Anti-FGFR3 antibody conjugates and medical uses thereof

Abstract

The invention relates to the field of biological pharmacy, in particular to a conjugate containing an anti-FGFR3 (Fibroblast Growth Factor Receptor 3) antibody and a linker-load, and a corresponding pharmaceutical composition, a preparation method and application thereof.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceuticals, in particular to a conjugate containing an anti-FGFR3 antibody and a linker-load substance, as well as a corresponding pharmaceutical composition, a preparation method and uses thereof. Background Art

[0002] Fibroblast growth factor (FGF) and its tyrosine kinase receptor (FGFR) play an important role in embryonic development, maintaining homeostasis of various tissues, wound healing processes and metabolic functions. In humans, there are FGFRs (FGFR1-4) and FGFs (FGF 1-14 and FGF 16-23) with high homology. FGFR contains an extracellular region that contains three immunodomains D1, D2 and D3, a single transmembrane region and a split cytoplasmic kinase part.

[0003] Dysregulation of FGFR1-4 signaling has been implicated in several types of cancer. Genomic FGFR mutations, including gene amplification, chromosomal translocations, and activating mutations, induce aberrant activation of the FGF pathway and promote neoplastic transformation. In particular, amplification of FGFR3 has been associated with the development of solid tumors such as brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, and intrahepatic cholangiocarcinoma. In addition, missense FGFR mutations have been found in several types of cancer, and S249C of FGFR3 can enhance FGF-driven signaling and tumor cell proliferation. FGFR3 fusion proteins act as cancer drivers by altering the constitutive activation of the kinase domain. Known FGFR3 fusion partners are ACC3, BAIAP2L1, AES, ELAVL3, JAKMIP1, TNIP2, and WHSC1.

[0004] To date, efforts have been made to develop therapeutic approaches targeting FGFR3.

[0005] B701 (Vofatamab) is a human immunoglobulin G1 monoclonal antibody against FGFR3 that has been clinically tested to confirm its anti-tumor activity and the possibility of being used in combination with docetaxel. When the anti-FGFR3 monoclonal antibody B-701 is administered, it specifically binds to and inhibits wild-type and mutant FGFR3 to inhibit FGFR3 phosphorylation, thereby inhibiting FGFR3 activation and FGFR3-mediated signaling pathways. Therefore, in tumors expressing FGFR3, cell proliferation is inhibited and apoptosis is induced.

[0006] Antibodies and ADCs targeting FGFR3 entered the clinical stage, but further development was stopped due to lack of efficacy. There is still a need in the art to develop new ADCs targeting FGFR3. Summary of the invention

[0007] In the first aspect of the present invention, the present invention provides an antibody drug conjugate (ADC) having a structure of formula (I):

[0008]

[0009] in,

[0010] A is an anti-FGFR3 antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is modified to bind to (Gly) in the compound of formula (I) n Partial connection, wherein the antibody or antigen-binding fragment comprises CDRs: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 6;

[0011] z is an integer from 1 to 20; preferably from 1 to 4; especially 2;

[0012] opSu is or a mixture thereof;

[0013] R 0 It is C 1-10 alkyl;

[0014] n is any integer from 2 to 20;

[0015] k1 and k2 are independently integers from 1 to 7;

[0016] i is an integer from 1 to 100;

[0017] j is an integer from 1 to 100;

[0018] P1 and P2 are independently loads.

[0019] In some embodiments, the ligation process between the modified antibody (or antigen-binding fragment) and the compound of formula (I) is catalyzed by a ligase.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises CDRs: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0021] In some embodiments, the anti-FGFR3 antibody or antigen-binding fragment binds to human FGFR3 and monkey FGFR3 with a KD value of less than 10 nM.

[0022] In some embodiments, the anti-FGFR3 antibody or antigen-binding fragment does not bind to mouse FGFR3.

[0023] In some embodiments, the cargo is a cytotoxin or a fragment thereof, with optional derivatization to connect the cargo and the linker;

[0024] The cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indol-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10 and analogs, halichondrin B. eribulin, indole-3-oxoacetamide, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone one), mitoguazone, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquone, meturedepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin antramycin, bleomycins, actinomycin C, carabicin, carminomycin, cardinophyllin, carminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin,Mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric adriamycin, rodorubicin, rufocromomycin, streptozocin, zinostatin, zorubicin, trichothecene, T-2 toxin, verracurin A, bacillocporin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethyl folic acid acid), methotrexate, pteroxin, trimetrexate, edatrexate, fludarabine, 6-mercaptopurine, tiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitors and proteasome inhibitors; and / or,

[0025] Selected from vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepines (pyrrolobenzodiazepine), exatecan and its derivatives; and / or

[0026] Selected from auristatins, in particular MMAE, MMAF or MMAD; and / or

[0027] Selected from exatecan and its derivatives; such as DX8951f; and / or

[0028] Selected from DXd-(1) and DXd-(2); preferably DXd-(1).

[0029] In some embodiments, the support has a structure of formula (II):

[0030]

[0031] in,

[0032] a is 0 or 1;

[0033] The carbon atoms marked with p1* and p2* are asymmetric centers, and the asymmetric centers are in S configuration, R configuration or racemic form;

[0034] L 1 Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2;

[0035] M is -CH2-, -NH- or -O-;

[0036] L 2 C 1-3 Alkylene;

[0037] R 1 and R 2 are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 In some embodiments, the carrier is selected from:

[0038]

[0039] In some embodiments, the ADC is selected from:

[0040]

[0041]

[0042] In some embodiments, the antibody or antigen-binding fragment comprises: a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:7; and / or a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0043] In some embodiments, the antibody or antigen-binding fragment comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:7; and / or a VL domain comprising the amino acid sequence of SEQ ID NO:8.

[0044] In some embodiments, the antibody or antigen-binding fragment comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:7; and a VL domain comprising the amino acid sequence of SEQ ID NO:8.

[0045] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and / or a light chain constant domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:11.

[0046] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and / or a light chain constant domain comprising the amino acid sequence of SEQ ID NO:11.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:9; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:11.

[0048] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:10; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:11.

[0049] In some embodiments, wherein the antibody or antigen structure fragment comprises a C-terminal modification of the heavy chain and / or a C-terminal modification of the light chain. In some embodiments, the antibody, Sp and the recognition sequence of the ligase donor substrate are sequentially connected; Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS; the recognition sequence of the ligase donor substrate is LPXTGJ, wherein X can be any single amino acid, natural or non-natural; J does not exist, or is an amino acid fragment comprising 1-10 amino acids.

[0050] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 or 13; and / or a light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0051] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:12; and a light chain comprising the amino acid sequence of SEQ ID NO:14.

[0052] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:13; and a light chain comprising the amino acid sequence of SEQ ID NO:14.

[0053] In some embodiments, the drug-antibody ratio (DAR) of the antibody drug conjugate is an integer or non-integer of 1-20, particularly an integer or non-integer of 2 to 8. In some embodiments, the value of DAR is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range between any two values ​​(including the final value).

[0054] In a second aspect, the present invention provides a pharmaceutical composition comprising the ADC of the present disclosure and at least one pharmaceutically acceptable carrier.

[0055] In some embodiments, the pharmaceutical composition comprises an alkylating agent. In some embodiments, the alkylating agent comprises temozolomide or a derivative thereof.

[0056] In a third aspect, the present invention provides a pharmaceutical combination comprising the ADC of the present disclosure and an alkylating agent.

[0057] In some embodiments, the drug combination comprises an ADC of the present disclosure and temozolomide or a derivative thereof. In some embodiments, the drug combination comprises the ADC and temozolomide. In some embodiments, the drug combination further comprises a pharmaceutically acceptable carrier.

[0058] In a fourth aspect, the present invention provides a kit comprising the ADC, pharmaceutical composition or pharmaceutical combination of the present disclosure.

[0059] In some embodiments, the kit comprises a first packaging unit comprising a conjugate having the structure of Formula (I), a second packaging unit comprising an alkylating agent; and

[0060] Optionally, instructions for administering the conjugate and the alkylating agent to a subject.

[0061] In some embodiments, the subject has a disease. In some embodiments, the disease is a FGFR3-mediated disease. In some embodiments, the alkylating agent is temozolomide or a derivative thereof.

[0062] In some embodiments, the disease is a tumor. In some embodiments, the disease includes FGFR3 positive tumors. In some embodiments, the disease includes a tumor that overexpresses FGFR3 or a tumor with FGFR3 changes. In some embodiments, the disease includes a tumor with FGFR3 fusion (such as TACC3 fusion, intracellular fusion) or a tumor with FGFR3 gene mutation (such as Y373C, G380R, S371C, S249C or R248C). In some embodiments, the disease is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing's tumor (Ewing's tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma, multiple myeloma. In some embodiments, the disease is selected from: brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct cancer. In some embodiments, the disease is glioblastoma, bladder cancer, or multiple myeloma.

[0063] In a fifth aspect, the present invention provides use of the ADC, the drug combination or the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating a disease; wherein the disease is a FGFR3-mediated disease.

[0064] In some embodiments, the disease is a tumor. In some embodiments, the disease includes FGFR3 positive tumors. In some embodiments, the disease includes a tumor overexpressing FGFR3 or a tumor with FGFR3 changes. In some embodiments, the disease includes a tumor with FGFR3 fusion (such as TACC3 fusion, intracellular fusion) or a tumor with FGFR3 gene mutation (such as Y373C, G380R, S371C, S249C or R248C). In some embodiments, the disease is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma, multiple myeloma. In some embodiments, the disease is selected from: brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct carcinoma. In some embodiments, the disease is glioblastoma, bladder cancer or multiple myeloma.

[0065] In a sixth aspect, the present invention provides a method of treating a subject suffering from a disease or reducing the likelihood of disease progression, comprising administering the conjugate, drug combination, pharmaceutical composition or kit, wherein the disease is a FGFR3-mediated disease.

[0066] In some embodiments, the disease is a tumor. In some embodiments, the disease includes FGFR3 positive tumors. In some embodiments, the disease includes a tumor overexpressing FGFR3 or a tumor with FGFR3 changes. In some embodiments, the disease includes a tumor with FGFR3 fusion (such as TACC3 fusion, intracellular fusion) or a tumor with FGFR3 gene mutation (such as Y373C, G380R, S371C, S249C or R248C). In some embodiments, the disease is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma, multiple myeloma. In some embodiments, the disease is selected from: brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct carcinoma. In some embodiments, the disease is glioblastoma, bladder cancer or multiple myeloma.

[0067] In some embodiments, the conjugate and the alkylating agent are administered simultaneously as part of the same pharmaceutical formulation. In some embodiments, the conjugate and the alkylating agent are administered simultaneously as part of different pharmaceutical formulations. In some embodiments, the alkylating agent is temozolomide or a derivative thereof.

[0068] In some embodiments, the conjugate and the alkylating agent are administered at different times.

[0069] In another aspect, the present invention provides the use of an effective amount of the conjugate in the preparation of a medicament for treating a subject suffering from cancer, in combination with an effective amount of an alkylating agent.

[0070] In some embodiments, the conjugate and the alkylating agent are administered simultaneously as part of the same pharmaceutical formulation. In some embodiments, the conjugate and the alkylating agent are administered simultaneously as part of different pharmaceutical formulations. In some embodiments, the alkylating agent is temozolomide or a derivative thereof.

[0071] In some embodiments, the conjugate and the alkylating agent are administered at different times.

[0072] In some embodiments, the disease includes FGFR3 positive tumors. In some embodiments, the disease includes tumors that overexpress FGFR3 or tumors with FGFR3 changes. In some embodiments, the disease includes tumors with FGFR3 fusions (such as TACC3 fusions, intracellular fusions) or tumors with FGFR3 gene mutations (such as Y373C, G380R, S371C, S249C or R248C). In some embodiments, the disease is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland carcinoma, papillary carcinoma, Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma, multiple myeloma. In some embodiments, the disease is selected from: brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct carcinoma. In some embodiments, the disease is glioblastoma, bladder cancer or multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The SDS-PAGE results of the antibodies prepared in Example 3 are shown.

[0074] Figure 2 The specificity of A9 for FGFR3 in affinity ELISA analysis is shown.

[0075] Figure 3 Shown is a comparison of the specificity of A9 and A9Q affinity for FGFR3 in affinity ELISA analysis.

[0076] Figure 4 Shown is a comparison of the specificity of A9 and ADC19 affinity for FGFR3 in affinity ELISA analysis.

[0077] Figure 5 The specificity of A9 for FGFR3 positive cells in the cell binding assay is shown.

[0078] Figure 6.1Figure 2 shows the specificity comparison of the affinity of A9 and A9Q for FGFR3 positive cells in the cell binding assay. Figure 6.2 Cell binding analysis of ADC20 is shown.

[0079] Figure 7.1 and Figure 7.2 In vivo efficacy testing in a glioblastoma PDX model is shown.

[0080] Figure 8.1-8.4 In vivo efficacy testing in the CDX model of bladder cancer is shown.

[0081] Fig. 9 In vivo efficacy testing in the multiple myeloma CDX model is shown.

[0082] Fig.10 The internalization activity of ADC20 is shown.

[0083] Fig.11 The bystander killing effect of ADC19 and ADC20 is shown.

[0084] Figure 12.1 and Figure 12.2 In vivo efficacy testing in a glioblastoma PDX model is shown. DETAILED DESCRIPTION

[0085] Specific embodiments are provided below to illustrate the technical contents of the present disclosure. Those skilled in the art can easily understand other advantages and effects of the present disclosure through the contents disclosed in the specification. The present disclosure can also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present disclosure.

[0086] definition

[0087] Unless otherwise defined below, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. The technology used herein refers to the technology commonly understood in the art, including variants and equivalent substitutes obvious to those skilled in the art. Although the following terms are believed to be easily understood by those skilled in the art, in order to better illustrate the present disclosure, the following definitions are listed. When a trade name appears in this article, it refers to the corresponding commodity or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0088] When a certain amount, concentration or other value or parameter is proposed in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it should be understood that this is equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is explicitly described. Unless otherwise indicated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the statement "i is an integer of 1-20" means that i is any integer of 1-20, for example, i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar statements such as j, k1, k2, n, k and z should also be understood in a similar manner. "Conservative amino acid substitution" refers to replacing an amino acid residue with another amino acid residue of a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of classes of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0089] Unless the context clearly dictates otherwise, singular forms such as "a", "an" and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0090] When used in conjunction with a numerical variable, the terms "about" and "approximately" generally indicate that the value of the variable and all values ​​of the variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or within a larger range.

[0091] The term "optional" or "optionally" means that the subsequently described event may or may not occur, but does not necessarily occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0092] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude other uncited elements, steps, or ingredients. The expression "consisting of excludes any element, step, or ingredient not specified. The expression "consisting essentially of" indicates that the scope is limited to the specified elements, steps, or ingredients, plus the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0093] As used herein, the term "antibody" is used in a broad manner, particularly including complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody can be any subtype (e.g., IgG, IgE, IgM, IgD and IgA) or subclass, and can be derived from any suitable species. In some embodiments, the antibody is human or mouse. The antibody can also be a fully human antibody, a humanized antibody or a chimeric antibody prepared by a recombinant method.

[0094] Monoclonal antibody is used herein to refer to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a few possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the characteristic of the antibody being derived from a substantially homogeneous antibody population and should not be construed as requiring some specific method for producing the antibody.

[0095] Complete antibodies or full-length antibodies basically comprise an antigen-binding variable region and a constant region of a light chain (CL) and a constant region of a heavy chain (CH), which may include CH1, CH2, CH3 and CH4, depending on the subtype of the antibody. Antigen-binding variable region (also referred to as a fragment variable region, an Fv fragment) generally comprises a variable region of a light chain (VL) and a variable region of a heavy chain (VH). The constant region may be a constant region with a native sequence (such as a constant region with a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes the target antigen and interacts with it. The constant region may be recognized by the immune system and interacts with it.

[0096] Antibody fragments can include a portion of a complete antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementary determining regions (CDRs). Fab fragments are antibody fragments obtained by papain digestion of full-length immunoglobulins, or fragments with the same structure produced by, for example, recombinant expression. Fab fragments include a light chain (including VL and CL) and another chain, wherein the other chain includes a variable domain (VH) of a heavy chain and a constant region domain (CH1) of a heavy chain. F(ab')2 fragments are antibody fragments obtained by pepsin digestion of immunoglobulins at pH 4.0-4.5, or fragments with the same structure produced by, for example, recombinant expression. F(ab')2 fragments essentially include two Fab fragments, wherein each heavy chain portion includes several additional amino acids, including cysteine ​​that forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment that contains half of a F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments can contain multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments can include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), can produce an antibody that immunospecifically binds to an antigen.

[0097] Antibodies according to the present disclosure can be prepared using techniques known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed by a suitable culture system (e.g., E. coli or mammalian cells). For example, engineering can refer to the introduction of a ligase-specific recognition sequence at its terminus.

[0098] As used herein, the term "antibody-drug conjugate" is referred to as "conjugate".

[0099] Small molecule compounds refer to molecules of comparable size to organic molecules commonly used in medicine. The term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, the molecular weight of a small molecule compound can be, for example, about 100 to about 2000Da, about 200 to about 1000Da, about 200 to about 900Da, about 200 to about 800Da, about 200 to about 700Da, about 200 to about 600Da, about 200 to about 500Da.

[0100] A spacer is a structure located between different structural modules, which can separate the structural modules in space. The definition of a spacer is not limited to whether it has a certain function or whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, amino acid derivatives or analogs. "Spacer sequence" refers to an amino acid sequence used as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids such as GA, or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-cleaving spacer is a covalent assembly specifically used to associate the breaking of two chemical bonds after the activation of the protective part in the precursor: when stimulated, the protective part (such as a cleavable sequence) is removed, which produces a series of decomposition reactions, resulting in the release of smaller molecules in a time sequence. Examples of self-cleaving spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0101] The term "alkyl" refers to a saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups include straight chain alkyl groups, branched chain alkyl groups, or cyclic alkyl groups (cycloalkyl groups) or partially cyclic alkyl groups (e.g., cycloalkyl-linear alkyl groups and cycloalkyl-branched alkyl groups). Alkyl groups can contain 1-10 carbon atoms and refer to C 1-10 Alkyl groups, such as C 1-6 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, C3 alkyl, C4 alkyl, C 3-6Alkyl. Non-limiting examples of linear alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc. Non-limiting examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.

[0102] The terms "cyclic alkyl" and "cycloalkyl" have the same meaning and are used interchangeably herein. Cyclic alkyl groups may include monocyclic or polycyclic (e.g., having 2 or more fused rings) groups. In polycyclic cycloalkyl groups, two or more rings may be fused or bridged or spiroed together. The ring-forming carbon atoms of the cyclic alkyl group may be optionally substituted with oxo (i.e., C(O)). The cyclic alkyl group may have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3-10 ). Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, and bicyclo[2.1.1]hexyl. In some embodiments, the cyclic alkyl group is C 3-6 Monocyclic or bicyclic alkyl, preferably C 3-6 Monocyclic cyclic alkyl, especially cyclopropyl.

[0103] The term "partially cyclic" means that the group contains one or more cyclic parts and one or more non-cyclic (i.e., linear or branched) parts. Partially cyclic alkyl groups can include cyclic alkyl-linear alkyl groups and cyclic alkyl-branched alkyl groups. Partially cyclic alkyl groups can have 4, 5, 6, 7, 8, 9 or 10 carbon atoms (C 3-10 ), including ring-forming carbon atoms and non-ring-forming carbon atoms. Examples of some cyclic alkyl groups include, but are not limited to, C 3-9 Cyclic alkyl - C1 alkyl, C 3-8 Cyclic alkyl - C2 alkyl, C 3-7 Cyclic alkyl - C3 linear alkyl, C 3-6 Cyclic alkyl - C4 linear alkyl, C 3-5 Cyclic alkyl - C5 linear alkyl, C 3-4 Cyclic alkyl - C6 linear alkyl, C3 cyclic alkyl - C7 linear alkyl, C 3-7 Cyclic alkyl - C3 branched alkyl, C 3-6 Cyclic alkyl - C4 branched alkyl, C 3-5 Cyclic alkyl - C5 branched alkyl, C 3-4Cyclic alkyl-C6 branched alkyl and C3 cyclic alkyl-C7 branched alkyl. In some embodiments, part of the cyclic alkyl is C 3-9 Cyclic alkyl - C1 alkyl, preferably C 3-6 Cyclic alkyl-C 1-2 Alkyl, C 3-6 Cyclic alkyl - C1 alkyl, more preferably C 3-4 Cyclic alkyl-C 1-2 Alkyl groups, especially C 3-4 Cyclic alkyl - C1 alkyl, especially cyclopropyl-methyl.

[0104] A divalent group refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of a corresponding monovalent group. A divalent group has two attachment sites that are attached to the rest of the molecule, wherein the two attachment sites may be on the same atom of the divalent group or on two different atoms.

[0105] "Alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group. Alkylene includes linear, branched, cyclic or partially cyclic groups. Examples of linear alkylene include, but are not limited to, methylene (-CH2-), -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched alkylene include, but are not limited to -CH(CH3)-, -CH(C2H5)-, -CH(CH3)-CH2-, -CH(C3H7)-, -CH(C2H5)-CH2-, -C(CH3)2-CH2-, -(CH(CH3))2-, -CH(CH3)-(CH2)2-, -CH2-CH(CH3)-CH2-, -CH(C4H9)-, -C(CH3)(C3H7)- , -C(C2H5)2-, -CH(C3H7)-CH2-, -CH(C2H5)-CH(CH3)-, -CH(C2H5)-(CH2)2-, -CH2-CH(C2H5)-CH2 -, -C(CH3)2-(CH2)2-, -CH2-C(CH3)2-CH2-, -CH(CH3)-(CH2)3-, -CH2-CH(CH3)-(CH2)2-, -CH(C5H 11)-, -C(C2H5)(C3H7)-, -C(CH3)(C4H9)-, -CH(C4H9)-CH2-, -C(C2H5)2-CH2-, -C (CH3)(C3H7)-CH2-, -CH(C2H5)-CH(C2H5)-, -CH(CH3)-CH(C3H7)-, -C(CH3)2-C( CH3)2-, -CH(C3H7)-(CH2)2-, -CH2-CH(C3H7)-CH2-, -CH(C2H5)-C(CH3)2-, -C(CH3)2-CH(CH3)-CH2-, -CH(CH3)-C(CH3)2-CH2-, -CH(C2H5)-CH(CH3)-CH2-, -CH( CH3)-CH(C2H5)-CH2-, -CH(CH3)-CH2-CH(C2H5)-, -CH(CH3)-C(CH3)2-CH2-, -(CH(CH3))3-, -C(CH3)2-(CH2)3-, -CH(C2H5)-(CH2)3-, -CH2-CH(C2H5)-(CH2)2-, -CH2-CH(CH3)-CH(CH3)-CH2-, -(CH(CH3))2-(CH2)2-, -CH(CH3)-(CH2)2-CH(CH3)-, -(CH2)2-CH(CH3)-(CH2)2-, -CH2-CH(CH3)-(CH2)3-, -CH(CH3)-(CH2)4-, etc.

[0106] The terms "cyclic alkylene" and "cycloalkylene" have the same meaning and are used interchangeably herein. Examples of cyclic alkylene include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, as well as divalent polycyclic alkyl groups containing fused, spiro, or bridged rings. In some embodiments, the cyclic alkylene is C 3-6 Cyclic alkylene, especially C 3-4 Cyclic alkylene, especially cyclopropyl.

[0107] Partial cyclic alkylene groups may include divalent groups in which the two attachment sites to the rest of the molecule may both be on one or more linear or branched alkyl moieties, or both on one or more cyclic alkyl moieties, or on a cyclic alkyl moiety and a linear or branched alkyl moiety, respectively. Examples of partial cyclic alkylene groups include, but are not limited to: (1) cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, and divalent polycyclic alkyl groups containing fused, spiro, or bridged rings, each of which is independently substituted with one or more linear or branched alkyl groups; (2) linear or branched alkylene groups, each of which is independently substituted with one or more cyclic alkyl groups; and (3) groups formed by combining one or more cyclic alkylene groups and one or more linear or branched alkylene groups, as long as a chemically stable structure is formed. In some embodiments, the partial cyclic alkylene group is C 3-9 Cyclic alkyl-C1 alkylene, preferably C 3-6 Cyclic alkyl-C 1-2 Alkylene, C 3-6 Cyclic alkyl-C1 alkylene, more preferably C 3-4 Cyclic alkyl-C 1-2 Alkylene, especially C 3-4 Cyclic alkyl-C1 alkylene, especially cyclopropyl-methylene.

[0108] As used herein, the expressions “antibody-drug conjugate”, “ADC” and “antibody-drug conjugate” have the same meaning.

[0109] Compound of formula (III)

[0110] In some embodiments, the linker-load intermediate of the ADC has the structure of formula (III):

[0111]

[0113] in,

[0114] opSu is or a mixture thereof;

[0115] R 0 It is C 1-10 alkyl;

[0116] n is any integer from 2 to 20;

[0117] k1 and k2 are independently integers from 1 to 7;

[0118] i is an integer from 1 to 100;

[0119] j is an integer from 1 to 100;

[0120] P1 and P2 are independently a support having a structure of formula (II).

[0121] In one embodiment, R 0 It is C 1-6 In a preferred embodiment, R 0 It is C 1-3 In a particular embodiment, R 0 It's methyl.

[0122] In one embodiment, n is an integer from 2 to 5. In a specific embodiment, n is 3.

[0123] In one embodiment, k1 and k2 are independently 1 or 3 or 5. In a specific embodiment, k1 and k2 are independently 5.

[0124] In one embodiment, i is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 2 to 8. In a specific embodiment, i is 4.

[0125] In one embodiment, j is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 8 to 12, especially 8 or 12. In a particular embodiment, j is 12.

[0126] In one embodiment, the compound of formula (III) has the structure of formula (III-1)

[0127]

[0128] in,

[0129] P1, P2, R 0 , opSu, n, i and j are as defined in formula (III).

[0130] In one embodiment, the compound of formula (III) is selected from:

[0131]

[0132] Preparation of compounds of formula (III)

[0133] In one embodiment, the compound of formula (III) can be synthesized by connecting a linker to a load or by connecting a series of suitable building blocks. Such building blocks can be easily designed by retrosynthetic analysis and any reaction known in the art can be used.

[0134] In one embodiment, the present invention provides a compound having the structure of formula (IV):

[0135]

[0136] in,

[0137] k is an integer from 1 to 7;

[0138] P is a support having a structure of formula (II), wherein the structure of formula (II) is as defined above.

[0139] In some embodiments, k is about 1, about 2, about 3, about 4, about 5, about 6, or about 7. In one embodiment, k is 1 or 3 or 5. In a specific embodiment, k is 5.

[0140] The compound of formula (III) can be synthesized using a compound of formula (II) / (IV) and other necessary building blocks, using a synthesis method similar to that disclosed in EP2907824A (e.g., the synthesis method of formula (2) or (2b) of EP2907824A). Suitable building blocks include, but are not limited to, linker-load intermediate 2 and linker-load intermediate 1.

[0141]

[0142] Then, the maleimide group of formula (IV) can react with the thiol group on another building block. The resulting thiosuccinimide is unstable under physiological conditions and is prone to reverse Michael addition reaction, resulting in the breakage of the connection site. In addition, when another thiol compound is present in the system, thiosuccinimide may also undergo thiol exchange with the other thiol compound. Both reactions will cause the load to fall off and produce toxic side effects. Then the thiosuccinimide is subjected to a ring-opening reaction. Then the compound of formula (III) can be obtained.

[0143] The method of the ring-opening reaction can be found in WO2015165413A1. Compounds containing the ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain a high purity and defined composition, regardless of the efficiency of the succinimide ring-opening reaction.

[0144] In the present disclosure, when applied to linker-loaded substances (linker-small molecule intermediates), the ring-opened succinimide structure no longer undergoes reverse Michael addition reaction or thiol exchange, and thus the product is more stable.

[0145] Contains the portion of the ligase acceptor and donor substrate recognition sequences

[0146] In one embodiment, (Gly) of the compound of formula (III) nThe part is a recognition sequence of a ligase donor substrate, which facilitates the enzymatic coupling of the compound of formula (III) with the antibody or antigen binding fragment under the catalysis of the ligase. The antibody or antigen binding fragment is optionally modified and comprises a corresponding recognition sequence of a ligase acceptor substrate.

[0147] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from natural transpeptidase, non-natural transpeptidase, variants thereof and combinations thereof. Non-natural transpeptidase can be but not limited to those obtained by engineering of natural transpeptidase. In a preferred embodiment, the ligase is selected from natural sortase, non-natural sortase and combinations thereof. The types of natural sortase include sortase A, sortase B, sortase C, sortase D, plant lactobacillus (L.plantarum) sortase, etc. (US20110321183A1). The type of ligase corresponds to the ligase recognition sequence, so as to realize the specific conjugation between different molecules or structural fragments.

[0148] In one embodiment, (Gly) of the compound of formula (III) n The antibody or antigen-binding fragment is optionally modified to contain a corresponding recognition sequence for a ligase donor substrate.

[0149] In some embodiments, the ligase is a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, and Lactobacillus plantarum sortase.

[0150] In a specific embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence of the ligase donor substrate can be the typical recognition sequence of the enzyme, LPXTG, where X can be any single amino acid, natural or unnatural. In another specific embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ, where X can be any single amino acid, natural or unnatural; J is absent or is an amino acid fragment comprising 1-10 amino acids, optionally labeled. In one embodiment, J is absent. In another embodiment, J is an amino acid fragment comprising 1-10 amino acids, where each amino acid is independently any natural or unnatural amino acid. In another embodiment, J is (Gly) m , wherein m is an integer from 1 to 10. In another specific embodiment, the recognition sequence of the ligase donor substrate is LPETG. In another specific embodiment, the recognition sequence of the ligase donor substrate is LPETGG.

[0151] In one embodiment, the ligase is sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN. In another embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG.

[0152] In another embodiment, the ligase is sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, wherein J is as defined above. In another embodiment, the ligase is sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be LAXTG.

[0153] In another embodiment, the ligase is sortase A from Lactobacillus plantarum and the corresponding donor substrate recognition sequence can be LPQTSEQ.

[0154] The ligase recognition sequence may also be other novel recognition sequences of a transpeptidase optimized by artificial screening.

[0155] Conjugates and their preparation

[0156] Furthermore, compounds having a cargo-bearing portion comprising a ligase recognition sequence (compounds of formula (III)) can be conjugated to anti-FGFR3 or antigen-binding fragments comprising a ligase recognition sequence.

[0157] In another aspect, the present invention provides a conjugate having the structure of formula (I):

[0158]

[0159] in,

[0160] A is an anti-FGFR3 antibody or an antigen-binding fragment thereof;

[0161] z is an integer from 1 to 20;

[0162] P1, P2, R 0 , opSu, n, k1, k2, i and j are as defined above.

[0163] In one embodiment, the antibody or antigen-binding fragment is modified to bind to (Gly) in the compound of formula (III). n Partially connected.

[0164] In one embodiment, z is about 1-20. In some embodiments, z is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range between any two values ​​(including the final values).

[0165] In one embodiment, the compound of formula (I) is selected from:

[0166]

[0167]

[0168] In one embodiment, the compound of formula (I) is selected from:

[0169]

[0170]

[0171] Antibodies or antigen-binding fragments

[0172] In one embodiment, the antibody or antigen-binding fragment (shown as "A" in formula (I)) is an anti-FGFR3 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises CDRs: a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:1; a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:2 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:2; a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:3 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:3; a light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:4 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:4; a light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:5 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:5; and a light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:6 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO:6.

[0173] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6.

[0174] In some embodiments, the anti-FGFR3 antibody or antigen-binding fragment binds to human FGFR3 and / or monkey FGFR3 with a KD value of less than 10 nM. In some embodiments, the anti-FGFR3 antibody or antigen-binding fragment binds to human FGFR3 and / or monkey FGFR3 with a KD value of about 9.9 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4.4 nM, about 3.9 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.7 nM, about 0.5 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, or a range between any two values ​​(including the final value).

[0175] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:7; and / or a light chain variable domain (VL) comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain comprising an amino acid sequence having at least about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 7, and the antibody or antigen-binding fragment comprises a light chain variable domain comprising an amino acid sequence having at least about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment comprises the VH of SEQ ID NO: 7 and / or the VL of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8.

[0176] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH) comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and / or a light chain constant domain (CL) comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain constant domain comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and a light chain constant domain comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant domain of SEQ ID NO: 9 and a light chain constant domain of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises the heavy chain constant domain of SEQ ID NO:10 and the light chain constant domain of SEQ ID NO:11.

[0177] In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13; and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:14. In some embodiments, the antibody or antigen-binding fragment comprises: a heavy chain comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; and / or a light chain comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% (range between any two values ​​(including the final value)) sequence identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain of SEQ ID NO: 12 and a light chain comprising an amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain of SEQ ID NO:13 and a light chain comprising the amino acid sequence of SEQ ID NO:14.

[0178] The above sequence is as follows (according to Kabat classification):

[0179] SEQ ID NO: 1: SYDMS

[0180] SEQ ID NO:2:GIYSGDGSIYYADSVKG

[0181] SEQ ID NO:3:DGPMNEESRFDY

[0182] SEQ ID NO:4:SGSSSNIGNNYVS

[0183] SEQ ID NO:5:ADSKRPS

[0184] SEQ ID NO:6:ASWDYSLSGYV

[0185] SEQ ID NO:7:

[0186]

[0187] SEQ ID NO:8:

[0188]

[0189] SEQ ID NO:9:

[0190]

[0191] SEQ ID NO:10:

[0192]

[0193] SEQ ID NO:11:

[0194]

[0195] SEQ ID NO:12:

[0196]

[0197] SEQ ID NO:13:

[0198]

[0199] SEQ ID NO:14:

[0200]

[0201] In one embodiment, the antibody or its antigen-binding fragment may include terminal modifications. Terminal modifications refer to modifications of the C-terminus or N-terminus of the antibody heavy chain or light chain, for example, comprising a ligase recognition sequence. In another embodiment, the terminal modifications may further include a spacer Sp, which comprises 2-100 amino acids, wherein the antibody, Sp and the ligase recognition sequence are sequentially connected. In a preferred embodiment, Sp is a spacer sequence containing 2-20 amino acids. In a specific embodiment, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS, particularly GA.

[0202] In some embodiments, the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 15 and / or a light chain of SEQ ID NO: 16. In some embodiments, the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 15 and a light chain of SEQ ID NO: 16. In some embodiments, the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 15 and a light chain of SEQ ID NO: 14. In some embodiments, the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 16.

[0203] SEQ ID NO:15:

[0204]

[0205] SEQ ID NO:16:

[0206]

[0207] Preparation of conjugates

[0208] The conjugates of the present disclosure (i.e., compounds of formula (I)) can be prepared by any method known in the art. In some embodiments, the conjugates are prepared by site-specific conjugation of an antibody or antigen-binding fragment catalyzed by a ligase with a compound of formula (III), wherein the antibody or antigen-binding fragment thereof is modified with a ligase recognition sequence.

[0209] The antibody or its Fab is connected to the compound of formula (III) by the ligase specific recognition sequence of the substrate. The recognition sequence depends on the specific ligase used. In one embodiment, the antibody or its Fab is an antibody in which the C-terminus of the light chain and / or the C-terminus of the heavy chain introduces a terminal modification based on the recognition sequence, and under the catalysis of a wild-type or optimized engineered ligase or any combination thereof, and under suitable catalytic reaction conditions, the antibody or its Fab is conjugated to the compound of formula (I).

[0210] In a specific embodiment, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme:

[0211]

[0212] The triangle represents a portion of the antibody; the pentagon represents a portion of the compound of formula (III); and G n Representative (Gly) n n, X and J are as defined above. n When conjugated, G n is the corresponding recognition sequence of the receptor substrate. The upstream peptide bond of glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate is G n The free N-terminus of LPXTG is connected to generate a new peptide bond. The resulting amino acid sequence is LPXTG n . Sequence G n and LPXTGJ are as defined above.

[0213] The compound of formula (III) disclosed in the present invention has a defined structure, a defined composition and high purity, so when the conjugation reaction is carried out with an antibody, less impurities are introduced or no other impurities are introduced. When such an intermediate is used for a ligase-catalyzed site-specific conjugation with a modified antibody containing a ligase recognition sequence, a homogeneous ADC with highly controllable quality is obtained.

[0214] Metabolism of the conjugate in a physiological environment

[0215] When a part or the whole of the linker is cleaved in tumor cells, the cargo is released. Since the linker is cleaved at the connection position with the anti-tumor compound, the anti-tumor compound is released with its inherent structure and exhibits its inherent anti-tumor effect.

[0216] In one embodiment, the compound of formula III) comprises a GGFG (Gly-Gly-Phe-Gly) moiety that can be cleaved by lysosomal enzymes (such as cathepsin B and / or cathepsin L).

[0217] In one embodiment, the compound of formula (III) comprises a self-excision spacer. In one embodiment, the self-excision spacer is an acetal or heteroacetal. In one embodiment, the -GGFG-NH-CH2-O- portion contained in the compound of formula (III) represents a combination of a restriction enzyme site and a self-excision spacer, which will be cut in the cell and release the target molecule (such as an anti-tumor compound).

[0218] Pharmaceutical compositions and pharmaceutical products

[0219] Another object of the present disclosure is to provide a pharmaceutical composition comprising the conjugate of the present disclosure and at least one pharmaceutically acceptable carrier.

[0220] The pharmaceutical composition of the present disclosure can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or curing human or animal symptoms. For example, various suitable dosage forms, especially injections, such as lyophilized powder for injection, injection or sterile powder for injection can be prepared according to the route of administration.

[0221] The term "pharmaceutically acceptable" means that it does not produce excessive toxicity, irritation or allergic response when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages and disadvantages, and is effective for the intended use.

[0222] The term pharmaceutically acceptable carrier refers to those carrier materials that are pharmaceutically acceptable and do not interfere with the biological activity and characteristics of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline, etc. Pharmaceutically acceptable carriers also include carrier materials that make the composition close to physiological conditions, such as pH regulators, buffers, toxicity regulators, etc., as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with the therapeutic active ingredient. Such drug carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil and sesame oil. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline and solutions of glucose in water or glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol (propylene, glycol), water, ethanol, etc. If necessary, the composition may also include a small amount of wetting agent, emulsifier or pH buffer, such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tension regulators such as sodium chloride or dextrose are also considered. Such compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated as a suppository using conventional adhesives and carriers such as triglycerides. Oral preparations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective amount of the antibody, preferably in purified form, and an appropriate amount of carrier to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0223] In one embodiment, the pharmaceutical composition of the present disclosure has an integer or non-integer drug to antibody ratio (DAR) of about 1 to about 20, such as about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4. In a specific embodiment, the conjugate of the present disclosure has a DAR of about 4.

[0224] Treatments and uses

[0225] The conjugates of the present disclosure can be used to treat FGFR3-mediated diseases.

[0226] Therefore, in another aspect, the present invention also provides the use of the conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease mediated by FGFR3, in particular a FGFR3-positive tumor, more particularly brain cancer, bladder cancer, urothelial carcinoma, cervical cancer or intrahepatic bile duct carcinoma.

[0227] In some embodiments, the disease comprises a tumor that overexpresses FGFR3 or a tumor that has a FGFR3 gene mutation. In some embodiments, the disease is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, nipple cancer, In some embodiments, the disease is selected from brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, or intrahepatic bile duct cancer. In some embodiments, the disease is glioblastoma. In a preferred embodiment, the conjugate of the present invention formed by conjugating an anti-FGFR3 antibody and a small molecule cytotoxin can specifically bind to FGFR3 on the surface of tumor cells and selectively kill tumor cells expressing FGFR3.

[0228] The dosage of the conjugate administered to a subject can be adjusted to a considerable extent. The dosage can be varied according to the specific route of administration and the needs of the subject, and can be determined by a health care professional.

[0229] Beneficial Effects

[0230] The novel small molecule topoisomerase I inhibitors provided by the present disclosure, whether used alone or as a component of ADC, can show higher activity, stability and physicochemical properties than the prior art.

[0231] The antibody-drug conjugate of the present invention uses a specially designed linker-load to achieve good efficacy and bystander killing effects. At the same time, it has a lower DAR, so it can reduce side effects and improve the therapeutic index, which is particularly important for bystander killing. The antibody-drug conjugate of the present invention is more stable in its structure (such as an open-ring succinimide structure).

[0232] The present disclosure utilizes a linker with a unique structure and uses a ligase to catalyze the conjugation of a targeting molecule and a load. The conjugate disclosed in the present disclosure has good uniformity and high activity. In addition, the toxicity of the linker-load intermediate is much lower than that of the free load, so the manufacturing process of the drug is less harmful, which is conducive to industrial production.

[0233] The conjugates disclosed herein achieve at least one of the following technical effects:

[0234] (1) High inhibitory activity on target cells, or strong killing effect on target cells.

[0235] (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability, where chemical stability includes low cytotoxin shedding rate from the ADC, resulting in low off-target toxicity).

[0236] (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action).

[0237] (4) High specificity and good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects and a wide therapeutic window), etc.

[0238] (5) Highly modular design, simple assembly of multiple drugs.

[0239] Example

[0240] In order to more clearly illustrate the purpose and technical solutions, the present disclosure is further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present disclosure. Specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0241] Unless otherwise specified, the instruments and reagents used in the examples are commercially available and the reagents were used directly without further purification.

[0242] MS: Thermo Fisher Q Exactive Plus, Waters 2795-Quattro micro triple quadrupole mass spectrometer

[0243] HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0244] Semi-preparative HPLC: Lisure HP plus 50D

[0245] Flow cytometry: CytoFLEX S

[0246] HIC-HPLC: Butyl-HIC; Mobile phase A: 25mM PB, 2M (NH4)2SO4, pH 7.0; Mobile phase B: 25mM PB, pH 7.0; Flow rate: 0.8ml / min; Acquisition time: 25min; Injection volume: 20μg; Column temperature: 25℃; Detection wavelength: 280nm; Sample chamber temperature: 8℃.

[0247] SEC-HPLC: column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID×300mm, 5μm; mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2; flow rate: 0.5ml / min; acquisition time: 30min; injection volume: 50μl; column temperature: 25℃; detection wavelength: 280nm; sample plate temperature: 8℃.

[0248] In some cases, the order in which the above reaction schemes are carried out can be changed to promote the reaction or to avoid unwanted reaction products. The following examples are provided to more fully understand the present invention. These examples are illustrative only and should not be construed as limiting the present invention in any way.

[0249] Example 1 Preparation of Linker-Loader 1

[0250]

[0251] opSu is or a mixture thereof;

[0252] Preparation of intermediate MC-GGFG-DXd

[0253] The intermediate MC-GGFG-DXd is commercially available or can be prepared according to the method described in EP2907824. This compound is used to prepare linker-load 1.

[0254] Preparation of linker-load intermediate 1

[0255]

[0256] Linker-load intermediate 1 can be synthesized by conventional solid phase peptide synthesis using Rink-amide-MBHA-resin. The amino acids in the linker unit are protected using Fmoc. The coupling reagent is selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the product is cleaved from the resin using TFA / TIS / H2O solution. The product is purified by preparative HPLC, lyophilized and stored for later use. LCMS m / z: [MH] - =1382.6.

[0257] Preparation of linker-carrier 1

[0258] The linker-load intermediate 1 and MC-GGFG-DXd (molar ratio ~1:2) were weighed, dissolved in water and DMF, respectively, and then mixed thoroughly to obtain a mixture, and the mixture was reacted at 0-40°C for 0.5-30 hours. Once the reaction was completed, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued at 0-40°C for another 0.2-20 hours. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the linker-load 1. LCMS m / z: [(M+3H) / 3] + =1163.3.

[0259] Example 2 Preparation of Linker-Loader 2

[0260] Preparation of intermediate 11

[0261]

[0262] Step A: N-(2-bromo-5-fluorophenyl)acetamide: To a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL) was added concentrated H2SO4 (3 mL) at room temperature, followed by addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) in portions. The mixture was stirred for 3 hours and then poured into 2000 mL of ice-water. A precipitate was formed, which was collected by filtration and dried under vacuum at room temperature to give N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ 7.68 (dd, J = 8.9, 6.0 Hz, 1H), 7.61 (ddd, J = 10.7, 5.3, 3.1 Hz, 1H), 7.02 (ddd, J = 8.9, 8.0, 3.1 Hz, 1H), 2.11 (s, 3H). LCMS m / z 232.0(M+H).

[0263] Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) was added dropwise n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) at -78°C over 1 hour. After completion, the mixture was stirred under N2 for 0.5 hour. A solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was then added dropwise at -78°C over 0.5 hour and the mixture was stirred at -78°C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated NH4Cl aqueous solution at 0°C. Extracted with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2SO4 and concentrated. The mixture was triturated with (PE / EA=1:1, 100 mL) for 10 minutes, filtered, and the filter cake was collected and dried in vacuo to give N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. LCMS m / z 206.1 (M-18+H), 246.1 (M+Na).

[0264] Step C: N-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL) was added silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) and the mixture was stirred at 30°C for 6 hours. The mixture was filtered on Celite and washed with CH2Cl2 (100 mL), the filtrate was concentrated and purified by FCC (EA / PE=0-40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g) as a light yellow solid. LCMS m / z 222.1 (M+H).

[0265] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirring mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) at 0°C was added 1-butyl nitrite (8.48 g, 63.28 mmol) followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 hours. After completion, the mixture was acidified with HCl (2N) to adjust to pH = 3. The mixture was extracted with ethyl acetate (200 mL x 3), washed with brine (100 mL x 2), dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 minutes, filtered, and the filter cake was collected and dried in vacuo to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. LCMS m / z 251.1 (M+H).

[0266] Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12 g, 47.96 mmol) in acetic anhydride (90 mL) and THF (90 mL) was added 10% Pd / C (1 g) and the mixture was stirred at 25 °C in H2 atmosphere for 16 hours. After cooling to 0 °C, Et3N (20 mL) was added dropwise and the mixture was stirred at 0 °C for 1 hour. Filtered on celite, the filtrate was poured into ice-water (500 mL). Extracted with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2SO4 and concentrated. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 minutes, filtered, and the filter cake was collected and dried in vacuo to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g) as a yellow solid. LCMS m / z 279.1 (M+H).

[0267] Step F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g, 28.39 mmol) in MeOH (150 mL) was added aqueous HCl (aq) (2N, 150 mL) and the mixture was stirred at 50°C for 7 hours. After cooling to 0°C, saturated aqueous NaHCO3 was added dropwise to adjust to pH = 8. Extracted with ethyl acetate (200 mL x 3), washed with brine (200 mL x 2), dried over Na2SO4 and concentrated under reduced pressure to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (6.0 g) as a yellow solid. 1 H NMR (400MHz, chloroform-d) δ6.57(s,3H),6.18(td,J=11.1,2.4Hz,2H),4.52(dt,J=13.3,5.0Hz,1H),3.13(ddd,J=17.5,1 3.0,4.6Hz,1H),3.00–2.81(m,1H),2.69(dtd,J=9.4,4.6,2.5Hz,1H),2.09(s,3H),1.79(qd,J=13.0,4.3Hz,1H). LCMS m / z 237.1(M+H).

[0268] Step G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide: NCS (2.26 g, 16.93 mmol) was added to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diethylamide (4.0 g, 16.93 mmol) in DMF (80 mL) at 0 °C and the mixture was stirred at room temperature for 16 hours. The mixture was poured into 200 mL of ice-water. A precipitate was formed, which was collected by filtration and dried under vacuum at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (4.0 g) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ8.11(d,J=8.0Hz,1H),7.71(s,2H),6.62(d,J=11.9Hz,1H),4.53(ddd,J=13.0,8.0 ,4.7Hz,1H),3.18–3.04(m,1H),2.91(ddd,J=17.5,12.4,4.8Hz,1H),2.21–2.08(m,1H),1.99–1.83(m,4H). LCMS m / z 271.0(M+H).

[0269] Step H: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: To N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4. To a mixture of 2-nitro-4-nitro-2-thiazolyl-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol) and o-cresol (10 mL) were added, and the mixture was heated at reflux under N2 for 24 h. The solvent was removed under reduced pressure, and the mixture was purified by FCC (THF / CH2Cl2=0-60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4.1 g) as a brown solid. LCMS m / z 498.1 (M+H).

[0270] Step I: (9S)-1-Amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione: A mixture of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of concentrated aqueous HCl was stirred at 70 °C under N2 for 36 hours. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. LCMS (ESI) m / z 456.1 (M+H).

[0271] Preparation of Intermediate 12 (12-1, 12-2)

[0272]

[0273] 12-1 and 12-2 were prepared by preparative HPLC from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (intermediate 11) as a TFA salt.

[0274] Table 1

[0275]

[0276] The conditions of the above HPLC: equipment Agilent 1200; chromatographic column: Waters XBridge C18 4.6*50mm, 3.5um; flow rate: 2.0mL / min; gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00min-1.50min-2.50min-2.52min-3.00min; temperature: 40°C; phase: A: acetonitrile, B: H2O (0.05% TFA); wavelength: 214nm / 254nm.

[0277] Preparation of linker-carrier 2

[0278]

[0279] Synthesis of 13 (Step A)

[0280] Weigh 4.33g Fmoc-Gly-Gly-OH and 6.84g Pb(OAc)4 and add to a 500ml single-necked round-bottom flask. Under a nitrogen atmosphere, add anhydrous THF / toluene (120 / 40ml) and stir to dissolve. Then add 1.16mL pyridine to the reaction system. Heat the reaction system to 80°C and reflux for 5 hours under a nitrogen atmosphere. Take a sample and monitor the reaction by HPLC.

[0281] The reaction system was cooled to room temperature, filtered, and the filter cake was washed with EA three times. The filtrates were combined and concentrated to dryness. Column chromatography (PE:EA=100:0-50:100) was performed to obtain about 2000 mg of the target product as a white solid with a yield of 44%.

[0282] Synthesis of 15 (Step B)

[0283] 200 mg of 13 was weighed and added to a 100 ml single-necked round-bottom flask. Then 15 ml of THF was added and stirred to dissolve. Then 14 (312 mg, 3.0 eq) and TsOH·H2O (15 mg, 0.15 eq) were added to the reaction system. The reaction system was allowed to react overnight at room temperature. Samples were taken and detected by TLC (PE / EA=1:1) to monitor the reaction. The raw material was basically disappeared, and new spots were detected.

[0284] Saturated sodium bicarbonate solution was added to quench the reaction. EA was used for 3 extractions. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (PE:EA=5:1-1:1) to obtain about 80 mg of the target product as a colorless oil in a yield of 29%. LCMS m / z: [M+H] + =501.1

[0285] Synthesis of 16 (Step C)

[0286] 200 mg of 15 was weighed and added to a 100 ml single-necked round-bottom flask. 10 ml of EtOH and 5 ml of EA were then added to completely dissolve. Then, under a nitrogen atmosphere, 40 mg of palladium carbon was added to the reaction system, and the reaction system was ventilated with hydrogen three times. The reaction system was kept under a hydrogen atmosphere and stirred at room temperature for 0.5 hours. Samples were taken and detected by TLC (DCM / MeOH=10:1) to monitor the reaction. The raw material was substantially disappeared, and new spots were detected.

[0287] The reaction system was filtered and the filter cake was washed 3 times with EA. The filtrates were combined and concentrated to dryness to obtain 200 mg of the product as a white solid with a yield of 100%. The product can be used directly in the next reaction without purification. LCMS m / z: [MH] - =409.4.

[0288] Synthesis of 21 (Step D)

[0289] Step D-1

[0290] Weigh 2.0g of dichlororesin and put it into a peptide synthesis tube. Add DCM (10ml) and allow it to swell at room temperature for 30 minutes. Remove the solvent by vacuum suction. Wash the resin twice with DCM, each wash volume is 7mL, and the time length is 1 minute. Remove the solvent by vacuum suction. Then weigh 16 (200mg) and add it to a 50ml centrifuge tube. Add DCM (about 10ml). Dissolve the solid by shaking. Add to the above resin. Stir to soak all the resin in the solution (if there is resin attached to the tube wall, use a small amount of DCM to rinse the tube wall). Stir for 4-5 hours. After the reaction is complete, add an appropriate amount of methanol. Stir for 30 minutes. Remove the solvent by vacuum suction. Wash the resin with DMF once, methanol once, DMF once, methanol once, DMF twice, each wash volume is 10mL, and the time length is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin detection. The resin was colorless and transparent, and the solution was light yellow, indicating that the next coupling step was possible.

[0291] Step D-2

[0292] Deprotection was performed twice, each time 10 mL of a ready-made 20% piperidine / DMF solution was added and reacted for 10 minutes. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash volume was 10 mL, and the time length was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0293] Add 563 mg of Fmoc-Phe-OH and 197 mg of HOBt to a 50 mL centrifuge tube. Then add about 7 mL of DMF. Dissolve the solid by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to obtain an activated reaction solution.

[0294] 3 molar equivalents of activated reaction solution are added to the resin. Stir so that the resin is fully immersed in the solution (if there is resin attachment on the tube wall, a small amount of DCM is used to rinse the tube wall). Stir 2-3 hours. After the reaction is completed, remove the solvent by vacuum suction. The resin is washed twice with DMF, methanol wash once, DMF wash once, methanol wash once, DMF wash twice successively, and each washing volume is 10mL, and the time length is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin and carry out ninhydrin detection. The resin is colorless and transparent, and the solution is light yellow, which shows that the next step coupling can be carried out.

[0295] Step D-3

[0296] Deprotection was performed twice, each time 10 mL of a ready-made 20% piperidine / DMF solution was added and reacted for 10 minutes. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash volume was 10 mL, and the time length was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0297] Add 531 mg of Fmoc-GG-OH and 197 mg of HOBt to a 50 mL centrifuge tube. Then add about 10 mL of DMF. Dissolve the solid by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to obtain an activated reaction solution.

[0298] 3 molar equivalents of activated reaction solution are added to resin.Stir to immerse resin in solution completely (if there is resin attachment on the tube wall, a small amount of DCM is used to rinse the tube wall).Stir 2-3 hours.After the reaction is completed, remove the reaction solution by vacuum suction.Resin is washed twice with DMF, methanol wash once, DMF wash once, methanol wash once, DMF wash twice successively, each washing volume is 10mL, and the time length is 1 minute.Remove solvent by vacuum suction.Take a small amount of dry resin and carry out ninhydrin detection.Resin is colorless and transparent, and solution is light yellow, shows that next step coupling can be carried out.

[0299] Step D-4

[0300] Deprotection was performed twice, and 10 mL of ready-made 20% piperidine / DMF solution was added each time and reacted for 10 minutes. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each washing volume was 10 mL, and the time length was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue. Then, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube and about 10 mL of DMF was added. The solid was dissolved by vibration. Then 0.24 mL of DIEA was added to the resin. Stirring was performed to completely immerse the resin in the solution (if there was resin attached to the tube wall, a small amount of DCM was used to rinse the tube wall). Stirring was performed for 2-3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. Resin is washed twice with DMF, washed once with methanol, washed once with DMF, washed once with methanol, washed twice with DMF, each washing volume is 10mL, and the time length is 1 minute. Remove solvent by vacuum suction. Take a small amount of dry resin and carry out ninhydrin detection. Resin is colorless and transparent, and the solution is light yellow, indicating that the next step coupling can be carried out.

[0301] Step D-5

[0302] The resin was washed twice with 10 mL of methanol. The solvent was then completely removed by vacuum suction. The resin was poured out and weighed. A lysis buffer was prepared in a 250 mL conical flask, wherein: the ratio of TFE / DCM was 80% / 20%, and the volume was 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and shaken thoroughly. The resin was completely soaked in the lysis buffer and lysed at room temperature for 2-3 hours. The lysis buffer was then filtered using a simple filter made of a syringe, and the resin was washed with 1-2 ml of DCM and discarded. Then 150 mL of pre-cooled anhydrous ether was added to the lysis buffer, shaken thoroughly, and then left to stand for 20-30 minutes. Using a 50 mL centrifuge tube, the above system was centrifuged in a centrifuge at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasound, centrifuged at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was placed in a centrifuge tube, air dried overnight, and then subjected to preparative purification to afford 125 mg of the product as a white solid in a 40% yield. LCMS m / z: [MH] - =641.5.

[0303] Synthesis of 22 (Step E)

[0304] Weigh 150 mg of raw material 21 and 55 mg of TSTU and add to 10 mL single-necked round-bottom flask, add anhydrous DMF (3 mL) and stir for 20 minutes under nitrogen atmosphere. Then 18 mg 12-1 and 20 μ l DIEA are added to the reaction system successively. Under nitrogen atmosphere, stir at room temperature for 2-8 hours. Take a sample and detect by HPLC to monitor the reaction. The raw material peak disappears completely, and a new peak is detected.

[0305] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 22 mg of the product in the form of a light yellow solid. LCMS m / z: [M+H] + =1081.0.

[0306] Synthesis of Linker-Loader 2 (Step F)

[0307] Weigh 22 (30 mg) and add to a 10 ml single-necked round-bottom flask, add purified water (2 ml). Stir to dissolve. A DMF solution (2 ml) containing the linker-load intermediate 1 (19.5 mg) is added to the reaction system and stirred. After reacting overnight, the reaction is monitored using HPLC until all raw materials are converted into intermediates. An appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction is directly added to the reaction mixture, and reacted for another 0.2-20 hours at 0-40 ° C. The reaction is monitored by HPLC until all intermediates are consumed and then quenched by acetic acid solution.

[0308] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 25 mg of the linker-load 2 as a light yellow solid. LCMS m / z: [(M+3H) / 3] + =1194.4

[0309] Example 3 Construction of Antibodies and ADCs

[0310] 1. Preparation of anti-FGFR3 antibodies

[0311] In each mammalian expression system, the anti-FGFR3 antibody consists of two vectors, one for the heavy chain and one for the light chain. The anti-FGFR3 antibody was prepared by co-transfecting the above vectors using the Expi293 transient mammalian expression system (Gibco, A14635, Carlsbad, CA, USA). The culture supernatant was purified using a HiTrap Mabselect SuRe (GE Healthcare Life Sciences, 11-0034-93, Uppsala, Sweden) protein purification system (GE Healthcare Life Sciences, Uppsala, Sweden). Ultra centrifugal filters (Merck Millipore, MA, USA) were used for concentration. Figure 1 ) and SEC-HPLC were used to analyze the characteristics of the high-purity antibody, and the results showed that the purity of the obtained antibody exceeded 98.5%.

[0312] The anti-FGFR3 antibodies thus obtained are shown in the following table. The CDRs are highlighted with underlines and the constant regions are shown in italics.

[0313] Table 2

[0314]

[0315] Note: The upstream peptide bond of GG in the LPETGG sequence is cleaved by sortase A, and the resulting intermediate is ligated to the free N-terminus of G3 to generate a new peptide bond.

[0316] 2. SPR analysis

[0317] The binding affinity (KD value) of anti-FGFR3 antibodies was measured using Biacore 3000. Human (FGFR3-IIIb and -IIIc, R&D systems, 1264-FR-050), mouse (R&D systems, 710-MF-050) and cynomolgus monkey FGFR3 protein (Sino Biological, 90313-C02H) were coated with an amine coupling kit (GE Healthcare Life Sciences, BR100050, Uppsala, Sweden). KD (Ka and Kd) values ​​were evaluated according to concentration, as shown in the table below.

[0318] Table 3

[0319]

[0320] NB indicates that the antibody does not bind to FGFR3.

[0321] 3. ADC Preparation

[0322] The linker-load intermediate is conjugated to the antibody in a site-specific manner by a ligase to form an ADC. The conjugation reaction method can be found in WO2015165413A1. The resulting ADC is shown in the following table.

[0323] Table 4

[0324] Name of ADC Linker-Payload Antibody ADC19 Linker-Load 1 A9 ADC20 Linker-Patent 2 A9

[0325] 4. Characterization of ADC

[0326] 1)SEC-HPLC

[0327] Chromatographic column: TSKgel G3000SWXL 7.8mm ID*30cm, 5μm; mobile phase: 2*PBS:methanol=9:1 (V / V); gradient: 100% isocratic, flow rate: 1.0ml / min; running time is 15 minutes, 280nm is selected as the detection wavelength to analyze and detect the purity of ADC drugs.

[0328] 2) HIC-HPLC

[0329] Chromatographic column: Proteomix HIC Butyl-NP5 4.6*100mm, 5μm Non-Porous; Mobile phase: 1.5M ammonium sulfate + 20mM phosphate buffered saline (pH 7.0) as mobile phase A, 20mM phosphate buffered saline (pH 7.0): isopropanol = 7:3 (V / V) as mobile phase B; Flow rate: 0.8ml / min; Phase B increased from 15% to 100% in 8 minutes; 280nm was selected as the detection wavelength to detect the DAR distribution and calculate the average DAR value of the ADC drug.

[0330] The results are shown in Table 5.

[0331] Table 5

[0332]

[0333] Table 5 shows that the residual free drug of both samples was less than 50 ppm, indicating that the shedding of the cargo (cytotoxin) was very low.

[0334] Example 4 Affinity ELISA Analysis

[0335] 1) 1 μg / mL human FGFR3 (R&D systems, 1264-FR-050), cynomolgus monkey FGFR3 (SinoBiological, 90313-C02H) protein or mouse FGFR3 (R&D systems, 710-MF-050) were coated on 96-well plates at 4°C overnight. The plates were blocked in 3% skim milk containing anti-FGFR3 antibody and incubated at room temperature for 1 h. After washing with PBST (0.1%), anti-human Fab antibody conjugated with horseradish peroxidase (HRP) (ThermoScientific, 31482, Waltham, MA, USA) was added at a ratio of 1:3000. After washing, the plate was treated with TMB solution (Thermo Scientific, N301, Waltham, MA, USA) as HRP substrate, and the reaction was stopped with STOP solution (Cell Signaling Technology, #7002, Danvers, MA, USA).

[0336] The absorbance of each well was measured at a wavelength of 450 nm.

[0337] The analysis results are as follows Figure 2-3 shown. Figure 2 Antibody A9 was shown to be highly specific for FGFR3 in both humans and cynomolgus monkeys. Figure 3 A9 and A9Q were shown to have similar affinities for human FGFR3.

[0338] 2) ADC affinity

[0339] The affinity of ADC binding to human FGFR3 (Sinobiological, 16044-H08H) was tested by ELISA (similar to the method described above).

[0340] Figure 4 The formation of ADCs was shown to have little effect on the potency of the antibodies (EC50s for antibody A9 and ADC ADC19 were 0.04772 nM and 0.04272 nM, respectively).

[0341] Example 5 Cell Binding Analysis of Antibodies

[0342] 1) Analyze the cell surface binding efficiency of anti-FGFR3 antibodies using flow cytometry. 5 Up to 5.0×10 5FGFR3 overexpressing or FGFR3 non-expressing cells were incubated with anti-FGFR3 antibodies for 1 hour at 4°C. After washing twice with flow cytometry staining buffer, the cells were stained with Alexa Fluor488-conjugated goat anti-human IgG cross-adsorbed secondary antibody (Invitrogen, A-11013, Carlsbad, CA, USA) diluted 1:200 in staining buffer at 4°C for 30 minutes. The mean fluorescence intensity was analyzed by flow cytometry.

[0343] The results of this analysis are Figure 5 and 6.1 As shown, AMB-BT-0050T is a cell that is positive for FGFR3 expression, while AMB-BT-0013T is a cell that is negative for FGFR3 expression. AMB-BT-0050T and AMB-BT-0013T were sampled from patients with brain cancer (glioblastoma).

[0344] Figure 5 The results showed that the binding affinity of antibody A9 to FGFR3-positive cells was significantly higher than that to FGFR3-negative cells, indicating that the antibody is highly specific to FGFR3. Figure 6.1 Both A9 and A9Q were shown to bind to AMB-BT-0050T cells.

[0345] 2) Analyze the cell surface binding efficiency of ADC20 using flow cytometry. 5 Multiple myeloma KMS-11 cells (JCRB, JCRB1178) with overexpression of FGFR3 were incubated with a series of concentrations of ADC20 at 4 ° C for 1 hour. After washing twice with flow cytometry staining buffer, the cells were stained with Alexa Fluor 647-conjugated goat anti-human IgG cross-adsorbed secondary antibodies (Invitrogen, A-21445, Carlsbad, CA, USA) diluted 1:300 in staining buffer at 4 ° C for 30 minutes. Antibody A9Q and human IgG1κ isotype (CrownVivo, C0001) were used as controls. The mean fluorescence intensity was analyzed by flow cytometry.

[0346] The results of this analysis are Figure 6.2 As shown, it was shown that ADC20 and antibody A9Q had similar and significantly higher binding affinities for KMS-11.

[0347] Example 6. In vitro cytotoxicity assay

[0348] 1) 3D single sphere model is formed by separating cancer cells derived from glioblastoma patients. Two FGFR3 overexpressed patient-derived cells (AMB-BT-0050T, AMB-BT-0112T) and FGFR3 non-expressing source cells (AMB-BT-0013T) sampled from patients with glioblastoma are incubated overnight to form a single sphere (3D), followed by processing ADC and incubating for one week, and then quantifying the sphere size and volume. The results are shown in the table below, indicating that for conjugates ADC19 and ADC20, the cytotoxicity to FGFR3 positive cells is significantly higher than that to FGFR3 negative cells, so ADC is highly specific to FGFR3.

[0349] Table 6.1

[0350]

[0351] GBM is the abbreviation for glioblastoma, and PDCs is the abbreviation for patient-derived cells.

[0352] 2) A 3D single sphere model was formed by the bladder cell line RT112 (DSMZ, ACC418) overexpressing FGFR3. RT112 was inoculated in a cell sphere culture plate and incubated overnight to form a single sphere (3D), followed by treatment with ADC and incubation for one week, and then quantification of sphere size and volume. The results are shown in Table 6.2 below, indicating that the conjugates ADC19 and ADC20 have significant cytotoxicity to FGFR3-positive bladder cancer cells.

[0353] Table 6.2

[0354]

[0355] Example 7 In vivo efficacy testing in glioblastoma PDX models

[0356] 1) To evaluate survival in an orthotopic mouse model of brain tumors with target expression, AMB-BT-0050T patient-derived cells were subcultured and 2.0x10 5cells were mixed with culture medium. 7-week-old female BALB / c nude mice were used for intracranial transplantation. The prepared patient-derived cells were injected into the mouse brain by stereotactic intracranial injection at a depth of 3.2 mm, 1.7 mm to the left and 0.5 mm above the bregma. The mice were housed with a 12-hour light / 12-hour dark cycle and had free access to food and water. The treatment was administered as follows. TMZ (temozolomide) was administered orally daily for a total of 5 injections. Starting from the 7th day after model establishment, each group was administered ADC once by intravenous injection (single injection) or once a week for four weeks (multiple injections). When a 20% weight loss or neurological symptoms (lethargy, ataxia, and seizures) were observed, the mice were killed, and the results are as follows. Figure 7.1 The survival rate of mice was evaluated by MST (median survival time) and ILS (extended life span); MST, the time point at which the probability of survival is equal to 50%; ILS (extended life span), ILS (%) = [(median survival time of the treatment group) / (median survival time of the control group) - 1] x 100.

[0357] The results showed that anti-FGFR3 ADC could inhibit the progression of brain tumors and prolong survival.

[0358] 2) To further evaluate survival in an orthotopic mouse model of brain tumors with target expression, mice were grouped and treated with (1) vehicle, (2) TMZ (temozolomide), 20 mg / kg; (3) ADC20 20 mg / kg; (4) a combination of TMZ and ADC20 in the AMB-BT-0050T PDX model. TMZ was administered orally daily for a total of 3 injections. ADC was administered once by intravenous injection. The results are shown in Figure 7.2 The survival rate of mice was evaluated by MST and ILS.

[0359] The results showed that both monotherapy with ADC20 and the combination of ADC20 and TMZ could inhibit brain tumor progression and prolong survival. The combination of ADC and TMZ (standard of care for GBM) could significantly prolong survival.

[0360] Example 8 In vivo efficacy testing in the bladder cancer CDX model

[0361] To evaluate the in vivo antitumor efficacy of ADC19 and ADC20 in tumor-bearing mice of FGFR3-overexpressing bladder cancer CDX models, several FGFR3-overexpressing bladder cancer CDX models were used.

[0362] 8.1) Collect RT112 cells in the exponential growth phase and count them for tumor inoculation. Use 0.2 mL of matrix gel buffer (PBS:Matrigel=1:1) to mix 10x10 6The cells were injected subcutaneously into the right abdomen of 6-8 week old SPF female BALB / c nude mice.

[0363] The tumor diameter was measured with a caliper and the formula V = 0.5axb was used. 2 (where a is the long diameter of the tumor and b is the short diameter of the tumor) to calculate the tumor volume. When the average tumor volume is about 100-300mm 3 At 14:00, mice were randomly divided into vehicle group, ADC19 5 mg / kg group and ADC20 5 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given the solvent of the ADC drug at the same frequency and route of administration. The tumor volume of each group of mice was measured twice a week. The experiment ended on day 27, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%.

[0364] Figure 8.1 The changes in tumor volume of tumor-bearing BALB / c nude mice treated with the following are shown: (1) vehicle; (2) ADC19 5 mg / kg, once a week (QW), 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 7.1 shows that at the end day (day 27), the average tumor volume of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group was 48 mm 3 and 32mm 3 ; TGI were 106.35% and 107.08% respectively.

[0365] Table 7.1

[0366]

[0367] a. Mean ± SEM; measured on the final day;

[0368] b. TGI (%) = [1-(T 27 -T0) / (V 27 -V0)]×100%. T0 is the average tumor volume of the treatment group on the first day of administration, T 27 is the average tumor volume of the treatment group on the 27th day after administration; V0 is the average tumor volume of the vehicle group on the first day of administration, V 27 is the average tumor volume of the vehicle group on day 27 after administration.

[0369] The results showed that both ADC19 and ADC20 had good anti-tumor efficacy in the FGFR3-overexpressing RT112 bladder cancer CDX model.

[0370] 8.2) To evaluate the dose-dependent antitumor efficacy in bladder cancer, BALB / c nude mice carrying RT112 cells were randomly divided and treated with the following: solvent; ADC20 8 mg / kg single dose and ADC20 8 mg / kg, QW, 2 times. The experiment ended on day 33, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%. The ADC group continued to be observed until day 84.

[0371] Figure 8.2 The changes in tumor volume of tumor-bearing BALB / c nude mice treated with the following are shown: (1) vehicle; (2) ADC20 8 mg / kg, single dose; (3) ADC20 8 mg / kg, QW, 2 times. Table 7.2 shows that at the end day (day 33), the average tumor volume of the ADC20 single dose group and the ADC20 repeated dose group was 36 mm 3 and 16mm 3 ; TGI were 106.34% and 107.65%, respectively. And complete response (CR) was produced in the repeated dose group.

[0372] Table 7.2

[0373]

[0374] The results showed that in the FGFR3-overexpressing RT112 bladder cancer CDX model, single or repeated doses of ADC20 had good anti-tumor efficacy, and repeated doses of ADC20 produced CR in this bladder cancer CDX model.

[0375] 8.3) Collect SW780 cells in the exponential growth phase and count them for tumor inoculation. Use 0.2 mL of matrix gel buffer (PBS:Matrigel=1:1) to inoculate 10x10 6 Cells were injected subcutaneously into the right flank of 6-8 week old SPF female NOD SCID mice.

[0376] The tumor diameter was measured with a caliper and the formula V = 0.5axb 2 Calculate tumor volume. When the average tumor volume is about 100-300mm 3At 14:00, mice were randomly divided into vehicle group, ADC19 5 mg / kg group and ADC20 5 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given the solvent of the ADC drug at the same frequency and route of administration. The tumor volume of each group of mice was measured twice a week. The experiment ended on day 20, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%.

[0377] Figure 8.3 The changes in tumor volume of tumor-bearing NOD SCID mice treated with the following are shown: (1) vehicle; (2) ADC19 5 mg / kg, QW, 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 7.3 shows that at the end day (day 20), the average tumor volume of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group was 1,109 mm 3 and 526mm 3 ; TGI were 40.80% and 76.66% respectively.

[0378] Table 7.3

[0379]

[0380] The results showed that both ADC19 and ADC20 had potential anti-tumor efficacy in the FGFR3-overexpressing SW780 bladder cancer CDX model.

[0381] 8.4) Collect UM-UC1 cells in the exponential growth phase and count them for tumor inoculation. Use 0.1 mL of matrix gel buffer (PBS:Matrigel=1:1) to mix 10x10 5 The cells were injected into the right flank of 6-8 week old SPF female Balb / c nude mice.

[0382] The tumor diameter was measured with a caliper and the formula V = 0.5axb was used. 2 Calculate tumor volume. When the average tumor volume is about 100-300mm 3At 14:00, mice were randomly divided into a vehicle group and an ADC20 8 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given the solvent of the ADC drug at the same frequency and route of administration. The tumor volume of each group of mice was measured twice a week. The experiment ended on day 17, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%.

[0383] Figure 8.4 The changes in tumor volume of tumor-bearing Balb / c nude mice treated with the following: (1) vehicle; (2) ADC20 8 mg / kg, QW, 2 times. Table 7.4 shows that at the end day (day 17), the average tumor volume of the ADC20 8 mg / kg group was 402 mm 3 ; TGI is 82.71%.

[0384] Table 7.4

[0385]

[0386] The results showed that ADC20 had antitumor efficacy in the FGFR3-overexpressing UM-UC1 bladder cancer CDX model.

[0387] Example 9 In vivo efficacy testing in multiple myeloma CDX model

[0388] To evaluate the in vivo antitumor efficacy of ADC19 and ADC20 in FGFR3-overexpressing multiple myeloma CDX model-bearing mice, KMS11 cells in the exponential growth phase were collected and counted for tumor inoculation. 10x10 6 Cells were injected subcutaneously into the right flank of 6-8 week old SPF female CB17.SCID mice.

[0389] The tumor diameter was measured with a caliper and the formula V = 0.5axb 2 Calculate tumor volume. When the average tumor volume is about 100-300mm 3At 14:00, mice were randomly divided into vehicle group, ADC19 5 mg / kg group and ADC20 5 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given the solvent of the ADC drug at the same frequency and route of administration. The tumor volume of each group of mice was measured twice a week. The experiment ended on day 32, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%.

[0390] Fig. 9 The changes in tumor volume of CB17.SCID mice bearing tumors treated with the following are shown: (1) vehicle; (2) ADC19 5 mg / kg, QW, 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 8 shows that at the end day (day 32), the average tumor volume of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group was 9 mm 3 and 11mm 3 ; TGI were 108.41% and 108.31% respectively.

[0391] Table 8

[0392]

[0393] The results showed that both ADC19 and ADC20 had good anti-tumor efficacy in the KMS11 multiple myeloma CDX model with FGFR3 overexpression.

[0394] Example 10 Internalization activity of ADC

[0395] Multiple myeloma cells KMS-11 (JCRB, JCRB1178) with good viability were treated with cell digestion solution (accutase), collected, and suspended in serum-free RPMI medium. 5 cells were diluted 1000-fold with LIVE / DEAD TMFixable near-infrared dead cell staining dye was stained in the dark at room temperature for 30 minutes. Wash twice with cold FACS buffer to remove excess dye, and incubate the cells with a test drug solution at a final concentration of 1 nM on ice for 30 minutes. After surface binding, the antibody-cell mixture was washed with cold FACS buffer and serum-free RPMI medium to remove excess antibody. The antibody-bound cells were then incubated in a 37°C CO2 incubator for 0 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours for antibody internalization. After incubation, the antibody-bound cells were washed with flow cytometry staining buffer and fluorescently labeled with ice-cold goat anti-human IgG-IgG cross-adsorbed secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A-11013, Carlsbad, CA, USA) diluted 1:300 in staining buffer at 4°C for 30 minutes. After fluorescent labeling, the antibody-cell mixture was washed twice more and analyzed by flow cytometry.

[0396] The MFI data were normalized to the MFI at 0 min, which was defined as 100%. The results were analyzed using GraphPad Prism 9. Fig.10 As shown, ADC20 exhibited internalization activity comparable to that of A9Q.

[0397] Example 11 Bystander Killing Effect of ADC20 in RT112 / U87MG

[0398] Far-Red-labeled FGFR3-positive RT112 cells and CFSE-labeled FGFR3-negative U87MG cells were seeded in 96-well round-bottom plates, with 1.0 × 10 cells of each cell type. 4 Cells. The cells were incubated overnight to allow them to stably adhere to the plate, then treated with ADC or PBS and incubated for one week. After incubation, the cells were centrifuged at 2300rpm for 2min, and the supernatant was then discarded. The cells were washed once with PBS, detached and resuspended in a flow cytometry staining buffer containing LIVE / DEAD staining dye. Finally, the total amount of FGFR3 positive and FGFR3 negative cells and their viability were detected and analyzed by flow cytometer (Novocyte, Agilent).

[0399] like Fig.11 As shown, both ADC19 and ADC20 have a bystander killing effect, and ADC20 has a more effective bystander killing effect than ADC19.

[0400] Example 12 In vivo efficacy testing in glioblastoma PDX model

[0401] To evaluate the in vivo antitumor efficacy of ADC20 in FGFR3-overexpressing glioblastoma PDX models bearing mice, several FGFR3-overexpressing glioblastoma PDX models were used.

[0402] 1) AMB-BT-0039T cells (GBM, patient-derived cells) with FGFR3-TACC3 fusion in the exponential growth phase were collected and counted for tumor inoculation. 1x10 6 The cells were injected into the right abdomen of 6-8 week old SPF female BALB / c nude mice. The tumor diameter was measured with a caliper and the formula V = 0.5 axb 2 (where a is the long diameter of the tumor and b is the short diameter of the tumor) to calculate the tumor volume. When the average tumor volume is about 100-300mm 3 At 14:00, mice were randomly divided into a vehicle group and an ADC20 8 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given a solvent of the ADC drug at the same frequency and route of administration. The tumor volume of each group of mice was measured twice a week. The experiment ended on the 24th day, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1-(average tumor volume of the treatment group on the end day-average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the end day-average tumor volume of the vehicle group on the first day)] × 100%.

[0403] Figure 12.1 The changes in tumor volume of tumor-bearing BALB / c nude mice treated with the following: (1) vehicle; (2) ADC20 8 mg / kg, QW, 2 times. Table 9 shows that at the end day (day 24), the average tumor volume of the ADC20 8 mg / kg group was 100 mm 3 , TGI is 100.03%.

[0404] Table 9

[0405]

[0406] a. Mean ± SEM; measured on the final day;

[0407] b. TGI (%) = [1-(T 24 -T0) / (V 24 -V0)]×100%. T0 is the average tumor volume of the treatment group on the first day of administration, T 24 is the average tumor volume of the treatment group on the 24th day after administration; V0 is the average tumor volume of the vehicle group on the first day of administration, V 24 is the average tumor volume of the vehicle group on day 24 after administration.

[0408] 2) AMB-BT-0112T cells (GBM, patient-derived cells) with FGFR3-TACC3 fusion in the exponential growth phase were collected and counted for tumor inoculation. 1x10 6 The cells were injected into the right flank of 6-8 week old SPF female BALB / c nude mice.

[0409] The tumor diameter was measured with a caliper and the formula V = 0.5axb was used. 2 (where a is the long diameter of the tumor and b is the short diameter of the tumor) to calculate the tumor volume. When the average tumor volume is about 100-300mm 3 At 14:00, mice were randomly divided into a vehicle group and an ADC20 8 mg / kg group. The date of the first administration was defined as day 0. The vehicle group mice were given the solvent of the ADC drug at the same frequency and administration route. The tumor volume of each group of mice was measured twice a week.

[0410] Figure 12.2 The changes in tumor volume of tumor-bearing BALB / c nude mice treated with the following: (1) vehicle; (2) ADC20 8 mg / kg, QW, 2 times. Table 10 shows that at the end day (day 33), the average tumor volume of the ADC20 8 mg / kg group was 216 mm 3 , TGI is 95.08%.

[0411] Table 10

[0412]

[0413] Acknowledgements

[0414] This application was supported in part by the Korea Drug Development Fund funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (HN21C0803, Republic of Korea).

Claims

1. An antibody drug conjugate (ADC) having a structure of formula (I): in, A is an anti-FGFR3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is modified to bind to (Gly) in the compound of formula (I) n Partial connection, wherein the antibody or antigen-binding fragment comprises CDRs: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or having 1-3 conservative amino acid substitutions compared to SEQ ID NO: 6; z is an integer from 1 to 20; preferably from 1 to 4; especially 2; opSu is or a mixture thereof; R 0 It is C 1-10 alkyl; n is any integer from 2 to 20; k1 and k2 are independently integers from 1 to 7; i is an integer from 1 to 100; j is an integer from 1 to 100; P1 and P2 are independently loads.

2. The antibody drug conjugate of claim 1, wherein the linking process between the modified antibody or antigen-binding fragment and the compound of formula (I) is catalyzed by a ligase.

3. The antibody drug conjugate of claim 1 or 2, wherein the cargo is a cytotoxin or a fragment thereof, with optional derivatization to connect the cargo and the linker; The cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, compretin A-4 phosphate, compretin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vincristine, anhydrous vinblastine, dolastatin 10 and analogs, halichondrin B, eribulin, indole-3-oxoacetamide, podophyllotoxins, 7-diethylamino-3-(2' -benzoxazolyl)-coumarin (DBC), dermatophyll, lelicycin, camptothecins and their derivatives, mitoxantrone, mitoguanidine, nitrogen mustards, nitrosoureas, aziridines, benzodepa, carboquinone, metodepa, uredepa, danemycin, esperamicin, neocarcinostatisin, aclarubicin, actinomycin, anthramycin, bleomycin, actinomycin C, carrubicin, carminomycin, anticancer mycin, carminomycin, actinomycin D, daunorubicin, detox Star, doxorubicin, epirubicin, esorubicin, idarubicin, marseromycin, mitomycins, noramycin, olive mycin, peplomycin, porfibrinocin, puromycin, doxorubicin iron, rhodorubicin, streptozocin, streptozocin, netastatin, zorubicin, trichothecenes, T-2 toxin, verrucosporin A, bacilli, serpentine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethylfolate, methotrexate, pteropterin, trimethoprim-sulfamethoxazole azathioprine, edatrexate, fludarabine, 6-mercaptopurine, thiomipurine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, calutosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprolide acetate, protein kinase inhibitors and proteasome inhibitors; and / or Selected from vinca alkaloids, colchicine, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmycin, SN-38, scutellaria cyclopeptide analogs, derutec, duocarbamazine, calicheamicin, sentamycin, dolastatin, pyrrolobenzodiazepine Exatecan and its derivatives; and / or Selected from auristatins, in particular MMAE, MMAF or MMAD; and / or Selected from exatecan and its derivatives; such as DX8951f; and / or Selected from DXd-(1) and DXd-(2); preferably DXd-(1).

4. The antibody drug conjugate of claim 1 or 2, wherein the loading substance has a structure of formula (II): in, a is 0 or 1; The carbon atoms marked with p1* and p2* are asymmetric centers, and the asymmetric centers are in S configuration, R configuration or racemic form; L 1 Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2; M is -CH2-, -NH- or -O-; L 2 C 1-3 Alkylene; R 1 and R 2 are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 Alkoxy.

5. The antibody drug conjugate of claim 1 or 2, wherein the loading substance is selected from: and / or 6. The antibody drug conjugate of claim 1 or 2, which is selected from: and / or 7. The conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises: A VH domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and / or A VL domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

8.

8. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises: A heavy chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and / or A light chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

11.

9. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises: A heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13; and / or A light chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

14.

10. The antibody drug conjugate of any one of claims 1-6, wherein the ligase is a sortase.

11. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises a C-terminal modification of the heavy chain and / or a C-terminal modification of the light chain, the antibody, Sp and the recognition sequence of the ligase donor substrate are sequentially connected; Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS; the recognition sequence of the ligase donor substrate is LPXTGJ, wherein X can be any single amino acid, natural or non-natural; J is absent or is an amino acid fragment comprising 1-10 amino acids.

12. The antibody drug conjugate of any one of claims 1 to 6, wherein The modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 15, a light chain of SEQ ID NO: 16; and / or The modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 15, a light chain of SEQ ID NO: 14; and / or The modified antibody or antigen-binding fragment thereof comprises the heavy chain of SEQ ID NO:12 and the light chain of SEQ ID NO:

16.

13. The antibody drug conjugate according to any one of claims 1 to 6, wherein the drug to antibody ratio (DAR) of the antibody drug conjugate is an integer or non-integer of 1 to 20, in particular an integer or non-integer of 2 to 8.

14. The antibody drug conjugate of any one of claims 1-6, wherein the anti-FGFR3 antibody or antigen-binding fragment binds to human FGFR3 and monkey FGFR3 with a KD value of less than 10 nM.

15. A pharmaceutical composition comprising a preventively or therapeutically effective amount of the antibody drug conjugate of any one of claims 1 to 14, and at least one pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, comprising an alkylating agent, wherein the alkylating agent comprises temozolomide or a derivative thereof.

17. A pharmaceutical combination comprising the antibody drug conjugate according to any one of claims 1 to 14, and an alkylating agent.

18. The pharmaceutical combination of claim 17, wherein the alkylating agent is temozolomide or a derivative thereof.

19. A kit comprising the antibody drug conjugate of any one of claims 1 to 14, the pharmaceutical composition of claim 15 or 16, or the pharmaceutical combination of claim 17 or 18.

20. The kit of claim 19, comprising a first packaging unit comprising the conjugate according to any one of claims 1 to 14, a second packaging unit comprising an alkylating agent; and Optionally, instructions for administering the conjugate and the alkylating agent to a subject.

21. The kit of claim 20, wherein the disease is a FGFR3-mediated disease.

22. Use of the antibody drug conjugate of any one of claims 1 to 14, or the pharmaceutical composition of claim 15 or 16, or the pharmaceutical combination of claim 17 or 18, or the kit of any one of claims 19 to 21 in the preparation of a medicament for treating a disease; wherein the disease is a FGFR3-mediated disease.

23. The use of claim 22, wherein the disease comprises a tumor overexpressing FGFR3, a tumor having a FGFR3 gene fusion, or a tumor having a FGFR3 gene mutation.

24. The use according to claim 23, wherein the disease is selected from the group consisting of brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, multiple myeloma or intrahepatic bile duct carcinoma.

25. A method for treating a subject having a disease or preventing progression of a disease, comprising administering to the subject an antibody drug conjugate of any one of claims 1 to 14, or a pharmaceutical composition of claim 15 or 16, or a pharmaceutical combination of claim 17 or 18, or a kit of any one of claims 19 to 21, wherein the disease is a FGFR3-mediated disease.

26. The method of claim 25, wherein the disease comprises a tumor that overexpresses FGFR3 or a tumor that has a FGFR3 gene mutation.

27. The method of claim 25, wherein the disease is selected from the group consisting of brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, multiple myeloma, or intrahepatic bile duct carcinoma.

28. The method of claim 25, wherein the disease is glioblastoma, bladder cancer, or multiple myeloma.

29. The method of claim 25, wherein administering the conjugate and the alkylating agent simultaneously as part of the same pharmaceutical formulation; or administering the conjugate and the alkylating agent simultaneously as part of different pharmaceutical formulations; or The conjugate and the alkylating agent are administered at different times.

30. The method of claim 25, wherein the alkylating agent is temozolomide or a derivative thereof.

31. Use of an effective amount of a conjugate according to any one of claims 1 to 14 in the preparation of a medicament for treating a subject suffering from cancer, in combination with an effective amount of an alkylating agent.

32. The method of claim 31, wherein administering the conjugate and the alkylating agent simultaneously as part of the same pharmaceutical formulation; or administering the conjugate and the alkylating agent simultaneously as part of different pharmaceutical formulations; or The conjugate and the alkylating agent are administered at different times.

33. The use according to claim 31, wherein the alkylating agent is temozolomide or a derivative thereof.

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