1 H NMR (400 MHz, DMSO-d6) δ 9.06 (dd, J = 9.0, 2.8 Hz, 1H), 8.00 (d, J = 10.9 Hz, 1H), 7.54 (s, 1H), 6.75 (s, 1H), 5.82 (d, J = 8.0 Hz, 1H), 5.65 (s, 2H), 5.43 (dt, J = 77.8, 12.4 Hz, 3H), 4.17-3.91 (m, 1H), 3.83 (ddd, J = 18.0, 12.4, 5.6 Hz, 1H), 3.40-3.27 (m, 1H), 2.62 (s, 3H), 2.50-2.34 (m, MS (ESI) m / z: 540.3 [M+H] + .

[0320] Isomer 2:P4-1: White solid (8.4 mg, yield 16.6%). UPLC-MS, RT=3.86 min. 1 H NMR (400 MHz, DMSO-d6) δ 8.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58 (d, J = 8.0 Hz, 1H), 5.42 (s, 2H), 5.32 - 5.05 (m, 3H), 3.83 (dd, J = 26.8, 12.0 Hz, 1H), 3.61 (dd, J = 21.6, 12.0 Hz, 1H), 3.22-3.07 (m, 2H), 2.46-2.30 (m, 3H), 2.28-2.05 (m, 2H), 2.02-1.74 (m, 2H), 1.45 (d, J = 21.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H); MS (ESI) m / z: 540.3 [M+H] + .

[0321] Example 20: Synthesis of Examples LD2-3 to LD2-8 This specification provides a synthetic procedure for preparing LD2-8 from LD2-3, a conjugator-linker-payload shown in Table 6, whose components and complete structure are useful for the immunoconjugates of this disclosure. Example LD2-3 [ka]

[0322] Step 1 Benzyl(5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8,14,14-trimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-15-oato(LD2-3c)

[0323] LD2-3a (300 mg, 0.62 mmol, synthesized according to reported procedure: ACS Med.Chem.Lett.2019, 10, 1386-1392 and US9808537B2), LD2-3b (260 mg, 1.25 mmol), and a white suspension mixture in anhydrous THF (10 mL) on a 4 Šmolecular sieve were stirred at room temperature for 10 minutes. Sc(OTf)3 (368 mg, 0.75 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 hours. The yellow suspension mixture was filtered through a Celite pad and washed with  (30 mL). The bound organic layer was washed with saturated fatty acid. It was washed with NaHCO3 (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain the residue. The solution was purified using a silica gel column (MeOH / DCM = 0%~5%), and the fraction was concentrated under vacuum to obtain LD2-3c (274 mg, yield 69.8%) as a white solid. MS(ESI)m / z:652.6[M+Na] +

[0324] Step 2 Benzyl 3-(((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)methoxy)-2,2-dimethylpropanoate(LD2-3d)

[0325] Et2NH (477 mg, 5.53 mmol) was added to a solution of LD2-3c (274 mg, 0.44 mmol) in DMF (5 mL). The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated under vacuum and evaporated twice with toluene to obtain LD2-3d (275 mg, crude) as a brown oil. MS(ESI)m / z:430.4[M+Na] + .

[0326] Step 3 Benzyl(5S,8S,11S)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-8-isopropyl-11,17,17-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-oate(LD2-3f)

[0327] To a solution of LD2-3d (275 mg, crude) and LD2-3e (282 mg, 0.52 mmol, purchased from WuXi AppTec) in DMF (5 mL), HATU (198 mg, 0.52 mmol) and DIPEA (168 mg, 1.30 mmol) were added. The mixture was stirred at room temperature for 10 minutes. The mixture was purified by reverse-phase chromatography (C18, 60 g, 30%-70%), and the fraction was freeze-dried to obtain LD2-3f (370 mg, 91.5% yield) as a brown solid. MS(ESI)m / z:953.8[M+Na] +

[0328] Step 4 (5S,8S,11S,17R)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-17-fluoro-8-isopropyl-11,17-dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-acid (LD2-3g).

[0329] To a solution of compound LD2-3f (3.01 g, 3.21 mmol) in cosolvent DMF-MeOH (40 mL, 1:1, v:v), Pd / C (10%, 600 mg) was added. The mixture was stirred under an H2 atmosphere (15 psi) for 7 hours. The mixture was filtered through a Celite pad and concentrated to obtain compound LD2-3 g (2.50 g, crude) as a white solid. MS(ESI)m / z:863.7[M+Na]+

[0330] Step 5 (9H-Fluoren-9-yl)methyl((6S,9S,12S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,1 5-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)amino)-9-isopropyl-12,18,18-trimethyl-3,7,10,13,19-pentaoxo-16-oxa-2,8,11,14-tetraazanonadecane-6-yl)carbamate (LD2-3h).

[0331] To a solution of compound exatecan mesylate (1000 mg, 1.18 mmol, purchased from MedChemExpressCO.LTD) in DMF (20 mL), compound LD2-3 g (692 mg, 1.30 mmol), HATU (675 mg, 1.78 mmol), and DIEA (459 mg, 3.55 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH = 0%~20%) to obtain the title compound LD2-3h (1.32 g, yield 88.6%) as an off-white solid. MS(ESI)m / z:1282.1[M+Na] +

[0332] Step 6 (S)-2-amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-(((3-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide(LD2-3i)

[0333] To a solution of compound LD2-3h (1000 mg, 0.79 mmol) in DMF (20 mL), Et2NH (580 mg, 7.93 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under high vacuum to obtain compound LD2-3i (825 mg, crude) as an off-white solid, which was used directly without further purification. MS(ESI)m / z:1036.9[M+H] +

[0334] Step 7 Step 7: (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetamide)propanamide)-N1-((S)-1-(((S)-1-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydo Roxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (LD2-3).

[0335] To a solution of LD2-3j (15 mg, 0.067 mmol) in dry DMF (1.0 mL), HATU (26 mg, 0.067 mmol) and DIEA (0.017 mL, 0.097 mmol) were added and the mixture was stirred at room temperature for 15 minutes. Next, LD2-3i (50 mg, 0.048 mmol) was added to the mixture and the mixture was stirred at room temperature for 10 minutes. The resulting solution was purified by preparative HPLC (Method: Column XBridgePrepC18OBD5um19*150 mm, Mobile phase: A-Water (0.1% TFA):B-Acetonitrile, Flow rate: 20 mL / min). The fraction was lyophilized to obtain LD2-3 (37 mg, yield 50.9%) as a yellow solid. MS(ESI)m / z:1266.7[M+Na] + Example LD2-4 [ka]

[0336] Step 1 Benzyl(S)-11-benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentazahenicosan-21-oate(LD2-4c)

[0337] A 4Å molecular sieve was placed over a solution of LD2-4a (250 mg, 0.40 mmol) and LD2-3b (83 mg, 0.40 mmol) in THF (5 mL). The mixture was stirred at room temperature for 10 minutes, then Sc(OTf)3 (195 mg, 0.40 mmol) was added, and the mixture was further reacted at room temperature for another 16 hours. The suspension mixture was filtered through a Celite pad, the cake was washed with THF (10 mL), and the filtrate was quenched with saturated NaHCO3 (10 mL) and extracted with siRNA (30 mL x 2). After separation, the bound organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain the residue, which was further purified by silica gel column chromatography (A-DCM, B-MeOH, MeOH / DCM = 0%~5%) to obtain LD2-4c (90 mg, yield 29.2%) as a white solid. MS(ESI)m / z:800.5[M+Na] +

[0338] Step 2 (S)-11-benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentazahenicosan-21-acid (LD2-4d)

[0339] A solution of LD2-4c (80 mg, 0.10 mmol) in MeOH (3 mL) was mixed with wet Pd / C (20 mg). The black suspension was purged three times with an H2 balloon, and then reacted at room temperature for 2 hours under an H2 balloon. After the reaction was complete, the black suspension was filtered off using a Celite pad, the cake was washed with MeOH, and the combined organic layer was concentrated under vacuum to obtain LD2-4d (61 mg, yield 84.8%). MS(ESI)m / z:710.4[M+Na] + Step 3 (9H-Fluoren-9-yl)methyl((S)-7-benzyl-17-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)carbamate(LD2-4f)

[0340] A mixture of LD2-4d (60 mg, 0.087 mmol) and HATU (33 mg, 0.087 mmol) in DMF (2 mL) was mixed with DIEA (43 μL, 34 mg, 0.26 mmol). The mixture was reacted at room temperature for 10 minutes. Exatecan mesylate (46 mg, 0.087 mmol) was added, and the mixture was reacted at the same temperature for a further 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was purified using preparative HPLC (method, column: XBridge Prep C18 OBD 5 μm 19*150 mm, mobile phase: A-water (0.1% formic acid): B-acetonitrile, flow rate: 20 mL / min) to obtain LD2-4f (85 mg, yield 88.2%). MS(ESI)m / z:1105.5[M+H] +

[0341] Step 4 3-(((S)-13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)-N-((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]indolidino[1,2-b]quinoline-1-yl)-2,2-dimethylpropanamide (LD2-4g)

[0342] To a solution of LD2-4f (85 mg, 0.062 mmol) in DMF (2 mL), Et2NH (64 μL, 46 mg, 0.62 mmol) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the mixture was concentrated under vacuum to obtain LD2-4 g (86 mg, crude) as a yellow solid. MS(ESI)m / z:883.5[M+H] +

[0343] Step 5 (9H-Fluoren-9-yl)methyl((6S,15S)-15-benzyl-25-(((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]indolidino[1,2-b]quinoline-1-yl) Mino)-24,24-dimethyl-3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (LD2-4i).

[0344] To a solution of LD2-4g (86mg, crude) and LD2-4h (43mg, 0.079 mmol, purchased from WuXiAppTec) in DMF (1.5mL), DIEA (26μL, 21mg, 0.16 mmol) was added. The mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the mixture was purified by preparative HPLC (FA) (Method: Column: XBridgePrepC18OBD 5μm 19*150mm, Mobile phase: A-Water (0.1% formic acid): B-Acetonitrile, Flow rate: 20mL / min). The fraction was lyophilized to obtain LD2-4i (70mg, yield 62.6%) as a white powder. MS(ESI)m / z:1410.7[M+H] +

[0345] Step 6 (S)-2-amino-N1-((S)-7-benzyl-17-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide(LD2-4j)

[0346] To a solution of LD2-4i (70 mg, 0.050 mmol) in DMF (1 mL), Et2NH (51 μL, 36 mg, 0.50 mmol) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the mixture was concentrated under vacuum to obtain LD2-4j (71 mg, crude) as a yellow solid. MS(ESI)m / z:1188.2[M+H] +

[0347] Step 7 (S)-N 1 -((S)-7-benzyl-17-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide(LD2-1)

[0348] To a solution of LD2-4k (19 mg) in DMF (3 mL), HATU (34 mg, 0.088 mmol) and DIEA (10 μL, 7.6 mg, 0.059 mmol) were added. The resulting yellow solution was stirred at room temperature for 5 minutes, and then LD2-4j (71 mg, crude) was added. The mixture was stirred at room temperature for 60 minutes. After the reaction was complete, the mixture was purified by preparative HPLC (FA) (Method: Column: XBridgePrepC18OBD 5 μm 19 * 150 mm, Mobile phase: A - Water (0.1% formic acid): B - Acetonitrile, Flow rate: 20 mL / min). The fraction was lyophilized to obtain LD2-4 (32 mg, yield 26.3%) as a white powder. MS(ESI)m / z:1381.1[M+H] + Example LD2-5 [ka] LD2-5 (30 mg, 50.7% yield) was synthesized according to the synthesis procedure for LD2-4. MS(ESI)m / z:1408.1[M+Na] + Example LD2-6 [ka]

[0349] Step 1 N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine(LD2-6b).

[0350] A mixture of LD2-6a (4.10 g, 4.92 mmol, purchased from MedChemExpress CO.LTD) in MeOH (50 mL), THF (100 mL), and DCM (20 mL) was mixed with wet Pd / C (400 mg, 10% purity). The black suspension was purged three times with an H2 balloon and then stirred at room temperature for 1 hour. The black suspension was filtered through a Celite pad and washed with MeOH (200 mL). The combined organic layer was concentrated under vacuum to obtain LD2-6b (3.65 g, 99.8% yield) as an off-white solid. MS(ESI)m / z:743.6[M+H] +

[0351] Step 2 (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-((2-(benzyloxy)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (LD2-6d).

[0352] To a solution of LD2-6b (3.65 g, 4.92 mmol) and LD2-6c (1.66 g, 4.92 mmol) in DMF (50 mL), HATU (1.87 g, 4.92 mmol) and DIEA (1.59 g, 12.29 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was purified by FCC (MeOH / DCM = 0%~10%), and the fraction was concentrated under vacuum to obtain LD2-6d (3.80 g, yield 86.9%) as an off-white foamy solid. MS(ESI)m / z:890.7[M+H] +

[0353] Step 3 N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-cerylglycine(LD2-6e)

[0354] A mixture of LD2-6d (3.80 g, 4.27 mmol) in MeOH (150 mL) and DCM (50 mL) was mixed with wet Pd / C (400 mg, 10% purity). The black suspension was purged three times with an H2 balloon and then stirred at room temperature for 40 minutes. The black suspension was filtered through a Celite pad and washed with MeOH (150 mL). The bound organic layer was concentrated under vacuum to obtain LD2-6e (3.30 g, 96.6% yield) as an off-white solid. MS(ESI)m / z:800.7[M+H] +

[0355] Step 4 (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-((Acetoxymethyl)amino)-3-oxopropoxy)-6-(Methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (LD2-6f)

[0356] To a solution of LD2-6e (3.30 g, 4.13 mmol) in DMF (30 mL), Pb(OAc)4 (2.74 g, 6.19 mmol), Cu(OAc)2 (74.9 mg, 0.41 mmol), and HOAc (247.8 mg, 4.13 mmol) were added. The resulting dark-colored mixture was purged three times with an N2 balloon and then stirred at 65°C for 40 minutes, at which point the mixture turned a deep blue color. The mixture was diluted with siRNA (300 mL), washed with brine (100 mL x 3), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue. This residue was purified by FCC (MeOH / DCM = 0-10%), and the fraction was concentrated under vacuum to obtain LD2-6f (2.81 g, yield 83.4%) as a pale yellow solid. MS(ESI)m / z:836.6[M+Na] +

[0357] Step 5 (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((3-(benzyloxy)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (LD2-6h)

[0358] A white suspension mixture of LD2-6f (300 mg, 0.37 mmol), LD2-3b (154 mg, 0.74 mmol), and 4 Å molecular sieves (200 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 minutes. Sc(OTf)3 (218 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 hours. The yellow suspension mixture was filtered through a Celite pad and washed with ethyl acetate. The bound organic layer was washed with saturated fatty acid. It was washed with NaHCO3 (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This was purified by silica gel column (MeOH / DCM = 0%~5%), and the fraction was concentrated under vacuum to obtain LD2-6h (275 mg, yield 77.5%) as a white foamy solid. MS(ESI)m / z:984.8[M+Na] +

[0359] Step 6 (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-14,14-dimethyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxa-4,7,10-triazapentadecane-15-euic acid (LD2-6j)

[0360] To a solution of LD2-6h (275 mg, 0.29 mmol) in MeOH (10 mL), wet Pd / C (55 mg, 10% purity) was added. The black suspension was purged three times with an H2 balloon and then stirred at room temperature for 2 hours. The mixture was filtered using a syringe head, washed with MeOH (15 mL), and concentrated under vacuum to obtain LD2-6j (230 mg, crude) as a white foamy solid. MS(ESI)m / z:894.6[M+Na] +

[0361] Step 7 (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-3-(((3-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (LD2-6k)

[0362] A mixture of LD2-6j (230 mg, crude), exatecan mesylate (140 mg, 0.26 mmol), and HATU (100 mg, 0.26 mmol) in DMF (5 mL) was mixed with DIEA (102 mg, 0.79 mmol). The resulting brown mixture was stirred at room temperature for 1 hour. The mixture was diluted with  (20 mL), washed with brine (20 mL x 3), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue. This was purified by FCC (MeOH / DCM = 0%~3%) and concentrated under vacuum to obtain LD2-6k (325 mg, yield 95.6%) as an off-white foamy solid. MS(ESI)m / z:1289.9[M+H] +

[0363] Step 8 (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutanamide)-3-(((3-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LD2-6l)

[0364] Et2NH (523 mg, 5.06 mmol) was added to a solution of LD2-6k (325 mg, 0.25 mmol) in DMF (5 mL). The mixture was stirred at room temperature for 20 minutes. LC-MS indicated completion of the reaction, and the reaction product was then concentrated under vacuum to obtain the crude product. The crude product was dissolved in MeOH (6 mL), K2CO3 (174.7 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then H2O (2 mL) was added to the mixture and the mixture was stirred at room temperature for 30 minutes. The mixture was acidified with saturated brine. Acidification with KHSO4 at 0°C to pH=3 was performed, the mixture was filtered, and purified by preparative HPLC (0.1% FA). The fraction was lyophilized to obtain LD2-6 l (140 mg, yield 59.7%) as a pale yellow solid. MS (ESI) m / z: 927.4 [M+H] + 1H NMR (400 MHz, d6-DMSO) δ 9.56 (s, 1H), 8.39 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.54 (dd, J = 13.2, 7.2 Hz, 1H), 5.43 (s, 2H), 5.18 (dd, J = 41.6, 18.8 Hz, 2H), 5.09 - 5.02 (m, 1H), 4.96 (s, 1H), 4.62 (dd, J = 10.0, 6.8Hz, 1H), 4.56 - 4.44 (m, 2H), 4.19 (d, J = 7.6 Hz, 1H), 3.82 (dd, J = 10.8, 6.8 Hz, 1H), 3.61 (dd, J = 11.6, 6.4 Hz, 2H), 3.17-3.05 (m, 4H), 2.94 (t, J = 8.0 Hz, 1H), 2.39 (s, 3H), 2.11 (dt, J = 21.3, 7.6 Hz, 2H), 2.03 - 1.93 (m, 2H), 1.92 - 1.78 (m, 3H), 1.12 (d, J = 8.0 Hz, 6H), 0.87 (dd, J = 13.0, 6.6 Hz, 9H).

[0365] Step 9 Methyl 4-(5-(methylthio)-1,2,4-thiadiazole-3-yl)benzoate (LD2-60)

[0366] A solution of compound LD2-6m (100 mg, 0.47 mmol) in toluene (4 mL) and H2O (1 mL) is prepared by adding compound LD2-6n (110 mg, 0.57 mmol), K2CO3 (168 mg, 0.95 mmol), and Pd(dppf)Cl2. . DCM (35 mg, 0.047 mmol) was added. The mixture was heated under an N2 atmosphere for 110°C. oThe mixture was stirred in 1C for 3 hours. The mixture was filtered through a Celite pad, diluted with siRNA (100 mL), and washed with brine (50 mL x 4). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by flash column chromatography (elution at PE / siRNA = 0%~40%). Compound LD2-6o (56 mg, yield 44.4%) was obtained as an off-white solid. MS(ESI)m / z:267.1[M+H] +

[0367] Step 10 4-(5-(methylthio)-1,2,4-thiadiazole-3-yl)benzoic acid (LD2-6p)

[0368] To a solution of compound LD2-6o (54 mg, 0.20 mmol) in MeOH (3 mL) and H2O (1 mL), LiOH (17 mg, 0.41 mmol) was added. The mixture was stirred at room temperature for 2 hours. The mixture was adjusted to pH 7 and purified by preparative HPLC (FA conditions) to obtain compound LD2-6p (36 mg, yield 70.3%) as a white solid. MS(ESI)m / z:253.1[M+H] +

[0369] Step 11 4-(5-(methylsulfonyl)-1,2,4-thiadiazole-3-yl)benzoic acid (LD2-6q)

[0370] To a solution of compound LD2-6p (35 mg, 0.14 mmol) in DCM (3 mL) and THF (3 mL), m-CPBA (96 mg, 0.55 mmol) was added. The mixture was stirred at room temperature for 16 hours. The mixture was concentrated and purified by preparative HPLC (method: column: XBridgePrepC18OBD5um19*150 mm, mobile phase: A-water (0.1% TFA): B-acetonitrile, flow rate: 20 mL / min). Compound LD2-6q (12 mg, 99% purity) was obtained as a white solid. MS(ESI)m / z:284.8[M+H] +

[0371] Step 12 (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((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]indolidino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(4-(5-(methylsulfonyl)-1,2,4-thiadiazole-3-yl)benzamide)butanamide)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LD2-6)

[0372] To a solution of compound LD2-6q (7.4 mg, 0.026 mmol) in DMF (2 mL), HATU (9 mg, 0.024 mmol) and DIEA (5.6 mg, 0.043 mmol) were added. The mixture was stirred at room temperature for 30 minutes. Compound LD2-6l (20 mg, 0.022 mmol) was added to the mixture and stirred at room temperature for 15 minutes. The reaction product was purified by preparative HPLC (method: column: XBridgePrepC18OBD5um19*150 mm, mobile phase: A-water (0.1% TFA): B-acetonitrile, flow rate: 20 mL / min) to obtain compound LD2-6 (7.6 mg, yield 29.5%) as a white solid. MS(ESI)m / z:1193.5[M+H] + Example LD2-7 [ka] LD2-7 (32 mg, yield 50.9%) was synthesized according to step 7 of LD2-3. MS(ESI)m / z:1303.0[M+H] + Example LD2-8 [ka]

[0373] Step 1 Methyl(R)-3-(((benzyloxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanoate (LD2-8b)

[0374] LD2-8a (2.00 g, 5.68 mmol) and K2CO3 (863 mg, 6.24 mmol) were added to DMF (10 mL), followed by the dropwise addition of CH3I (1.61 g, 11.35 mmol) at 0°C. The resulting mixture was stirred at 0°C for 20 minutes, then heated to 25°C and stirred further at 25°C for 60 minutes. The reaction process was monitored by TLC (PE / EA) and LCMS. After the reaction was complete, the reaction mixture was diluted with EA (80 mL) and washed with brine (30 mL x 3) and H2O (30 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain methyl ester of LD2-8b (2.08 g, quantified) as a pale yellow solid. MS(ESI)m / z:267.2[M-Boc+H] + .

[0375] Step 2 Benzyl tert-butyl(4-hydroxybutan-1,2-diyl)(R)-dicarbamate(LD2-8c)

[0376] LD2-8b (1.00 g, 2.73 mmol) was dissolved in MeOH (15 mL), followed by the addition of LiBH4 (2 M stock solution in THF, 6.80 mL) at 0°C. The resulting mixture was stirred at 25°C for 2 hours. The reaction process was monitored by LC-MS and TLC. After the reaction was complete, saturated aqueous NH4Cl (10 mL) was added to quench the reaction product. The reaction mixture was diluted with H2O (80 mL) and extracted with EA (50 mL x 3). The bound organic layer was washed with brine (40 mL x 2) and water (40 mL x 2), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and further purified by flash column chromatography (PE / EA) to obtain LD2-8c (760 mg, yield 82.3%) as a white solid. MS(ESI)m / z:239.2[M-Boc+H]+ .

[0377] Step 3 Benzyl tert-butyl (4-(((4-nitrophenoxy)carbonyl)oxy)butane-1,2-diyl)(R)-dicarbamate (LD2-8e)

[0378] LD2-8c (300 mg, 0.89 mmol) and LD2-8d (405 mg, 1.33 mmol) were dissolved in DMF (5 mL), followed by the addition of DIEA (229 mg, 1.77 mmol). The resulting mixture was stirred at 25°C for 1.5 hours. After complete reaction, the reaction mixture was diluted with EA (100 mL) and washed with brine (35 mL x 2) and water (35 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain LD2-8e as a white solid (371 mg, yield 83.1%). MS(ESI)m / z:404.4[M-Boc+H] + .

[0379] Step 4 (9H-Fluoren-9-yl)methyl tert-butyl (4-(((3-(dimethylamino)-3-oxopropyl)carbamoyl)oxy)butan-1,3-diyl)(S)-dicarbamate (LD2-8g)

[0380] LD2-8e (420 mg, 0.83 mmol) and LD2-8f (149 mg, 1.67 mmol) were dissolved in DMF (5 mL), followed by the addition of NaHCO3 aqueous solution (1 M, 5 mL). The resulting mixture was stirred at 25°C for 2.5 hours. After the reaction was complete, the reaction mixture was concentrated and purified by flash column chromatography (DCM / MeOH) to obtain LD2-8 g as a pale yellow solid (365 mg, yield 96.5%). MS(ESI)m / z:354.4[M-Boc+H] + .

[0381] Step 5 (R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecane-15-acid (LD2-8h)

[0382] LD2-8g (360mg, 0.79 mmol) was dissolved in MeOH (18mL), followed by the addition of Pd / C (wet base, 108mg). The resulting mixture was stirred at room temperature under H2 (15psi) for 2 hours. After the reaction was complete, the reaction mixture was filtered and concentrated under reduced pressure to obtain LD2-8h as a clear syrup (252mg, 99.4% yield). The crude product was used directly in the next step without purification. MS(ESI)m / z:320.3[M+H] +

[0383] Step 6 (R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecane-15-acid (LD2-8j)

[0384] LD2-8h (250 mg, 0.78 mmol) and LD2-8i (243 mg, 1.57 mmol) were dissolved in a mixed solvent of ACN (8 mL) and NaHCO3 aqueous solution (1 M, 16 mL). The resulting mixture was stirred at 0°C for 1 hour, and then stirred further at 25°C until the reaction was complete. The reaction mixture was then acidified with aqueous solution. It was acidified with KHSO4 (20 mL) aqueous solution and extracted with EA (35 mL x 3). The bound organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude yellow oil, which was purified by flash column chromatography to obtain LD2-8j (280 mg, yield 89.6%) as a white solid. MS (ESI) m / z: 422.3 [M+Na] + 1H NMR (400 MHz, d6-DMSO) δ 12.48 (s, 1H), 7.03-7.01 (m, 2H), 6.99 (s, 2H), 4.08-4.03 (m, 3H), 3.86-3.83 (m, 2H), 3.14-3.11 (m, 2H), 2.35 (t, J=7.2 Hz, 2H), 2.16-2.09 (m, 1H), 1.9LD2-8.84 (m, 1H), 1.32 (s, 9H).

[0385] Step 7 (R)-4-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl((8S,11S,14S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-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]indolidino[1,2-b]quinoline-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4-oxa-6,9,12,15-tetraazaoctadecane-18-yl)carbamate (LD2-8l).

[0386] LD2-8l (32 mg, yield 50.9%) was synthesized according to step 7 of LD2-3. MS(ESI)m / z:1418.1[M+H] +

[0387] Step 8 (R)-4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl((8S,11S,14S)-14-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-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]indolidino[1,2-b]quinoline-1-yl)amino)-11-isopropyl-2,2,8-trimethyl-1,7,10,13,16-pentaoxo-4-oxa-6,9,12,15-tetraazaoctadecane-18-yl)carbamate(LD2-8)

[0388] LD2-8 (22 mg, 0.016 mmol) was dissolved in a mixed solvent of DCM (2 mL), followed by the addition of ZnBr2 (151 mg, 0.67 mmol). The resulting suspension was stirred at 40°C for 12 hours. After the reaction was complete, the reaction mixture was filtered and concentrated. The residue was diluted in a mixed solvent of CH3CN / 0.1% FA aqueous solution and purified by preparative HPLC (method: column: XBridgePrepC18OBD5um19*150 mm, mobile phase: A-water (0.1% TFA): B-acetonitrile, flow rate: 20 mL / min) to obtain compound LD2-8 (13 mg, yield 61.7%) as a white solid. MS(ESI)m / z:1318.1[M+H] +

[0389] Example 21. Preparation of immunoconjugate Antibodies (concentration 0.5-25 mg / mL, PBS buffer pH 6.0-8.5) in conjugation buffer were incubated at reducing temperature (0-40°C) for 10 minutes. 8-15 equivalents. TECP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to stand at reducing temperature for 1-8 hours. Organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker toxin stock (LD2-3, see Table 6) (2-25 equivalents, 10 mM stock in organic solvent) were added stepwise to a reaction buffer (PBS buffer, pH 6.0-9.0) containing anti-FGFR2B antibody (BGAh9179 for BGA3457, BGAh9239 for BGA9823) (1-20 mg / mL) at 0-37°C for 0.5-48 hours. The solution was transferred to a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with sucrose, trehalose, and optional additives such as Tween® 20, 60, 80) for buffer exchange (spin desalination column, ultrafiltration, and dialysis).

[0390] For the preparation of the immunoconjugate, please also refer to Example 23. Example 22. Measurement of BGA3457DAR (drug-antibody ratio)

[0391] The drug-antibody ratio (DAR) of ADCs was determined by LCMS or HIC.

[0392] LC-MS method: LC-MS analysis was performed under the following measurement conditions. LC-MS system: VanquishFlexUHPLC and OrbitrapExploris240 mass spectrometer Column: MAbPac® RP, 2.1*50mm, 4μm, 1,500Å, ThermoScientific® Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) aqueous solution Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient Programs: 25%B~25%B (0 min~2 min), 25%B~50%B (2 min~18 min), 50%B~90%B (18 min~18.1 min), 90%B~90%B (18.1 min~20 min), 90%B~25%B (20 min~20.1 min), 25%B~25%B (20.1 min~25 min) Injection sample volume: 1 or 2 μg MS parameters: Intact and denatured MS data were acquired in HMR mode with R=15k and deconvolved using the ReSpect algorithm and sliding window integration in Thermo Scientific® BioPharma Finder® 4.0 software.

[0393] HIC method: HPLC analysis was performed under the following measurement conditions. HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280nm Column: Tosoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5M ammonium sulfate, 50mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient Programs: 0%B~0%B (0 min~2 min), 0%B~100%B (2 min~15 min), 100%B~100%B (15 min~16 min), 100%B~0%B (16 min~17 min), 0%B~0%B (17 min~20 min) Amount of sample injected: 20 μg

[0394] Example 23. Preparation of DAR8 immunoconjugate To prepare an immunoconjugate ("DAR8") containing 8 units of drug (toxin) per antibody, antibodies in conjugation buffer (concentration 0.5–25 mg / mL, PBS buffer pH 6.0–8.5) were incubated at reducing temperature (0–40°C) for 10 minutes and then incubated at 8–15 equivalents. TECP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to stand at reducing temperature for 1–8 hours. After cooling the reducing mixture to 0–25°C, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0–25% v / v) and linker-payload stock (10–25 equivalents, 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0–25°C for 1–3 hours, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was transferred to a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with sucrose, trehalose, and optional additives such as Tween® 20, 60, 80) for buffer exchange (spin desalination column, ultrafiltration, and dialysis).

[0395] Maleimide hydrolysis after bonding After the conjugation step, the immunoconjugate was subjected to buffer exchange to replace the buffer with a ring-opening buffer (pH 7.0-9.0, PBS, borate, or Tris buffer), and the solution was left to stand at 22 or 37°C for 5-48 hours. The maleimide ring-opening process was monitored by reducing LC-MS. Once the hydrolysis of the conjugated maleimide was complete, the resulting immunoconjugate was subjected to buffer exchange by dialysis to a basic Tris pH 8.0-8.5 buffer or an acidic histidine acetate pH 5.0-6.5 buffer.

[0396] Characterization of Immunoconjugates Immunoconjugates were prepared using the DAR8 profile according to the procedure described above. All immunoconjugates were characterized by the following analytical methods. The DAR of the immunoconjugates was determined according to the method described in Example 22.

[0397] SEC method for determining the purity of immunoconjugates HPLC analysis was performed under the following measurement conditions. HPLC System: Waters H-Class UPLC System Detector: Measurement wavelength: 280nm Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6x150mm, Waters Column temperature: Room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropyl alcohol, pH 7.0 Gradient program: 10 minutes of isocratic elution, flow rate 0.3 mL / min Amount of sample injected: 20 μg

[0398] The SEC purity of the constructed immunoconjugate was >95%.

[0399] Example 23. Evaluation of the hydrophobicity of immunoconjugates Immunoconjugates with higher hydrophobic properties exhibit slower retention times from HIC (hydrophobic interaction column) chromatography. DAR8 (antibody loaded with eight drug units) was the exemplary immunoconjugate peak for this comparison.

[0400] HIC method 1: HPLC analysis was performed under the following measurement conditions. HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280nm Column: Tosoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5M ammonium sulfate, 50mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient Programs: 0%B~0%B (0 min~2 min), 0%B~100%B (2 min~15 min), 100%B~100%B (15 min~16 min), 100%B~0%B (16 min~17 min), 0%B~0%B (17 min~20 min) Amount of sample injected: 20 μg

[0401] HIC method 2: HPLC analysis was performed under the following measurement conditions. HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280nm Column: MABPac HIC-10, 5μm, 4.6×10mm (Thermo) Column temperature: 25℃ Mobile phase A: 1.5M ammonium sulfate, 50mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20% B to 20% B (0 min to 1 min), 0% B to 0% B (1 min to 35 min), 20% B to 20% B (35 min to 40 min) Flow rate: 0.5mL / min Sample preparation: The sample was diluted from the initial mobile phase to 0.5 mg / mL. [Table 18] Both ADCs shown in Table 16 have an SEC purity of over 95%.

[0402] Example 25. Evaluation of plasma stability and PK profile analysis of immunoconjugates Plasma immunoconjugate and total therapeutic antibody (Ab) concentrations were determined under the following measurement conditions. Assay: Ligand-binding assay (ELISA) Capture reagent: FGFR2b ECD Detection reagent: Antitoxin antibody for immunoconjugates, anti-human IgG polyclonal antibody for total antibodies.

[0403] Immunoconjugates were intravenously administered to mice with tumors expressing FGFR2b (a SNU-16 human gastric cancer xenograft model in BALB / c nude mice) or to non-tumor mice. Blood samples were collected 0, 2, 4, 8, 24, 72, and 168 hours after the first administration, and plasma was separated by centrifugation (4°C, 3000×g, 7 min). The concentrations of immunoconjugates and total therapeutic antibodies were measured using the in-house developed Meso Scale Discovery (MSD) ligand-binding method. Briefly, ECD of FGFR2b was used as the capture reagent, and biotin-labeled antitoxin antibody or goat anti-human kappa antibody was used as the detection reagent for immunoconjugate and total antibody measurements, respectively.

[0404] The PK profiles of BGA3457 and BGA9823 were evaluated in tumor-carrying mice and hFcRn mice (Figures 17A-17B). BGA3457 and BGA9823 showed good plasma stability in human (Figure 17A) and mouse (Figure 17B) serum samples. Figures 17A-17B show low release of free payload (<1%) and stable DAR values ​​of 8 at all time points. BGA3457 showed nonlinear PK of 1-10 mg / kg in mice, suggesting target-mediated clearance (TMDD) (Figures 18A-18B). BGA8723, a comparison drug, showed nonlinear PK of 1-10 mg / kg in mice, but linear PK of 1-15 mg / kg in patients. This suggests that TMDD is less severe in humans (data not shown). As shown in Figure 19, BGA3457 exhibited favorable PK profiles in monkey serum at 1, 3, and 10 mpk, respectively.

[0405] While the present invention has been described with reference to currently preferred embodiments, it should be understood that various modifications are possible without departing from the scope of the disclosure. Unless otherwise clearly evident from the context, any step, element, embodiment, feature, or aspect of this disclosure may be used in conjunction with any other. All publications, patents, patent applications, accession numbers, etc., cited herein are incorporated herein by reference in whole for all purposes to the same extent as any individual publication, patent, patent application, or accession number is explicitly and individually indicated to be incorporated by whole for all purposes.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human FFFR2b, comprising heavy chain complementarity-determining regions (HCDRs) 1, HCDRs 2, and HCDRs 3, and light chain complementarity-determining regions (LCDRs) 1, LCDRs 2, and LCDRs 3, HCDR1 is sequence IYWX １ Includes N (Sequence ID 77), HCDR2 is sequence IYPENX ２ DTNYX ３ Contains GKFKG (Sequence ID 78), HCDR3 contains the sequence GGFDY (SEQ ID NO: 5), LCDR1 contains the sequence RASENIYSNLA (SEQ ID NO: 6), LCDR2 is sequence TATNLAX ４ (Sequence ID 79) LCDR3 contains the sequence QHFYGILYT (SEQ ID NO: 8), X １ is M or L, X ２ is G or A, X ３ is S or N, X ４ The antibody or its antigen-binding fragment is D or E.

2. An antibody or antigen-binding fragment thereof that specifically binds to human FFFR2b, which includes HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, HCDR1 contains the sequence DTYIH (sequence number 15), HCDR2 contains the sequence RIDPAX １ GNTMFASEFQG (SEQ ID NO: 80), and HCDR3 contains the sequence SKIHYDYDEGFAY (SEQ ID NO: 17), LCDR1 contains the sequence RASESVDDYGYSFLH (SEQ ID NO: 18), LCDR2 contains the sequence RASNLES (SEQ ID NO: 19), LCDR3 contains the sequence QQSNQNPRT (SEQ ID NO: 20), X １ The antibody or its antigen-binding fragment, wherein is E or N.

3. The antibody or its antigen-binding fragment is (i) Heavy chain variable region, (a) HCDR1 having sequence IYWLN (Sequence ID 3), (b) HCDR2 having sequence QIYPENADTNYSGKFKG (Sequence ID 4), (c) HCDR3 having sequence GGFDY (sequence number 5), The heavy chain variable region includes, (ii) Light chain variable region, (d) LCDR1 having sequence RASENIYSNLA (sequence number 6), (e) LCDR2 having sequence TATNLAE (sequence number 7), (f) LCDR3 having sequence QHFYGILYT (sequence number 8), The light chain variable region includes, The antibody or antigen-binding fragment according to claim 1, comprising:

4. The antibody or its antigen-binding fragment is (i) Heavy chain variable region, (a) HCDR1 having sequence DTYIH (sequence number 15), (b) HCDR2 having sequence RIDPAEGNTMFASEFQG (Sequence ID 16), (c) HCDR3 having the sequence SKIHYDYDEGFAY (SEQ ID NO: 17), The heavy chain variable region includes, (ii) Light chain variable region, (d) LCDR1 having sequence RASESVDDYGYSFLH (Sequence ID 18), (e) LCDR2 having sequence RASNLES (sequence number 19), (f) LCDR3 having sequence QQSNQNPRT (sequence number 20), The light chain variable region includes, The antibody or antigen-binding fragment according to claim 2, comprising:

5. (i) A heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to that of SEQ ID NO: 9, and a light chain variable region (VL) containing an amino acid sequence at least 95% identical to that of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 21, and a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:

22. The antibody or antigen-binding fragment according to claim 1 or 2, comprising:

6. The antibody or antigen-binding fragment according to claim 5, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in SEQ ID NO: 9 and / or 10 and / or SEQ ID NO: 21 and / or 22 are inserted, deleted, or substituted.

7. The antibody or its antigen-binding fragment may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or F(ab'). ２ A fragment, which is an antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment has reduced glycosylation, is not glycosylated, or is low-fucosylated.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment comprises an increase in the bisecting GlcNac structure.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, comprising the Fc domain of IgG1.

11. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10, further comprising a pharmaceutically acceptable carrier.

12. An isolated nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid described in claim 12.

14. A host cell comprising the nucleic acid according to claim 12, or the vector according to claim 13.

15. A process for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in claim 14 in a culture medium, and recovering the antibody or an antigen-binding fragment thereof from the culture medium.

16. A purified composition comprising an anti-human FGFR2b antibody or its antigen-binding fragment produced by the process described in claim 15.

17. An immunoconjugate, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a cytotoxic agent.

18. formula: Ab-(C-L-(D) ｍ ) ｎ 、 or comprising a pharmaceutically acceptable salt, solvate, or hydrate thereof, Ab is the antibody or its antigen-binding fragment, C is a conjugator, L is a linker, D is a cytotoxic agent, m is an integer from 1 to 8. The immunoconjugate according to claim 17, wherein n is 1 to 10.

19. The immunoconjugate according to claim 18, wherein m is 1.

20. The immunoconjugate according to claim 18 or 19, wherein n is 3, 4, 5, 6, 7, 8, 9, or 10.

21. C is represented by the formulas (C-I), (C-Ia), (C-Ib), (C-II), (C-III), (C-IIIa), (C-IIIb), or (C-IV): 【Chemistry 33-1】 【Chemistry 33-2】 It has, * indicates a coupling in which the conjugator connects to Ab, according to any one of claims 18 to 20.

22. L is represented by equations (LI), (L-II), or (L-III): 【Transformation 34】 It has, Su is a hydrophilic residue, The immunoconjugate according to any one of claims 18 to 21, wherein * indicates a coupling in which the linker connects to the conjugator.

23. Su is 【Chemistry 35】 The immunoconjugate according to any one of claims 18 to 22.

24. Su is 【Transformation 36】 The immunoconjugate according to any one of claims 18 to 23.

25. The cytotoxic agent (D) is 【Chemistry 37】 An immunoconjugate according to any one of claims 18 to 24, selected from the group consisting of the following.

26. The cytotoxic agent (D) is 【Transformation 38】 An immunoconjugate according to claim 25, selected from the group consisting of the following.

27. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 39-1】 【Chemistry 39-2】 It is represented by one of the following: The immunoconjugate according to any one of claims 18 to 26, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof.

28. The immunoconjugate according to claim 27, wherein n is 3, 4, 5, 6, 7, 8, 9, or 10.

29. The immunoconjugate according to claim 28, wherein n is approximately 8.

30. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 40-1】 【Chemistry 40-2】 It is represented by one of the following: The immunoconjugate according to any one of claims 18 to 26, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof.

31. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 41】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

32. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 42】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

33. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 43】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

34. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 44】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

35. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 45】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

36. The aforementioned immunoconjugate is given by the following formula: 【Chemistry 46】 Represented by or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof, where n is approximately 8, and the Ab is, (i) A heavy chain variable region (VH) containing the amino acid of SEQ ID NO: 9, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 10, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 22, An immunoconjugate according to any one of claims 18 to 27, including the immunoconjugate described in any one of claims 18 to 27.

37. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 17 to 36 and a pharmaceutically acceptable carrier.

38. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a pharmaceutical composition according to claim 11 or 37, or an immunoconjugate according to any one of claims 17 to 36, and instructions for use thereof.

39. The kit according to claim 38, wherein the antibody or its antigen-binding fragment forms a complex with FGFR2b, which is detected by an assay including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and / or Western blotting.

40. A method for treating small cell lung cancer or gastric cancer, comprising administering to a patient in need of treatment an effective amount of the pharmaceutical composition according to claim 11 or 37, the purified composition according to claim 16, or the immunoconjugate according to any one of claims 17 to 36.

41. A method for treating a tubulin inhibitor-resistant tumor, comprising administering an effective amount of the pharmaceutical composition according to claim 11 or 37, the purified composition according to claim 16, or the immunoconjugate according to any one of claims 17 to 36 to a subject in which FGFR2b is accumulated or overexpressed in a biological sample.

42. Use of the pharmaceutical composition according to claim 11 or 37, the purified composition according to claim 16, or the immunoconjugate according to any one of claims 17 to 36 in the manufacture of a pharmaceutical product for treating small cell lung cancer or gastric cancer in a patient.

43. A pharmaceutical composition according to claim 11 or 37, a purified composition according to claim 16, or an immunoconjugate according to any one of claims 17 to 36, for use in the treatment of a patient's small cell lung cancer or gastric cancer.

44. The pharmaceutical composition, the purified composition, or the immunoconjugate according to any one of claims 40 to 43, administered in combination with at least one other therapeutic agent.

45. The method, use, or immunoconjugate according to claim 44, wherein the other therapeutic agent is a chemotherapeutic agent.

46. The other therapeutic agent comprises at least one immune checkpoint inhibitor, according to the method, use, or immunoconjugate of claim 44.

47. The method, use, or immunoconjugate according to claim 46, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody.

48. The method, use, or immunoconjugate according to claim 47, wherein the anti-PD1 antibody is tislerizumab.

49. A method for producing an immunoconjugate according to any one of claims 17 to 36, (i) Culturing host cells transformed with an isolated nucleic acid comprising an anti-human FGFR2b antibody or a sequence encoding an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acids of SEQ ID NO: 10, or a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acids of SEQ ID NO: 22, (ii) Expressing the antibody or its antigen-binding fragment, (iii) Recovering the expressed antibody or its antigen-binding fragment, (iv) Conjugate at least one cytotoxic agent (D) to the antibody or its antigen-binding fragment using a linker (L) and a conjugator (C) so that an immunoconjugate is formed, The method, including the method described above.

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