Anti-FGFR3 Antibody Conjugate and Its Medical Use

Abstract

Provided are conjugates containing anti-FGFR3 antibodies and linker-payloads, as well as corresponding pharmaceutical compositions, preparation methods and uses, in the field of biopharmaceuticals, in particular.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceuticals, particularly to conjugates containing anti-FGFR3 antibodies and linker-payloads, and corresponding pharmaceutical compositions, preparation methods and uses thereof.

Background Art

[0002] Fibroblast growth factor (FGF) and its tyrosine kinase receptor (FGFR) play important roles in embryonic development, maintenance of homeostasis in 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 including three immunodomains as D1, D2 and D3, a single transmembrane region and a split cytoplasmic kinase portion.

[0003] Abnormal regulation of signal transduction by FGFR1-4 is associated with several types of cancer. Genomic FGFR mutations, including gene amplification, chromosomal translocation and activating mutations, induce abnormal activation of the FGF pathway and promote tumor transformation. In particular, amplification of FGFR3 is associated with the development of solid tumors such as brain cancer, bladder cancer, urothelial cancer, cervical cancer, and intrahepatic cholangiocarcinoma. Additionally, missense FGFR mutations have been found in several types of cancer, and FGF-driven signal transduction and tumor cell proliferation can be enhanced by S249C of FGFR3. FGFR3 fusion proteins constitutively activate the kinase domain as a cancer driver alteration. Known FGFR3 fusion partners are TACC3, BAIAP2L1, AES, ELAVL3, JAKMIP1, TNIP2, and WHSC1.

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

[0005] B701 (Bofatumumab) is a human immunoglobulin G1 monoclonal antibody against FGFR3, and clinical trials have been conducted to confirm whether it exhibits anti-tumor activity and the possibility of combination with docetaxel. When the anti-FGFR3 monoclonal antibody B-701 is administered, it specifically binds to and inhibits both wild-type FGFR3 and mutant FGFR3, inhibits FGFR3 phosphorylation, thereby inhibiting FGFR3 activation and the FGFR3-mediated signal transduction pathway. Thereby, cell proliferation is inhibited and apoptosis is induced in FGFR3-expressing tumors.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] Antibodies and ADCs targeting FGFR3 have entered the clinical stage, but further development has been discontinued due to lack of efficacy. The need to develop new ADCs targeting FGFR3 still exists in the art.

Means for Solving the Problems

[0009] In a first aspect of the present invention, formula (I):

[0010] [Chemistry]

[0011] (wherein, A is an anti-FGFR3 antibody or an antigen-binding fragment, and the antibody or antigen-binding fragment is modified for connection to the (Gly) n moiety in the compound of formula (I), and the antibody or antigen-binding fragment comprises a heavy-chain CDR1 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 1, a heavy-chain CDR2 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 2 or SEQ ID NO: 2, a heavy-chain CDR3 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 3 or SEQ ID NO: 3, a light-chain CDR1 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO: 4, a light-chain CDR2 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 5 or SEQ ID NO: 5, and a light-chain CDR3 comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 6 or SEQ ID NO: 6; z is an integer from 1 to 20; preferably from 1 to 4; particularly 2; opSu is

[0012] [Chemistry]

[0013] or a mixture thereof; R 0 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 payloads) An antibody-drug conjugate (ADC) having the structure of

[0014] In some embodiments, the connection process between the modified antibody (or antigen-binding fragment) and the compound of formula (I) is catalyzed by a ligase.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises 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.

[0016] In some embodiments, the KD value of the anti-FGFR3 antibody or antigen-binding fragment that binds to human FGFR3 and monkey FGFR3 is less than 10 nM.

[0017] In some embodiments, the anti-FGFR3 antibody or antigen-binding fragment does not bind to mouse FGFR3.

[0018] In some embodiments, the payload is a cytotoxin or fragment thereof, with optional derivatization to connect the payload and the linker; The cytotoxin is selected from taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, 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, mitoguazone, nitrogen mustards, nitrosoureas, aziridines, Benzodepa, carbocons, methredepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, calminomycin, cardinophilin, calminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, adriamycin iron, rhodrubicin, lufocromomycin, streptozocin, dinostatin, zorubicin, trichothecene, T-2 toxin, Velcalin A , bacillosporin A, angizidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethyl folic acid, methotrexate, pteropterin, trimethoprim, edatrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, floxuridine, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, a protein kinase inhibitor and a proteasome inhibitor selected from the group consisting of; and / or vinblastins, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, Doxorubicin , Duocarmycin , SN-38, a cryptophycin analog, deruxtecan, duocarmycin, calicheamicin, centanamycin, Dostaratin , a pyrrolobenzodiazepine, exatecan and derivatives thereof selected from; and / or auristatins, particularly selected from MMAE, MMAF or MMAD; and / or exatecan and its derivatives, such as selected from DX8951f; and / or DXd-(1) and DXd-(2); preferably selected from DXd-(1).

[0019] In some embodiments, the payload is of formula (II):

[0020]

Chemical formula

[0021] (wherein a is 0 or 1; The carbon atoms marked with p1* and p2* respectively are asymmetric centers, and the asymmetric centers are in S configuration, R configuration or racemic; L 1 is selected from unsubstituted, or C substituted with one substituent selected from halogen, -OH and -NH2 1~6 alkylene; M is -CH2-, -NH- or -O-; L 2 is 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) has the structure of.

[0022] In some embodiments, the payload is

[0023]

Chemical formula

[0024] selected from.

[0025] In some embodiments, the ADC is

[0026]

Chemical formula

[0027] is selected from.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises a VH domain comprising an amino acid sequence having at least 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 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 and comprises.

[0029] 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 and comprises.

[0030] 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 and comprises.

[0031] 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 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 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and comprises.

[0032] 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 and comprises.

[0033] 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.

[0034] 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.

[0035] In some embodiments, 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. In some embodiments, the antibody, Sp, and the recognition sequence of the ligase donor substrate are sequentially linked; Sp is GA, GGGGS (SEQ ID NO: 17) , GGGGSGGGGS (SEQ ID NO: 18) and GGGGSGGGGSGGGGS (SEQ ID NO: 19) is a spacer sequence selected from; the recognition sequence of the ligase donor substrate is LPXTGJ (SEQ ID NO: 20) wherein X may be any single natural or non-natural amino acid; J is absent or an amino acid fragment containing 1 to 10 amino acids.

[0036] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity to 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 to the amino acid sequence of SEQ ID NO: 14 is included.

[0037] 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 is included.

[0038] 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 is included.

[0039] In some embodiments, the antibody-drug conjugate has a drug-to-antibody ratio (DAR) that is an integer or non-integer from 1 to 20, particularly from 2 to 8. In some embodiments, the value of the 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 terminal values).

[0040] In a second aspect, there is provided a pharmaceutical composition comprising the ADC of the present disclosure and at least one pharmaceutically acceptable carrier.

[0041] In some embodiments, the pharmaceutical composition comprises an alkylating agent. In some embodiments, the alkylating agent comprises temozolomide or a derivative thereof.

[0042] In a third aspect, there is provided a pharmaceutical combination comprising the ADC of the present disclosure and an alkylating agent.

[0043] In some embodiments, the pharmaceutical combination comprises the ADC of the present disclosure and temozolomide or a derivative thereof. In some embodiments, the pharmaceutical combination comprises an ADC and temozolomide. In some embodiments, the pharmaceutical combination further comprises a pharmaceutically acceptable carrier.

[0044] In a fourth aspect, there is provided a kit comprising the ADC, pharmaceutical composition, or pharmaceutical combination of the present disclosure.

[0045] 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 optionally, instructions for administering the conjugate and the alkylating agent to a subject comprising.

[0046] In some embodiments, the subject has a disease. In some embodiments, the disease is an FGFR3-mediated disease. In some embodiments, the alkylating agent is temozolomide or a derivative thereof.

[0047] In some embodiments, the disease is a tumor. In some embodiments, the disease includes an FGFR3-positive tumor. In some embodiments, the disease includes a tumor that overexpresses FGFR3 or a tumor with an alteration in FGFR3. In some embodiments, the disease includes a tumor with an FGFR3 fusion (e.g., a TACC3 fusion, an intracellular fusion) or a tumor with an FGFR3 gene mutation (e.g., Y373C, G380R, S371C, S249C, or R248C). In some embodiments, the disease is selected from the group consisting of 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, 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 cancer, cervical cancer, or intrahepatic cholangiocarcinoma. In some embodiments, the disease is glioblastoma, bladder cancer, or multiple myeloma.

[0048] A fifth aspect provides the use of an ADC of the present disclosure, a pharmaceutical combination or pharmaceutical composition of the present disclosure, in the manufacture of a medicament for treating a disease, wherein the disease is an FGFR3-mediated disease.

[0049] In some embodiments, the disease is a tumor. In some embodiments, the disease comprises an FGFR3-positive tumor. In some embodiments, the disease comprises a tumor that overexpresses FGFR3 or a tumor with an alteration in FGFR3. In some embodiments, the disease comprises a tumor with an FGFR3 fusion (e.g., a TACC3 fusion, an intracellular fusion) or a tumor with an FGFR3 gene mutation (e.g., Y373C, G380R, S371C, S249C, or R248C). In some embodiments, the disease is selected from the group consisting of 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, 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 cancer, cervical cancer, or intrahepatic bile duct cancer. In some embodiments, the disease is glioblastoma, bladder cancer, or multiple myeloma.

[0050] In a sixth aspect, a method of treating a subject having a disease or reducing the likelihood of disease progression, the method comprising administering a conjugate, pharmaceutical combination, pharmaceutical composition, or kit, wherein the disease is an FGFR3-mediated disease, is provided.

[0051] In some embodiments, the disease is a tumor. In some embodiments, the disease includes an FGFR3-positive tumor. In some embodiments, the disease includes a tumor that overexpresses FGFR3 or a tumor with an alteration in FGFR3. In some embodiments, the disease includes a tumor with an FGFR3 fusion (e.g., a TACC3 fusion, an intracellular fusion) or a tumor with an FGFR3 gene mutation (e.g., Y373C, G380R, S371C, S249C, or R248C). In some embodiments, the disease is selected from the group consisting of 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, 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 cancer, cervical cancer, or intrahepatic cholangiocarcinoma. In some embodiments, the disease is glioblastoma, bladder cancer, or multiple myeloma.

[0052] 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.

[0053] In some embodiments, the conjugate and the alkylating agent are administered at different times.

[0054] In another aspect, provided is the use of an effective amount of a conjugate for the production of a medicament for treating a subject having cancer, which is used in combination with an effective amount of an alkylating agent.

[0055] 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.

[0056] In some embodiments, the conjugate and the alkylating agent are administered at different times.

[0057] In some embodiments, the disease includes an FGFR3-positive tumor. In some embodiments, the disease includes a tumor that overexpresses FGFR3 or a tumor associated with an alteration of FGFR3. In some embodiments, the disease includes a tumor associated with an FGFR3 fusion (e.g., a TACC3 fusion, an intracellular fusion) or a tumor associated with an FGFR3 gene mutation (e.g., Y373C, G380R, S371C, S249C, or R248C). In some embodiments, the disease is selected from the group consisting of 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, 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 cancer, cervical cancer, or intrahepatic cholangiocarcinoma. In some embodiments, the disease is glioblastoma, bladder cancer, or multiple myeloma.

Brief Description of the Drawings

[0058]

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Figure 12.2

Mode for Carrying Out the Invention

[0059] To explain the technical content of the present disclosure, specific embodiments are provided below. Those skilled in the art can easily understand other advantages and effects of the present disclosure through the content disclosed in this specification. The present disclosure can also be implemented or applied through other different specific embodiments. Various modifications and variations can be made by those skilled in the art without departing from the spirit of the present disclosure.

[0060] Definitions Unless otherwise defined below, all scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art. The techniques used in this specification refer to techniques generally understood in the art, including obvious variations and equivalent alternatives by those skilled in the art. The following terms are considered to be easily grasped by those skilled in the art, but the following definitions are provided to better explain the present disclosure. When a trade name exists in this specification, it refers to the corresponding product or its active ingredient. All patents, patent application publications, and publications referred to in this specification are incorporated herein by reference.

[0061] If a particular quantity, concentration, or other value or parameter is recited in the form of a range, preferred range, or preferred upper or lower limit, it is to be understood as specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, whether or not the range is explicitly recited. Unless otherwise stated, numerical ranges recited herein are intended to include the endpoints of the range as well as all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions, such as j, k1, k2, n, k, and z, are to be understood in a similar manner. "Conservative amino acid substitutions" refer to the replacement of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids having side chains of 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.

[0062] Unless the context clearly dictates otherwise, singular forms such as "a" and "the" include the plural. The expressions "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0063] When used in connection with numerical variables, the terms "about" and "approximately" generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within the 95% confidence interval for the mean) or within ±10% of the specified value, or within a wider range.

[0064] The term "optional" or "optionally" means that the event described subsequently may occur but does not necessarily occur, and the description includes the case where the event or situation occurs or does not occur.

[0065] The expressions "comprising", "including", "containing" and "having" are open-ended and do not exclude additional unrecited elements, steps or components. The expression "consisting of" excludes any unspecified element, step or component. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps or components, plus optionally existing elements, steps or components that do not substantially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0066] As used herein, the term "antibody" is used in a broad sense and particularly includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they have the desired biological activity. Antibodies may be of any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and may be derived from any suitable species. In some embodiments, the antibody is of human or mouse origin. Antibodies may also be fully human antibodies, humanized antibodies or chimeric antibodies prepared by recombinant methods.

[0067] As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for minor possible natural variations. A monoclonal antibody is highly specific for a single antigenic site. The term “monoclonal” refers to the fact that the characteristics of the antibody are derived from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method for producing the antibody.

[0068] An intact antibody or full-length antibody essentially comprises, in addition to the antigen-binding variable region, a light chain constant region (CL) and a heavy chain constant region (CH) which may include CH1, CH2, CH3 and CH4 depending on the antibody subtype. The antigen-binding variable region (also known as the fragment variable region, Fv fragment) typically includes a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region having a native sequence (e.g., a constant region having a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0069] An antibody fragment may comprise a portion of an intact antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, an Fd fragment consisting of VH and CH1 domains, an Fv fragment, a single-domain antibody (dAb) fragment, and an isolated complementarity-determining region (CDR). A Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin or a fragment having the same structure produced, for example, by recombinant expression. A Fab fragment comprises a light chain (including VL and CL) and another chain, the other chain comprising the variable domain of the heavy chain (VH) and the constant region domain of the heavy chain (CH1). An F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin at pH 4.0 to 4.5 or a fragment having the same structure produced, for example, by recombinant expression. An F(ab')2 fragment essentially comprises two Fab fragments, and each heavy chain portion contains a few additional amino acids including a cysteine that forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment comprising one half of an F(ab')2 fragment (one heavy chain and one light chain). An antibody fragment may comprise a plurality of chains joined together, for example, via disulfide bonds and / or via a peptide linker. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, and other fragments including modified fragments. An antibody fragment typically comprises at least or about 50 amino acids, typically at least or about 200 amino acids. An antigen-binding fragment can include any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of a corresponding region), can result in an antibody that immunospecifically binds to an antigen.

[0070] Antibodies according to the present disclosure can be prepared using techniques well 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 mimetics) can be expressed by a suitable culture system (e.g., Escherichia coli (E. coli) or mammalian cells). The manipulation can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0071] As used herein, the term "antibody-drug conjugate" is referred to as "conjugate".

[0072] A small molecule compound refers to a molecule having a size equivalent to that of an organic molecule commonly used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides, etc. Typically, the molecular weight of a small molecule compound can be, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, about 200 to about 500 Da.

[0073] A spacer is a structure that is positioned between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or whether it can be cleaved or disassembled in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, and the non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" refers to an amino acid sequence that functions as a spacer, and examples thereof include a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, etc., or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc., but are not limited thereto. A self-destructing spacer is a covalent assembly designed to relate the cleavage of two chemical bonds after activation of a protecting moiety in a precursor, and upon stimulation, the protecting moiety (e.g., a cleavable sequence) is removed, thereby generating a cascade of disassembly reactions leading to the sequential release of smaller molecules over time. Examples of self-destructing spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0074] The term "alkyl" refers to a saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms and connected to the rest of the molecule through a single bond. Alkyl groups include straight-chain alkyl, branched-chain alkyl, or cyclic alkyl (cycloalkyl) or partially cyclic alkyl (e.g., cycloalkyl-straight-chain alkyl and cycloalkyl-branched-chain alkyl). Alkyl groups may contain 1 to 10 carbon atoms, which is C 1~10 alkyl group, for example, C 1~6 alkyl group, C 1~4 alkyl group, C 1~3 alkyl group, C 1~2 alkyl, C3 alkyl, C4 alkyl, C 3~6Refers to alkyl. Non-limiting examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc. Non-limiting examples of branched-chain 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.

[0075] The terms "cyclic alkyl" and "cycloalkyl" have the same meaning and are used interchangeably herein. A cyclic alkyl group can include monocyclic or polycyclic (e.g., having two or more fused rings) groups. In a polycyclic cycloalkyl, two or more rings can be fused or bridged together or can be spiro. The ring-forming carbon atoms of a cyclic alkyl group can be optionally substituted by oxo (i.e., C(O)). A cyclic alkyl group can 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 is a monocyclic or bicyclic cyclic alkyl, preferably a C 3~6 monocyclic cyclic alkyl, particularly cyclopropyl. 3~6

[0076] ​The term "partially cyclic" refers to a group in which the radical contains one or more cyclic moieties and one or more acyclic (i.e., straight-chain or branched-chain) moieties. Partially cyclic alkyl groups can include cyclic alkyl-straight-chain alkyl groups and cyclic alkyl-branched-chain 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 partially cyclic alkyl groups include, but are not limited to, C 3~9 cyclic alkyl-C1 alkyl groups, C 3~8 cyclic alkyl-C2 alkyl groups, C 3~7 cyclic alkyl-C3 straight-chain alkyl groups, C 3~6 cyclic alkyl-C4 straight-chain alkyl groups, C 3~5 cyclic alkyl-C5 straight-chain alkyl groups, C 3~4 cyclic alkyl-C6 straight-chain alkyl groups, C3 cyclic alkyl-C7 straight-chain alkyl groups, C 3~7 cyclic alkyl-C3 branched-chain alkyl groups, C 3~6 cyclic alkyl-C4 branched-chain alkyl groups, C 3~5 cyclic alkyl-C5 branched-chain alkyl groups, C 3~4 cyclic alkyl-C6 branched-chain alkyl groups, and C3 cyclic alkyl-C7 branched-chain alkyl groups. In some embodiments, the partially cyclic alkyl is a C 3~9 cyclic alkyl-C1 alkyl group, preferably a C 3~6 cyclic alkyl-C 1~2 alkyl group, a C 3~6 cyclic alkyl-C1 alkyl group, more preferably a C 3~4 cyclic alkyl-C 1~2 alkyl group, particularly a C 3~4 cyclic alkyl-C1 alkyl group, particularly cyclopropyl-methyl.

[0077] A divalent radical refers to a group obtained from the corresponding monovalent radical by removing one hydrogen atom from a carbon atom having a free valence electron. A divalent radical has two connecting sites that are connected to the remainder of the molecule, and the two connecting sites may be on the same atom or two different atoms of the divalent radical.

[0078] "Alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group. The alkylene group includes linear, branched, cyclic or partially cyclic groups. Examples of linear alkylene groups include, but are not limited to, methylene (-CH2-), -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched alkylene groups include -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., but are not limited thereto.

[0079] The terms "cyclic alkylene" and "cycloalkylene" have the same meaning and are used interchangeably herein. Examples of cyclic alkylene groups 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, cyclic alkylene is C 3~6 cyclic alkylene group, particularly C 3~4 cyclic alkylene group, particularly Cyclopropylidene is.

[0080] The partial cyclic alkylene group can contain a divalent radical where the two connection sites connected to the rest of the molecule can both be on one or more linear or branched alkyl moieties, or both can be on one or more cyclic alkyl moieties, or each can be on a cyclic alkyl moiety and a linear or branched alkyl moiety. Examples of partial cyclic alkylene groups are: (1) cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, and divalent polycyclic alkyl groups containing fused, spiro, or bridged rings, each independently substituted by one or more linear or branched alkyl groups; (2) linear or branched alkylene groups each independently substituted by 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, provided that chemically stable structures are formed, but not limited thereto. In some embodiments, the partial cyclic alkylene is a C 3~9 cyclic alkyl-C1 alkylene group, preferably a C 3~6 cyclic alkyl-C 1~2 alkylene group, a C 3~6 cyclic alkyl-C1 alkylene group, more preferably a C 3~4 cyclic alkyl-C 1~2 alkylene group, particularly a C 3~4 cyclic alkyl-C1 alkylene group, particularly cyclopropyl-methylene.

[0081] As used herein, the expressions "antibody conjugate drug", "ADC", and "antibody-drug conjugate" have the same meaning.

[0082] The compound of formula (III) In some embodiments, the linker-payload intermediate of the ADC is of formula (III):

[0083]

Chemical formula

[0084] (wherein, opSu is

[0085] [Chemical formula]

[0086] or a mixture thereof; R 0 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, payloads having the structure of formula (II)) having the structure of.

[0087] In one embodiment, R 0 is C 1~6 alkyl. In a preferred embodiment, R 0 is C 1~3 alkyl. In a particular embodiment, R 0 is methyl.

[0088] In one embodiment, n is an integer from 2 to 5. In a particular embodiment, n is 3.

[0089] In one embodiment, k1 and k2 are, independently, 1, or 3 or 5. In a particular embodiment, k1 and k2 are independently 5.

[0090] 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 particular embodiment, i is 4.

[0091] In one embodiment, j is independently an integer from 1 to 20, preferably from 1 to 12, more preferably from 8 to 12, particularly 8 or 12. In a particular embodiment, j is 12.

[0092] In one embodiment, the compound of formula (III) is of formula (III-1)

[0093]

Chemical formula

[0094] (wherein, P1, P2, R 0 , opSu, n, i and j are as defined in formula (III)) has the structure of.

[0095] In one embodiment, the compound of formula (III) is

[0096]

Chemical formula

[0097] selected from the group consisting of.

[0098] Preparation of the compound of formula (III) In one embodiment, the compound of formula (III) can be synthesized by connecting a linker to a payload or by connecting a continuum of suitable building blocks. Such building blocks can be readily designed by retrosynthetic analysis and any reaction known in the art can be used.

[0099] In one embodiment, formula (IV):

[0100]

Chemical formula

[0101] (wherein, k is an integer from 1 to 7; P is a payload having the structure of formula (II), and the structure of formula (II) is as defined above) Compounds having the structure of are provided.

[0102] 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 particular embodiment, k is 5.

[0103] The compound of formula (III) can be synthesized using a method similar to the synthetic methods disclosed in European Patent Application No. 2907824A (for example, the synthetic methods of formula (2) or (2b) of European Patent Application No. 2907824A) using the compound of formula (II) / (IV) and other necessary building blocks. Suitable building blocks include, but are not limited to, linker-payload intermediate 2 and linker-payload intermediate 1.

[0104]

Chemical formula

[0105] Next, the maleimide group of formula (IV) therein can be reacted with a thiol group on another building block. The resulting thiosuccinimide is unstable under physiological conditions and tends to undergo a retro-Michael addition leading to cleavage at the connecting site. Further, if another thiol compound is present in the system, the thiosuccinimide may also undergo thiol exchange with other thiol compounds. Both of these reactions cause payload dropout and result in toxic side effects. The thiosuccinimide is then subjected to a ring-opening reaction. The compound of formula (III) can then be obtained.

[0106] The method of the ring-opening reaction can be found in WO 2015 / 165413 A1. Compounds containing the ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain them with high purity and defined composition, regardless of the efficiency of the succinimide ring-opening reaction.

[0107] In the present disclosure, when applied in a linker-payload (linker-small molecule intermediate), the ring-opened succinimide structure no longer undergoes either a Michael addition or a thiol exchange, so the product is more stable.

[0108] A moiety comprising recognition sequences of ligase acceptor and donor substrates In one embodiment, the (Gly) of the compound of formula (III) n The moiety is a recognition sequence of a ligase donor substrate that promotes the enzyme-catalyzed coupling of the compound of formula (III) with an antibody or antigen-binding fragment under the catalysis of a ligase. The antibody or antigen-binding fragment is optionally modified and contains the corresponding recognition sequence of the ligase acceptor substrate.

[0109] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of native transpeptidases, non-native transpeptidases, variants thereof, and combinations thereof. The non-native transpeptidase enzyme may be obtained by manipulation of native transpeptidases, but is not limited thereto. In a preferred embodiment, the ligase is selected from the group consisting of native sortases, non-native sortases, and combinations thereof. Species of native sortases include sortase A, sortase B, sortase C, sortase D, sortase L. plantarum, etc. (US Patent Application No. 20110321183 A1). The type of ligase corresponds to the ligase recognition sequence and is thereby used to achieve specific conjugation between different molecules or structural fragments.

[0110] In one embodiment, the (Gly) of the compound of formula (III)n The portion is a recognition sequence of a ligase acceptor substrate; and the antibody or antigen-binding fragment is optionally modified and contains the corresponding recognition sequence of the ligase donor substrate.

[0111] In some embodiments, the ligase is a sortase selected from sortase A, sortase B, sortase C, sortase D, and sortase L. plantarum.

[0112] In a particular embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence of the ligase donor substrate may be the typical recognition sequence LPXTG of the enzyme (SEQ ID NO: 21) where X may be any single natural or non-natural amino acid. In yet another particular embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ, where X may be any single natural or non-natural amino acid; J is absent or is an amino acid fragment containing 1 to 10 amino acids, optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, and each amino acid is independently any natural or non-natural amino acid. In another embodiment, J is (Gly) m where m is an integer from 1 to 10. In yet another particular embodiment, the recognition sequence of the ligase donor substrate is LPETG (SEQ ID NO: 22) In another particular embodiment, the recognition sequence of the ligase donor substrate is LPETGG (SEQ ID NO: 23) In one embodiment, the ligase is sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN

[0113] In one embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG (SEQ ID NO: 24) In another embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG (SEQ ID NO: 25) In another embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG

[0114] In yet another embodiment, the ligase may be sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In another embodiment, the ligase may be sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be LAXTG (SEQ ID NO: 26) and may be.

[0115] In yet another embodiment, the ligase may be sortase A from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ (SEQ ID NO: 27) and may be.

[0116] The ligase recognition sequence may also be any other completely novel recognition sequence for transpeptidase optimized by manual screening.

[0117] Conjugates and their preparation Furthermore, a payload-bearing compound (compound of formula (III)) having a portion comprising a ligase recognition sequence may be conjugated with an anti-FGFR3 or antigen-binding fragment thereof comprising a ligase recognition sequence.

[0118] In yet another aspect, formula (I):

[0119]

Chemical formula

[0120] (wherein A is an anti-FGFR3 antibody or an antigen-binding fragment thereof; z is an integer from 1 to 20; P1, P2, R 0 , opSu, n, k1, k2, i and j are as defined above) A conjugate having the structure is provided.

[0121] In one embodiment, the antibody or antigen-binding fragment is modified to connect to the (Gly) moiety in the compound of formula (III). n moiety.

[0122] In one embodiment, z is from about 1 to 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 terminal values).

[0123] In one embodiment, the compound of formula (I) is

[0124]

Chemical formula

[0125] selected from the group consisting of.

[0126] In one embodiment, the compound of formula (I) is

[0127]

Chemical formula

[0128] selected from.

[0129] The antibody or antigen-binding fragment In one embodiment, the antibody or antigen-binding fragment (shown as "A" in formula (I)) is an anti-FGFR3 antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 2 or SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 3 or SEQ ID NO: 3, a light chain CDR1 (LCDR1) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO: 4, a light chain CDR2 (LCDR2) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 5 or SEQ ID NO: 5, and a light chain CDR3 (LCDR3) comprising an amino acid sequence having 1 to 3 conservative amino acid substitutions compared to SEQ ID NO: 6 or SEQ ID NO: 6.

[0130] 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.

[0131] In some embodiments, the KD value of the anti-FGFR3 antibody or antigen-binding fragment that binds to human FGFR3 and / or monkey FGFR3 is less than 10 nM. In some embodiments, the KD value of the anti-FGFR3 antibody or antigen-binding fragment that binds to human FGFR3 and / or monkey FGFR3 is 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 terminal values).

[0132] 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 to 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 to 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 terminal values)) sequence identity to 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 terminal values)) sequence identity to 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.

[0133] 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 to 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 to 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 terminal values)) sequence identity to 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 terminal values)) sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises the heavy chain constant domain of SEQ ID NO: 9 and the 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.

[0134] 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 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 about 90% sequence identity to 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 terminal values)) 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 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 terminal values)) sequence identity to 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 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: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 14.

[0135] The above sequences are listed below (split according to Kabat): SEQ ID NO: 1: SYDMS

[0136] SEQ ID NO: 2: GIYSGDGSIYYADSVKG

[0137] SEQ ID NO: 3: DGPMNEESRFDY

[0138] SEQ ID NO: 4: SGSSSNIGNNYVS

[0139] SEQ ID NO: 5: ADSKRPS

[0140] SEQ ID NO: 6: ASWDYSLSGYV

[0141] SEQ ID NO: 7:

[0142] [Chemical]

[0143] Array number 8:

[0144] [Chemical]

[0145] Array number 9:

[0146] [Chemical]

[0147] Array number 10:

[0148] [Chemical]

[0149] Array number 11:

[0150] [Chemical]

[0151] Array number 12:

[0152] [Chemical]

[0153] Array number 13:

[0154] [Chemical]

[0155] Array number 14:

[0156] [Chemistry]

[0157] In one embodiment, the antibody or its antigen-binding fragment may include a terminal modification. The terminal modification refers to a modification at the C-terminus or N-terminus of the heavy or light chain of the antibody, including, for example, a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp containing 2 to 100 amino acids, and the antibody, Sp, and ligase recognition sequence are sequentially linked. In a preferred embodiment, Sp is a spacer sequence containing 2 to 20 amino acids. In a particular embodiment, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, particularly GA.

[0158] In some embodiments, the modified antibody or its antigen-binding fragment includes the heavy chain of SEQ ID NO: 15 and / or the light chain of SEQ ID NO: 16. In some embodiments, the modified antibody or its antigen-binding fragment includes the heavy chain of SEQ ID NO: 15 and the light chain of SEQ ID NO: 16. In some embodiments, the modified antibody or its antigen-binding fragment includes the heavy chain of SEQ ID NO: 15 and the light chain of SEQ ID NO: 14. In some embodiments, the modified antibody or its antigen-binding fragment includes the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 16.

[0159] SEQ ID NO: 15:

[0160] [Chemistry]

[0161] SEQ ID NO: 16:

[0162] [Chemistry]

[0163] Preparation of Conjugate The conjugates of the present disclosure (i.e., the compounds of formula (I)) can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by ligase-catalyzed site-specific conjugation of an antibody or antigen-binding fragment and a compound of formula (III), and the antibody or antigen-binding fragment thereof is modified by a ligase recognition sequence.

[0164] The antibody or antigen-binding fragment thereof and the compound of formula (III) are linked to each other via a ligase-specific recognition sequence of the substrate. The recognition sequence depends on the particular ligase used. In one embodiment, the antibody or antigen-binding fragment thereof is an antibody having a recognition sequence-based terminal modification introduced at the C-terminus of the light chain and / or the C-terminus of the heavy chain, and the antibody or antigen-binding fragment thereof is under the catalysis of a wild-type or optimized engineered ligase or any combination thereof, and under suitable catalytic reaction conditions, Formula (III) is conjugated to the compound of.

[0165] In a particular embodiment, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme:

[0166]

Chemical formula

[0167] The triangle represents a part of the antibody; the pentagon represents a part of the compound of formula (III); G n represents the (Gly) n part. n, X and J are as defined above. When conjugated with the corresponding recognition sequence of the acceptor substrate G n , the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate is linked to the free N-terminus of G n to form a new peptide bond. The resulting amino acid sequence is LPXTG n (SEQ ID NO: 28) . The sequences G n and LPXTGJ are as defined above.

[0168] The compounds of formula (III) of the present disclosure have a defined structure, defined composition and high purity. As a result, when the conjugation reaction with an antibody is carried out, fewer impurities are introduced or no other impurities are introduced. When such an intermediate is used for ligase-catalyzed site-specific conjugation with a modified antibody containing a ligase recognition sequence, a homogeneous ADC with highly controllable quality can be obtained.

[0169] Metabolism of the conjugate in the physiological environment When part or all of the linker is cleaved in tumor cells, the payload is released. When the linker is cleaved at the connection position to the anti-tumor compound, the anti-tumor compound is released in its original structure and exhibits its original anti-tumor effect.

[0170] In one embodiment, the GGFG (Gly-Gly-Phe-Gly ; SEQ ID NO: 29 ) moiety contained by the compound of formula III) can be cleaved by lysosomal enzymes (such as cathepsin B and / or cathepsin L).

[0171] In one embodiment, the compound of formula (III) contains a self-immolative spacer. In one embodiment, the self-immolative spacer is an acetal or heteroacetal. In one embodiment, the -GGFG-NH-CH2-O- moiety contained by the compound of formula (III) represents a combination of a restriction enzyme site and a self-immolative spacer, which is cleaved in cells to release the molecule of interest (such as an anti-tumor compound).

[0172] Pharmaceutical compositions and pharmaceutical preparations 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.

[0173] The pharmaceutical composition of the present disclosure may be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or curing symptoms in humans or animals. For example, various suitable dosage forms can be prepared according to the administration route, which are particularly injections, such as freeze-dried powders for injection, injection solutions, or sterile powders for injection.

[0174] The term "pharmaceutically acceptable" means that when contacted with the tissues of a patient within the scope of ordinary medical judgment, it does not cause excessive toxicity, irritation or allergic reactions, etc., has a reasonable benefit-risk ratio, and is effective for the intended use.

[0175] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline and the like. Pharmaceutically acceptable carriers also include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffering agents, and toxicity adjusters, etc., and include sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, etc. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with the active ingredient for treatment. Such pharmaceutical carriers may be sterile liquids, such as water and oils, and the oils include oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Solutions of glucose in saline and water or glycerol can also be used as liquid carriers, especially for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, corn, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, and ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, and tonicity adjusters, such as sodium chloride or dextrose are also contemplated. Such compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained release formulations, etc. The composition may be formulated as a suppository using conventional binders and carriers, such as triglycerides. Oral formulations 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, E. W. Martin, which is incorporated herein by reference. Such compositions include a clinically effective dosage of the antibody, preferably in purified form, together with a suitable amount of carrier to provide a suitable dosage form for the patient. The formulation should be suitable for the mode of administration. The parent formulation may be enclosed in an ampoule, disposable syringe or multi-dose vial made of glass or plastic.

[0176] In one embodiment, the pharmaceutical composition of the present disclosure has a drug-to-antibody ratio (DAR) that is an integer or non-integer from about 1 to about 20, such as from about 1 to about 10, from about 1 to about 8, from about 1 to about 6, or from about 1 to about 4. In a particular embodiment, the conjugate of the present disclosure has a DAR of about 4.

[0177] Methods of Treatment and Use The conjugates of the present disclosure are useful for the treatment of FGFR3-mediated diseases.

[0178] Thus, in yet another aspect, there is also provided the use of the conjugates of the present disclosure or the pharmaceutical compositions of the present disclosure in the production of a medicament for treating an FGFR3-mediated disease. FGFR3-mediated diseases are particularly FGFR3-positive tumors, more particularly brain cancer, bladder cancer, urothelial cancer, cervical cancer, or intrahepatic cholangiocarcinoma.

[0179] In some embodiments, the disease includes tumors that overexpress FGFR3 or tumors associated with FGFR3 gene mutations. In some embodiments, the disease is selected from the group consisting of 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, bronchial 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. In some embodiments, the disease is selected from brain cancer, bladder cancer, urothelial cancer, cervical cancer, or intrahepatic bile duct cancer. In some embodiments, the disease is glioblastoma. In a preferred embodiment, the conjugate of the present disclosure formed by the conjugation of an anti-FGFR3 antibody and a small molecule cytotoxin specifically binds to FGFR3 on the surface of tumor cells and can selectively kill FGFR3-expressing tumor cells.

[0180] The dosage of the conjugate administered to a subject can be adjusted to a considerable extent. The dosage can vary according to the particular route of administration and the needs of the subject and can be subject to the judgment of a medical professional.

[0181] Beneficial effects The novel small molecule topoisomerase I inhibitor provided in the present disclosure, used alone or as a component of an ADC, may exhibit higher activity, stability, and physicochemical properties than the prior art.

[0182] The antibody-drug conjugate of the present invention uses a specially designed linker-payload to achieve high efficacy and bystander killing effect. At the same time, it has a lower DAR, thus reducing side effects and increasing 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 the ring-opened succinimide structure.

[0183] The present disclosure utilizes a linker with a unique structure and uses a ligase to catalyze the conjugation of the targeting molecule and the payload. The conjugate of the present disclosure has good homogeneity and high activity. Furthermore, the toxicity of the linker-payload intermediate is much lower than that of the free payload, so the drug production process is less harmful, which is advantageous for industrial manufacturing.

[0184] The conjugate of the present disclosure achieves at least one of the following technical effects: (1) High inhibitory activity against target cells, or strong killing effect on target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability; chemical stability includes a low off-target toxicity-linked low shedding rate of cytotoxins from the ADC). (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action). (4) High specificity and good safety (low toxicity to non-target normal cells or tissues, and / or smaller side effects, wider treatment window), etc. (5) Highly modular design, simple assembly of multiple drugs.

Example

[0185] To more clearly illustrate the objectives and technical solutions, the present disclosure will be further described below with reference to specific embodiments. It should be understood that the embodiments are not intended to limit the scope of the present disclosure. Specific experimental methods not mentioned in the following embodiments were carried out in accordance with conventional experimental methods.

[0186] Unless otherwise stated, the equipment and reagents used in the examples are commercially available. The reagents can be used directly without further purification. MS: Thermo Fisher Q Exactive Plus, Waters 2795 - Quattro micro triple quadrupole mass spectrometer HPLC: Waters 2695, Agilent 1100, Agilent 1200 Semi - preparative HPLC: Lisure HP plus 50D Flow cytometry: CytoFLEX S HIC - HPLC: Butyl - HIC; Mobile phase A: 25 mM PB, 2 M (NH4)2SO4, pH 7.0; Mobile phase B: 25 mM PB, pH 7.0; Flow rate: 0.8 ml / min; Acquisition time: 25 min; Injection volume: 20 μg; Column temperature: 25 °C; Detection wavelength: 280 nm; Sample chamber temperature: 8 °C. SEC - HPLC: Column: TSK - gel G3000 SWXL, TOSOH 7.8 mm ID × 300 mm, 5 μm; Mobile phase: 0.2 M KH2PO4, 0.25 M KCl, pH 6.2; Flow rate: 0.5 ml / min; Acquisition time: 30 min; Injection volume: 50 μl; Column temperature: 25 °C; Detection wavelength; 280 nm; Sample tray temperature: 8 °C.

[0187] In some cases, the order of executing the above reaction schemes may be changed to facilitate the reaction or to avoid unwanted reaction products. The following examples are provided so that the present invention can be more fully understood. These examples are merely illustrative and should not be construed as limiting the present invention in any way.

[0188] (Example 1) Preparation of Linker-Payload 1

[0189]

Chem.

[0190] opSu is

[0191]

Chem.

[0192] or a mixture thereof.

[0193] Preparation of Intermediate MC-GGFG-DXd Intermediate MC-GGFG-DXd is either commercially available or prepared according to the procedure described in European Patent No. 2907824. This compound is used to prepare Linker-Payload 1.

[0194] Preparation of Linker-Payload Intermediate 1

[0195]

Chem.

[0196] Linker-Payload Intermediate 1 can be synthesized by conventional solid-phase polypeptide synthesis using Rink-amide-MBHA resin. The amino acids in the linking units were protected using Fmoc. The coupling reagent was selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the product was cleaved from the resin using a TFA / TIS / H2O solution. The product was purified by preparative HPLC, lyophilized and stored for use. LCMS m / z: [M-H] - = 1382.6.

[0197] Preparation of Linker-Payload 1 Weighed and taken Linker-Payload Intermediate 1 and MC-GGFG-DXd (molar ratio of about 1:2), dissolved them in water and DMF respectively, then thoroughly mixed them to obtain a mixture, and reacted this mixture at 0 - 40 °C for 0.5 - 30 hours. After 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 carried out at 0 - 40 °C for an additional 0.2 - 20 hours. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and freeze-dried to obtain Linker-Payload 1. LCMS m / z: [(M + 3H) / 3] + = 1163.3.

[0198] (Example 2) Preparation of Linker-Payload 2 Preparation of Intermediate 11

[0199] [Chemical formula]

[0200] Step A: N-(2-Bromo-5-fluorophenyl)acetamide: Concentrated H2SO4 (3 mL) was added dropwise to a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL), followed by 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) added portionwise at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice water. A precipitate formed, which was collected by filtration and dried in vacuo at room temperature to obtain N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 1H NMR (400 MHz, 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).

[0201] Process 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 n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) dropwise at -78 °C over 1 hour. After completion, the mixture was stirred under N2 for 0.5 hour. Next, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise at -78 °C over 0.5 hour, and the mixture was stirred from -78 °C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated aqueous NH4Cl at 0 °C. Extracted with ethyl acetate (500 mL × 3), washed with brine (250 mL × 2), dried over Na2SO4, and concentrated. The mixture was triturated with (PE / EA = 1:1, 100 mL) for 10 minutes, filtered, the 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).

[0202] Process 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) were 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 through celite, 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 pale yellow solid. LCMS m / z 222.1 (M + H).

[0203] Project D: N-(3-Fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: 0 o To a stirred mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) of C was added 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 h. After completion, the mixture was acidified with HCl (2N) to adjust the pH = 3. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (100 mL × 2), dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 min, filtered, the cake was collected, and dried in vacuo to obtain 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).

[0204] Project E: N,N'-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-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 under a H2 atmosphere at 25 °C for 16 h. After cooling to 0 °C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0 °C for 1 h. Filtered through celite, the filtrate was poured into ice water (500 mL). Extracted with ethyl acetate (500 mL × 3), washed with brine (250 mL × 2), dried over Na2SO4, and concentrated. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 min, filtered, the cake was collected, and dried in vacuo to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide (7.9 g) as a yellow solid. LCMS m / z 279.1 (M+H).

[0205] Process F: N,N'-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g, 28.39 mmol) in MeOH (150 mL) was added aqueous HCl (2 N, 150 mL), and the mixture was stirred at 50 °C for 7 h. After cooling to 0 °C, saturated aqueous NaHCO3 was added dropwise to adjust the pH to 8. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (200 mL × 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)diacetamide (6.0 g) as a yellow solid. 1 1H NMR (400 MHz, chloroform-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00 - 2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). LCMS m / z 237.1(M+H).

[0206] Procedure G: N-(8-Amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL) was added NCS (2.26 g, 16.93 mmol) portionwise at 0 °C, and the mixture was stirred at room temperature for 16 h. The mixture was poured into 200 mL of ice water. A precipitate formed, which was collected by filtration and dried in vacuo at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 8.0 Hz, 1H), 7.71 (s, 2H), 6.62 (d, J = 11.9 Hz, 1H), 4.53 (ddd, J = 13.0, 8.0, 4.7 Hz, 1H), 3.18 - 3.04 (m, 1H), 2.91 (ddd, J = 17.5, 12.4, 4.8 Hz, 1H), 2.21 - 2.08 (m, 1H), 1.99 - 1.83 (m, 4H). LCMS m / z 271.0(M+H).

[0207] Process 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 a mixture of N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g, 14.78 mmol) in toluene (400 mL) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-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), 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).

[0208] Process 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 aqueous concentrated HCl was stirred at 70 °C under N2 for 36 h. 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).

[0209] Preparation of Intermediate 12 (12-1, 12-2)

[0210] [Chemical formula]

[0211] 12-1 and 12-2 were prepared as TFA salts 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) by preparative HPLC.

[0212] [Table 1]

[0213] Preparation of Linker-Payload 2

[0214] [Chemical formula]

[0215] Synthesis of 13 (Step A) Weighed and taken 4.33 g of Fmoc-Gly-Gly-OH and 6.84 g of Pb(OAc)4 and added them to a 500 ml one-neck round-bottom flask. Added anhydrous THF / toluene (120 / 40 ml) under a nitrogen atmosphere and stirred for dissolution. Next, 1.16 mL of pyridine was added to the reaction system. The reaction system was heated to 80 °C and refluxed for 5 hours under a nitrogen atmosphere. Samples were taken and the reaction was monitored by HPLC detection.

[0216] The reaction system was cooled to room temperature, filtered, and the filter cake was washed 3 times with EA. The filtrates were combined and concentrated to dryness. Column chromatography was performed (PE:EA = 100:0 → 50:100), and approximately 2000 mg of the target product in white solid was obtained in a 44% yield.

[0217] Synthesis of 15 (Step B) Weighed and taken 200 mg of 13 and added it to a 100 ml one-neck round-bottom flask. Next, 15 ml of THF was added and stirred for dissolution. Then 14 (312 mg, 3.0 equivalents) and TsOH·H2O (15 mg, 0.15 equivalents) were added to the reaction system. The reaction system was reacted overnight at room temperature. Samples were taken and the reaction was monitored by TLC (PE / EA = 1:1) detection. The raw materials basically disappeared and new spots were detected.

[0218] Saturated sodium bicarbonate solution was added to quench the reaction. Extraction was carried out 3 times with EA. 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 approximately 80 mg of the target product in colorless oil in a 29% yield. LCMS m / z: [M+H] + = 501.1

[0219] Synthesis of 16 (Step C) 15 of 200 mg was weighed and taken, and added to a 100 ml one-neck round-bottom flask. Next, 10 ml of EtOH and 5 ml of EA were added with complete dissolution. Next, 40 mg of palladium carbon was added to the reaction system under a nitrogen atmosphere, and the reaction system was purged three times with hydrogen gas. The reaction system was kept under a hydrogen atmosphere and stirred at room temperature for 0.5 h. A sample was taken and detected by TLC (DCM / MeOH = 10:1) to monitor the reaction. The raw materials basically disappeared and new spots were detected.

[0220] The reaction system was filtered, and the filter cake was washed three times with EA. The filtrates were combined and concentrated to dryness to obtain 200 mg of the product as a white solid in 100% yield. The product could be used directly in the next reaction without purification. LCMS m / z: [M-H] - = 409.4.

[0221] Synthesis of 21 (Step D) Step D-1 2.0 g of dichloro resin was weighed and taken and placed in a polypeptide synthesis tube. DCM (10 ml) was added and allowed to swell at room temperature for 30 min. The solvent was removed by vacuum aspiration. The resin was washed twice with DCM, using a volume of 7 mL and a time length of 1 min for each wash. The solvent was removed by vacuum aspiration. Next, 16 (200 mg) was weighed and taken and added to a 50 ml centrifuge tube. DCM (about 10 ml) was added. The solid was dissolved by shaking and added to the above resin. Stirring was carried out to immerse all the resin in the solution (if there was resin adhering to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 4 - 5 h. After the reaction was completed, an appropriate amount of methanol was added. Stirring was carried out for 30 min. The solvent was removed by vacuum aspiration. The resin was washed successively with DMF once, methanol once, DMF once, methanol once and DMF twice, using a volume of 10 mL and a time length of 1 min for each wash. The solvent was removed by vacuum aspiration. A small amount of dry resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was qualified for the next coupling step.

[0222] Process D-2 A 10 mL commercially available 20% piperidine / DMF solution was added and deprotection was carried out twice by reacting for 10 minutes for each run. After the reaction was completed, the solution was removed by vacuum aspiration. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. The solvent was removed by vacuum aspiration. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0223] 563 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Next, approximately 7 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added. It was activated for 10 - 30 minutes to obtain an activated reaction solution.

[0224] 3 molar equivalents of the activated reaction solution were added to the resin. Stirring was carried out to completely immerse the resin in the solution (if there was resin adhering to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the solvent was removed by vacuum aspiration. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. The solvent was removed by vacuum aspiration. A small amount of dry resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was qualified for the next coupling step.

[0225] Process D-3 A 10 mL commercially available 20% piperidine / DMF solution was added and deprotection was carried out twice by reacting for 10 minutes for each run. After the reaction was completed, the solution was removed by vacuum suction. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. 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.

[0226] 531 mg of Fmoc-GG-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Next, approximately 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added. It was activated for 10 - 30 minutes to obtain an activated reaction solution.

[0227] 3 molar equivalents of the activated reaction solution were added to the resin. Stirring was carried out to completely immerse the resin in the solution (if there was resin adhering to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was qualified for the next coupling step.

[0228] Step D-4 A 10 mL commercially available 20% piperidine / DMF solution was added and deprotection was carried out twice by reacting for 10 minutes for each run. After the reaction was completed, the solution was removed by vacuum suction. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of the dried resin was taken for ninhydrin detection. Both the resin and the solution were dark blue. Next, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube and approximately 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIEA was added to the resin. Stirring was carried out to completely immerse the resin in the solution (if there was resin adhering to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was sequentially washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, using a volume of 10 mL and a time length of 1 minute for each wash. The solvent was removed by vacuum suction. A small amount of the dried resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was qualified for the next coupling step.

[0229] Step D-5 The resin was washed twice with 10 mL of methanol. Next, the solvent was thoroughly removed by vacuum suction. The resin was poured out and weighed. The dissolution buffer was prepared in a 250 mL conical flask with a TFE / DCM ratio of 80% / 20% and a volume 7 - 8 times the mass of the peptide resin. The dissolution buffer was added to the peptide resin and shaken well. The resin was completely immersed in the dissolution buffer and dissolution was carried out at room temperature for 2 - 3 hours. The dissolution buffer was then filtered off using a simple filter made from a syringe, the resin was washed with 1 - 2 mL of DCM and discarded. Next, 150 mL of pre-cooled anhydrous ether was added to the dissolution buffer, shaken well, and then left to stand for 20 - 30 minutes. Using a 50 mL centrifuge tube, the above system was centrifuged at 3500 rpm for 3 minutes in a centrifuge, the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasonic waves, centrifuged at 3500 rpm for 3 minutes, 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 obtain 125 mg of the product in the form of a white solid in a 40% yield. LCMS m / z: [M-H] - = 641.5。

[0230] Synthesis of 22 (Step E) 150 mg of raw material 21 and 55 mg of TSTU were weighed and taken, added to a 10 mL one-neck round-bottom flask, anhydrous DMF (3 mL) was added under a nitrogen atmosphere, and stirred for 20 minutes. Next, 18 mg of 12-1 and 20 μL of DIEA were sequentially added to the reaction system. Stirring was carried out at room temperature for 2 - 8 hours under a nitrogen atmosphere. Samples were taken and detected by HPLC to monitor the reaction. The raw material peak completely disappeared and a new peak was detected.

[0231] The reaction system was subjected to preparative purification, the target product was collected and freeze-dried to obtain approximately 22 mg of the product in the form of a yellowish solid. LCMS m / z: [M+H] + = 1081.0。

[0232] Synthesis of Linker-Payload 2 (Step F) Weighed and took 22 (30 mg), added it to a 10 ml one-neck round-bottom flask, and added purified water (2 ml). Stirring was carried out for dissolution. A DMF solution (2 ml) containing the linker-payload intermediate 1 (19.5 mg) was added to the reaction system and stirred. After reacting overnight, the reaction was monitored using HPLC until all of the raw materials were converted to the intermediate. 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 carried out at 0 - 40 °C for an additional 0.2 - 20 hours. The reaction was monitored by HPLC until all of the intermediate was consumed, and then quenched with an acetic acid solution.

[0233] The reaction system was subjected to preparative purification, the target product was collected, and freeze-dried to obtain about 25 mg of linker-payload 2 as a yellowish solid. LCMS m / z: [(M + 3H) / 3] + = 1194.4

[0234] (Example 3) Construction of Antibody and ADC 1. Production of Anti-FGFR3 Antibody The anti-FGFR3 antibody consists of two vectors as the heavy chain and light chain respectively in each mammalian expression system. Using the Expi293 transient mammalian expression system (Gibco, A14635, Carlsbad, CA, USA), the anti-FGFR3 antibody was produced via co-transfection of the above vectors. After transfection, the culture supernatant was purified using an AKTA protein purification system (GE Healthcare Life Sciences, Uppsala, Sweden) together with HiTrap Mabselect SuRe (GE Healthcare Life Sciences, 11-0034-93, Uppsala, Sweden). After purification, concentration was carried out using an Amicon® Ultra Centrifugal Filter (Merck Millipore, MA, USA). When analyzing the characteristics of the high-purity antibody using SDS-PAGE (see Figure 1) and SEC-HPLC, it was shown that the purity of the obtained antibody was higher than 98.5%.

[0235] The anti-FGFR3 antibodies thus obtained are shown in the following table. The CDRs are emphasized with underlines and the constant regions are shown in italics.

[0236] [Table 2]

[0237] 2. SPR analysis The binding affinity (KD value) of the anti-FGFR3 antibodies was measured using a Biacore 3000. Human (FGFR3-IIIb and -IIIc, R&D systems, 1264-FR-050), mouse (R&D systems, 710-MF-050), and cynomolgus FGFR3 proteins (Sino Biological, 90313-C02H) were coated using an amine coupling kit (GE Healthcare Life Sciences, BR100050, Uppsala, Sweden). As shown in the following table, the KD (Ka and Kd) values were evaluated according to the concentration.

[0238] [Table 3]

[0239] 3. Preparation of ADC Linker-payload intermediates were conjugated to the antibodies in a site-specific manner by ligase to form ADCs. The method of the conjugation reaction can be found in International Publication No. WO 2015 / 165413 A1. The obtained ADCs are as listed in the following table.

[0240] [Table 4]

[0241] 4. Characterization of ADC 1) SEC-HPLC Chromatography column: TSKgel G3000SWXL 7.8 mm I.D. * 30 cm, 5 μm; Mobile phase: 2 * PBS: methanol = 9:1 (V / V); Gradient: Uniform concentration of 100%, Flow rate: 1.0 ml / min; Running time was 15 minutes, and 280 nm was selected as the detection wavelength to analyze and detect the purity of the ADC drug.

[0242] 2) HIC-HPLC Chromatography column: Proteomix HIC Butyl-NP5 4.6 * 100 mm, 5 μm Non-Porous; Mobile phase: 1.5 M ammonium sulfate + 20 mM phosphate buffered saline (pH 7.0) as mobile phase A, 20 mM phosphate buffered saline (pH 7.0): isopropanol = 7:3 (V / V) as mobile phase B; Flow rate: 0.8 ml / min; Phase B was increased from 15% to 100% within 8 minutes; 280 nm was selected as the detection wavelength to detect the DAR distribution and calculate the averaged DAR value of the ADC drug.

[0243] The results are shown in Table 5 (Table 5).

[0244]

Table 5

[0245] Table 5 (Table 5) shows that the residual free drug for both samples is lower than 50 ppm, indicating that the payload (cytotoxin) dropout is very low.

[0246] (Example 4) Affinity ELISA analysis 1) Each of 1 microgram per milliliter of human FGFR3 (R&D Systems, 1264 - FR - 050), cynomolgus monkey FGFR3 (Sino Biological, 90313 - C02H) protein, or mouse FGFR3 (R&D Systems, 710 - MF - 050) was coated on a 96 - well plate at 4°C overnight respectively. The plates were blocked in 3% skim milk containing anti - FGFR3 antibody and incubated at room temperature for 1 hour. After washing with PBST (0.1%), anti - human Fab antibody conjugated horseradish peroxidase (HRP) (Thermo Scientific, 31482, Waltham, MA, USA) was added at a ratio of 1:3000. After washing, the plates were treated with TMB solution (Thermo Scientific, N301, Waltham, MA, USA) as the HRP substrate, and the reaction was stopped with STOP solution (Cell Signaling Technology, #7002, Danvers, MA, USA).

[0247] The absorbance for each well was detected at a wavelength of 450 nm.

[0248] The results of this analysis are shown in Figures 2 - 3. Figure 2 shows that antibody A9 has high specificity for FGFR3 in both human and cynomolgus monkey. Figure 3 shows that A9 and A9Q have similar affinities for human FGFR3.

[0249] 2) Affinity of ADC The affinity of the ADC that binds to human FGFR3 (Sinobiological, 16044 - H08H) is tested by ELISA (similar to the above method).

[0250] Figure 4 shows that the formation of ADC does not substantially affect the efficacy of the antibody (the EC50 of antibody A9 and ADC ADC19 are 0.04772 nM and 0.04272 nM respectively).

[0251] (Example 5) Cell - binding analysis of antibody 1) The cell surface binding efficiency of the anti-FGFR3 antibody was analyzed using flow cytometry. Approximately 3.0×10 5 ~5.0×10 5 cells overexpressing FGFR3 or not expressing FGFR3 were incubated with the anti-FGFR3 antibody at 4°C for 1 hour. After washing twice with Flow Cytometry Staining Buffer, the cells were stained with Alexa Fluor 488 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.

[0252] The results of this analysis are shown in Figures 5 and 6.1. AMB-BT-0050T is a cell with positive FGFR3 expression, and AMB-BT-0013T is a cell with negative FGFR3 expression. AMB-BT-0050T and AMB-BT-0013T were collected from patients suffering from brain cancer (glioblastoma).

[0253] Figure 5 shows that antibody A9 has a significantly higher binding affinity for FGFR3-positive cells than for FGFR3-negative cells, indicating that the antibody is highly specific for FGFR3. Figure 6.1 shows that both A9 and A9Q bind to AMB-BT-0050T cells.

[0254] 2) The cell surface binding efficiency of ADC20 was analyzed using flow cytometry. 1.0×10 5Multiple myeloma KMS-11 cells with FGFR3 overexpression (JCRB, JCRB1178) were incubated with serial 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 antibody (Invitrogen, A-21445, Carlsbad, CA, USA) diluted 1:300 in staining buffer at 4°C for 30 minutes. Antibody A9Q and human IgG1 kappa isotype (CrownVivo, C0001) were used as controls. The mean fluorescence intensity was analyzed by flow cytometry.

[0255] The results of this analysis are shown in Figure 6.2, which indicates that ADC20 and antibody A9Q have similar and significantly high binding affinities for KMS-11.

[0256] (Example 6) In vitro cytotoxicity assay 1) A 3D single spheroid model was formed by isolating cancer cells derived from glioblastoma patients. Two types of patient-derived cells with FGFR3 overexpression (AMB-BT-0050T, AMB-BT-0112T) and cells derived from FGFR3 non-expression (AMB-BT-0013T) collected from patients with glioblastoma were incubated overnight to form single spheroids (3D), followed by treatment and incubation with ADC for 1 week, and then the spheroid size and volume were quantified. The results are shown in the following table, which indicates that for both conjugate ADC19 and ADC20, the cytotoxicity against FGFR3-positive cells is significantly higher than that against FGFR3-negative cells, thus indicating that ADC is highly specific for FGFR3.

[0257] [Table 6]

[0258] 2) A 3D single spheroid model was formed from the bladder cell line RT112 (DSMZ, ACC418) that overexpresses FGFR3. RT112 was seeded in a cell spheroid culture plate and incubated overnight to form single spheroids (3D), followed by treatment and incubation with ADC for 1 week, and then the spheroid size and volume were quantified. The results are shown in Table 6.2 (Table 7) below, which indicates that both conjugate ADC19 and ADC20 were significantly cytotoxic to FGFR3-positive bladder cancer cells.

[0259]

Table 7

[0260] (Example 7) In Vivo Efficacy Test in Glioblastoma PDX Model 1) To evaluate survival in an orthotopic mouse model of brain tumors with target expression, patient-derived cells from AMB-BT-0050T were subcultured and 2.0×10 5 cells were mixed with the medium. Seven-week-old female BALB / c nude mice were used for intracranial transplantation. The prepared patient-derived cells were injected into the mouse brain at a depth of 3.2 mm at a position 1.7 mm to the left and 0.5 mm above bregma by stereotactic intracranial injection. The mice were housed on a 12-hour light / 12-hour dark cycle and had free access to food and water. Therapeutic agent administration was as follows. TMZ (temozolomide) was injected 5 times daily via oral administration. ADC was administered to each group via intravenous injection once (single injection) or once a week for 4 weeks (multiple injections) starting on day 7 after model establishment. Mice were sacrificed when either 20% weight loss or neurological symptoms (somnolence, ataxia, and seizures) were observed, and the results are shown in Figure 7.1. Mouse survival was evaluated through MST (median survival time) and ILS (increase in life span); MST, the time point when the probability of survival is equal to 50%; ILS (increase in life span), ILS (%) = [(median survival time of the treatment group) / (median survival time of the control group) - 1] × 100.

[0261] The results indicate that the anti-FGFR3 ADC was able to inhibit the progression of brain tumors and prolong the survival time.

[0262] 2) To further evaluate the survival rate in an orthotopic mouse model of brain tumors with target expression, in the AMB-BT-0050T PDX model, 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. TMZ was infused three times daily via oral administration. The ADC was administered once via intravenous injection. The results are shown in Figure 7.2. The survival of mice was evaluated through MST and ILS.

[0263] The results indicate that both monotherapy with ADC20 and the combination of ADC20 with TMZ were able to inhibit the progression of brain tumors and prolong the survival time. The combination of ADC and TMZ (standard treatment for GBM) can significantly prolong the survival time.

[0264] (Example 8) In Vivo Efficacy Test in a Bladder Cancer CDX Model To evaluate the in vivo antitumor efficacy of ADC19 and ADC20 in mice bearing a bladder cancer FGFR3-high CDX model, several types of FGFR3-overexpressing bladder cancer CDX models were used.

[0265] 8.1) RT112 cells in the exponential growth stage were collected and counted for tumor inoculation. Using 0.2 mL of matrix gel buffer (PBS:Matrigel = 1:1), 10×10 6 cells were injected subcutaneously into the right flank of 6- to 8-week-old SPF female BALB / c nude mice.

[0266] The tumor diameter was measured with calipers, and the tumor volume was calculated according to the formula V = 0.5a × b 2 (where a is the major axis of the tumor and b is the minor axis of the tumor). The average tumor volume was approximately 100 - 300 mm3 When it became so, the mice were randomized into the vehicle group, the ADC19 5 mg / kg group, and the ADC20 5 mg / kg group. The day of the first administration was defined as day 0. The vehicle of the ADC drug was given to the mice in the vehicle group at the same frequency and route of administration. The tumor volumes of the mice in each group were measured twice a week. The experiment was terminated on day 27, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1 - (average tumor volume of the treatment group at the end day - average tumor volume of the treatment group on day 1) / (average tumor volume of the vehicle group at the end day - average tumor volume of the vehicle group on day 1)] × 100%.

[0267] Figure 8.1 shows the changes in tumor volume of tumor-bearing BALB / c nude mice treated with (1) vehicle; (2) ADC19 5 mg / kg, QW, 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 7.1 (Table 8) shows that at the end day (day 27), the average tumor volumes of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group were 48 mm 3 and 32 mm 3 respectively; and the TGI was 106.35% and 107.08% respectively, indicating that

[0268]

Table 8

[0269] The results indicate that both ADC19 and ADC20 have excellent antitumor efficacy in the FGFR3 overexpressing RT112 bladder cancer CDX model.

[0270] ​8.2) To evaluate the dose-dependent antitumor efficacy in bladder cancer, BALB / c nude mice bearing RT112 cells were randomized and grouped, and treated with solvent; ADC20 at a single dose of 8 mg / kg and ADC20 at 8 mg / kg, QW, for 2 times. The experiment was terminated on the 33rd 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 1st day) / (average tumor volume of the vehicle group on the end day - average tumor volume of the vehicle group on the 1st day)] × 100%. The ADC group was observed until the 84th day.

[0271] Figure 8.2 shows the changes in tumor volume of tumor-bearing BALB / c nude mice treated with (1) vehicle; (2) ADC20 at a single dose of 8 mg / kg; (3) ADC20 at 8 mg / kg, QW, for 2 times. Table 7.2 (Table 9) shows that on the end day (the 33rd day), the average tumor volumes of the ADC20 single-dose group and the ADC20 repeated-dose group were 36 mm 3 and 16 mm 3 respectively; the TGI was 106.34% and 107.65% respectively, and it was shown that a complete response (CR) was induced in the repeated-dose group.

[0272]

Table 9

[0273] The results indicate that both the single dose or repeated dose of ADC20 have excellent antitumor efficacy in the FGFR3-overexpressing RT112 bladder cancer CDX model, and the repeated dose of ADC20 induced CR in this bladder cancer CDX model.

[0274] 8.3) SW780 cells in the exponential growth stage were collected and counted for tumor inoculation. Using 0.2 mL of matrix gel buffer (PBS:Matrigel = 1:1), 10×10 6 cells were injected subcutaneously into the right flank of 6 - 8-week-old SPF female NOD SCID mice.

[0275] The tumor diameter was measured with calipers, and the tumor volume was calculated according to the formula V = 0.5a×b 2 When the average tumor volume reached approximately 100 - 300 mm 3 the mice were randomized into a vehicle group, an ADC19 5 mg / kg group, and an ADC20 5 mg / kg group. The first administration day was defined as day 0. The vehicle of the ADC drug was given to the mice in the vehicle group with the same frequency and administration route. The tumor volume of the mice in each group was measured twice a week. The experiment was terminated 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 day 1) / (average tumor volume of the vehicle group on the end day - average tumor volume of the vehicle group on day 1)]×100%.

[0276] Figure 8.3 shows the tumor volume changes of tumor-bearing NOD SCID mice treated with (1) vehicle; (2) ADC19 5 mg / kg, QW, 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 7.3 (Table 10) shows that on the end day (day 20), the average tumor volumes of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group were 1,109 mm 3 and 526 mm 3 respectively; and the TGI was 40.80% and 76.66% respectively.

[0277]

Table 10

[0278] The results indicate that both ADC19 and ADC20 have potential anti-tumor efficacy in the FGFR3 overexpressing SW780 bladder cancer CDX model.

[0279] 8.4) UM-UC1 cells in the exponential growth stage were collected and counted for tumor inoculation. Using 0.1 mL of matrix gel buffer (PBS:Matrigel = 1:1), 10×10 5 cells were injected subcutaneously into the right flank of 6- to 8-week-old SPF female Balb / c nude mice.

[0280] Tumor diameter was measured with calipers, and tumor volume was calculated according to the formula V = 0.5a × b 2 . When the average tumor volume reached approximately 100 - 300 mm 3 , the mice were randomized into a vehicle group and an ADC20 8 mg / kg group. The day of the first administration was defined as day 0. Mice in the vehicle group were given the solvent of the ADC drug at the same frequency and route of administration. The tumor volume of mice in each group was measured twice a week. The experiment was terminated 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 day 1) / (average tumor volume of the vehicle group on the end day - average tumor volume of the vehicle group on day 1)] × 100%.

[0281] Figure 8.4 shows the changes in tumor volume of tumor-bearing Balb / c nude mice treated with (1) vehicle; (2) ADC20 8 mg / kg, QW, 2 times. Table 7.4 (Table 11) shows that on the end day (day 17), the average tumor volume of the ADC20 8 mg / kg group was 402 mm 3 ; and the TGI was 82.71%.

[0282]

Table 11

[0283] The results indicate that ADC20 has antitumor efficacy in the FGFR3-overexpressing UM-UC1 bladder cancer CDX model.

[0284] (Example 9) In Vivo Efficacy Test in a Multiple Myeloma CDX Model To evaluate the in vivo antitumor efficacy of ADC19 and ADC20 in mice bearing a multiple myeloma FGFR3 overexpression CDX model, KMS11 cells in the exponential growth stage were collected and counted for tumor inoculation. Using 0.2 mL of matrix gel buffer (PBS:Matrigel = 1:1), 10×10 6 cells were injected subcutaneously into the right flank of 6 - 8 week-old SPF female CB17.SCID mice.

[0285] Tumor diameter was measured with calipers, and tumor volume was calculated according to the formula V = 0.5a×b 2 . When the average tumor volume reached approximately 100 - 300 mm 3 , the mice were randomized into a vehicle group, an ADC19 5 mg / kg group, and an ADC20 5 mg / kg group. The first administration day was defined as day 0. Mice in the vehicle group were given the solvent of the ADC drug with the same frequency and route of administration. The tumor volume of mice in each group was measured twice a week. The experiment was terminated 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 day 1) / (average tumor volume of the vehicle group on the end day - average tumor volume of the vehicle group on day 1)]×100%.

[0286] Figure 9 shows the tumor volume changes of tumor-bearing CB17.SCID mice treated with (1) vehicle; (2) ADC19 5 mg / kg, QW, 2 times; (3) ADC20 5 mg / kg, QW, 2 times. Table 8 (Table 12) shows that on the end day (day 32), the average tumor volumes of the ADC19 5 mg / kg group and the ADC20 5 mg / kg group were 9 mm 3 and 11 mm 3 respectively; the TGI was 108.41% and 108.31% respectively.

[0287]

Table 12

[0288] The results indicate that both ADC19 and ADC20 have excellent antitumor efficacy in the FGFR3-overexpressing KMS11 multiple myeloma CDX model.

[0289] (Example 10) Internalization activity of ADC Multiple myeloma cells KMS-11 (JCRB, JCRB1178) in good survival condition were treated with Accutase, collected, and suspended in serum-free RPMI medium. 1.0×10 5 cells were stained with the LIVE / DEAD™ Fixable Near-IR Dead Cell Stain dye diluted 1000-fold for 30 minutes in the dark at room temperature, washed twice with cold FACS buffer to remove excess dye, and the cells were incubated on ice for 30 minutes with the tested drug solution at a final concentration of 1 nM. After surface binding, the antibody-cell mixture was washed with cold FACS buffer and serum-free RPMI medium to remove excess antibody. Next, the cells bound to the antibody were incubated in a 37°C CO2 incubator for 0 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours respectively for antibody internalization. After incubation, the cells bound to the antibody were washed with Flow Cytometry Staining Buffer and fluorescently labeled with an ice-cold goat anti-human IgG cross-adsorbed secondary antibody conjugated with Alexa Fluor 488 diluted 1:300 in staining buffer (Invitrogen, A-11013, Carlsbad, CA, USA) for 30 minutes at 4°C. After fluorescent labeling, the antibody-cell mixture was washed again twice and analyzed by flow cytometry.

[0290] The MFI data was normalized to the MFI at 0 minutes defined as 100%. The results were analyzed by GraphPad Prism 9. As shown in Figure 10, ADC20 shows internalization activity equivalent to A9Q.

[0291] (Example 11) Bystander killing effect of ADC20 in RT112 / U87MG Far-Red-labeled FGFR3-positive RT112 cells and CFSE-labeled FGFR3-negative U87MG cells were seeded on 96-well round-bottom plates at 1.0×10 4 cells for each cell type. The cells were incubated overnight for stable adhesion to the plates, followed by treatment and incubation with ADC or PBS for one week. After incubation, the cells were centrifuged at 2300 rpm for 2 minutes, and then the supernatant was discarded. The cells were washed once with PBS, detached, and resuspended in flow cytometry staining buffer containing LIVE / DEAD staining dye. Finally, the total amounts of FGFR3-positive and FGFR3-negative cells and their viability were detected and analyzed by a flow cytometer (Novocyte, Agilent).

[0292] As shown in Figure 11, both ADC19 and ADC20 have a bystander killing effect, and ADC20 has a more effective bystander killing effect than ADC19.

[0293] (Example 12) In Vivo Efficacy Test in Glioblastoma PDX Model To evaluate the in vivo antitumor efficacy of ADC20 in mice bearing glioblastoma FGFR3-high PDX models, several types of FGFR3-overexpressing glioblastoma PDX models were used.

[0294] 1) AMB-BT-0039T cells (GBM, patient-derived cells) with FGFR3-TACC3 fusion in the exponential growth stage were collected and counted for tumor inoculation. Using 0.1 mL of matrix gel buffer (PBS:Matrigel = 1:1), 1×10 6 cells were injected subcutaneously into the right flank of 6-8-week-old SPF female BALB / c nude mice. The tumor diameter was measured with calipers, and the tumor volume was calculated according to the formula V = 0.5a×b 2 (where a is the major axis of the tumor and b is the minor axis of the tumor). The average tumor volume was about 100-300 mm 3When it became [a certain state], the mice were randomized into the vehicle group and the ADC20 8 mg / kg group. The first administration day was defined as day 0. The vehicle of the ADC drug was given to the mice in the vehicle group at the same frequency and administration route. The tumor volume of the mice in each group was measured twice a week. The experiment was terminated on day 24, 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 day 1) / (average tumor volume of the vehicle group on the end day - average tumor volume of the vehicle group on day 1)] × 100%.

[0295] Figure 12.1 shows the changes in tumor volume of tumor-bearing BALB / c nude mice treated with (1) vehicle; (2) ADC20 8 mg / kg, QW, 2 times. Table 9 (Table 13) shows that on the end day (day 24), the average tumor volume of the ADC20 8 mg / kg group was 100 mm 3 and the TGI was 100.03%.

[0296]

Table 13

[0297] 2) AMB-BT-0112T cells (GBM, patient-derived cells) having FGFR3-TACC3 fusion in the exponential growth stage were collected and counted for tumor inoculation. Using 0.1 mL of matrix gel buffer (PBS:Matrigel = 1:1), 1×10 6 cells were injected subcutaneously into the right flank of 6- to 8-week-old SPF female BALB / c nude mice.

[0298] The tumor diameter was measured with calipers, and the tumor volume was calculated according to the formula V = 0.5a × b 2 (where a is the long diameter of the tumor and b is the short diameter of the tumor). When the average tumor volume was about 100 - 300 mm 3When this was achieved, the mice were randomized into the vehicle group and the ADC20 8 mg / kg group. The first dosing day was defined as day 0. The vehicle of the ADC drug was given to the mice in the vehicle group at the same frequency and route of administration. The tumor volumes of the mice in each group were measured twice a week.

[0299] Figure 12.2 shows the tumor volume changes of tumor-bearing BALB / c nude mice treated with (1) vehicle; (2) ADC20 8 mg / kg, QW, twice. Table 10 (Table 14) shows that at the end day (day 33), the average tumor volume of the ADC20 8 mg / kg group was 216 mm 3 and the TGI was 95.08%.

[0300] [Table 14]

[0301] Acknowledgements This application is partially supported by the Korea Drug Development Fund, which was 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. Equation (I): 【Chemistry 1】 (In the formula, A is an anti-FGFR3 antibody or its antigen-binding fragment, and the antibody or antigen-binding fragment contains (Gly) in the compound of formula (I). n The antibody or antigen-binding fragment is connected to a portion and comprises: a heavy chain CDR1 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 1; a heavy chain CDR2 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 2 or SEQ ID NO: 2; a heavy chain CDR3 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 3 or SEQ ID NO: 3; a light chain CDR1 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO: 4; a light chain CDR2 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 5 or SEQ ID NO: 5; and a light chain CDR3 containing an amino acid sequence having 1 to 3 conserved amino acid substitutions compared to SEQ ID NO: 6 or SEQ ID NO: 6; z is an integer between 1 and 4; Each occurrence of opSu is independent. 【Chemistry 2】 and; R 0 is C 1～10 It is alkyl; n is any integer between 2 and 20; k1 and k2 are independent integers between 1 and 7; i is an integer between 1 and 100; j is an integer between 1 and 100; P1 and P2 are independent of each other, given by equation (II): 【Transformation 3】 (In the formula, a is either 0 or 1; The carbon atoms marked with p1* and p2* respectively are chiral centers; L1 is selected from C1-6 alkylenes, either unsubstituted or substituted with one substituent selected from halogens, -OH, and -NH2; M is -CH2-, -NH-, or -O-; L2 is C1-3 alkylene; R1 and R2 are independently selected from hydrogen, C1-6 alkyl groups, halogens, and C1-6 alkoxy groups. (A payload having the structure of...) An antibody-drug conjugate (ADC) having the following structure.

2. The aforementioned payload, 【Chemistry 4】 An antibody-drug conjugate according to claim 1, selected from the following. 【Request Item 3】 【Chemistry 5】 An antibody-drug conjugate according to claim 1, selected from the following.

4. The anti-FGFR3 antibody or its antigen-binding fragment comprises a VH domain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 7; and / or VL domain containing an amino acid sequence having at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 The antibody-drug conjugate according to claim 1, comprising:

5. The anti-FGFR3 antibody or its antigen-binding fragment comprises a heavy chain constant domain containing 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 Light chain constant domain containing an amino acid sequence having at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 11 The antibody-drug conjugate according to claim 1, comprising:

6. The anti-FGFR3 antibody or its antigen-binding fragment is a heavy chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 or 13; and / or A light chain containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

14. The antibody-drug conjugate according to claim 1, comprising:

7. The modified antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO: 15 and the light chain of SEQ ID NO: 16, and / or The modified antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO:

16. The amino acid sequence of each light chain of the anti-FGFR3 antibody or its antigen-binding fragment is modified by the deletion of GG in the LPETGG sequence, and the T in the resulting LPET sequence is linked by peptide bonds. 【Transformation 6】 It is connected to, thereby forming the amino acid sequence LPETG3. The antibody-drug conjugate according to claim 1.

8. Below formula: 【Transformation 7】 (In the equation, z is 2) The antibody-drug conjugate according to claim 1, having the following characteristics.

9. Each heavy chain of the anti-FGFR3 antibody or its antigen-binding fragment has the sequence of SEQ ID NO: 12, and each light chain of the anti-FGFR3 antibody or its antigen-binding fragment has the sequence of SEQ ID NO: 16, The amino acid sequence of each light chain of the anti-FGFR3 antibody or its antigen-binding fragment is modified by the deletion of GG in the LPETGG sequence, and the T in the resulting LPET sequence is linked by peptide bonds. 【Transformation 8】 It is connected to, thereby forming the amino acid sequence LPETG3. The antibody-drug conjugate according to claim 8.

10. Below formula: 【Chemistry 9】 (In the equation, z is 2) The antibody-drug conjugate according to claim 1, having the following characteristics.

11. Each heavy chain of the anti-FGFR3 antibody or its antigen-binding fragment has the sequence of SEQ ID NO: 12, and each light chain of the anti-FGFR3 antibody or its antigen-binding fragment has the sequence of SEQ ID NO: 16, The amino acid sequence of each light chain of the anti-FGFR3 antibody or its antigen-binding fragment is modified by the deletion of GG in the LPETGG sequence, and the T in the resulting LPET sequence is linked by peptide bonds. 【Chemistry 10】 It is connected to, thereby forming the amino acid sequence LPETG3. The antibody-drug conjugate according to claim 10.

12. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 11, and at least one pharmaceutically acceptable carrier.

13. A pharmaceutical composition according to claim 12 for treating a disease of which a tumor is present, wherein the tumor is selected from tumors that overexpress FGFR3 and tumors that have an FGFR3 gene mutation.

14. The pharmaceutical composition according to claim 13, wherein the disease is selected from brain cancer, bladder cancer, urothelial carcinoma, cervical cancer, multiple myeloma, and intrahepatic cholangiocarcinoma.

15. The pharmaceutical composition according to claim 13, wherein the disease is glioblastoma, bladder cancer, or multiple myeloma.

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