Antibody-drug conjugates, their production methods and medical uses

Abstract

Provided is an antibody-drug conjugate, as well as a production method and pharmaceutical use thereof. Specifically, the present invention relates to an anti-TROP-2 antibody-drug conjugate and pharmaceutical use thereof, the antibody-drug conjugate comprising an anti-TROP-2 antibody or an antigen-binding fragment thereof linked to an exatecan derivative via a linker, and the antibody-drug conjugate or a pharma- ceutical acceptable salt or solvate thereof has a remarkable antitumor effect and good safety.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-TROP-2 antibody conjugate and its medical use. [Background technology]

[0002] As genomic oncology, proteomics and signaling pathway studies are conducted in greater detail, the interactions between oncogenes and tumor inhibitor genes in tumor cells and their effects on the tumor microenvironment will become more clear, allowing the design of novel antitumor therapeutic regimens directed against specific molecular targets in tumors.

[0003] Molecular targeting therapy of tumor is a new treatment method different from traditional surgery, radiotherapy and chemotherapy, and has the advantage that drugs usually only bind to the corresponding target site, and directly affect the function of the target site molecule or affect the physical or chemical effector molecule carried to achieve the action of killing or inhibiting target cells.Because the target site is clear, this kind of drug usually has very high selectivity, can effectively kill or inhibit target cells, and has no or little toxic side effects on normal tissue cells.Therefore, the development of molecular targeting drugs has become the focus of tumor clinical research.

[0004] Human trophoblast cell surface antigen 2 (TROP-2) is a cell surface glycoprotein encoded by the TACSTD2 gene. TROP-2 consists of 323 amino acids, including 26 amino acids in the signal peptide, 248 amino acids in the extracellular domain, 23 amino acids in the transmembrane domain, and 26 amino acids in the cytoplasmic domain. The TROP-2 extracellular domain contains four heterogeneous N-linked glycosylation sites, and the apparent molecular weight increases by 11-13 KD after glycosylation. In the TACSTD gene family, the extracellular domain has a characteristic thyroglobulin (TY) sequence, which is usually thought to be involved in the proliferation, invasion, and metastasis of cancer cells.

[0005] A large amount of clinical studies and literature reports have revealed that TROP-2 is overexpressed in multiple epithelial cancers, including gastric cancer, lung cancer, colon cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer. In contrast, TROP-2 is weakly or not expressed in normal adult tissues, and is expressed in small amounts only in cells in the epithelial region, and the expression level is lower than that in carcinomas, suggesting that TROP-2 is involved in tumor formation. Overexpression of TROP-2 in tumor tissues is closely related to poor prognosis and metastasis of cancer cells, and affects the overall survival rate of patients. Therefore, TROP-2 has become a target that has attracted attention in tumor molecular targeting therapy.

[0006] Several studies have been reported on the antitumor effects of anti-hTROP-2 antibodies: US Patent No. 5,840,854 reported the cytotoxicity of an anti-hTROP-2 monoclonal antibody (BR110) conjugated to a cytotoxin against human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.

[0007] U.S. Patent No. 6,653,104 discloses an antibody (RS7), which was tested in an in vivo model using a radioactively labeled antibody and showed antitumor activity in a nude mouse xenograft model, but no antitumor effect was reported for the naked antibody alone.

[0008] U.S. Patent No. 7,420,040 further reported that isolated monoclonal antibodies produced by hybridoma cell lines AR47A6.4.2 or AR52A301.5 obtained from mice immunized with human ovarian cancer tissue bind to hTROP-2 and exhibit anti-tumor activity in nude mouse xenograft models.

[0009] CN102827282A discloses a human-derived anti-TROP-2 genetically engineered antibody IgG and its application, and the results of in vitro tests show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

[0010] CN104114580A discloses an antibody (particularly a humanized antibody) that specifically reacts with hTROP-2 and has anti-tumor activity in vivo, as well as a hybridoma producing the antibody, a conjugate of the antibody with a drug, a pharmaceutical composition for diagnosing or treating tumors, a method for detecting tumors, and a kit for detecting or diagnosing tumors.

[0011] However, it is difficult to find monoclonal antibodies with high affinity, high specificity and strong cytotoxicity or tumor killing / inhibiting / regression activity, so there is still a need to develop Trop-2 antibodies and other immunotherapeutic agents that have excellent therapeutic effects, are safe, and suitable for human patients. Summary of the Invention

[0012] An object of the present invention is to provide an antibody-drug conjugate represented by general formula (I) or a pharma- ceutically acceptable salt or solvate thereof: [ka] Where: L is -(CR1 R 2 ) m -[X1-(CR 1 R 2 ) n- X2] t -(CR 1 R 2 ) r - and R 1 or R 2 are each independently selected from hydrogen, deuterium, a hydroxy group, an amino group, an alkyl group, a halogen, a halogenated alkyl group, a deuterated alkyl group, or a hydroxyalkyl group, and preferably R 1 or R 2 is hydrogen, X1 or X2 are each independently selected from a bond, N, O or S, preferably X1 or X2 are a bond or O; m, n, r or t are each independently selected from 1, 2, 3 or 4, preferably m, n, r or t are each independently selected from 1 or 2; R 3 or R 4 are each independently selected from hydrogen, halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclo group, an aryl group, or a heteroaryl group; Or R 3 and R 4 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclo group, y is 1 to 20, preferably 1 to 10, more preferably 2 to 8, and further preferably 4, 6, or 8; The mAb is an anti-TROP-2 antibody or an antigen-binding fragment thereof.

[0013] In a preferred embodiment of the present invention, R 1 or R 2 are each independently hydrogen, deuterium, a hydroxyl group, an amino group, or C 1-3 Alkyl groups, halogens, C 1-3 Halogenated alkyl groups, C 1-3 Deuterated alkyl group or C 1-3hydroxyalkyl groups, preferably R 1 or R 2 is hydrogen, X1 or X2 are each independently selected from a bond, N, O or S, preferably X1 or X2 are a bond or O; m, n or r are each independently 1, 2, 3 or 4, preferably m, n or r are each independently selected from 1 or 2; R 3 or R 4 are each independently hydrogen, halogen, or C 1-3 Halogenated alkyl groups, C 1-3 Deuterated alkyl groups, C 3-6 Cycloalkyl groups, 4-8 membered heterocyclo groups, C 5-10 aryl group or 4- to 8-membered heteroaryl group, preferably R 3 or R 4 are each independently hydrogen or C 3-6 cycloalkyl groups, Or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 It forms a cycloalkyl group or a 4- to 8-membered heterocyclo group.

[0014] In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises an HCDR1 as shown in SEQ ID NO:3, an HCDR2 as shown in RIDPXDSETHYNQKFKD, and an HCDR3 as shown in SEQ ID NO:5, and an LCDR1 as shown in SEQ ID NO:6, an LCDR2 as shown in SEQ ID NO:7, and an LCDR3 as shown in SEQ ID NO:8; X is selected from R, Y, Q, L, T, I, F, E or A.

[0015] In a preferred embodiment of the invention, the TROP-2 antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:9 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:10 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:12 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:13 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:14 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:15 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:16 and HCDR3 shown in SEQ ID NO:5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8, or It includes an HCDR1 shown in SEQ ID NO:3, an HCDR2 shown in SEQ ID NO:17, and an HCDR3 shown in SEQ ID NO:5, as well as an LCDR1 shown in SEQ ID NO:6, an LCDR2 shown in SEQ ID NO:7, and an LCDR3 shown in SEQ ID NO:8.

[0016] In a preferred embodiment of the present invention, in the antibody-drug conjugate according to the present invention or a pharma- ceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from a mouse-derived antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

[0017] In a preferred embodiment of the present invention, in the antibody-drug conjugate according to the present invention, or a pharma- ceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4, or a mutant thereof.

[0018] In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4 or a mutant thereof.

[0019] In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:1.

[0020] In a preferred embodiment of the present invention, in the antibody-drug conjugate according to the present invention or a pharma- ceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain or λ chain, or a mutant thereof.

[0021] In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain, In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:2.

[0022] In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from those set forth in the sequences SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

[0023] In a preferred embodiment of the invention, the antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof according to the invention, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:19, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

[0024] In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises: A heavy chain variable region as set forth in SEQ ID NO:18 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:20 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:21 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:22 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:23 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:24 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:25 and a light chain variable region as set forth in SEQ ID NO:19; or A heavy chain variable region as set forth in SEQ ID NO:26 and a light chain variable region as set forth in SEQ ID NO:19; or It comprises a heavy chain variable region shown in SEQ ID NO:27 and a light chain variable region shown in SEQ ID NO:19.

[0025] In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain selected from those set forth in the sequence SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0026] In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a light chain selected from those set forth in the following sequences: the light chain set forth in SEQ ID NO:29, or one having 80%, 85%, 90%, 95% or 99% identity thereto.

[0027] In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises: A heavy chain as set forth in SEQ ID NO:28 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:30 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:31 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:32 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:33 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:34 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:35 and a light chain as set forth in SEQ ID NO:29; or A heavy chain as set forth in SEQ ID NO:36 and a light chain as set forth in SEQ ID NO:29; or It comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:29.

[0028] In a preferred embodiment of the present invention, the antibody drug conjugate or a pharma- ceutically acceptable salt or solvate thereof is selected from the antibody drug conjugate shown in general formula (II) or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer, mesomeric, racemic, enantiomer, diastereomer, or mixture form thereof: [ka] X1 or X2 is a bond or O; m is 0 or 1, and t is 1 or 2.

[0029] In a preferred embodiment of the present invention, the antibody drug conjugate or a pharma- ceutically acceptable salt or solvate thereof is selected from the antibody drug conjugate shown in general formula (III) or a pharma- ceutically acceptable salt or solvate thereof: [ka] the mAb is selected from the anti-TROP-2 antibodies or antigen-binding fragments thereof described above, y is selected from 2 to 10, preferably 4 to 10, and more preferably 4, 6, 8 or 10.

[0030] In a preferred embodiment of the invention, the antibody drug conjugate or a pharma- ceutically acceptable salt or solvate thereof is selected from the following structures: [ka] [ka] [ka] [ka] [ka] where y is as defined in claim 1.

[0031] The present invention further provides a method for producing an antibody-drug conjugate represented by general formula (I) or a pharma- ceutically acceptable salt or solvate thereof, which comprises: [ka] The method includes the steps of reducing the mAb and then conjugating it with a compound represented by general formula (F) to obtain a compound represented by general formula (I), where L is as defined above.

[0032] the mAb is selected from the anti-TROP-2 antibodies or antigen-binding fragments thereof described above, y is an integer of 1 to 20, preferably 4 to 10, and more preferably 4, 6, 8 or 10.

[0033] In another aspect, the present invention provides a pharmaceutical composition, which comprises an antibody-drug conjugate according to the present invention or a pharma- ceutically acceptable salt or solvate of said antibody-drug conjugate, and one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0034] In another aspect, the present invention provides a pharmaceutical use, the present invention relates to the use of an anti-TROP-2 antibody drug conjugate or a pharma- ceutically acceptable salt or solvate of said antibody drug conjugate, or a pharmaceutical composition thereof, for treating or preventing a TROP-2 mediated disease or condition.

[0035] In another aspect, the present invention further provides an application of the antibody-drug conjugate according to general formula (I) or a pharma- ceutically acceptable salt or solvate of said antibody-drug conjugate, or a pharmaceutical composition thereof, in the manufacture of a drug for treating a disease associated with human TROP-2.

[0036] In a more preferred embodiment of the present invention, the disease associated with human TROP-2 is a cancer in which TROP-2 is highly expressed, and the cancer is selected from the group consisting of triple-negative breast cancer, small cell lung cancer, urothelial carcinoma, human cerebral astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrodysplasia bone, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, and gestational cancer. The cancer is selected from gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma, malignant fatty tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

[0037] The antibody-drug conjugate of the present invention or a pharma- ceutically acceptable salt or solvate thereof can specifically bind to a target antigen, has high endocytosis efficiency, a long in vivo half-life, ensures safety, and exhibits significant tumor killing.

[0038] The antibody-drug conjugate of the present invention and its pharma- ceutically acceptable salt or solvate have remarkable antitumor effects and good safety, as well as good metabolic activity in the body, a long duration of efficacy in the body, and a broad future of clinical application.

[0039] 1. Terminology In order to make the present invention more readily understandable, certain technical and scientific terms are specifically defined below. All other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art of the present invention, unless otherwise clearly and explicitly defined elsewhere in this specification.

[0040] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0041] The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked via interchain disulfide bonds. The immunoglobulin heavy chain constant regions differ in amino acid composition and sequence, and therefore in their antigenicity. This allows immunoglobulins to be divided into five classes, or may be called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Igs of the same class may be divided into various subclasses depending on the amino acid composition of their hinge regions and the number and position of the heavy chain disulfide bonds, for example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are divided into κ or λ chains depending on the constant region. Each of the five classes of Igs may have either κ or λ chains.

[0042] In the present invention, the antibody light chain variable region may further comprise a light chain constant region, and the light chain constant region comprises a κ or λ chain or a mutant thereof derived from human or mouse.

[0043] In the present invention, the antibody heavy chain variable region may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or mouse-derived IgG1, IgG2, IgG3, IgG4, or a mutant thereof.

[0044] Approximately 110 amino acids near the N-terminus of antibody heavy and light chains are variable regions (V regions) with large sequence variation, while other amino acids near the C-terminus are constant regions (C regions) with relatively stable sequences. The variable regions include three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conservative sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments according to the invention are numbered and positioned according to the known Kabat numbering conventions and Kabat or ABM definition conventions (http: / / bioinf.org.uk / abs / ).

[0045] The term "TROP-2" includes any variant or isotype of TROP-2 naturally expressed by cells. The antibodies of the invention can cross-react with TROP-2 obtained from non-human species. Alternatively, the antibodies may be specific for human TROP-2 and may not show cross-reactivity with other species. TROP-2 or any variant or isotype thereof may be isolated from cells or tissues that naturally express them or may be produced by recombinant techniques using techniques common in the art and those techniques described herein. Preferably, the anti-TROP-2 antibody targets human-derived TROP-2 with a normal glycosylation mode.

[0046] The term "recombinant human antibody" includes human antibodies that are produced, expressed, generated or isolated by recombinant methods, such techniques and methods being well known in the art, e.g., 1. Antibodies isolated from transgenic, transchromosomal animals (e.g., mice) containing human immunoglobulin genes or hybridomas produced therefrom; 2. Host cells transformed to express an antibody, e.g., an antibody isolated from a transfectoma; 3. Antibodies isolated from recombinant combinatorial human antibody libraries, and 4. Antibodies produced, expressed, generated or isolated by any process, such as by splicing human immunoglobulin gene sequences into other DNA sequences.

[0047] Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also contain subsequent rearrangements and mutations, such as those that occur during antibody maturation.

[0048] The term "mouse-derived antibody" in the present invention refers to a monoclonal antibody against human TROP-2 produced based on the knowledge and skill in the art. During production, a TROP-2 antigen is injected into a test subject and a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, said mouse TROP-2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa, lambda chain or a mutant thereof, or may further comprise a heavy chain constant region of a mouse-derived IgG1, IgG2, IgG3 or IgG4 or a mutant thereof.

[0049] The term "human antibody" includes antibodies having variable and constant regions with human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific induction in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0050] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto a human antibody variable region framework. Humanized antibodies can overcome the drawback of chimeric antibodies that induce strong immune response reactions due to the large amount of mouse protein components they carry. To avoid reduced immunogenicity and reduced activity, minimal back mutations may be made to the human antibody variable region to maintain activity.

[0051] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody, and can reduce the immune response induced by mouse-derived antibodies. To establish a chimeric antibody, first, a hybridoma secreting a specific mouse-derived monoclonal antibody is established, and then the variable region gene is cloned from the mouse hybridoma cell, and if necessary, the constant region gene of a human antibody is cloned, and the mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector, and finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody may be selected from the heavy chain constant region of human-derived IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and preferably includes the heavy chain constant region of human-derived IgG1, IgG2, or IgG4, or an IgG1 heavy chain constant region with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity using amino acid mutations.

[0052] The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which usually contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Usually, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the parental binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parent antibody's binding affinity for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

[0053] The term "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0054] The term "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of an antibody, which are held together by two or more disulfide bonds via the hydrophobic interaction of the CH3 domain.

[0055] The term "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0056] The term "F(ab')2 fragment" contains two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments held together via disulfide bonds between the two heavy chains.

[0057] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0058] The term "multispecific antibodies" is used in the broadest sense to cover antibodies with specificity for multiple epitopes, including, but not limited to, antibodies comprising a heavy chain variable domain VH and a light chain variable domain VL, the VH-VL unit having specificity for multiple epitopes, antibodies with two or more VL and VH domains, each VH-VL unit binding to a different target or a different epitope on the same target, antibodies with two or more single variable domains, each single variable domain binding to a different target or a different epitope on the same target, full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments linked together covalently or non-covalently, etc.

[0059] The term "single-chain antibody" refers to a single-chain recombinant protein consisting of the heavy chain variable region VH and the light chain variable region VL of an antibody linked via a connecting peptide segment, and is the minimum antibody fragment having a complete antigen-binding site.

[0060] The term "domain antibody fragment" refers to an immunologically functional immunoglobulin fragment that contains only a heavy or light variable region chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

[0061] The term "binds to TROP-2" refers to the ability to interact with human TROP-2.

[0062] The term "antigen-binding site" refers to the three dimensional spatial site recognized by an antibody or antigen-binding fragment of the invention.

[0063] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes may be formed from adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed with adjacent amino acids are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost after denaturing solvent treatment. Epitopes usually contain at least 3-15 amino acids in a unique conformation. Methods for determining which epitope a given antibody binds are well known in the art and include immunoblot and immunoprecipitation detection assays. Methods for determining the conformation of an epitope include techniques in the art and described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance.

[0064] The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. Typically, when human TROP-2 is used as the analyte and an antibody is used as the ligand, and the antibody binds to an epitope on an antigen by surface plasmon resonance (SPR) technology, the antibody binds to an epitope on an antigen by about 10 -7 The equilibrium dissociation constant (K D ) and the affinity with which it binds to the predetermined antigen is at least twice the affinity with which it binds to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, etc.). The term "antibody that recognizes an antigen" is used interchangeably herein with the term "antibody that specifically binds."

[0065] "Cross-reactivity" refers to the ability of an antibody of the invention to bind to TROP-2 from a different species. For example, an antibody of the invention that binds to human TROP-2 can also bind to TROP-2 from another species. Cross-reactivity is measured by specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or by binding or functional interaction with cells that physiologically express TROP-2. Methods for determining cross-reactivity include standard binding assays described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0066] The terms "inhibition" or "blocking" are used interchangeably and cover both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-TROP-2 antibody compared to a ligand not contacted with an anti-TROP-2 antibody.

[0067] "Growth inhibition" (eg, with respect to a cell) is intended to include any measurable decrease in cell proliferation.

[0068] "Immune response induction" and "immune response enhancement" are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a particular antigen. The term "induction" as opposed to CDC or ADCC induction refers to the stimulation of a specific direct cell killing mechanism.

[0069] "ADCC" or antibody-dependent cell-mediated cytotoxicity refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors, via the Fc segment that recognizes the antibody. Modifications, enhancements or reductions to the Fc segment on IgG reduce or eliminate the ADCC effector function of the antibody. The modifications refer to mutations in the heavy chain constant region of the antibody.

[0070] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the art, such as Cold Spring's Antibody Laboratory Technical Guidelines, Chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or a fragment thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods. Antigen-binding fragments can likewise be produced by conventional methods. The antibodies or antigen-binding fragments described in the invention use genetic engineering methods to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from Immunoglobulin Magazine, 2001 ISBN 012441351.

[0071] The engineered antibody or antigen-binding fragment of the present invention can be produced and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended conventional technique, the mammalian expression system causes glycosylation of the antibody, especially at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing an antibody that specifically binds to a human-derived antigen. Positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture fluid secreting the antibody can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can also be removed by conventional methods, e.g., molecular sieves, ion exchange. The resulting product must be immediately frozen, e.g., at -70°C, or lyophilized.

[0072] The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) according to the present invention refers to an antibody obtained from a single clonal cell line, which is not limited to a eukaryotic, prokaryotic or phage clonal cell line. The monoclonal antibody or antigen-binding fragment can be recombinantly obtained using hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or other conventional technology.

[0073] The terms "administration", "giving" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid. "Administration", "giving" and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of a reagent with a cell and contact of a reagent with a body fluid, where the body fluid contacts the cell. "Administration", "giving" and "treatment" are further intended to refer to in vitro and ex vivo treatment of, for example, a cell with a reagent, diagnostic, binding composition or another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0074] The term "treatment" refers to the internal or external administration of a therapeutic agent, such as any one of the antibodies of the present invention, to a patient having one or more disease symptoms, the therapeutic agent being known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the patient or population being treated in an amount that effectively relieves one or more disease symptoms, by inducing regression of such symptoms or inhibiting the progression of such symptoms to a clinically measurable extent. The amount of the therapeutic agent that effectively relieves any specific disease symptom (also called a "therapeutically effective amount") may vary depending on several factors, such as the disease state, age and weight of the patient, and the ability of the drug to elicit the required therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical detection method that a physician or other specialized medical practitioner would normally use to evaluate the severity or progression of the condition. It is possible that an embodiment of the present invention (e.g., a method of treatment or product) may be ineffective in alleviating the target disease symptoms suffered by any patient, but may be determined based on any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, that it should alleviate the target disease symptoms in a statistically significant number of patients.

[0075] The term "consisting essentially of" or variations thereof, as used throughout the specification and claims, refers to the inclusion of all the elements or groups of elements, and optionally, other elements that are similar or different in nature to the elements, which do not significantly alter the basic or novel characteristics of a given dosage regimen, method, or composition.

[0076] The term "naturally occurring" as applied to an object according to the present invention refers to the fact that the object can be found in nature. For example, a polypeptide sequence or a polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified in a laboratory by man, is naturally occurring.

[0077] The term "effective amount" encompasses an amount that is sufficient to ameliorate or prevent a symptom or sign of a medical condition. Effective amount also refers to an amount that is sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or administration protocol that avoids significant side effects or toxic effects.

[0078] The term "exogenous" refers to a material that is produced outside an organism, cell, or body, as appropriate.

[0079] The term "endogenous" refers to a substance that is produced inside a cell, organism, or body, as the context requires.

[0080] The term "homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, if, in optimal alignment of sequences, 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. Generally, comparison is performed when two sequences are aligned to obtain the maximum percentage of homology.

[0081] The terms "cell," "cell line," and "cell culture" are interchangeable, and all such designations include their progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that not all progeny may be exactly identical in DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened from the originally transformed cell are included. "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, and the description includes cases where the event or circumstances have or have not occurred. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but do not necessarily, be present.

[0082] The term "pharmaceutical composition" is meant to contain one or more antibodies or antigen-binding fragments thereof described herein, as well as other components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism and facilitate absorption of the active ingredient(s), thereby exerting a biological activity.

[0083] The term "pharmaceutical acceptable salt" refers to a salt of the antibody-drug conjugate of the present invention, which is safe and effective when used in a mammalian body and has the desired biological activity. The antibody-drug conjugate of the present invention contains at least one amino group, and therefore can form a salt with an acid, and non-limiting examples of pharmaceutical acceptable salts include hydrochloride, hydrobromide, hydrofluoric acid, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbic acid, oxalate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0084] The term "solvate" refers to an antibody-drug conjugate compound of the invention that forms a pharma-ceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, and ethyl acetate.

[0085] The term "cytotoxic agent" as used herein refers to a substance that inhibits the function of cells and / or causes cell death or destruction.

[0086] The term "microtubulin inhibitor" refers to a class of compounds that exert an antitumor effect by disrupting the cell mitotic process by inhibiting the polymerization of microtubulin or promoting the assembly of microtubulin. Non-limiting examples include maytansines, calicheamicin, taxanes, vincristine, colchicine, dolastatins / auristatins / monomethylauristatin E (MMAE) / monomethylauristatin F (MMAF).

[0087] The term "linker" refers to a chemical module comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug. Non-limiting examples of linkers include arylene groups, heteroarylene groups, PEG, polymethyleneoxy groups, succinate esters, succinamides, diglycolate esters, malonates, and hexanamides.

[0088] The term "drug antibody ratio" (DAR) is represented by y, i.e., the average cytotoxic drug number for each antibody in general formula (A). The drug antibody ratio range in the present invention may be 1-20 cytotoxic drugs (D) per antibody. The antibody-drug conjugate of general formula (A) is a collection of antibodies to which a range (1-20) of cytotoxic drugs are conjugated. The drug antibody ratio (DAR) in the antibody-drug conjugate from the conjugation reaction can be characterized by common means, such as mass spectrometry, HPLC, and ELISA. These means allow the quantitative distribution of the y value of the antibody-drug conjugate to be measured.

[0089] The present invention further includes various deuterated forms of the compound of formula (I). Each available hydrogen atom connected to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art can synthesize the deuterated forms of the compound of formula (I) by referring to relevant literature. When preparing the deuterated forms of the compound of formula (I), commercially available deuterated starting materials can be used, or they can be synthesized using conventional techniques with deuterated reagents, including but not limited to deuterated borane, tritium borane tetrahydrofuran solution, deuterated lithium aluminum, deuterated iodoethane and deuterated iodomethane.

[0090] In some embodiments of the invention, the cytotoxic drug is conjugated to the N-terminal amino group and / or the ε-amino group of a lysine residue of the ligand via a linking unit, and in other embodiments of the invention, the cytotoxic drug is conjugated to a mercapto group of the ligand via a linking unit. Generally, the number of drug molecules that can be conjugated to an antibody in a conjugation reaction will be less than the theoretical maximum.

[0091] The loading of the ligand-cytotoxic drug conjugate can be controlled using the following non-limiting methods: (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling reaction time and temperature; (3) Selecting different reaction reagents.

[0092] The antibody-drug conjugate of the present invention or a pharma- ceutically acceptable salt or solvate thereof has a remarkable antitumor effect and good safety. [Brief description of the drawings]

[0093] [Figure 1] 1H-NMR chart of compound D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] The present invention will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the examples of the present invention are generally performed according to conventional conditions, such as Cold Spring's antibody technology experimental guidelines, molecular cloning guidelines, or according to the conditions suggested by the manufacturers of raw materials or products. Reagents for which no specific source is specified are common reagents purchased on the market. Example 1: HU6DL mutant design experiment

[0095] The CDR sequences of the heavy and light chains of the humanized antibody HU6DL disclosed in Patent WO 2020228604 are shown in Table 1 below, and HCDR2 contains N 54 D 55 S 56 The presence of this motif makes it prone to glycosylation.

[0096] [Table 1]

[0097] Site-directed mutation of N54 was performed by computer-aided techniques to reduce potential glycosylation risk without affecting its antigen binding and its thermal stability, resulting in mutants of antibody HU6DL, HU6DL.R54, HU6DL.Y54, HU6DL.Q54, HU6DL.L54 HU6DL.T54, HU6DL.I54, HU6DL.F54, HU6DL.E54 and HU6DL.A54, with the corresponding heavy chain HCDR2 sequences as follows:

[0098] [Table 2]

[0099] The corresponding heavy and light chain variable regions are as follows: [ka] [ka] [ka]

[0100] The designed heavy and light chain variable region sequences were linked to IgG1 heavy chain constant region and light chain constant region sequences, respectively, to obtain the linked human IgG1 heavy chain constant region sequence: [ka]

[0101] The concatenated human kappa chain constant region sequences are as follows: [ka]

[0102] After ligation, the resulting exemplary heavy and light chain sequences are as follows: [ka] [ka] [ka] [ka] [ka]

[0103] cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each of the above humanized antibodies, and the mutants expressing HU6DL protein were transiently transfected into HEK293 cells. The purity of the antibodies was detected by molecular exclusion chromatography technology, and the concentration and purity are shown in Table 3 below.

[0104] [Table 3] Example 2: Affinity study of HU6DL mutants for Trop-2 antigen

[0105] Purpose of the Test: The ELISA sandwich, ie "antigen-antibody-HRP-labeled secondary antibody", evaluates the differences in affinity levels of the different anti-Trop-2 mutants to the Trop-2 antigen.

[0106] Experimental steps: Trop-2, his-tag protein (SinoBiologics, Cat:10428-H08H) was diluted to 1ug / mL in DPBS pH7.4, and added to a high affinity 96-well plate (Corning, Cat:3590) at 100 μL / well, incubated at 4℃ overnight, and the next day, the Trop-2 antigen solution was shaken off and added to PBS pH7.4 (PBST) solution containing 0.05% Tween 20 at 200 μL / well, washed 3 times, blocked with 200 μL / well of 2% BSA (dissolved in PBST) at 37℃ for 1 h, washed 3 times with PBST, and then incubated in a 10x, 8x gradient (starting concentration 10 nM-1x10 -6 Candidate antibodies diluted in 0.5% BSA as negative control were added at 100 μL / well, blocked for 1 h at 37°C, washed 3 times with PBST, and secondary antibody solution of sheep anti-human IgG, Fc-HRP (abcam, cat:ab97225) diluted at 1:10000 was added at 100 μL / well, blocked for 1 h at 37°C, washed 3 times with PBST. TMB (CST, Cat: 7004P6) substrate was added at 100 μL / well and incubated at room temperature for 3 min until the solution in the well with the highest antibody concentration turned deep blue. Stop solution (CST, Cat: 7002P6) was added at 50 μL / well to stop the reaction, and the optical density (OD) was read at 450 nm.

[0107] Data Processing: The EC values ​​of the affinity of each HU6DL mutant for the antigen were calculated using the logarithm of the concentration of the candidate antibody as the x-axis coordinate and the OD450 absorbance value as the y-axis coordinate, using the GraphPad PRISM 8.0 log(agonist) vs. response-viable slope(four parameters) equation. 50 The affinity of the HU6DL mutant for the human TROP-2 antigen (EC 50 ) are as shown in Table 4 below,

[0108] [Table 4]

[0109] Test conclusion: The above data demonstrate that the HU6DL mutant of the present invention has good affinity for the human TROP-2 antigen. Example 3: Experiment on affinity of HU6DL mutant to tumor cells

[0110] Purpose of the Test: The differences in affinity levels of HU6DL mutants for tumor cell lines expressing the Trop-2 antigen are assessed by flow cytometry.

[0111] Experimental Reagents: Gastric cancer cells NCI-N87 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCHu130); Non-small cell lung cancer cells HCC827 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCHu153); Bladder cancer cells SW780 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCHu219); Bladder cancer cells RT4 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCHu226);

[0112] Experimental steps: Tumor cells in good growth condition were digested with Accutase (Sigma, cat: A6964) digestion solution to prepare a single cell suspension in 2% FBS (diluted with DBPS, pH 7.4) solution, and the cell density was adjusted to 1x10 6The solution was adjusted to 1000 nM-1x10 cells / mL. 100 μL / well was evenly distributed into a 96-well V-bottom plate, centrifuged at 300 g × 5 min, 4°C, and the supernatant was discarded. -6 Add 100 μL / well of candidate antibody solution diluted with 0.01 nM and incubate at 4°C for 1 h. Centrifuge at 300g for 5 min at 4°C, wash twice, and add 5 μL / 10 6 A solution of mouse anti-human IgG Fc, PE-labeled secondary antibody (Biolegend, cat:409304) diluted with the cell ratio was added at 100 μL / well, incubated at 4°C for 1 h, centrifuged at 300g×5 min at 4°C, washed twice, and added 70 μL 2% FBS solution to resuspend the cells, and the mean fluorescence intensity (MFI) of the PE channel was detected with a ZE5 flow cytometer (Bio-Rad, ZE5).

[0113] Data Processing: The EC50 of affinity of each candidate antibody for tumor cells was calculated using the log(agonist) vs. response-viable slope(four parameters) equation in GraphPad PRISM 8.0 with the log(agonist) vs. response-viable slope(four parameters) of mutant antibody concentration as the x-axis coordinate and MFI as the y-axis coordinate. 50 was calculated and is shown in Table 5 below:

[0114] [Table 5]

[0115] Test conclusion: The above data demonstrated that the HU6DL mutant of the present invention has good affinity for all of NCI-N87, HCC827, SW780 and RT4 tumor cells. Example 4: Experiments on HU6DL mutant-mediated TROP2 endocytosis

[0116] Purpose of the Test: The antibody endocytosis activity of the HU6DL mutant in tumor cell lines expressing the Trop-2 antigen is evaluated by flow cytometry.

[0117] Experimental steps: Gastric cancer cells NCI-N87 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCHu130) in good growth condition were digested with Accutase (Sigma, cat:A6964) digestion solution, and a single cell suspension was prepared in 2% FBS (diluted with DBPS, pH 7.4) solution. The cell density was adjusted to 1x10 7 The cells were adjusted to 100 μL / mL. 100 μL / well of the candidate antibody solution was evenly distributed into a 96-well V-bottom plate, with a final concentration of 20 μg / mL added, mixed evenly, and incubated at 4°C for 1 h. Centrifuge at 300g for 5 min at 4℃ and extract 5 μL / 10 6 Add 100 μL / well of mouse anti-human IgG Fc, PE-labeled secondary antibody (Biolegend, cat:409304) diluted to the cell ratio and incubate at 4°C for 1 h. Centrifuge at 300g for 5 min at 4°C, wash twice, resuspend the cell pellet in 1 mL of pre-warmed complete medium, divide into 4 equal parts, and name them as 0 min, blank, 30 min, and 120 min groups. Remove 0 min and blank and place on ice, and place other groups in a 37°C incubator. Perform endocytosis for 30 min and 120 min, respectively. Remove the corresponding groups at the appropriate time points and place on ice for 5 min to pre-cool. Centrifuge all treatment groups, discard the supernatant (4°C, 1500 rpm x 5 min), wash once with FACS buffer, discard the supernatant, add 250 μL of strip buffer to all treatment groups except the 0 min group, incubate at room temperature for 8 min, centrifuge and discard the supernatant (4°C, 1500 rpm x 5 min), wash twice with FACS buffer, remove the supernatant, and add 80 μL of strip buffer to each group. The cells were resuspended by adding μL of 2% FBS, and the fluorescent signals of the samples to be detected were detected using a ZE5 flow cytometer (Bio-Rad, ZE5).

[0118] Data Processing: Calculate the endocytosis efficiency of each candidate antibody according to this formula:

number

[0119] [Table 6]

[0120] Test conclusion: The above data demonstrated that the HU6DL mutant-mediated TROP-2 protein of the present invention has good endocytosis activity in gastric cancer cells NCI-N87. Example 5: Detection of HU6DL mutant impurities

[0121] Experimental Objective: To detect and compare the content and change level of impurity peaks in the antibody after mutation using capillary electrophoresis equipment based on Maurice-nrCE-SDS method.

[0122] Experimental steps 1. Sample preparation: (1) Liquid exchange and concentration of the sample: If the protein concentration of the sample is lower than 5 mg / ml or the salt concentration of the sample buffer is high, liquid exchange and concentration of the sample are required to ensure that the protein concentration is about 5 mg / ml and the salt concentration in the sample is less than 50 mM. (2) Processing of non-reduced CE samples: Samples were placed in EP tubes, and the protein yield of each sample was 50 μg. 1 μl of 10 kD internal standard (Protein Simple, 046-144), 2.5 μl of 250 mM IAM (Sigma, I1149-5G), and 1 × sample buffer (Protein Simple, 046-567) were added to a final volume of 50 μl. (3) After shaking to mix evenly, the mixture was heated to 70°C and incubated for 10 min, then removed, placed on ice for 5 min incubation, cooled, and centrifuged at 12000 rpm for 5 min. After centrifugation, 35 μl of the supernatant was taken and transferred to a 96-well plate that matched the instrument, then centrifuged at 1000 rpm for 5 min, and the 96-well sample plate was placed in Maurice (Protein Simple) to load the samples and prepare for analysis. 2.On-machine detection

[0123] Turn on the instrument and software, perform self-inspection of the instrument according to the instrument operation procedure, install the capillary cartridge, prepare the corresponding reagents and place them in the corresponding positions of the instrument. Set the corresponding parameters according to the instrument operation procedure and perform non-reduced CE analysis. Set the sample sequence and edit the corresponding sequence according to the sample name, with the number of samples in each sequence being no more than 48. After completing the sequence editing, click start to start the sequence detection.

[0124] The contents of the main peak and impurity peaks of the sample were calculated using the following formula:

[0125]

number

[0126] [Table 7]

[0127] The experimental results showed that sample HU6DL T54 had good purity with the main peak content reaching 93.62% and no impurities detected. Example 6 Preparation of Compound 1 [ka]

[0128] Step 1: 2a (2 g, 17.2 mmol) was dissolved in 75 mL acetonitrile, and potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol), and tetrabutylammonium iodide (620 mg, 1.68 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (20 ml). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using developing solvent system C to give product 5a (3.2 g, yield: 90.1%).

[0129] Step 2: 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were placed in a reaction flask, 6 mL of tetrahydrofuran was added, argon gas was replaced three times, the temperature was lowered to 0-5 ° C in an ice-water bath, tert-butoxide (164 mg, 1.46 mmol) was added, the ice bath was removed, the temperature was raised to room temperature, and the mixture was stirred for 40 minutes, 15 mL of ice water was added, and the mixture was extracted with ethyl acetate (40 mL × 2) and chloroform (20 mL × 5), and the organic phases were combined and concentrated. The resulting residue was dissolved in 6 mL of dioxane, 3 mL of water was added, sodium bicarbonate (73.8 mg, 0.879 mmol) and chloroformate-9-fluorenylmethyl (190 mg, 0.734 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 30 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL×3). The organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified using silica gel column chromatography with developing solvent system C to obtain product 5b 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid benzyl ester (73 mg, yield: 19.4%).

[0130] MS m / z (ESI): 515.0 [M+1].

[0131] Step 3, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (18 mg, content 10%, dry type) was added, hydrogen gas was replaced three times, and the reaction was carried out at room temperature with stirring for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid (20 mg), which was directly used in the next step without purification.

[0132] MS m / z (ESI): 424.9 [M+1].

[0133] Step 4, 1b (15 mg, 28.2 μmol) was added to the reaction flask, 1.5 mL of N,N-dimethylformamide was added, argon gas was replaced three times, and the temperature was lowered to 0-5 °C in an ice-water bath, one drop of triethylamine was added, crude 5c (20 mg, 47.1 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25.4 mg, 86.2 μmol) was added, and the reaction was carried out for 40 minutes while stirring in an ice bath. 15 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL × 3), and the organic phase was combined. The organic phase was washed with saturated sodium chloride solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified using thin layer chromatography with developing solvent system B to obtain the title product 5d (9H-fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (23.7 mg, yield: 78.9%).

[0134] MS m / z (ESI): 842.1[M+1].

[0135] Step 5: 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, 1.5 mL of toluene was added, and the mixture was concentrated under reduced pressure, and this was repeated twice. The residue was triturated with 4.5 mL of n-hexane, and the mixture was allowed to stand, after which the supernatant liquid was decanted to retain the solid. The solid residue was concentrated under reduced pressure and dried on an oil pump to obtain the crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (23 mg), which was used directly in the next step reaction without purification.

[0136] MS m / z (ESI): 638.0[M+18].

[0137] Step 6: Crude 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N,N-dimethylformamide, argon gas was replaced three times, and the temperature was lowered to 0-5°C in an ice-water bath. 4g (31.8 mg, 67.3 μmol) in 0.5 mL of N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27.8 mg, 94.3 μmol) was added, and the mixture was reacted for 10 minutes while stirring in an ice bath. The ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 1 hour, and the reaction produced compound 5. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm, mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain products 5-A and 5-B (3.6mg, 2.6mg).

[0138] MS m / z (ESI): 1074.4 [M+1].

[0139] Compound 5-A in a single three-dimensional configuration (short retention time): UPLC analysis: retention time 1.14 minutes, purity: 85% (Cromatogram analyzer: ACQUITY UPLC BEHC18 1.7 um 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-Acetonitrile).

[0140] 1 H NMR (400 MHz, DMSO-d6): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 2H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 2H), 2.80-2.62 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H).

[0141] Compound 5-B with single stereo configuration (long retention time): UPLC analysis: retention time 1.16 minutes, purity: 89% (Cromatogram analyzer: ACQUITY UPLC BEHC18 1.7 um 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-Acetonitrile).

[0142] 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

[0143] For the preparation of other intermediates, see Intermediate 5.

[0144] Under conditions of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 μmol) was added to an aqueous PBS buffer solution of antibody HU6DL.R54 (0.05 M PBS buffer solution with pH=6.5, 7.3 ml, 13.8 mg / ml, 0.681 μmol), placed in a water bath oscillator, and reacted for 3 hours with shaking at 37°C. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 14.0 ml. 3.3 ml of the solution was removed and used for the next reaction.

[0145] Compound 5-A (5.0 mg, 2.75 μmol) was dissolved in 0.15 mL DMSO, added to the above 3.3 ml solution, placed on a water bath oscillator, and reacted for 3 hours with shaking at 25 ° C. to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain compound 1 in PBS buffer (1.45 mg / mL, 17 mL), which was frozen and stored at 4 ° C.

[0146] The average value y was measured using the ultraviolet method. A cuvette containing sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after removing the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0147] Data Processing: By constructing a standard curve, the absorbance under 280 nm wavelength was measured to determine the antibody content Cmab, and the absorbance under 370 nm wavelength was determined to determine the small molecule content CDrug.

[0148]

number

[0149] The drug loading of the product was measured by the above method, and a sample of compound 1 (y=4) was obtained by UV-HPLC purification.

[0150] For the preparation of Compounds 2 to 9, please refer to Compound 1. Example 7 Synthesis of Compound 10

[0151] Synthesis of compound a [ka]

[0152] Raw material a-1 (4.1 g, 9.71 mmol) and raw material a-2 (4.3 g, 8.09 mmol, containing 4% amino group isomer impurity) were taken and placed in a 250 mL reaction flask, and under nitrogen gas protection, DCM (54 mL), MeOH (18 mL) were added, and the mixture was stirred and cooled to 0°C. DMTMM (3.6 g, 12.1 mmol), triethylamine (2.5 g, 24.2 mmol) were added, and the mixture was kept at 0°C and stirred for 1 h. The HPLC central control unit indicated that raw material a-2 had reacted completely, and the reaction liquid was evaporated to dryness under reduced pressure (<25°C). MTBE (120 mL) was added and the mixture was stirred and beaten (mud-like substance). The solution was poured out and filtered, and 120 mL of MTBE-beaten (solid) was further added to the mud-like substance, and the mixture was filtered. The filter cake was then washed with water (60 The crude product was dissolved in dichloromethane and methanol, wet sampled, and subjected to column chromatography twice (eluent DCM:MeOH=40:1-20:1) to obtain pure compound a (6.2 g, 7.37 mmol), with a purity of 99.3% and a yield of 91%.

[0153] Synthesis of compound b [ka]

[0154] Compound a (5.7 g, 6.77 mmol) was taken and placed in a 500 mL three-neck flask, and under nitrogen gas protection, dry THF (114 mL) was added, stirred to dissolve, and cooled to about -10°C. DBU (3.09 g, 20.31 mmol) was added, and the internal temperature was maintained at -10 to -5°C during the dropwise addition. The dropwise addition was completed in 5 min. After the dropwise addition was completed, the internal temperature was maintained at -10 to -5°C and the reaction was continued for 2.5 h, at which point a solid precipitated.

[0155] The internal temperature was cooled to -20°C, and MTBE (114 mL) was added while maintaining the internal temperature at -20 to -10°C. The product was completely precipitated, filtered, and the filter cake was washed with MTBE (57 mL x 2). After drying, 6 g of crude compound b was obtained, which was stored at -78°C and waited.

[0156] Synthesis of compound e [ka]

[0157] Compound c (651 mg, 1 mmol) was dissolved in 10 mL of DCM, and stirring was started while cooling in an ice bath, and DBU (456 mg, 3 mmol) was added dropwise. After reacting in an ice bath for one hour, the reaction was completed, and compound d (257 mg, 1 mmol) and HATU (420 mg, 1.1 mmol) were added in sequence, and after stirring in an ice bath for 30 minutes, LCMS showed the reaction was completed, and the reaction solution was concentrated under 25 °C, and the residue was purified by reverse phase column of a column apparatus (ACN in H2O, 50% product) to obtain compound e as a red-brown solid, 130 mg, 19% yield.

[0158] MS:691.3 [M+23].

[0159] Synthesis of compound f [ka]

[0160] Compound e (130 mg, 0.19 mmol) was dissolved in DCM, anisole (62 mg, 0.57 mmol) and dichloroacetic acid (245 mg, 1.9 mmol) were added, and the reaction was stirred at room temperature overnight for a total of 16 hours. When the sampling LC-MS central control system indicated that the raw material was completely consumed, the reaction was stopped, the reaction solution was concentrated under 25°C, and the residue was purified by reverse phase column of a column system (ACN / HO, 30% product) to give compound f as a pink solid, 53 mg, 54% yield.

[0161] (MS:519.2 [M+1].

[0162] Synthesis of Compound D [ka]

[0163] Compound f (23 mg, 0.044 mmol) and compound b (27 mg, 0.044 mmol) were dissolved in DCM (3 mL) and MeOH (1 mL) and cooled to -30 °C under nitrogen gas protection. DMTMM (20 mg, 0.067 mmol) was added, and the reaction was reacted for 1 h by controlling the temperature at -20 °C to -10 °C, and the sampling LC-MS central control unit indicated that the raw material was completely consumed. The temperature was controlled at -10 °C, and the reaction was quenched by adding 10 mL of water, and layered by adding 30 mL of DCM. The aqueous phase was extracted with DCM / MeOH = 10 / 1 (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 25 °C, and the residue was separated and purified (ACN / H2O / 0.05% FA) to obtain compound D as a white solid, 5.8 mg, yield 12%, HPLC purity 98.87%.

[0164] MS:1120.3 [M+1].

[0165] The hydrogen spectrum chart is shown in Figure 1.

[0166] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.239 mL, 1.70 μmol) was added to an aqueous PBS buffer solution of antibody HU6DL.R54 (0.05 M PBS buffer solution with pH=6.5, 7.3 ml, 13.8 mg / ml, 0.681 μmol), placed in a water bath oscillator, and reacted for 3 hours with shaking at 37°C. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 14.0 ml. 3.3 ml of the solution was removed and used for the next reaction.

[0167] Compound D (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL of DMSO, added to the above 3.3 ml solution, placed in a water bath oscillator, and reacted for 3 hours by shaking at 25 ° C., and the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain mAb2 antibody conjugate compound 10 in PBS buffer (1.35 mg / mL, 13 mL), which was frozen and stored at 4 ° C.

[0168] The average value y was measured using the ultraviolet method. A cuvette containing sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after removing the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0169] Data Processing: By constructing a standard curve, the absorbance under 280 nm wavelength was measured to determine the antibody content Cmab, and the absorbance under 370 nm wavelength was determined to determine the small molecule content CDrug.

[0170]

number

[0171] The drug loading of the product was measured by the above method, and a sample of compound 10 (y=4) was obtained by UV-HPLC purification.

[0172] For the preparation of compounds 11 to 18, please refer to compound 10. Example 8 Cell killing activity of antibody drug conjugates

[0173] The tumor cell killing effect of the antibody-drug conjugates of the present disclosure was evaluated using the Trop-2 positive cell line MDA-MB-468. Cultured MDA-MB-468 monolayer cells were digested with pancreatin, resuspended in medium, centrifuged, and counted to a cell density of 4×10 in complete medium.4 The antibody-drug conjugate working solution was diluted with complete medium in a 96-well V-bottom plate (Corning, cat:3894) starting at 10000 nM, diluted 10-fold, and 9 concentrations were added to the white 96-well plate after preparation was completed, and 50 μl was added per well to the two sets of wells. The cell plate was incubated at 37°C, 5% CO2 for overnight. The next day, the antibody-drug conjugate working solution was diluted with complete medium in a 96-well V-bottom plate (Corning, cat:3894) starting at 10000 nM, diluted 10-fold, and 9 concentrations were added to the white 96-well plate after preparation was completed, and 50 μl was added per well to the two sets of wells. The cell plate was incubated at 37°C, 5% CO2 for overnight. 2の The plates were placed in an incubator and cultured for 6 days. On the sixth day of the experiment, the plate was detected and read, and the plate was removed and equilibrated to room temperature. Then, 50 μl of CellTiter-Glo® cell viability detection reagent (Promega, Cat#: G7573) was added to each well, and the plate was shaken to mix uniformly, and then the plate was left in the dark for 20 minutes. The luminescence chemiluminescence signal value was detected at 490 nm and 500 ms / well using a microplate reader. The inhibition rate (%) was calculated based on the luminescence signal value. The formula is as follows:

number

[0174] Sample wells are cell wells treated with the test drug and control wells are cell wells treated with complete medium.

[0175] Curve fitting: Based on the inhibition rate (%) corresponding to each concentration, IC was calculated by curve fitting using the log(inhibitor) vs. response -- Variable slope (four parameters) equation in GraphPad Prism 6.0, with the concentration logarithm on the X-axis and the inhibition rate on the Y-axis. 50 To obtain the value, the calculation equation is:

number

[0176] [Table 8]

[0177] Cell killing IC 50 By comparing the results, the experimental results showed that, compared with the negative control, all of the antibody-drug conjugates of the present invention had a stronger cell-killing effect. Example 9 Pharmacokinetics of Antibody Drug Conjugates

[0178] Using a BALB / c mouse model, we evaluated the drug metabolism of anti-Trop-2 antibody-drug conjugate Tro-2 ADC in mice. BALB / c mice, 6-8 weeks old, with an average weight of 18-22 g, were randomly divided into three groups, with three animals in each group. The Trop-2 ADC-drug conjugates tested were administered in a single dose, IV, at 4 mpk, and blood was collected at 0.5, 2, 4, 8, 24, 48, 72, 96, 144, and 240 hours, respectively. The plasma was separated and frozen in a refrigerator at -20°C. Then, anti-ADC polyclonal antibodies were coated on a 96-well high affinity fully transparent plate (Corning, cat:3590), and diluted plasma samples were added to the plate. The secondary antibody of sheep anti-human IgG1 F(ab')2 fragment F(ab')2 HRP label was used to detect the Trop-2 ADC concentration in mouse plasma, and the PK parameters were analyzed using the non-atrioventricular model and intravascular administration model in PKSolver software. The details of the experimental results are shown in the table below.

[0179] [Table 9]

[0180] Taking into consideration the values ​​of parameters such as half-life t1 / 2, time to peak blood concentration Tmax, blood concentration Cmax, and clearance Cl_obs, the antibody-drug conjugate of the present invention exhibits favorable metabolic properties. Example 10 In vivo antitumor activity of antibody-drug conjugates

[0181] To further investigate the inhibitory effect of the antibody-drug conjugates on tumors formed in vivo, a mouse model of human gastric cancer NCI-N87 tumor cell xenografts of Trop-2 positive tumor cells was used to evaluate the in vivo tumor growth inhibitory effect of the candidate molecules. 6 NCI-N87 cells were subcutaneously injected into nude mice (Balb / c nude) that were 8 weeks old and weighed about 18 to 20 mg, and after 10 days, the tumor volume increased to an average of 160 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups and started to receive intravenous injections of the antibody-drug conjugate, with one injection every 2 weeks at a dose of 4 mg / kg. As a control, a human IgG1 isotype control antibody was used at a dose of 4 mg / kg. The control group or treatment group consisted of 5 mice each. The antitumor rate was calculated by measuring the tumor volume.

[0182]

number

[0183] [Table 10]

[0184] The experimental results showed that when injected once every two weeks at a dose of 4 mg / kg, the antibody-drug conjugates of the present invention all exhibited good antitumor effects, and that the antibody-drug conjugates of the present invention also exhibited good antitumor rates at low doses. Example 11 Dose-dependent in vivo antitumor activity of antibody-drug conjugates

[0185] To further investigate the inhibitory effect of the antibody-drug conjugates on tumors formed in vivo, a mouse model of human gastric cancer NCI-N87 tumor cell xenografts of Trop-2 positive tumor cells was used to evaluate the in vivo tumor growth inhibitory effect of the candidate molecules. 6 NCI-N87 cells were subcutaneously injected into nude mice (Balb / c nude) that were 6 weeks old and weighed about 18 to 22 mg, and after 8 days, the tumor volume was 175 mm on average. 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups and started to receive antibody-drug conjugates by intravenous injection, once every 2 weeks, at doses of 2 mg / kg and 1 mg / kg. As a control, a human IgG1 isotype control antibody was used at a dose of 2 mg / kg. The control group or treatment group consisted of 5 mice each. The antitumor rate was calculated by measuring the tumor volume.

[0186]

number

[0187] [Table 11]

[0188] The experimental results showed that after administration at doses of 2 mg / kg and 1 mg / kg once every two weeks for 28 days, the antibody-drug conjugate of the present invention showed good antitumor effects against both human head and neck cancer and gastric cancer cell NCI-N87 CDX tumor-bearing mouse models, and showed dose-dependent effects. Example 12: In vivo pharmacodynamics model study of antibody-drug conjugates against head and neck squamous cell carcinoma FaDu

[0189] To further study the inhibitory effect of the antibody-drug conjugates on tumors formed in vivo, a mouse model of human head and neck squamous cell carcinoma FaDu tumor cell xenografts of Trop-2 positive tumor cells was used to evaluate the in vivo tumor growth inhibitory effect of the candidate molecules. The cell suspension mixed with matrigel in the same ratio was added, and 5x10 cells were added per animal.6 FaDu cells were subcutaneously injected into nude mice (Balb / c nude) aged 6 weeks and weighing approximately 18 to 22 mg, and after 8 days, the tumor volume averaged 133 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups and started to receive antibody-drug conjugates by intravenous injection, with a single dose of 3 mg / kg or 1 mg / kg. As a control, a human IgG1 isotype control antibody was used with a dose of 3 mg / kg. The control group or treatment group consisted of 5 mice each. The antitumor rate was calculated by measuring the tumor volume.

[0190]

number

[0191] [Table 12]

[0192] The experimental results showed that after randomly dividing the subjects into groups and administering a single dose of 3 mg / kg or 1 mg / kg for 17 days, the two doses of 3 mg / kg and 1 mg / kg showed significant antitumor effects on both human head and neck squamous cell carcinoma FaDu CDX tumor-bearing mouse models, with the antitumor rates both reaching 200%. Example 13: In vivo pharmacodynamic model study of antibody-drug conjugates against lung cancer Calu-3

[0193] To further study the inhibitory effect of the antibody-drug conjugates on tumors formed in vivo, a mouse model of human lung cancer Calu-3 tumor cell xenografts of Trop-2 positive tumor cells was used to evaluate the in vivo tumor growth inhibitory effect of the candidate molecules. The cell suspension mixed with matrigel in the same ratio was added at 2x10 per animal. 6 Calu-3 cells were subcutaneously injected into nude mice (Balb / c nude) aged 6 weeks and weighing approximately 18 to 22 mg, and the tumor volume increased to an average of 148 mm after 8 days. 3When the tumor volume reached 10 mg / kg, the mice were divided into groups and started to receive intravenous injection of the antibody-drug conjugate at a dose of 10 mg / kg once. The control group was a human IgG1 isotype control antibody at a dose of 10 mg / kg. Each control group or treatment group had 5 mice. The tumor volume was measured to calculate the antitumor rate.

[0194]

number

[0195] [Table 13]

[0196] The experimental results showed that after a single administration in randomly divided groups at a dose of 10 mg / kg for 21 days, the antibody-drug conjugate of the present invention showed significant antitumor effect on the human lung cancer Calu-3 CDX tumor-bearing mouse model, with the antitumor rate reaching 100%.

Claims

1. An antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, 【Chemical 1】 where: L is -(CR １ R ２ ) ｍ -[X １ - (CR １ R ２ ) ｎ－ X ２ ] ｔ - (CR １ R ２ ) ｒ - and R １ or R ２ are each independently selected from hydrogen, deuterium, a hydroxy group, an amino group, an alkyl group, a halogen, a halogenated alkyl group, a deuterated alkyl group, or a hydroxyalkyl group, and preferably R １ or R ２ is hydrogen, X １ or X ２ are each independently selected from a bond, N, O or S, preferably X １ or X ２ is a bond or O, m, n, r or t are each independently selected from 1, 2, 3 or 4, preferably m, n, r or t are each independently selected from 1 or 2; R ３ or R ４ are each independently selected from hydrogen, halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclo group, an aryl group, or a heteroaryl group; Or, R ３ and R ４ together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclo group, y is 1 to 20, preferably 1 to 10, more preferably 2 to 8, and even more preferably 4, 6, or 8; mAb is an anti-TROP-2 antibody or antigen-binding fragment thereof; It comprises an HCDR1 represented by SEQ ID NO: 3, an HCDR2 represented by RIDPXDSETHYNQKFKD, and an HCDR3 represented by SEQ ID NO: 5, and an LCDR1 represented by SEQ ID NO: 6, an LCDR2 represented by SEQ ID NO: 7, and an LCDR3 represented by SEQ ID NO: 8, X is selected from the amino acid residues of T, R, Y, Q, L, I, F, E, or A; or a pharmaceutically acceptable salt or solvate thereof.

2. The TROP-2 antibody or antigen-binding fragment thereof, HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 13 and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 11 and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 12, and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 15 and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 16 and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising an HCDR1 set forth in SEQ ID NO: 3, an HCDR2 set forth in SEQ ID NO: 17, and an HCDR3 set forth in SEQ ID NO: 5, and an LCDR1 set forth in SEQ ID NO: 6, an LCDR2 set forth in SEQ ID NO: 7, and an LCDR3 set forth in SEQ ID NO:

8.

3. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof described in claim 1, characterized in that the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from a mouse-derived antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

4. The anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof; Preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a human IgG1, IgG2, or IgG4 heavy chain constant region; More preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region shown in SEQ ID NO: 48, or SEQ ID NO:

1. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 1 .

5. The anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human antibody κ chain, λ chain, or a variant thereof; Preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human antibody κ chain; More preferably, the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO:

2.

6. The anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the sequences set forth in SEQ ID NO: 23, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and / or a light chain variable region of SEQ ID NO: 19, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; Preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof is a heavy chain variable region set forth in SEQ ID NO: 23 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 18 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 20 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 21 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 24 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 25 and a light chain variable region set forth in SEQ ID NO: 19; or a heavy chain variable region set forth in SEQ ID NO: 26 and a light chain variable region set forth in SEQ ID NO: 19; or 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising a heavy chain variable region set forth in SEQ ID NO: 27 and a light chain variable region set forth in SEQ ID NO:

19.

7. The anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain selected from those set forth in the sequences SEQ ID NO: 33, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity thereto; and / or a light chain as set forth in SEQ ID NO: 29, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity thereto; Preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof is a heavy chain as set forth in SEQ ID NO: 33 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 28 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 30 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 31 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 32 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 34 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 35 and a light chain as set forth in SEQ ID NO: 29, or a heavy chain as set forth in SEQ ID NO: 36 and a light chain as set forth in SEQ ID NO: 29, or 2. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising a heavy chain as set forth in SEQ ID NO: 37 and a light chain as set forth in SEQ ID NO:

29.

8. It is selected from an antibody-drug conjugate shown in general formula (II) or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomeric, racemic, enantiomer, diastereomer, or mixture thereof, 【Chemistry 2】 X 1 or X 2 is a bond or O; m is 0 or 1, and 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein t is 1 or 2.

9. It is selected from an antibody-drug conjugate shown in general formula (III) or a pharmaceutically acceptable salt or solvate thereof: 【Chemistry 3】 wherein y is 1 to 10, preferably 4, 6, 8 or 10, or a pharmaceutically acceptable salt or solvate thereof.

10. The antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following structures: 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 10. The antibody-drug conjugate of claim 9, or a pharmaceutically acceptable salt or solvate thereof, wherein y is as defined in claim 1.

11. A method for producing an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, comprising: 【Chemistry 10】 The method comprises the steps of reducing the mAb and then conjugating it with a compound represented by general formula (F) to obtain a compound represented by general formula (I), wherein L, R 3 , R 4 , mAb, and y are as defined in claim 1.

12. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate of said antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.

13. Use of the antibody-drug conjugate of any one of claims 1 to 10 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 12, in the manufacture of a pharmaceutical for the treatment of a disease or symptom associated with TROP-2.

14. The disease associated with human TROP-2 is a cancer in which TROP-2 is highly expressed, and the cancer is selected from the group consisting of triple-negative breast cancer, small cell lung cancer, urothelial carcinoma, human cerebral astrocytoma, human pharyngeal carcinoma, adrenal tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, desmoplastic bone, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, 14. The use according to claim 13, wherein the cancer is selected from hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma, malignant fatty tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer and uterine cancer.

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