Anti-CD39 antibody-drug conjugates and uses thereof

Abstract

The present invention provides anti-CD39 antibody-drug conjugates or pharma- ceutically acceptable salts or solvates thereof, and pharmaceutical compositions comprising said conjugates or pharma- ceutically acceptable salts or solvates thereof, and their use as anti-cancer drugs, particularly in the manufacture of a medicament for treating a CD39-mediated disease or condition. The anti-CD39 antibody-drug conjugates provided herein significantly improve the efficacy of cytotoxin drugs and tumor killing activity in vivo and in vitro.

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate having a completely new structure, or a pharmaceutically acceptable salt or solvate thereof. Specifically, the present invention relates to an anti-CD39 antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition containing the antibody-drug conjugate, and pharmaceutical uses of the antibody-drug conjugate or pharmaceutical composition. [Background technology]

[0002] Purinergic signaling in the tumor microenvironment (TME) plays an important role in regulating immune responses. In solid tumors, tumor cell death, metabolic, hypoxic stress, and pro-inflammatory signals all lead to massive extracellular release and accumulation of intracellular ATP, far exceeding levels in healthy tissue (PloS One 2008, 3(7):e2599; Curr Opin Pharmacol. 2016, 29:17-25). Extracellular ATP binds to P2 purinergic receptors on immune cells, generating pro-inflammatory stimuli and positively regulating tumor cell killing (Trends Immunol. 2016, 37(7):427-39). However, tumor cells express CD39 (ectonucleoside triphosphate diphosphohydrolase 1, also known as nTPDase1) and CD73 (ecto-5'-nucleotidase), which convert ATP to adenosine, and adenosine binds to the A2A receptor on immune cells, inhibiting their activity and further enabling immune evasion by tumor cells.

[0003] In the tumor microenvironment, CD39 is highly expressed on regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and exhibits inhibitory effects on cytotoxic T cells (Blood. 2007, 110(4):1225-32; Cell Mol Immunol. 2017, 14(6):521-8; Cancer Res. 2016, 76(18):5241-52; J Immunother Cancer. 2016, 4:49). Elevated CD39 expression has also been observed on tumor cells of various hematological and solid tumors, including thyroid cancer, colorectal cancer, gastric cancer, renal cancer, prostate cancer, testicular cancer, breast cancer, ovarian cancer, melanoma, and lymphoma (Human Protein Atlas). It has also been reported that overexpression of CD39 is associated with poor cancer prognosis (Int J Clin Exp Pathol. 2015, 8(11):14757-14764). Therefore, CD39 is considered to be a highly potential therapeutic target. Currently, several monoclonal antibodies targeting CD39, such as the monoclonal antibodies IPH5201 and TTX-030, are in clinical research, but there is also a need to develop drug molecules with more effective therapeutic effects in clinical settings.

[0004] Antibody drug conjugates (ADCs) are targeted therapeutic approaches that utilize the antibody's targeting ability to tumor cell surface antigens by linking a monoclonal antibody or antibody fragment to a cytotoxic toxin via a stable linker. These toxins are efficiently delivered to tumor sites. ADC drugs consist of three components: an antibody, a linker, and a toxin. The antibody component determines the specificity of the ADC drug, including specific target binding and effective endocytosis. The type and chemical properties of the linker significantly affect the therapeutic index (the ratio of therapeutic effect to toxicity), as well as the number of toxins coupled, the in vivo release mechanism, and the pharmacokinetics of the ADC drug. The type of toxin determines the mechanism of action by which the ADC drug kills tumor cells, such as targeting tubulin to disrupt microtubule dynamics or targeting the DNA groove to disrupt the DNA double helix. The toxicity and chemical properties of the toxin itself also affect the number and stability of toxins bound to the ADC drug to some extent, and different types of tumor cells usually exhibit different sensitivities to different toxins, all of which affect the therapeutic index of the ADC drug. Therefore, the efficacy and safety of ADCs obtained by combining novel antibodies with different linkers and toxins cannot be determined and are difficult to predict.

[0005] Currently, many ADC drugs have been approved for commercial sale. For example, Kadcyla and Enhertu are ADC drugs formed by coupling a monoclonal antibody targeting HER2 with DM1 and Deruxtecan, respectively. I-394 (referred to as F1 in this application) is an anti-CD39 monoclonal antibody developed by Innate, Inc. The relevant patent publication number is WO2019096900A1, and it is currently undergoing phase 1 clinical trials. Currently, no ADC drugs targeting CD39 have been disclosed as being the subject of clinical research. Those skilled in the art are working to develop new and effective ADC drugs targeting CD39. Summary of the Invention

[0006] The present invention aims to provide an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, [Chemical formula] Among them, Ab is an anti-CD39 antibody or an antigen-binding fragment thereof, L is a linker, D is a cytotoxic molecule, "-" represents a bond, n is the average coupling number of the cytotoxic molecule coupled to the antibody, and is also the average value of the ratio (drug antibody ratio) of the cytotoxic molecule to the antibody, also called the DAR value, where 0 < n ≤ 10, preferably 0.8 ≤ n ≤ 5.5, and n may be an integer or a non-integer.

[0007] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CD39 antibody or an antigen-binding fragment thereof has the following CDR regions: HCDR1 whose amino acid sequence is shown in SEQ ID NO: 1 or includes the amino acid sequence shown in SEQ ID NO: 1, HCDR2 whose amino acid sequence is shown in SEQ ID NO: 2 or includes the amino acid sequence shown in SEQ ID NO: 2, HCDR3 whose amino acid sequence is shown in SEQ ID NO: 3 or includes the amino acid sequence shown in SEQ ID NO: 3, LCDR1 whose amino acid sequence is shown in SEQ ID NO: 4 or includes the amino acid sequence shown in SEQ ID NO: 4, LCDR2 whose amino acid sequence is shown in SEQ ID NO: 5 or includes the amino acid sequence shown in SEQ ID NO: 5, LCDR3 whose amino acid sequence is shown in SEQ ID NO: 6 or includes the amino acid sequence shown in SEQ ID NO: 6, and is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

[0008] In a preferred embodiment of the present invention, the anti-CD39 antibody or antigen-binding fragment thereof is an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CD39 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7, and / or a light chain variable region amino acid sequence set forth in SEQ ID NO: 8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8, or a pharmaceutically acceptable salt or solvate thereof.

[0009] In a preferred embodiment of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof is represented by general formula (I), wherein the anti-CD39 antibody or antigen-binding fragment thereof is a mouse antibody or a fragment thereof, and further comprises a heavy chain constant region of mouse-derived IgG1, IgG2, IgG3, or IgG4 or a mutant thereof, and / or a light chain constant region of mouse-derived κ or λ chain or a mutant thereof.

[0010] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CD39 antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, and further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, preferably a human IgG4 heavy chain constant region, and / or a light chain constant region of human κ or λ chain or a variant thereof, preferably a human λ light chain constant region, or a pharmaceutically acceptable salt or solvate thereof.

[0011] In a preferred embodiment of the present invention, the chimeric antibody or antigen-binding fragment thereof is an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the amino acid sequence of the heavy chain of the chimeric antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 9 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9, and / or the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 10 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10, or a pharmaceutically acceptable salt or solvate thereof.

[0012] In a preferred embodiment of the present invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof is represented by general formula (I), wherein the anti-CD39 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, and further comprises a heavy chain FR region of human-derived IgG1, IgG2, IgG3, or IgG4 or a variant thereof, preferably comprising the FR region of human germline heavy chain IGHV1-2*02 or a variant thereof, and / or a light chain FR region of human-derived κ or λ chain or a variant thereof, preferably comprising the FR region of human germline light chain IGKV1-33*01 or a variant thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0013] In a preferred embodiment of the present invention, the humanized antibody or antigen-binding fragment thereof is an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the amino acid sequence of the heavy chain variable region of the humanized antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 11, 12, 13, 14, or 15, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, 12, 13, 14, or 15, and / or the amino acid sequence of the light chain variable region of the humanized antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 16 or 17, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16 or 17, or a pharmaceutically acceptable salt or solvate thereof.

[0014] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the humanized antibody or antigen-binding fragment thereof has an amino acid sequence of a heavy chain variable region set forth in SEQ ID NO: 11 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, and an amino acid sequence of a light chain variable region set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, in which the amino acid sequence of its heavy chain variable region is set forth in SEQ ID NO: 15 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17.

[0015] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the humanized antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human-derived IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and preferably comprises a human IgG4 heavy chain constant region, more preferably an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the amino acid sequence of the human-derived IgG4 heavy chain constant region is set forth in SEQ ID NO: 18 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18.

[0016] In a preferred embodiment of the present invention, the humanized antibody or antigen-binding fragment thereof is an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the humanized antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human-derived κ or λ chain or a variant thereof, preferably a human-derived κ light chain constant region, and more preferably the amino acid sequence of the human-derived κ light chain constant region is set forth in SEQ ID NO: 19 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19, or a pharmaceutically acceptable salt or solvate thereof.

[0017] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the linker L is represented by general formula (L): [ka] Among them, L1 is [ka] Selected from L2 is [ka] wherein R1 and R2 are each independently selected from H, an alkyl group, a haloalkyl group, or a halogen; m is 1 to 8, preferably 2 to 5; and L2 is preferably [ka] and Or L2 is [ka] and L3 is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0018] In a preferred embodiment of the present invention, there is provided an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the linker L is [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate has the structure:

[0019] In a preferred embodiment of the present invention, the antibody-drug conjugate is represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic molecule D is selected from a chemotherapeutic agent, a DNA alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, an antibiotic, or a radioisotope cytotoxic agent.

[0020] In a preferred embodiment of the present invention, an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic molecule D is

Chemical formula

[0021] In a preferred embodiment of the present invention, an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, which comprises an antibody-drug conjugate represented by the general formula (II) or a pharmaceutically acceptable salt or solvate thereof,

Chemical formula

[0022] As can be understood from the common knowledge in the art, among the 20 common amino acids, only the lysine side chain has the activity to undergo a condensation reaction with the linker and forms an imino residue after the reaction. Therefore, as can be understood by combining the structure of the general formula (II) and the common knowledge in the art, in the general formula (II), the above linker is linked to the lysine side chain of the above Ab, and at the same time, the lysine side chain of the antibody reacts with the linker to form an imino group, and the number thereof is the same as the number of cytotoxic molecules, so it is described on the leftmost side within the square brackets of the general formula (II).

[0023] In a preferred embodiment of the present invention, it is an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, which contains an antibody-drug conjugate represented by the general formula (III) or a pharmaceutically acceptable salt or solvate thereof,

Chemical formula

[0024] As can be understood from the common general knowledge in the art, among the 20 common amino acids, the amino acid side chain that has the activity to carry out a condensation reaction with the linker and forms a -S- residue after the reaction is only the cysteine side chain. Therefore, (III) Combined with the structure of the general formula and the common general knowledge in the art, it can be understood that in the general formula (III) the above linker is linked to the cysteine side chain of the above Ab, and at the same time, the cysteine side chain of the antibody reacts with the linker to form a -S- residue, and the number thereof is the same as the number of cytotoxic molecules. Therefore, it is described on the leftmost side within the square brackets of the general formula (III)

[0025] In a preferred embodiment of the present invention, the antibody-drug conjugate described in the present invention or a pharmaceutically acceptable salt or solvate thereof includes, but is not limited to, the following:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0026] In a more preferred embodiment, the antibody-drug conjugate described in the present invention is as follows: (1) F314-MC-VC-PAB-MMAE, whose structure is shown in the following formula: [ka] wherein n is 3.8 to 4.2, preferably 3.9 to 4.1, and more preferably 4.0, and the linker is linked to a cysteine ​​side chain of the Ab; (2) The structure is F314-MC-MMAF shown in the following formula: [ka] wherein n is 1.3 to 1.7, preferably 1.4 to 1.6, and more preferably 1.5, and the linker is linked to a cysteine ​​side chain of the Ab; (3) The structure is F314-SMCC-DM1 shown in the following formula: [ka] wherein n is 4.8 to 5.2, preferably 4.9 to 5.1, and more preferably 5.0, and the linker is linked to a lysine side chain of the Ab; (4) The structure is F314-SPDB-DM4 shown in the following formula: [ka] wherein n is 3.6 to 4.0, preferably 3.7 to 3.9, and more preferably 3.8, and the linker is linked to a lysine side chain of the Ab; (5) The structure is F314-C4-VC-PAB-MMAE shown in the following formula: [ka] wherein n is 1.9 to 2.3, preferably 2.0 to 2.2, and more preferably 2.1, and the linker is linked to a lysine side chain of the Ab; (6) The structure is F314-C5-VC-PAB-MMAE shown in the following formula: [ka] wherein n is 0.5 to 3.5, preferably 0.8 to 3.0, and more preferably 0.8, 1.8, 2.3, or 3.0, and the linker is linked to a lysine side chain of the Ab; (7) The structure is F314-C6-VC-PAB-MMAE shown in the following formula: [ka] wherein n is 1.9 to 2.3, preferably 2.0 to 2.2, and more preferably 2.1, and the linker is linked to a lysine side chain of the Ab; In each formula, F314 represents the humanized monoclonal antibody F314, the amino acid sequence of whose heavy chain variable region is shown in SEQ ID NO: 14, the amino acid sequence of whose light chain variable region is shown in SEQ ID NO: 17, the amino acid sequence of whose heavy chain constant region is shown in SEQ ID NO: 18, and the amino acid sequence of whose light chain constant region is shown in SEQ ID NO: 19.

[0027] The present invention further provides a pharmaceutical composition comprising the above-described antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent, or vector.

[0028] The present invention further provides use of the above-mentioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising same, in the preparation of a medicament for treating a CD39-mediated disease or condition, wherein the disease or condition is preferably cancer, preferably lymphoma (e.g., follicular lymphoma, mantle cell lymphoma), multiple myeloma, thyroid cancer, colorectal cancer, gastric cancer, renal cancer, prostate cancer, testicular cancer, breast cancer, ovarian cancer, or melanoma.

[0029] The present invention further provides a method for treating and preventing a CD39-mediated disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising same, wherein the disease or condition is preferably cancer, preferably lymphoma (e.g., follicular lymphoma, mantle cell lymphoma), multiple myeloma, thyroid cancer, colorectal cancer, gastric cancer, renal cancer, prostate cancer, testicular cancer, breast cancer, ovarian cancer, or melanoma.

[0030] The anti-CD39 antibody-drug conjugates designed according to the present invention not only retain the affinity and endocytosis activity of monoclonal antibodies, but also achieve direct targeted killing of tumor cells through the coupled toxic molecules, significantly increasing the activity of ADCs compared to monoclonal antibodies. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows the in vitro killing activity of naked antibodies and different ADCs against CD39-positive tumor cells. [Figure 2] Figure 1 shows the in vivo antitumor activity of naked antibodies and different ADCs in mice inoculated with CD39-positive tumor cells. [Figure 3] Figure 1 shows the in vitro killing activity of naked antibodies and different ADCs against CD39-negative tumor cells. [Figure 4] Figure 1 shows the affinity activity of naked antibodies and different ADCs against CD39-positive tumor cells. [Figure 5] 1 shows the endocytic activity of naked antibodies and different ADCs. [Figure 6] 1 shows the in vitro killing activity of F314-C5-VC-PAB-MMAE with different DAR values ​​against CD39-positive tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0032] 1. Terminology In order that the present invention may be more readily understood, certain technical and scientific terms are defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0033] The terms "CD39," "CD39 antigen," and "CD39 protein" may be used interchangeably in the present invention. CD39 is also known as ectonucleoside triphosphate diphosphohydrolase 1 (gene: ENTPD1, ​​protein: NTPDase1; see www.ncbi.nlm.nih.gov / gene / 953). CD39 may also be referred to as ATPD enzyme and SPG64. These terms may be used interchangeably. Unless otherwise specified, the terms include any variants, subtypes, and species homologs of human CD39 naturally expressed by cells or expressed by cells transfected with the CD39 gene. In the present invention, the UniProtKB / Swiss-Prot accession number for the CD39 protein is P49961.1.

[0034] An "antibody drug conjugate" (ADC) of the present invention refers to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxin via a stable chemical linker compound.

[0035] The term "pharmaceutically acceptable salt" refers to a salt of the antibody-cytotoxic drug conjugate of the present invention, which is safe and effective when used in the mammalian body and retains its inherent biological activity, and the antibody-drug conjugate compound of the present invention contains at least one amino group and is therefore capable of forming a salt with an acid.

[0036] The term "solvate" refers to a pharmaceutically acceptable solvate formed by an antibody-drug conjugate compound of the invention with one or more solvent molecules.

[0037] The one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0038] The term "antibody" as used herein refers to an immunoglobulin having a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to differences in the amino acid composition and sequence order of the heavy chain constant regions. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes, namely Igm, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig species within the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains can be divided into κ or λ chains based on differences in the constant region. Each of the five types of IgG may have either κ or λ chains.

[0039] In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus varies significantly and constitutes the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and constitutes the constant region (C region). The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. 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 LCVR and HCVR regions of the antibodies or antigen-binding fragments described herein conform in number and position to the known Kabat numbering convention (e.g., LCDR1-3, HCDR1-3) or conform to the Chothia numbering convention.

[0040] The monoclonal antibody or mAb of the present invention refers to an antibody obtained from a single clonal cell line, including but not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or other conventional techniques.

[0041] The term "mouse antibody" in the present invention refers to an anti-human CD39 monoclonal antibody prepared in a mouse using knowledge and techniques in the art. In a preferred embodiment of the present invention, the mouse-derived CD39 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived κ or λ chain or a mutant thereof, or may further comprise a heavy chain constant region of a mouse-derived IgG1, IgG2, IgG3, IgG4 or a mutant thereof.

[0042] The term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response elicited by mouse antibodies. To construct a chimeric antibody, first, a hybridoma secreting a mouse-specific monoclonal antibody is constructed, and then the variable region genes are cloned from the mouse hybridoma cells and further cloned into the constant region genes of a human antibody for recombinant expression.

[0043] The term "humanized antibody," also known as humanized CDR-grafted antibody, refers to an antibody generated by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. This overcomes the strong immune response induced by chimeric antibodies due to the large amount of mouse protein components. Such framework sequences can be obtained from a consensus DNA database containing germline antibody gene sequences or from published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available online at www.mrccpe.com.ac.uk / vbase) and Kabat, E. A., 1991, Sequences of Proteins of Immunological Interest, 5th Edition. To avoid a loss of activity associated with reduced immunogenicity, minimal back mutations can be made to the human antibody variable region to maintain activity.

[0044] The term "antigen-binding fragment" as used herein refers to a Fab fragment, Fab' fragment, or F(ab')2 fragment that has antigen-binding activity, as well as an Fv or scFv fragment that binds to human CD39, and comprises one or more CDR regions selected from SEQ ID NOs: 1 to 6 of the antibody described herein. An Fv fragment comprises an antibody heavy chain variable region and a light chain variable region, but lacks a constant region and comprises the minimum antibody fragment containing all of the antigen-binding site. Generally, an Fv antibody further comprises a polypeptide linker between the VH and VL domains, and is capable of forming the structure necessary for antigen binding. Two antibody variable regions may be linked by different linkers to form a single polypeptide chain called a single-chain antibody or single-chain Fv (scFv).

[0045] The antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chain amino acid sequences can be cloned and recombined into the expression vector pcDNA3.4. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Positive clones can be expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium into which the antibodies are secreted can be purified by conventional techniques.

[0046] The term "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, when each position in two DNA molecules is occupied by adenine, the molecules are identical at that position. The percentage identity between two sequences is calculated by dividing the number of matching or homologous positions shared by the two sequences by the number of positions compared = 100. %It is a function multiplied by . For example, when sequences are optimally aligned, if 9 out of 10 positions in two sequences match or are homologous, the two sequences are 90% identical. Generally, two sequences are compared when aligned to obtain the maximum percentage identity. One skilled in the art can determine the number of base or amino acid changes that are expressed as a percentage sequence identity.

[0047] The terms "conservative modification" or "conservative substitution or substitution" refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, rigidity, etc.), allowing frequent changes without altering the biological activity of the protein. As known to those skilled in the art, a single amino acid substitution in a non-essential region of a polypeptide generally does not fundamentally alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). Furthermore, substitution of amino acids with similar structures or functions is unlikely to destroy biological activity. In the present invention, the above-mentioned antibody light chain or heavy chain variants are "conservative modifications" or "conservative substitutions or substitutions" of 0 to 10 amino acids in the light chain or heavy chain, and those skilled in the art can predict that the variants will have substantially the same activity as before the modification or substitution. The antibody light or heavy chain variants described in the present invention also include the results of backmutation, in which individual amino acids in the human template of the humanized antibody FR region are backmutated to the mouse amino acid at the corresponding position. Those skilled in the art can expect that this variant will have activity equivalent to or better than that of the humanized antibody before backmutation and the mouse antibody containing the same CDRs.

[0048] "Affinity" or "binding" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen), unless otherwise specified. The affinity of a molecule X for its binding partner Y is typically expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein, for example, using surface plasmon resonance (SPR) techniques such as instrumentation.

[0049] "Specific binding or specifically binds to," "specific for," "selectively binds to," and "selective for" a particular antigen (e.g., polypeptide target) or epitope of a particular antigen refer to binding that is measurably different from a nonspecific or selective interaction. Specific binding can be determined, for example, by determining binding to a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target (e.g., excess unlabeled target). As used herein, the term "kd" (sec-1) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also referred to as the k dissociation value. As used herein, the term "ka" (M-1 x sec-1) refers to the association rate constant of a specific antibody-antigen interaction. This value is also referred to as the k association value. As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. KD = kd / ka.

[0050] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount further refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0051] The term "vector" is used in the context of the drug of the present invention and refers to a system that can change the way a drug enters the human body and its distribution within the body, control the release rate of the drug, and deliver the drug to the target organ. The release and targeting system of the drug vector can reduce drug degradation and loss, decrease side effects, and improve bioavailability.

[0052] "Cytotoxic molecule" refers to any substance capable of causing a deleterious effect on cell growth or proliferation, such as the small molecule drug classes MMAE, MMAF, DM1 and DM4 in the present invention.

[0053] A "naked antibody" refers to an antibody or antigen-binding fragment thereof that does not have any toxic molecule bound to it.

[0054] "Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. This definition also includes antihormonal agents that regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth, and is always a form of systemic or whole-body treatment. It may itself be a hormone.

[0055] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.

[0056] The term "bond" means a covalent bond represented by "-".

[0057] [ka] means that it is linked to another group by a covalent bond. For example, in the present invention, an antibody and a linker, and a linker and a toxic molecule are linked by a covalent bond.

[0058] "Linker" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody to a drug moiety, an "extender unit" that connects an antibody to another linker member or drug moiety. In some embodiments, the linker unit may be an amino acid unit. In one such embodiment, the amino acid unit allows a protease to cleave the linker, thereby facilitating release of the drug from the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof after exposure to an intracellular protease (such as a lysosomal enzyme).

[0059] The "loading of an ADC" (drug / antibody ratio, DAR) can be controlled in different ways, for example, (i) by limiting the molar amount of drug-linker intermediate or linker reagent relative to the antibody, (ii) by limiting the time or temperature of the coupling reaction, (iii) by limiting cysteine ​​thiol-modifying moieties or reducing conditions, and (iv) by recombinantly manipulating the amino acid sequence of the antibody so that the number and position of cysteine ​​residues are altered to control the number and / or position of linker-drug attachments.

[0060] Several methods for preparing antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvates thereof. Using organic chemistry reactions, conditions, and reagents known to those skilled in the art, ADCs of general formula (I) can be prepared by several routes, including: (1) covalent reaction of a nucleophilic group on an antibody with a bivalent linker reagent to form Ab-L, followed by reaction with a drug moiety, D; and (2) covalent reaction of a nucleophilic group on a drug moiety with a bivalent linker reagent to form DL, followed by reaction with a nucleophilic group on an antibody.

[0061] SKOV-3 is a human ovarian cancer cell line that does not express human CD39 on the cell surface and is used to detect the in vitro killing activity of anti-CD39 antibody-drug conjugates against negative tumor cells.

[0062] DB is a human follicular lymphoma cell line that naturally expresses human CD39 on the cell surface and is used to detect the in vitro killing activity of anti-CD39 antibody-drug conjugates against CD39-positive tumor cells.

[0063] MOLP-8 is a human multiple myeloma cell line that naturally expresses human CD39 on the cell surface and is used to detect the affinity and endocytosis activity of anti-CD39 antibody-drug conjugates to positive tumor cells.

[0064] Mino is a human mantle cell lymphoma cell line that naturally expresses human CD39 on the cell surface. These cells are inoculated into CB17 / SCID mice to establish a mantle cell lymphoma model and to detect the in vivo antitumor effects of anti-CD39 antibody-drug conjugates.

[0065] Abbreviations: Linker Elements The structural formula of MC is [ka] is The structural formula of Val-Cit or "VC" is [ka] is The structural formula of PAB is [ka] is The structural formula of SPDB is [ka] is The structural formula of SMCC is [ka] is The structural formula of C4 is [ka] is The structural formula of C5 is [ka] is The structural formula of C6 is [ka] is Cytotoxic drugs: MMAE = monomethylauristatin E (MW718) [ka] MMAF = a variant of auristatin E (MMAE) with a phenylalanine (MW 731.5) at the C-terminus of the drug [ka] DM1 = N(2')-deacetyl-N(2')-(3-mercapto-1-oxypropyl)-maytansine [ka] DM4 = N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine [ka] Other structures: The structure of NC is [ka] is The structure of the PC [ka] is

[0066] II. Example The following examples are used to further illustrate the present invention, but are not intended to limit the scope of the present invention.

[0067] Experimental methods for which specific conditions are not specified in the Examples or Test Examples of the present invention generally follow common conditions or conditions recommended by the manufacturers of raw materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are commonly available commercially.

[0068] The DNA sequences encoding the CDRs, variable regions, or light and heavy chains of the anti-CD39 antibodies of the present invention can be designed based on the corresponding amino acid sequences, which is conventional in the art.

[0069] Example 1 Preparation of monoclonal antibodies 1.1 Preparation of the Novel Antibody of the Present Application Using molecular cloning technology, cDNAs of the amino acid sequences of the antibodies provided herein (as shown in Table 1, all antibodies in the table contain the same antibody CDR sequences) were first cloned into the expression vector pcDNA3.4, which contains a signal peptide. Plasmids containing the corresponding antibody heavy and light chain cDNA sequences, respectively, were then amplified in E. coli. The expression plasmids containing the heavy and light chains were then co-transfected into CHO-S cells. After cultivation, the supernatant was collected and purified using MabSelect PrismA, yielding the monoclonal antibodies provided herein. Verification confirmed that the sequences of the antibodies matched those in the table below.

[0070] [Table 1] JPEG0007762294000042.jpg228169JPEG0007762294000043.jpg211169JPEG0007762294000044.jpg211169JPEG0007762294000045.jpg211169JPEG0007762294000046.jpg211169JPEG0007762294000047.jpg2361691.2 Preparation of monoclonal antibody F1 Antibody F1, whose heavy chain amino acid sequence is set forth in SEQ ID NO: 37 and whose light chain amino acid sequence is set forth in SEQ ID NO: 38, was prepared by referring to the description in innate patent WO2019096900A1. Since no other anti-CD39 ADCs have been disclosed in the prior art, an ADC was prepared as a positive control group using the F1 monoclonal antibody, the linker used in the present invention, and the toxin.

[0071] Example 2: Obtaining cytotoxic molecules

[0072] 2.1 MC-VC-PAB-MMAE [ka] It was obtained commercially (product number SET0201, Levena Biopharma).

[0073] 2.2 MC-MMAF [ka] It was obtained commercially (product number SET0202, Levena Biopharma).

[0074] 2.3 SMCC-DM1 [ka] It was obtained commercially (product number SET0101, Levena Biopharma).

[0075] 2.4 SPDB-DM4 [ka] It was obtained commercially (product number SET0102, Levena Biopharma).

[0076] 2.5 Synthesis of VC-PAB-MMAE [ka] Fmoc-VC-PAB-MMAE was prepared according to the method disclosed in Patent WO2004 / 010957, Example 18. Fmoc was removed by stirring with 20% piperidine in DMF for 20 minutes, and pure VC-PAB-MMAE was obtained by HPLC.

[0077] 2.6 Synthesis of NC4-VC-PAB-MMAE [ka] VC-PAB-MMAE (0.18 mmol) was dissolved in 1 mL of anhydrous DMF (N,N-dimethylformamide), and DIEA (N,N-diisopropylethylamine) (0.44 mmol) and succinic anhydride (0.21 mmol) were added. The reaction mixture was stirred at room temperature for 10 minutes, and then dichloromethane (1 mL), N-hydroxysuccinimide (0.89 mmol), and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) (0.89 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes. The solvent was then removed by rotary evaporation under reduced pressure. After purification by HPLC, NC4-vc-PAB-MMAE (m / z: 1320.8) was obtained. [M+H] + was obtained.

[0078] 2.7 Synthesis of PC4-VC-PAB-MMAE [ka] VC-PAB-MMAE (0.18 mmol) was dissolved in 1 mL of anhydrous DMF (N,N-dimethylformamide), and DIEA (N,N-diisopropylethylamine, 0.44 mmol) and succinic anhydride (0.21 mmol) were added. After stirring the reaction mixture at room temperature for 10 minutes, dichloromethane (1 mL), pentafluorophenol (0.89 mmol), and EDC (0.89 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes. The solvent was then removed by rotary evaporation under reduced pressure. After purification by HPLC, PC4-VC-PAB-MMAE (m / z: 1389.9) was obtained. [M+H] + was obtained.

[0079] 2.8 Synthesis of NC5-VC-PAB-MMAE [ka] Referring to the synthesis of NC4-VC-PAB-MMAE in Example 2.6, glutaric anhydride was used instead of succinic anhydride to obtain NC5-VC-PAB-MMAE, m / z: 1334.8 [M+H] + was obtained.

[0080] 2.9 Synthesis of PC5-VC-PAB-MMAE [ka] PC5-VC-PAB-MMAE, m / z: 1403.9, was obtained by using glutaric anhydride instead of succinic anhydride in accordance with the synthesis of PC4-VC-PAB-MMAE in 2.7. [M+H] + was obtained.

[0081] 2.10 Synthesis of NC6-VC-PAB-MMAE [ka] VC-PAB-MMAE (0.18 mmol) was dissolved in 1 mL of anhydrous DMF, and DIEA (77 μL, 0.44 mmol) and adipic acid bis-N-hydroxysuccinimide (1 mmol) were added. The reaction mixture was stirred at room temperature for 20 minutes, and then purified by HPLC to give NC6-VC-PAB-MMAE, m / z: 1348.8. [M+H] + was obtained.

[0082] 2.11 Synthesis of PC6-VC-PAB-MMAE [ka] Referring to the synthesis of NC6-VC-PAB-MMAE in Example 2.10, bis-pentafluorophenyl adipate (synthesized by the literature method: Liu, Yijiang et al., Biomacromolecules (2015), 16(12), 3995-4003) was used instead of bis-N-hydroxysuccinimide adipate to obtain PC6-VC-PAB-MMAE, m / z: 1417.7. [M+H] + was obtained.

[0083] Example 3 Preparation of Coupling Drugs The amino acid sequences of the variable and constant regions of the heavy and light chains of the F314 antibody used in the following examples are shown in Table 1.

[0084] 3.1 Preparation of F314-MC-VC-PAB-MMAE Antibody reduction: F314 antibody was dissolved in PBS (pH 7±0.2) buffer at a concentration of 1 to 10 mg / mL, 3 to 10 molar equivalents of TCEP were added, and the reaction solution was incubated at 4°C to 37°C for 2 to 24 hours.

[0085] Coupling: The toxic molecule MC-VC-PAB-MMAE was dissolved in DMA (N,N-dimethylacetamide) at a concentration of 10 mg / mL. After complete dissolution, 6-10 molar equivalents of MC-VC-PAB-MMAE were added to the reduced antibody solution for coupling. The final volume content of DMA was controlled to approximately 5%. The reaction was allowed to proceed at 37°C for 0.5-5 hours. The DAR of the reaction product was monitored by HIC-HPLC over this time range and the reaction was terminated when the target DAR approached approximately 4.0. (Since the DAR of the product ADC changed with reaction time, the reaction product was monitored by HIC-HPLC and the reaction endpoint was determined according to the DAR of the target product ADC.)

[0086] Purification of the product: Excess cysteine ​​was added to the reaction mixture, and the mixture was left at room temperature for 30-60 minutes. The reaction mixture was then buffer-exchanged using an ultrafiltration tube with a molecular weight cutoff of 50 kDa. At the same time, DMA and unreacted toxic molecules were removed, and the final coupling product was stored in PBS (pH 7 ± 0.2) buffer. The average DAR of the conjugate F314-MC-VC-PAB-MMAE determined by HIC-HPLC was 4.0.

[0087] The structure of the conjugate F314-MC-VC-PAB-MMAE is shown in the following formula: [ka] Among them, the average value of DAR n is 4.0.

[0088] The average DAR was calculated as follows: average DAR = (0 x DAR0 area percent (%) + 2 x DAR2 area percent (%) + 4 x DAR4 area percent (%) + 6 x DAR6 area percent (%) + 8 x DAR8 area percent (%)) / 100.

[0089] The calculation results are as follows:

[0090] [Table 2]

[0091] 3.2 Preparation of F314-MC-MMAF With reference to the preparation of F314-MC-VC-PAB-MMAE in Example 3.1, the average DAR of the complex F314-MC-MMAF was measured using Ellman's reagent, using MC-MMAF instead of MC-VC-PAB-MMAE as the damaging molecule, and the average DAR was 1.5 (see the literature: Interaction of Nitric Oxide with 2-Thio-5-nitrobenzoic Acid: Implications for the Determination of Free Sulfhydryl Groups by Ellman's Reagent for the detection method and the calculation method of the average DAR).

[0092] The structure of the complex F314-MC-MMAF is shown in the following formula. [ka] Among them, the average value of DAR, n, is 1.5.

[0093] 3.3 Preparation of F314-SMCC-DM1 Coupling: The F314 antibody was dissolved in PBS (pH 7 ± 0.2) buffer at a concentration of 1–10 mg / mL, and the small molecule SMCC-DM1 was dissolved in DMA at a concentration of 10 mg / mL. After complete dissolution, 6–10 molar equivalents of SMCC-DM1 were added to the antibody solution for coupling. The final volume content of DMA was controlled to approximately 5%. The reaction was carried out at room temperature, protected from light, for 12–24 hours.

[0094] Purification of the product: The reaction system was buffer-exchanged using an ultrafiltration tube with a molecular weight cutoff of 50 kDa, while simultaneously removing DMA and unreacted toxic molecules. The final coupling product was stored in PBS (pH 7 ± 0.2) buffer. The average DAR of F314-SMCC-DM1 was measured to be 5.0 (see Yan Chen, Drug-to-Antibody Ratio (DAR) by UV / Vis Spectroscopy, for the detection method and calculation method of the average DAR).

[0095] The structure of the complex F314-SMCC-DM1 is shown in the following formula. [ka] Among them, the average value of DAR n is 5.0.

[0096] 3.4 Preparation of F314-SPDB-DM4 Referring to the preparation of F314-SMCC-DM1 in Example 3.3, the DAR value of F314-SPDB-DM4 was measured to be 3.8 using SPDB-DM4 as the toxic molecule instead of SMCC-DM1.

[0097] The structure of the complex F314-SPDB-DM4 is shown in the following formula. [ka] Among them, the average value of DAR, n, is 3.8.

[0098] 3.5 Preparation of F314-C4-VC-PAB-MMAE The F314 antibody was dissolved in PBS (pH 7 ± 0.2) buffer at a concentration of 1–10 mg / mL. NC4-VC-PAB-MMAE or PC4-VC-PAB-MMAE was dissolved in DMA at a concentration of 10 mg / mL. After complete dissolution, 6–10 molar equivalents of NC4-VC-PAB-MMAE or PC4-VC-PAB-MMAE solution were added to the antibody solution to allow coupling. The final volume content of DMA was controlled to approximately 5%. The reaction was allowed to proceed at room temperature for 2–24 hours, protected from light. The DAR of the reaction product was monitored by HIC-HPLC over this time range and the reaction was terminated when the DAR approached the target DAR. (Because the DAR of the product ADC changed with reaction time, the reaction product was monitored by HIC-HPLC and the reaction endpoint was determined according to the DAR of the target product ADC.) The reaction mixture was buffer exchanged using an ultrafiltration tube with a molecular weight cutoff of 50 kDa, while DMA and unreacted toxic molecules were removed. The final coupling product was stored in PBS (pH 7 ± 0.2) buffer. The average DAR of the conjugate F314-C4-VC-PAB-MMAE determined by HIC-HPLC was 2.1.

[0099] The structure of the conjugate F314-C4-VC-PAB-MMAE is shown in the following formula: [ka] Among them, the average value of DAR, n, is 2.1.

[0100] The average DAR was calculated as follows: average DAR = (0 x DAR0 area percent (%) + 1 x DAR1 area percent (%) + 2 x DAR2 area percent (%) + 3 x DAR3 area percent (%) + 4 x DAR4 area percent (%)) / 100.

[0101] The calculation results are as follows:

[0102] [Table 3] 3.6 Preparation of F314-C5-VC-PAB-MMAE Referring to the preparation of F314-C4-VC-PAB-MMAE in Example 3.5, the toxic molecule was replaced with NC5-VC-PAB-MMAE or PC5-VC-PAB-MMAE, and 2-fold, 6-fold, 7.5-fold, and 10-fold molar equivalents of the toxic molecule were added to the antibody solution for coupling, respectively, to prepare products with average DAR values ​​of 0.8, 1.8, 2.3, and 3.0, respectively.

[0103] The structure of the conjugate F314-C5-VC-PAB-MMAE is shown in the following formula: [ka] The mean values ​​n of the DAR are 0.8, 1.8, 2.3, and 3.0, respectively.

[0104] The average DAR was calculated as follows: average DAR = (0 x DAR0 area percent (%) + 1 x DAR1 area percent (%) + 2 x DAR2 area percent (%) + 3 x DAR3 area percent (%) + 4 x DAR4 area percent (%)) / 100.

[0105] The calculation results are as follows:

[0106] [Table 4] The mass spectrometry detection results were consistent with the HIC-HPLC results, and the molecular weight differences between each DAR component and the naked antibody (DAR0) measured by mass spectrometry were as follows:

[0107] [Table 5] 3.7 Preparation of F314-C6-VC-PAB-MMAE Referring to the preparation of F314-C4-VC-PAB-MMAE in Example 3.5, the toxic molecule was replaced with NC6-VC-PAB-MMAE or PC6-VC-PAB-MMAE, and the average DAR of the conjugate F314-C6-VC-PAB-MMAE measured using HIC-HPLC method was 2.1.

[0108] The structure of the conjugate F314-C6-VC-PAB-MMAE is shown in the following formula: [ka] Among them, the average value of DAR, n, is 2.1.

[0109] The average DAR was calculated as follows: average DAR = (0 x DAR0 area percent (%) + 1 x DAR1 area percent (%) + 2 x DAR2 area percent (%) + 3 x DAR3 area percent (%) + 4 x DAR4 area percent (%)) / 100.

[0110] The calculation results are as follows:

[0111] [Table 6] 3.8 Preparation of F1-SMCC-DM1 With reference to the preparation of F314-SMCC-DM1, F1-SMCC-DM1 was prepared by substituting F1 for F314 for the monoclonal antibody, and the average DAR n of F1-SMCC-DM1 measured was 5.5. [ka] Among them, the average value of DAR, n, is 5.5.

[0112] Example 4: In vitro killing activity of ADCs against positive tumor cells The ADC or antibody samples to be tested were diluted to an initial concentration of 6.67 μg / mL in 1640 + 10% FBS (Gibco) complete medium, followed by a 3-fold gradient dilution for a total of five concentration points. 100 μL / well of each was added to a 96-well cell plate. Zero-concentration control wells (i.e., complete medium without the ADC or antibody samples to be tested) were also included. DB cells (Nanjing Kebai Biotechnology Co., Ltd.) in logarithmic growth phase were harvested and diluted to 0.8–1.2 × 10 cells / well in 1640 + 10% FBS (Gibco) complete medium. 5 The solution was diluted to 100 cells / mL and added to the 96-well plate at 100 μL / well. The 96-well plate was placed in a CO2 incubator at 37°C for 60-96 hours, and 20 μL / well of CCK-8 (Dojindo Laboratories, CK04) was added to allow color development for 4-6 hours. The OD450nm reading was then detected using a microplate reader (Molecular Devices). The formula for calculating cell viability was: OD サンプルウェル / OD 対照ウェル ×100%.

[0113] As shown in Figure 1, the results showed that monoclonal antibodies F1 and F314 had no direct killing effect on antigen-positive tumor cells DB, whereas ADCs coupled to different toxins all had significant killing effect, and F314-SMCC-DM1, F314-SPDB-DM4, F314-MC-VC-PAB-MMAE, and F314-MC-MMAF had superior killing activity to F1-SMCC-DM1.

[0114] Example 5 In vivo antitumor activity of ADCs 5×10 6 Mino cells (ATCC) (supplemented with 50% Matrigel, Thermo Fisher Scientific) were inoculated into the right flank of CB17 / SCID mice (Shanghai SLAC, 6-8 weeks old), and tumor volumes were measured to reach an average of 90 mm. 3The blank control group and F314 group were divided into groups until they grew to 100 mm2, which was recorded as Day 0. Administration began on Day 0 in the blank control group and F314 group. Five animals per group were intraperitoneally injected, administered twice a week for six consecutive doses. The dose in the F314 group was 10 mg / kg, and the blank control group was injected with an equal volume of PBS. The F314-MC-VC-PAB-MMAE group, F314-C4-VC-PAB-MMAE group, F314-C5-VC-PAB-MMAE group, and F314-C6-VC-PAB-MMAE group were administered on Day 17 (mean tumor volume: 450 mm2). 3 The administration was started at 12:00 AM on the first day of the study, with four animals per group administered intravenously at a single dose of 88 μg / kg of the small molecule toxin MMAE. The length and width of the tumor were measured with a caliper, and the tumor volume was calculated using the formula TV = (length × width). 2 ) / 2, and the tumor volume was averaged for each group of mice to create a tumor growth curve. The results are shown in Figure 2. Under these experimental conditions, the F314 monoclonal antibody (calculated based on the mass of the F314 monoclonal antibody, with the antibody dose in the F314 group being higher than in the ADC-administered groups) showed no antitumor effect, but the four ADCs of the present invention exhibited significant antitumor effects, with tumors in all mice in the F314-C4-VC-PAB-MMAE, F314-C5-VC-PAB-MMAE, and F314-C6-VC-PAB-MMAE groups completely regressing.

[0115] Example 6 In vitro killing activity of ADCs against negative tumor cells Each ADC or antibody sample to be tested was diluted to an initial concentration of 6.67 μg / mL in McCoy's 5A + 10% FBS complete medium, followed by a 3-fold gradient dilution for a total of five concentration points. 100 μL / well of each was added to a 96-well cell plate. Zero-concentration control wells (i.e., complete medium without the ADC or antibody sample to be tested) were set up. SKOV-3 cells (ATCC) in logarithmic growth phase were harvested and 0.8–1.2 × 10 cells were cultured in McCoy's 5A + 10% FBS complete medium. 5The solution was diluted to 100 cells / mL and added to the 96-well plate at 100 μL / well. The 96-well plate was placed in a CO2 incubator at 37°C for 60-96 hours, and 20 μL / well of CCK-8 was added to allow color development for 4-6 hours. The OD450nm reading was then detected using a microplate reader. The formula for calculating cell viability was: OD サンプルウェル / OD 対照ウェル ×100%.

[0116] As shown in Figure 3, the results showed that the F314 monoclonal antibody and its ADCs coupled to different toxins (F314-MC-MMAF, F314-MC-VC-PAB-MMAE, F314-C4-VC-PAB-MMAE, F314-C5-VC-PAB-MMAE, and F314-C6-VC-PAB-MMAE) had no killing effect on the tumor cells SKOV-3, which do not express human CD39, indicating that the ADCs of the present invention have good targeting properties and are stable without causing nonspecific killing due to shedding of toxic molecules.

[0117] Example 7 Affinity activity of ADC against positive tumor cells The ADC or antibody samples to be tested were diluted with 1x PBS to an initial concentration of 20 μg / mL, then diluted 3-fold to a total of seven concentration points, and added at 50 μL per well to a 96-well V-bottom plate. At the same time, zero-concentration control wells (i.e., complete medium without the ADC or antibody samples to be tested) were also set up. MOLP-8 cells (DSMZ) were harvested and diluted to 8.0 × 10 6The cells were resuspended in 1:100 PBS and added to the 96-well plate at 50 μL per well. After mixing, the cells were incubated at 4°C for 0.5 hours. Washing: 100 μL per well of 1x PBS was added, centrifuged at 2000 rpm for 7 minutes, and the supernatant was discarded. 200 μL per well of 1x PBS was added, mixed thoroughly by pipetting, centrifuged at 2000 rpm for 7 minutes, and the supernatant was discarded. FITC-labeled goat anti-human H+L secondary antibody (Jackson 109-095-088) was diluted 1:100 and added at 100 μL per well. After mixing thoroughly, the cells were incubated at 4°C for 0.5 hours. The above washing step was repeated. 200 μL per well of 1x PBS was added to resuspend the cells, and the cells were loaded and analyzed on a flow cytometer.

[0118] As shown in Figure 4, the results show that both the F314 monoclonal antibody and the ADC of the present invention have affinity for MOLP-8 cells expressing CD39, and the affinity of the ADC did not change significantly compared to the naked antibody (i.e., the F314 monoclonal antibody).

[0119] Example 8 Endocytic Activity of ADCs ADC or antibody samples to be tested were diluted to 20 μg / mL in 1×PBS and added to 1.5 mL centrifuge tubes at 100 μL / tube. MOLP-8 cells were harvested and 1.0×10 7The cells were resuspended in 1x PBS at 1:100, added to the 1.5 mL centrifuge tubes at 100 μL per tube, mixed uniformly, and incubated at 4°C for 0.5 hours. Washing: 1 mL of 1x PBS was added, mixed uniformly by pipetting, and centrifuged at 2000 rpm for 7 minutes. The supernatant was discarded and the washing process was repeated once. 200 μL of 1x PBS was added to resuspend the cells, and the cells were divided into two equal portions. One portion was kept at 4°C, while the other was placed in a CO2 incubator at 37°C for 1 hour. The above washing step was repeated. FITC-labeled goat anti-human H+L secondary antibody (Jackson 109-095-088) was diluted 1:100, added at 100 μL per tube, mixed uniformly, and incubated at 4°C for 0.5 hours. The above washing step was repeated. The cells were resuspended in 200 μL of 1x PBS per tube, loaded into a flow cytometer, and analyzed. The formula for calculating % endocytosis is (MFI 4℃ -MFI 37℃ ) / MFI 4℃ ×100%.

[0120] The results, shown in Figure 5, show that the ADC retained the endocytic activity of the naked antibody.

[0121] Example 9 In vitro killing activity of F314-C5-VC-PAB-MMAE with different DAR values ​​against positive tumor cells The ADC or antibody samples to be tested were diluted to an initial concentration of 20 μg / mL with 1640 + 10% FBS complete medium, then diluted 10-fold to 2 μg / mL, and added to a 96-well cell plate at 100 μL / well. Zero-concentration control wells (i.e., complete medium without the ADC or antibody samples to be tested) were also set up. DB cells in the logarithmic growth phase were harvested and 0.8–1.2 × 10 cells were cultured in complete medium. 5 The solution was diluted to 100 μL / well and added to the 96-well plate. The 96-well plate was placed in a CO2 incubator at 37°C for 60-96 hours, and 20 μL / well of CCK-8 was added to allow color development for 4-6 hours. The OD450nm reading was then detected using a microplate reader. The cell viability percentage was calculated as follows: OD サンプルウェル / OD 対照ウェル×100%.

[0122] As shown in Figure 6, F314-C5-VC-PAB-MMAE, with a DAR ranging from 0.8 to 3.0, significantly improved the killing activity against positive tumor cells compared with the naked antibody F314 at the effective concentrations of 10 μg / mL and 1 μg / mL.

Claims

1. An antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, 【Chemistry 1】 Among them, L is a linker, D is a cytotoxic molecule; "-" is a bond, n is the average number of cytotoxic molecules coupled to the antibody, 0<n≦10, and n may be an integer or a non-integer; Ab is an anti-CD39 antibody or antigen-binding fragment thereof, comprising the following CDR regions: HCDR1, whose amino acid sequence is set forth in SEQ ID NO: 1; HCDR2, whose amino acid sequence is set forth in SEQ ID NO: 2; HCDR3, whose amino acid sequence is set forth in SEQ ID NO: 3; LCDR1, whose amino acid sequence is set forth in SEQ ID NO: 4; LCDR2, the amino acid sequence of which is set forth in SEQ ID NO: 5; and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 6; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

2. The anti-CD39 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO:7 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:7, and / or a light chain variable region amino acid sequence set forth in SEQ ID NO:8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

8. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

3. the anti-CD39 antibody or antigen-binding fragment thereof is a mouse antibody or antigen-binding fragment thereof, and further comprises a mouse-derived IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and / or a mouse-derived κ or λ light chain constant region; Alternatively, the anti-CD39 antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, and further comprises a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 of human origin, and / or a light chain constant region of κ or λ of human origin; or the amino acid sequence of the heavy chain of the chimeric antibody is set forth in SEQ ID NO: 9 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9, and / or the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 10 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10; The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or solvate thereof.

4. The anti-CD39 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, and further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, or IgG4 or the FR region of human germline heavy chain IGHV1-2*02, and / or a light chain FR region of human κ or λ chain or the FR region of human germline light chain IGKV1-33*01; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14 or 15, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, 12, 13, 14 or 15, and / or a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or 17, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 16 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 17 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

17. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

5. The humanized antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 of human origin, wherein the amino acid sequence of the human-derived IgG4 heavy chain constant region is set forth in SEQ ID NO: 18 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18; and / or the humanized antibody or antigen-binding fragment thereof further comprises a light chain constant region of κ or λ chain of human origin, wherein the amino acid sequence of the human-derived κ light chain constant region is set forth in SEQ ID NO: 19 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

19. The antibody-drug conjugate according to claim 4, or a pharmaceutically acceptable salt or solvate thereof.

6. The linker L is represented by the general formula (L): 【Chemistry 2】 Among them, L １ teeth, 【Transformation 3】 Selected from L ２ teeth 【Chemistry 4】 Among them, R １ and R ２ are each independently selected from H, an alkyl group, a haloalkyl group, or a halogen atom, and m is 1 to 8; or L ２ teeth 【Transformation 5】 or L ２ teeth 【Transformation 6】 and L ３ teeth, 【Transformation 7】 or The linker L is 【Transformation 8】 It is chosen from the structure The antibody-drug conjugate according to claim 1 or 4, or a pharmaceutically acceptable salt or solvate thereof.

7. the cytotoxic molecule D is selected from the group consisting of chemotherapeutic agents, DNA alkylating agents, tubulin inhibitors, topoisomerase inhibitors, antibiotics, or radioisotopes; or The cytotoxic molecule D is 【Chemistry 9】 It is chosen from the structure The antibody-drug conjugate according to claim 1 or 4, or a pharmaceutically acceptable salt or solvate thereof.

8. An antibody-drug conjugate represented by general formula (II) or a pharmaceutically acceptable salt or solvate thereof, 【Chemistry 10】 wherein m is 1 to 8, 0<n≦10, and Ab is as defined in claim 1 or 4; or An antibody-drug conjugate represented by general formula (III) or a pharmaceutically acceptable salt or solvate thereof, 【Chemistry 11】 wherein m is 1 to 8, 0<n≦8, and Ab is as defined in claim 1 or 4; or The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the following structures: 【Chemistry 12】 wherein 0<n≦8 and Ab is as defined in claim 1 or 4; 【Chemistry 13】 wherein 0<n≦10 and Ab is as defined in claim 1 or 4; 【Chemistry 14】 wherein 0<n≦10 and Ab is as defined in claim 1 or 4; or the antibody-drug conjugate is: 【Chemistry 15】 wherein 0<n≦8 and Ab is as defined in claim 1 or 4; 【Chemistry 16】 wherein 0<n≦10 and Ab is as defined in claim 1 or 4; 【Chemistry 17】 wherein 0<n≦10 and Ab is as defined in claim 1 or 4; [Chemistry 18] wherein 0<n≦10 and Ab is as defined in claim 1 or 4; or the antibody-drug conjugate is: (1) F314-MC-VC-PAB-MMAE, whose structure is shown in the following formula: 【Chemistry 19】 wherein n is 3.8 to 4.2; (2) F314-MC-MMAF, whose structure is shown in the following formula: 【Chemistry 20】 wherein n is 1.3 to 1.7; (3) F314-SMCC-DM1, whose structure is shown in the following formula: 【Chemistry 21】 wherein n is 4.8 to 5.2; (4) F314-SPDB-DM4, whose structure is shown in the following formula: 【Chemistry 22】 wherein n is 3.6 to 4.0; (5) F314-C4-VC-PAB-MMAE, whose structure is shown in the following formula: 【Chemistry 23】 wherein n is 1.9 to 2.3; (6) F314-C5-VC-PAB-MMAE, whose structure is shown in the following formula: 【Chemistry 24】 wherein n is between 0.5 and 3.5; or (7) F314-C6-VC-PAB-MMAE, whose structure is shown in the following formula: 【Chemistry 25】 wherein n is 1.9 to 2.3; In each formula, F314 represents humanized monoclonal antibody F314, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 14, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17, the amino acid sequence of its heavy chain constant region is shown in SEQ ID NO: 18, and the amino acid sequence of its light chain constant region is shown in SEQ ID NO:

19. The antibody-drug conjugate according to claim 1 or 4, or a pharmaceutically acceptable salt or solvate thereof.

9. The antibody-drug conjugate according to claim 8 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent, or vector. Pharmaceutical compositions.

10. 10. Use of the antibody-drug conjugate of claim 8, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating a CD39-mediated disease or condition.

11. The disease or condition is cancer, and the cancer is lymphoma, multiple myeloma, thyroid cancer, colorectal cancer, gastric cancer, renal cancer, prostate cancer, testicular cancer, breast cancer, ovarian cancer, or melanoma. The use according to claim 10.

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