Anti-PDL1 antibody-drug conjugate and use

Abstract

An antibody-drug conjugate (ADC) and a use thereof. The present invention relates to a novel anti-PDL1 ADC and a method and use thereof for treating diseases or conditions related to PDL1 expression.

Description

An anti-PDL1 antibody-drug conjugate and its application

[0001] This application claims priority to Chinese patent application No. CN202310513959.4 filed on May 9, 2023 and Chinese patent application No. CN202311311898.X filed on October 10, 2023. Technical Field

[0002] The present application belongs to the field of medicine and relates to an antibody-drug conjugate (ADC) and its use. Specifically, the present application relates to a novel anti-PDL1 antibody-drug conjugate and its method and use for treating diseases or conditions related to PDL1 expression. Background Art

[0003] In recent decades, immune checkpoint inhibitors, represented by PD-1 and PDL1 antibodies, have driven the rapid development of tumor immunotherapy, becoming an effective cancer treatment option alongside surgery, radiotherapy, chemotherapy, and targeted therapy. However, due to tumor heterogeneity, the complex regulatory mechanisms of PDL1 expression, and the poor penetration of large-molecule anti-PDL1 monoclonal antibodies into dense tumors, the clinical application of anti-PDL1 monoclonal antibodies has been significantly limited. The overall objective response rate (ORR) for patients is approximately 15%-30%, leaving a significant unmet clinical need.

[0004] In order to improve the response rate and treatment effect of cancer patients, anti-PD-1 monoclonal antibodies have begun to be explored in combination with chemotherapy, targeted therapy and radiotherapy, especially combined chemotherapy. Since chemotherapy can cause immunogenic cell death, more tumor antigens can be released, which can effectively promote the activation of the immune response. In addition, the number and activity of immunosuppressive cells can also be reduced. Therefore, chemotherapy is the main choice for immune combination. However, the systemic side effects caused by chemotherapy are still a very difficult problem. Since PDL1 is expressed in multiple tumor types and its expression in normal tissues is limited. Therefore, coupling highly active cytotoxic small molecule chemical drugs with anti-PDL1 monoclonal antibodies to produce bifunctional PDL1-targeted ADC drugs will be a very promising direction for drug research and development.

[0005] Currently, there are ADC drugs targeting PDL1 in the preclinical and Phase I trial exploration stages. SGN-PDL1V is a classic ADC platform technology product targeting PDL1 developed by Seagen. The payload drug (Payload) uses MMAE and is currently in Phase I clinical trials. MMAE (monomethyl auristatin E) is one of the most widely used payload drugs for ADCs. It mainly inhibits microtubule polymerization to effectively inhibit mitosis and kill cancer cells, but the quality window of this ADC product is relatively narrow. Therefore, this field urgently needs drugs with higher biological activity and lower toxicity, especially those that are effective against drug-resistant mutants, such as antibody-drug conjugates, to further improve efficacy and reduce side effects.

[0006] SUMMARY OF THE INVENTION

[0007] The present application relates to an antibody-drug conjugate of formula (I) as defined herein and a pharmaceutical composition comprising the antibody-drug conjugate. The present application is characterized by a method for treating or preventing a tumor or cancer associated with PDL1 expression, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate of formula (I) as defined herein, a prodrug, a pharmaceutically acceptable salt, a solvate, or a pharmaceutically acceptable salt solvate thereof. The method of the present application can be used to treat or prevent a tumor or cancer associated with PDL1 expression.

[0008] The first aspect of the present application relates to an antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure of formula (I):

[0009] TP-[L1-L2-L3-D] k

[0010] (I)

[0011] in:

[0012] TP is a targeting moiety that selectively binds to or recognizes PDL1;

[0013] L1 is an extension unit, which connects TP and L2;

[0014] L2 is an optional amino acid residue or a short peptide consisting of 2-10 amino acid residues;

[0015] L3 is a spacer element;

[0016] D is a bioactive molecule;

[0017] k represents any value between about 0.1 and about 10.0.

[0018] Another aspect of the present application relates to a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), a prodrug, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, diluent or excipient.

[0019] Yet another aspect of the present application relates to a method for treating a tumor or cancer related to PDL1 expression. The method includes administering an antibody-drug conjugate of formula (I), a prodrug thereof, a pharmaceutically acceptable salt, a solvate or a pharmaceutically acceptable salt thereof to a subject in need thereof, or a pharmaceutical composition comprising the same. The tumor or cancer related to PDL1 expression is selected from melanoma, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), head and neck cancer, breast cancer (e.g., triple negative breast cancer (TNBC)), ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, bladder cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer and glioma.

[0020] Another aspect of the present application relates to the use of the antibody-drug conjugate of formula (I) of the present invention, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition comprising the same in the preparation of a drug.

[0021] The present application particularly provides antibody-drug conjugates and compositions having improved efficacy and / or anti-tumor activity and lower toxicity compared to anti-PDL1-ADCs known in the prior art.

[0022] The details of the present application are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. Other features, objects and advantages of the present application will be apparent from the specification and claims. In the specification and the appended claims, the singular also includes the plural, unless the context clearly provides otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0023] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a graph showing the affinity of anti-PDL1 antibody-drug conjugates for PDL1-positive cells NCI-H292 and NCI-H441. (A) Affinity of the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR8) for NCI-H292 cells. (B) Affinity of the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR8) for NCI-H441 cells. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by coupling MAB20 and LP62 (whose structure is shown in the Examples); DAR represents the drug-loaded:antibody ratio; IgG1 represents human IgG1, κ isotype control.

[0025] Figure 2 shows the uptake of an anti-PDL1 antibody-drug conjugate into PDL1-positive NCI-H292 cells over time. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB20 and LP62; DAR represents the drug-to-antibody ratio.

[0026] Figure 3 is a graph showing the cytotoxic activity of the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR8). (A) Cytotoxic activity of MAB20-LP62 (DAR8) against the NCI-H292 cell line. (B) Cytotoxic activity of MAB20-LP62 (DAR8) against the BXPC3 cell line. (C) Cytotoxic activity of MAB20-LP62 (DAR8) against the NCI-H441 cell line. Note: MAB20 represents the anti-PDL1 antibody, the amino acid sequence of which is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB20 and LP62; DAR represents the drug-loaded:antibody ratio.

[0027] Figure 4 is a graph showing the cross-binding activity of anti-PDL1 antibody-drug conjugates against PDL1 derived from different species. (A) Binding of MAB20-LP62 (DAR8) to human PDL1 ECD. (B) Binding of MAB20-LP62 (DAR8) to cynomolgus monkey PDL1 ECD. (C) Binding of MAB20-LP62 (DAR8) to mouse PDL1 ECD. (D) Binding of MAB20-LP62 (DAR8) to rat PDL1 ECD. Note: MAB20 represents the anti-PDL1 antibody, the amino acid sequence of which is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 to LP62; DAR represents the drug-loaded:antibody ratio; IgG1 represents human IgG1, a kappa isotype control.

[0028] Figure 5 is a graph showing the bystander effect of the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR 8). (A) In vitro killing of MDA-MB-468 cells by the test molecule. (B) Bystander killing of MDA-MB-468 cells by the culture supernatant of HEK293 cells overexpressing PD-L1 incubated with the test molecule. Note: MAB20 represents the anti-PDL1 antibody, the amino acid sequence of which is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 and LP62; IgG1-LP62 represents the antibody-drug conjugate formed by conjugating IgG1 and LP62, with a DAR value of 8; DAR represents the drug-loaded:antibody ratio.

[0029] Figure 6 is a graph showing the in vitro killing of human macrophages by the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR8). Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is recorded in the sequence table; MAB20-LP62 represents the antibody-drug conjugate formed by coupling MAB20 and LP62; IgG1-LP62 represents the antibody-drug conjugate formed by coupling IgG1 and LP62; IgG1-VC-MMAE represents the antibody-drug conjugate formed by isotype control IgG1 prepared by a method similar to Example 3.2 and MC-VC-PABC-MMAE; SGN-PDL1V represents the reference ADC molecule as described in WO2021067776A1, which is an antibody-drug conjugate formed by coupling the SGN-PDL1V antibody portion (whose amino acid sequence is shown in the sequence table) to the linker-toxin MC-VC-PABC-MMAE; DAR represents the drug-load:antibody ratio.

[0030] Figure 7 is a graph showing the in vitro killing of human dendritic cells by the anti-PDL1 antibody-drug conjugate MAB20-LP62 (DAR8). Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is recorded in the sequence table; MAB20-LP62 represents the antibody-drug conjugate formed by coupling MAB20 and LP62; IgG1-VC-MMAE represents the antibody-drug conjugate formed by isotype control IgG1 prepared by a method similar to Example 3.2 and MC-VC-PABC-MMAE; SGN-PDL1V represents the reference ADC molecule as described in WO2021067776A1, which is an antibody-drug conjugate formed by coupling the SGN-PDL1V antibody portion (whose amino acid sequence is shown in the sequence table) to the linker-toxin MC-VC-PABC-MMAE; DAR represents the drug-loaded:antibody ratio.

[0031] Figure 8 is a graph showing the anti-tumor activity of anti-PDL1 antibody-drug conjugates in an NCG mouse model inoculated subcutaneously with the human breast cancer cell line MDA-MB-231. Note: MAB20 represents the anti-PDL1 antibody, the amino acid sequence of which is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 to LP62; MAB20-VC-MMAE represents the antibody-drug conjugate formed by conjugating MAB20 to the linker-toxin MC-VC-PABC-MMAE; MAB20-GGFG-Dxd represents the antibody-drug conjugate formed by conjugating MAB20 to the linker-toxin MC-GGFG-Dxd; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0032] Figure 9 is a graph showing the anti-tumor activity of anti-PDL1 antibody-drug conjugates in an NCG mouse model inoculated subcutaneously with the human pancreatic cancer cell line BxPC3. Note: MAB20 represents the anti-PDL1 antibody, the amino acid sequence of which is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 to LP62; MAB20-VC-MMAE represents the antibody-drug conjugate formed by conjugating MAB20 to the linker-toxin MC-VC-PABC-MMAE; MAB20-GGFG-DXD represents the antibody-drug conjugate formed by conjugating MAB20 to the linker-toxin MC-GGFG-DXD; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0033] Figure 10 is a graph showing the anti-tumor effect of anti-PDL1 antibody-drug conjugates in BALB / c nude mice bearing human non-small cell lung cancer LU6437 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by coupling MAB20 to LP62; SGN-PDL1V represents the reference ADC molecule described in WO2021067776A1, which is an antibody-drug conjugate formed by coupling the SGN-PDL1V antibody portion (whose amino acid sequence is shown in the sequence listing) to the linker-toxin MC-VC-PABC-MMAE; DAR represents the drug-loaded:antibody ratio.

[0034] Figure 11 is a graph showing the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PD-L1-positive liver cancer LD1-0011-200617 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 to LP62; DAR represents the drug-to-antibody ratio.

[0035] Figure 12 is a graph showing the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in NCG mice bearing human PD-L1-positive head and neck squamous cell carcinoma LD1-2023-411020 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB20 and LP62; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0036] Figure 13 shows the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in Balb / c nude mice bearing human PD-L1-positive cervical cancer LD1-0010-200614 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB20 and LP62; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0037] Figure 14 shows the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in Balb / c nude mice bearing human PD-L1-positive esophageal squamous cell carcinoma LD1-0015-362448 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 to LP62; DAR represents the drug-to-antibody ratio.

[0038] Figure 15 is a graph showing the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PD-L1-positive gastric cancer LD1-0017-200636 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 and LP62; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0039] Figure 16 shows the anti-tumor effect of an anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PD-L1-positive colorectal cancer LD1-2013-362125 PDX xenografts. Note: MAB20 represents the anti-PDL1 antibody, whose amino acid sequence is described in the sequence listing; MAB20-LP62 represents the antibody-drug conjugate formed by conjugating MAB20 and LP62; A1.3 represents the toxin compound A1.3 described in Example 1, which is the free drug payload; DAR represents the drug payload:antibody ratio.

[0040] Detailed Description of the Invention

[0041] The present disclosure provides a novel anti-PDL1 antibody-drug conjugate. The anti-PDL1 antibody-drug conjugate exhibits good affinity for PDL1-positive cells, can be effectively internalized into PDL1-positive cells, and even exhibits good cell-killing effects on tumor cells that are insensitive to anti-PDL1. Furthermore, the anti-PDL1 antibody-drug conjugate has a good bystander effect and lower toxicity (especially immunotoxicity). Furthermore, the anti-PDL1 antibody-drug conjugate exhibits excellent tumor inhibitory activity in various tumor models (even PDL1 low-expressing tumor models).

[0042] For the sake of clarity and not by way of limitation, the detailed description of the invention is divided into the following subsections:

[0043] 1. Definition;

[0044] 2. Antibody-drug conjugates;

[0045] 3. Instructions for use;

[0046] 4. Pharmaceutical preparations; and

[0047] 5. Products and kits.

[0048] 1. Definition

[0049] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0050] The terms "PD-L1," "PDL1," "CD274," "B7-H1," and "programmed cell death ligand 1" are used interchangeably herein and, unless otherwise indicated, include any variants, isoforms, and homologs of human PD-L1 that are normally expressed by cells or expressed on cells transfected with the PD-L1 gene.

[0051] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0052] A full-length antibody contains two heavy chains and two light chains. The variable regions of the heavy and light chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as “V H ” and “V L The variable regions in both chains typically contain three highly variable loops, termed complementarity determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs comprising HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the following conventions: Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of the constant region of their heavy chains. The five major classes, or isotypes, of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0053] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a bispecific antibody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized bispecific antibody (dsbispecific antibody), a single-chain Fv (scFv), an scFv dimer (a bivalent diabody), a multispecific antibody formed by a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which a parent antibody or parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted to a framework region from one or more different human antibodies.

[0054] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem., 252: 6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein the definitions include overlap or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or transplanted antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined in each of the above references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0055] Table 1: CDR Definition

[0056] 1 Residue numbering follows the nomenclature of Kabat et al. (supra).

[0057] 2 Residue numbering follows the nomenclature of Chothia et al. (supra).

[0058] 3 Residue numbering follows the nomenclature of MacCallum et al. (supra).

[0059] 4 Residue numbering follows the nomenclature of Lefranc et al. (supra).

[0060] 5 Residue numbering follows the nomenclature of Honegger and Plückthun (supra).

[0061] The phrase "variable domain residues are numbered as in Kabat" or "amino acid positions are numbered as in Kabat" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may comprise a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with regions of homology to the "standard" Kabat numbering sequence.

[0062] Unless otherwise indicated herein, the amino acid residues encompassing the CDRs of full-length antibodies are defined according to the Kabat nomenclature of Kabat et al., supra, and the residue numbering in an immunoglobulin heavy chain, e.g., an Fc region, is that of the EU index as described in Kabat et al., supra, except that the amino acid residues encompassing the CDRs of any consensus sequence are defined according to the Kabat nomenclature, with modifications based on experimental conditions. The "EU index as described in Kabat" refers to the residue numbering of a human IgG1 EU antibody.

[0063] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0064] The non-human (e.g., rodent) antibody of the "humanized" form is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor hypervariable region (HVR) are replaced by residues from the hypervariable region (donor antibody) with desired antigen-specificity, affinity and capacity from a non-human species (e.g., mouse, rat, rabbit or non-human primate). In some instances, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may include residues not found in the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will include substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0065] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also useful for preparing human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., an immunized xenomice) that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled (see, e.g., for XENOMOUSETM For human antibodies generated by human B cell hybridoma technology, see also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).

[0066] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after the sequences have been aligned (taking into account any conservative substitutions as part of the sequence identity). For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that achieves maximal alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0067] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage 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, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, a comparison is made when two sequences are aligned to give maximum homology.

[0068] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C H 1. C H 2 and C H 3 domains (collectively referred to as C H ) and the CHL (or CL ) domain.

[0069] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0070] A "CH1 domain" (also called "C1" of an "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0071] The "hinge region" is generally defined as the region corresponding to Glu216 to Pro230 of human IgG1 in IgG (Burton, Molec. Immunol., 22: 161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0072] The "CH2 domain" of the human IgG Fc region (also known as the "C2" domain) typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that carbohydrates can provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol., 22: 161-206 (1985).

[0073] The "CH3 domain" (also called the "C2" domain) comprises the region of residues in the Fc region that are C-terminal to the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence (typically at amino acid residue 446 or 447 in IgG)).

[0074] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed during, for example, the production or purification of an antibody or by recombinant engineering of nucleic acids encoding the heavy chain of the antibody. Therefore, the composition of a complete antibody can include an antibody population having all K447 residues removed, an antibody population not having the K447 residue removed, and an antibody population with and without an antibody mixture of the K447 residue. Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0075] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and spliced ​​forms of these receptors, the FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those identified in the future.

[0076] As used herein, the terms "specific binding," "specific recognition," and "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to the target with an affinity, avidity, readiness, and / or duration that is longer than binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the extent of binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In some embodiments, the dissociation constant (K) of an antibody that specifically binds to a target is less than about 10% of the extent of binding of the antibody to the target as measured by radioimmunoassay (RIA). D )≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤10 -12 In some embodiments, the antibody specifically binds to an epitope of a protein that is conserved among proteins from different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of an antibody or antigen binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE TM -Inspection and peptide scanning.

[0077] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include: radioactive isotopes, such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、C 60 and radioactive isotopes of Lu; chemotherapeutic agents; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0078] "Linker," "Linker unit," or "Linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently links an antibody or targeting moiety to a drug moiety.

[0079] As used herein, the terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a compound linked to an antibody (e.g., an anti-PDL1 antibody) or a derivative thereof, and are defined by the following general formula: Ab-(LD)k, where Ab = antibody portion (i.e., antibody or antigen-binding fragment), L = linker portion, D = drug moiety, and k = the number of drug moieties conjugated per antibody portion.

[0080] Among the antibody-drug conjugates currently on the market, there are two main ways to couple antibodies and linkers: (1) Lysine is the most common linking site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, there is a technology that can achieve site-specific coupling, that is, activating the carboxyl group of the linker with an activating group, and then forming an amide bond with the specific lysine ε-amino group in the antibody to complete the coupling. (2) The sulfhydryl group (SH) of the cysteine ​​of the antibody exists in the form of a disulfide bond. Opening the disulfide bond in the antibody can provide multiple free sulfhydryl groups as coupling sites. One method of coupling with the antibody sulfhydryl group is to react the free sulfhydryl group on the antibody with maleimide through Michael addition reaction, or a specific substrate can react with the free sulfhydryl group on the antibody through two Michael addition reactions to form a unique structural sulfur bridge bond.

[0081] As used herein, the three letter and one letter codes for amino acids are as described in J. boil. Chem. 1968, 243, 3558.

[0082] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0083] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which 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, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which 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, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, amino, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0084] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0085] As used herein, the term "cyano" refers to -CN.

[0086] As used herein, the term "sulfonic acid" refers to the group -SO3H.

[0087] As used herein, the term "carboxy" refers to -C(O)OH.

[0088] As used herein, the term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0089] The term "alkenyl" refers to an alkyl compound containing at least one carbon-carbon double bond in the molecule, wherein the alkyl group is as defined above and has 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) carbon atoms (i.e., C 2-10 The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (i.e., C 2-6 The alkenyl group may be substituted or unsubstituted. When substituted, the substituent is preferably selected from one or more of alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0090] The term "alkynyl" refers to an alkyl compound containing at least one carbon-carbon triple bond in the molecule, wherein the alkyl group is as defined above and has 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) carbon atoms (i.e., C 2-10 The alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (i.e., C 2-6 Alkynyl). Alkynyl may be substituted or unsubstituted. When substituted, the substituent is preferably selected from one or more of alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0091] As used herein, the term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0092] The present invention also includes various deuterated forms of formula (I). Each available hydrogen atom connected to the carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula (I) with reference to relevant literature. When preparing the deuterated form of formula (I), commercially available deuterated starting materials can be used, or they can use conventional techniques to adopt deuterated reagents to synthesize, and the limiting examples of deuterated reagents include: deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane etc.

[0093] As used herein, the term "DAR" refers to the drug loading:antibody ratio, which represents the average number of cytotoxic drugs loaded per antibody and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 0-12, preferably 1-10, cytotoxic drugs (D) attached per antibody (Ab). In embodiments of the present invention, DAR is expressed as k, which can be, by way of example and not limitation, an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average number of drugs per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0094] In one embodiment of the present invention, the cytotoxic drug is conjugated to the opened interchain cysteine ​​thiol-SH groups of the antibody and / or to the site-directed mutagenesis cysteine ​​thiol-SH groups via a linker unit. Generally speaking, the number of drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than or equal to the theoretical maximum.

[0095] The loading capacity of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

[0096] (1) Control the molar ratio of linker-loaded drug, reducing agent and monoclonal antibody,

[0097] (2) Control reaction time, temperature and pH value,

[0098] (3) Select different reaction reagents and contents.

[0099] As used herein, the term "solvate" or "solvate" refers to a pharmaceutically acceptable solvate formed between the ligand-drug conjugate of the present invention and one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, and dimethylacetamide (DMAC).

[0100] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of an antibody-drug conjugate. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. Counterions can be any organic or inorganic module that stabilizes the charge of the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In the case where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions may be present. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0101] "Pharmaceutically acceptable solvate" refers to the combination of one or more solvent molecules and ADC or its salt. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0102] As used herein, " treatment (treatment or treating) " is the method for obtaining beneficial or desired result (including clinical outcome).For the purpose of the application, beneficial or desired clinical outcome includes but is not limited to one or more of the following: alleviate one or more symptoms caused by the disease, reduce the degree of the disease, stabilize the disease (for example, prevent or delay the deterioration of the disease), prevent or delay the spread of the disease (for example, metastasis), prevent or delay the recurrence of the disease, delay or slow down the progress of the disease, improve the disease state, provide the alleviation (partial or complete) of the disease, reduce the dosage of one or more other drugs required for the treatment of the disease, delay the progress of the disease, increase or improve the quality of life, increase weight gain and / or prolong survival." treatment " also encompasses the pathological consequences (such as, tumor volume) that reduce cancer. The method of the application contemplates any one or more of these treatment aspects.

[0103] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0104] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or to a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to a decrease, reduction, or prevention of an activity, function, and / or amount, as compared to a reference. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

[0105] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy and / or non-diseased sample. In certain embodiments, a reference can be obtained from an untreated sample. In certain embodiments, a reference is obtained from a non-diseased or untreated sample of an individual. In some instances, a reference is obtained from one or more healthy individuals who are not the individual or patient.

[0106] As used herein, "delaying the development of a disease" means postponing, hindering, slowing, slowing, stabilizing, inhibiting, and / or retarding the development of a disease (e.g., cancer). The delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay can actually include prevention, in that the individual does not develop the disease. For example, advanced cancer (e.g., the development of metastases) may be delayed.

[0107] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0108] As used herein, "inhibiting" a function or activity is reducing the function or activity compared to conditions that are identical except for the condition or parameter of interest, or alternatively compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of the tumor in the absence of the antibody.

[0109] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0110] An "effective amount" of an agent is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. The specific dosage may vary depending on one or more of the following: the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0111] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist of the present invention can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the substance / molecule, agonist or antagonist are offset by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0112] A "prophylactically effective amount" refers to an amount effective, measured at dosages and for the period of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactic effective amount will be less than the therapeutically effective amount because the prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0113] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in such form as to permit the biological activity of one or more active ingredients to be effective and that contains no other components that are unacceptably toxic to the individual to whom the formulation is to be administered. Such formulations may be sterile.

[0114] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or vehicle conventional in the art used with a therapeutic agent, which together constitutes a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for use in the formulation employed.

[0115] It should be understood that in the event of any inconsistency between a chemical name and a chemical structure in this application, the chemical structure shall prevail.

[0116] It should be understood that embodiments of the application described herein include "consisting of" and / or "consisting essentially of.

[0117] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X."

[0118] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, which means that the method is for treating cancer that is different from type X.

[0119] The term "about XY" as used herein has the same meaning as "about X to about Y".

[0120] As used herein, the term "about" when used to modify an amount of an ingredient or reactant of the invention refers to variations in the numerical amount that may occur, for example, in typical measurements and liquid handling procedures used to prepare concentrates or actual use solutions; accidental errors in such procedures; differences in the manufacture, source, or purity of ingredients used to prepare the compositions or practice the methods; etc. The term "about" also includes amounts that vary due to different equilibrium conditions relative to the composition obtained from a particular starting mixture. Whether or not modified by the term "about," the claims include equivalent amounts of the amounts. In one embodiment, the term "about" means within 10% of the reported value, preferably within 5% of the reported value.

[0121] Those skilled in the art will understand that when a numerical value is stated in a claim, whether or not it is preceded by "about," the actual value of the numerical value may fluctuate by 10% (±10%) above or below the stated value, preferably by 5% (±5%) above or below the stated value.

[0122] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0123] 2. Antibody-drug conjugates

[0124] Anti-PD-L1 antibodies can be coupled to cytotoxic or cytostatic moieties (including pharmaceutically compatible salts thereof) to form antibody-drug conjugates (ADCs). Particularly suitable moieties for coupling to antibodies are cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-PD-L1 antibodies can be coupled to cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cytostatic or cell-killing agents, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin).

[0125] The anti-PD-L1 antibody can be coupled to a prodrug converting enzyme. The prodrug converting enzyme can be recombinantly fused to the antibody or chemically coupled thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0126] The techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known. (See, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al., eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, e.g., PCT Publication No. WO 89 / 12624.)

[0127] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleavage agent). Such a therapeutic agent is attached to the antibody with a cleavable linker that is sensitive to cleavage in the intracellular environment or tumor microenvironment of cancer cells expressing PD-L1, such that it is cleaved from the antibody when the conjugate is in that environment (e.g., in an endosome or, for example, due to pH sensitivity or protease sensitivity, in a lysosomal environment, in a caveolar environment, or in a tumor microenvironment).

[0128] Typically, ADC includes a linker region between a therapeutic agent and an anti-PD-L1 antibody. As described above, typically, the linker is cleavable under intracellular conditions or in a tumor microenvironment, such that the cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., in a lysosome or endosome or pit) or in a tumor microenvironment. The linker can be, for example, a peptidyl linker cleaved by an intracellular peptidase or protease (including a lysosomal or endosomal protease). Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. The cleavage agent can include cathepsins B and D and plasmin, etc. (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most typical is a peptidyl linker that can be cleaved by an enzyme present in a cell expressing PD-L1 or in a tumor microenvironment. For example, a peptidyl linker (e.g., a linker comprising a Phe-Leu or Gly-Phe-Leu-Gly peptide) that can be cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, can be used. Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In a specific embodiment, the peptidyl linker that can be cleaved by an intracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent typically decays upon conjugation, and the serum stability of the conjugate is generally high.

[0129] The cleavable linker can be pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers (e.g., hydrazones, semicarbazones, thiothiocarbamides, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). Such linkers are relatively stable under neutral pH conditions (such as those in blood), but are unstable when they are below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0130] Other linkers (e.g., disulfide linkers) are cleavable under reducing conditions. Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB, and SMPT. See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

[0131] The linker can also be a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12). The linker can also be a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0132] The linker can also be a non-cleavable linker, such as a maleimido-alkylene- or maleimido-aryl linker that is directly attached to the therapeutic agent (e.g., a drug). The active drug-linker is released by degradation of the antibody.

[0133] Typically, the linker is substantially insensitive to the circulatory environment, meaning that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of ADC are cleaved when the ADC is present in plasma.

[0134] Whether a linker is substantially insensitive to the circulatory environment can be determined, for example, by independently incubating (a) ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample to the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0135] The anti-PDL1 antibody can be coupled to the linker via heteroatoms of the antibody. These heteroatoms can be present on the antibody in its natural state or can be introduced into the antibody. In some aspects, the anti-PDL1 antibody will be coupled to the linker via the nitrogen atom of a lysine residue. In other aspects, the anti-PDL1 antibody will be coupled to the linker via the sulfur atom of a cysteine ​​residue. The cysteine ​​residue can be a naturally occurring residue or a residue engineered into the antibody. Methods for coupling linkers and drug-linkers to antibodies via lysine and cysteine ​​residues are known in the art.

[0136] Exemplary antibody-drug conjugates also include antibody-drug conjugates based on camptothecin (i.e., the drug component is a camptothecin drug). Camptothecin is a topoisomerase inhibitor that has been shown to have anti-cancer activity. Typically, the antibody-drug conjugate based on camptothecin is included in a linker between the camptothecin drug and the anti-PDL1 antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released by degradation of the antibody). The synthesis and structure of exemplary camptothecin drug linkers are described in PCT / US19 / 025968, which is incorporated herein by reference in its entirety and for all purposes.

[0137] Other exemplary antibody-drug conjugates include maytansinoid antibody-drug conjugates (i.e., the drug component is a maytansinoid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is a benzodiazepine (e.g., pyrrolo[1,4]benzodiazepine dimer (PBD dimer), indolebenzodiazepine dimer, and oxazolidinebenzodiazepine dimer)).

[0138] Useful classes of cytotoxic agents conjugated to anti-PDL1 antibodies include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, chemosensitizers, etc. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposide, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include camptothecins (e.g., isitecan, DXD, etc.), auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., enediynes and lexitropsin), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, and cyclomorpholino-doxorubicin.

[0139] The cytotoxic agent can be a chemotherapeutic agent such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analog, a calicheamicin, a maytansine, an analog of Aplysia 10, a rhizobactin, or a sea anemone toxin.

[0140] The cytotoxic agent may also be an auristatin. The auristatin may be an auristatin E derivative, such as an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include auristatin T, AFP, MMAF, and MMAE. The synthesis and structure of various auristatins are described, for example, in US2005-0238649 and US2006-0074008.

[0141] The present invention discloses an antibody-drug conjugate and its preparation method and application, in particular, a conjugate of an anti-PD L1 antibody and a new tumor-sensitive topoisomerase (TOP) I inhibitor and its pharmaceutical composition, preparation method and application. Through anti-tumor experiments, it was found that the obtained antibody-drug conjugate has excellent activity, such as strong anti-tumor activity, can significantly improve the survival rate of tumor-bearing animals, and significantly reduce toxicity, with less burden on the body of experimental animals, greatly reducing the minimum effective dose of small molecule drugs when used alone, expanding their therapeutic window, and is expected to be used in the development of therapeutic drugs for various diseases (such as tumors, etc.), with good application prospects and value.

[0142] The anti-PDL1 antibody-drug conjugate of the present invention is a new type of targeted PDL1 antibody-drug conjugate (ADC), which is mainly composed of three parts, namely a targeted PDL1 monoclonal antibody, a small molecule drug (payload) and a tumor microenvironment-sensitive linker (Linker), wherein the loaded drug is a new type of topoisomerase (TOP) I inhibitor that is more sensitive to tumors. After the anti-PDL1 antibody-drug conjugate of the present invention enters the body, it can specifically bind to PDL1 highly expressed on tumor cells through its antibody part. A part of it releases the loaded drug in the tumor microenvironment to kill cancer cells, and the other part enters the cancer cells after PDL1-mediated drug internalization. In the special environment inside the tumor, the peptide linker of the anti-PDL1 antibody-drug conjugate of the present invention is cleaved to efficiently release the payload, and the loaded small molecule drug exerts a cytotoxic effect and can kill cancer cells at a specific point. Due to its high lipophilicity, part of the payload can exert a "bystander effect" and enter the cells around the cancer cells to exert its effect. At the same time, PDL1-ADC has the potential to block the PD-1 / PDL1 immune checkpoint. These anti-cancer mechanisms are also highly effective against antigen-low-expressing and negative tumor cells. The anti-PD-1 antibody-drug conjugates of the present invention are expected to be ideal therapeutics for patients who have a suboptimal response to, or whose disease progresses after, treatment with PD-1 / PD-1 inhibitors.

[0143] The anti-PDL1 antibody-drug conjugate of the present invention has the following characteristics:

[0144] (1) Adopt a new generation of linker types that are more sensitive to the tumor microenvironment to ensure the stability of ADC drugs in the blood circulation and to efficiently release the loaded small molecule drugs after entering the tumor cells or in the tumor microenvironment.

[0145] (2) The use of a new generation of payload drugs (Payloads) can effectively kill cancer cells with different PDL1 expression levels.

[0146] (3) Adopting a new generation of antibody conjugation technology to ensure the consistency and accuracy of the antibody conjugation site, making its half-life and immunogenicity similar to that of the antibody, and retaining the PD-1 / PDL1 immune checkpoint blocking function of the antibody part.

[0147] In some aspects, the present disclosure provides an antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by formula (I):

[0148] TP-[L1-L2-L3-D] k

[0149] (I)

[0150] in:

[0151] TP is a targeting moiety that selectively binds to or recognizes PDL1;

[0152] L1 is a stretching unit that connects TP and L2, wherein TP is connected to L1 through an active group (e.g., a thiol or amino group, etc.), preferably wherein L1 is coupled to an open interchain cysteine ​​thiol-SH group and / or a site-directed mutated cysteine ​​thiol-SH group in TP;

[0153] L2 is an optional amino acid residue or a short peptide consisting of 2-10 amino acid residues;

[0154] L3 is a spacer element;

[0155] D is a bioactive molecule;

[0156] k represents any value between 0.1 and 10.0.

[0157] When referring to antibody-drug conjugates, the subscript k represents the drug loading and, depending on the context, can represent the number of drug-linker molecules attached to a single antibody molecule and, therefore, be an integer value; or can represent the average drug loading and, therefore, can be an integer or non-integer value. The average drug loading represents the average number of drug-linker molecules per antibody in the population. Typically, but not always, when referring to an antibody, such as a monoclonal antibody, we are referring to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, the average drug loading is an important quality attribute because it determines the amount of drug that can be delivered to the target cell. The percentage of unconjugated antibody molecules in the composition is included in the average drug loading value.

[0158] In preferred aspects of the invention, when referring to a composition comprising a population of antibody-drug conjugate compounds, the average drug loading is from 1 to about 16, preferably from about 2 to about 14, more preferably from about 2 to about 10.

[0159] For MMAE and camptothecin ADCs, such as those exemplified herein, the preferred average drug load is about 2, 4, or 8, and a particularly preferred average drug load is about 8. In one embodiment, the preferred average drug load for MMAE ADCs is 2 or 4. In one embodiment, the preferred average drug load for camptothecin ADCs is 4 or 8. In exemplary embodiments, the drug-linker is coupled to a cysteine ​​residue of a reduced interchain disulfide. In some aspects, the actual drug load of individual antibody molecules in the antibody-drug conjugate compound population is from 1 to 10 (or from 6 to 10 or from 6 to 8), with the predominant drug load being 8.

[0160] In some embodiments of Formula (I), the TP is an antibody or an antigen-binding fragment thereof.

[0161] In some embodiments of Formula (I), the antigen-binding fragment is selected from a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0162] In some embodiments of Formula (I), the TP is a humanized antibody or an antigen-binding fragment thereof.

[0163] In some embodiments of Formula (I), the TP comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3 comprised by SEQ ID NO:7, and the light chain variable region (VL) comprises a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3 comprised by SEQ ID NO:8.

[0164] In some embodiments of Formula (I), the amino acid sequence of the HCDR1 is SEQ ID NO: 1, the amino acid sequence of the HCDR2 is SEQ ID NO: 2, the amino acid sequence of the HCDR3 is SEQ ID NO: 3, and the amino acid sequence of the LCDR1 is SEQ ID NO: 4, the amino acid sequence of the LCDR2 is SEQ ID NO: 5, and the amino acid sequence of the LCDR3 is SEQ ID NO: 6.

[0165] In some embodiments of Formula (I), the TP comprises an Fc sequence of human IgG.

[0166] In some embodiments of Formula (I), the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0167] In some embodiments of Formula (I), the TP comprises a heavy chain variable region (VH), and the amino acid sequence of the heavy chain variable region (VH) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:7.

[0168] In some embodiments of Formula (I), the TP comprises a light chain variable region (VL), and the amino acid sequence of the light chain variable region (VL) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:8.

[0169] In some embodiments of Formula (I), the TP comprises a heavy chain (HC), and the amino acid sequence of the heavy chain (HC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:9.

[0170] In some embodiments of Formula (I), the TP comprises a light chain (LC), and the amino acid sequence of the light chain (LC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:10.

[0171] In some embodiments of Formula (I), the TP is a multispecific antibody.

[0172] In some embodiments of Formula (I), the TP is an afucosylated antibody.

[0173] In some embodiments of Formula (I), the Fc region comprises a C-terminal lysine.

[0174] In some embodiments of Formula (I), the Fc region comprises a deletion of the C-terminal lysine.

[0175] In some embodiments of Formula (I), the L1 is selected from

[0176] wherein L1 is connected to TP via an S atom at position a and to L2 at position b, m is any integer between 1 and 10, n is any integer between 1 and 10, and X and Y are each independently C, O, S or N.

[0177] In some embodiments of formula (I), said L1 is

[0178] Wherein, m is selected from 1, 2, 3, 4, 5,

[0179] Wherein L1 is connected to TP via an S atom at the a position and is connected to L2 at the b position.

[0180] In some embodiments of formula (I), said L1 is

[0181] Wherein L1 is connected to TP via an S atom at the a position and is connected to L2 at the b position.

[0182] In some embodiments, L1 is linked to the cysteine ​​(more specifically, the sulfhydryl group of the cysteine) of the TP at the α position.

[0183] In some embodiments of Formula (I), said L2 is selected from

[0184] Wherein L2 is connected to L1 at the c position and to L3 at the d position.

[0185] In some embodiments of Formula (I), said L2 is

[0186] Wherein L2 is connected to L1 at the c position and to L3 at the d position.

[0187] In some embodiments of formula (I), L3 is a single bond or a group selected from the following:

[0188] Wherein L3 is connected to L2 at the e position and to D at the f position,

[0189] Z is selected from hydrogen, halogen, C1-C 20 Alkyl, C1-C 20 Haloalkyl, cyano, sulfonyl, carboxyl, C1-C 20 Alkoxy, C2-C 10 Unsaturated alkyl groups, natural amino acids, non-natural amino acids, and short peptides composed of the above amino acids.

[0190] In some embodiments of Formula (I), Z is selected from:

[0191] wherein Z is connected to the rest of L3 at the g position, z1-z9 are each independently an integer between 0 and 10, and T is NH2 or OH.

[0192] In some embodiments of Formula (I), D is a cytotoxic agent.

[0193] In some embodiments of Formula (I), said D is selected from

[0194] Wherein D is connected to L3 at the h position.

[0195] In some embodiments of Formula (I), the antibody-drug conjugate is selected from:

[0196] wherein Ab represents an antibody or antigen-binding fragment thereof as defined in the preceding embodiments.

[0197] In some embodiments of formula (I), k is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 .0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or a range between any two thereof. In further embodiments, k is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or a range between any two thereof. In further embodiments, k is 4.0, 6.0, or 8.0.

[0198] 3. Usage

[0199] The anti-PDL1 antibody-drug conjugates of the present invention can be used to treat cancer. Some such cancers show detectable PDL1 levels measured at the protein (e.g., by immunoassay using one of the exemplified antibodies) or mRNA levels. Some such cancers show elevated PDL1 levels relative to non-cancerous tissue of the same type (preferably from the same patient). Exemplary PDL1 levels on treatable cancer cells are 5000-500,000 PDL1 molecules per cell, but higher or lower levels can be treated. Optionally, the level of PDL1 in the cancer is measured before treatment.

[0200] Examples of cancers associated with PDL1 expression and suitable for treatment include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, prostate cancer, pancreatic cancer, and glioma. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat melanoma. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat NSCLC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat SCLC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat head and neck cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat breast cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat TNBC. Triple-negative breast cancer is a term for cancers that lack detectable estrogen and progesterone receptors and lack HER2 / neu overexpression. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat pancreatic cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat ovarian cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat urothelial carcinoma. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat HCC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat esophageal cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat cervical cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat renal cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat prostate cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat pancreatic cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used to treat glioma. The treatment can be applied to patients with these types of primary or metastatic tumors. The treatment can also be applied to patients who are refractory to conventional treatment or who have relapsed after responding to such treatment.

[0201] The antibody-drug conjugate of the present invention is administered with an effective regimen, which means a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or improves at least one sign or symptom of cancer. If the patient already has cancer, the regimen may be referred to as a treatment-effective regimen. If the patient has an elevated risk of developing cancer relative to the general population, but has not yet experienced symptoms, the regimen may be referred to as a prevention-effective regimen. In some cases, treatment or prevention efficacy may be observed in individual patients relative to historical controls or past experience in the same patient. In other cases, treatment or prevention efficacy in preclinical or clinical trials may be demonstrated relative to a control population of untreated patients.

[0202] Exemplary dosages of the antibody-drug conjugate can be 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 20 mg / kg), for example, 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg or 3 mg / kg to 7.5 mg / kg of subject body weight, or 0.1-20 mg / kg body weight or 0.5-5 mg / kg body weight (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg) or as a fixed dose of 10-1500 mg or 200-1500 mg. In some methods, the patient is administered a dose of at least 1 mg / kg, at least 1.5 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 8 mg / kg, at least 10 mg / kg, once a week, every three weeks or longer. The dosage will depend on factors such as frequency of administration, the patient's condition and response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the condition is acute or chronic.

[0203] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration can also be directly localized into the tumor. Preferably, administration is administered into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can be performed, for example, by infusion for a period of time (e.g., 30-90 min) or by a single bolus injection.

[0204] The frequency of administration depends on factors such as the half-life of the conjugate in the circulation, the patient's condition, and the route of administration. The frequency can be once a day, once a week, once a month, once a quarter, or at irregular intervals in response to changes in the patient's condition or the progression of the cancer being treated. In a continuous treatment process, an exemplary frequency of intravenous administration is between twice a week and once a quarter, but it can also be administered at a higher or lower frequency. In a continuous treatment process, other exemplary frequencies of intravenous administration are between once a week or three times every four weeks, but it can also be administered at a higher or lower frequency. For subcutaneous administration, an exemplary frequency of administration is once a day to once a month, but it can also be administered at a higher or lower frequency.

[0205] The quantity of the administered dosage depends on the nature of the cancer (e.g., whether acute symptoms or chronic symptoms are present) and the reaction of the disease to treatment. For acute conditions or acute exacerbations of chronic conditions, between 1 and 10 dosages are generally sufficient. Sometimes, a single bolus dose (optionally in a separated form) is sufficient for acute conditions or acute exacerbations of chronic conditions. For recurrence or acute exacerbations of acute conditions, repeated treatment is possible. For chronic conditions, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, or six-month intervals for at least 1, 5, or 10 years or the patient's lifespan.

[0206] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dose for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, prior to use, the antibody can be in lyophilized form for construction with a suitable vehicle (e.g., sterile, pyrogen-free water). The concentration of the antibody in the liquid formulation can be, for example, 1-100 mg / ml, such as 10 mg / ml.

[0207] Treatment with the antibody-drug conjugates of the present invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, or other effective treatments for the condition being treated. Useful classes of other agents that can be administered with the antibody-drug conjugates for PDL1 as described herein include, for example, antibodies to other receptors expressed on cancer cells, anti-tubulin agents (e.g., auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono-, bis-, and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, leximycins, nitrosoureas, cisplatin, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0208] Treatment with the antibody-drug conjugate, optionally in combination with any of the other agents or regimens described above, can increase the median progression-free survival or overall survival time of patients with tumors (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), esophageal cancer, gastric cancer, colorectal cancer, and cervical cancer), especially in the case of recurrent or refractory tumors, by at least 30% or 40%, but preferably by 50%, 60% to 70%, or even 100% or more, compared to the same treatment (e.g., chemotherapy) but without the antibody-drug conjugate. Additionally or alternatively, a treatment including the antibody-drug conjugate (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) in patients with a tumor by at least 30% or 40%, but preferably by 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) but without the antibody-drug conjugate.

[0209] 4. Pharmaceutical preparations

[0210] While it is possible for the antibody-drug conjugates described herein to be used (eg, administered) alone, it is often preferable to present them as a composition or formulation.

[0211] In one aspect, the composition is a pharmaceutical composition (eg, formulation, preparation, medicament) comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0212] In one aspect, the composition is a pharmaceutical composition comprising at least one antibody-drug conjugate as described herein and one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavorings, and sweeteners.

[0213] In one aspect, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

[0214] Suitable carriers, diluents, excipients, and the like can be found in standard pharmaceutical literature. See, for example, Handbook of Pharmaceutical Additives, 2nd edition (M. Ash and I. Ash, eds.), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA, Remington's Pharmaceutical Sciences, 20th edition, Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0215] Another aspect of the present invention relates to a method for preparing a pharmaceutical composition comprising mixing at least one [11C]-radiolabeled antibody-drug conjugate or antibody-drug conjugate-like compound as defined herein with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art (e.g., carriers, diluents, excipients, etc.). If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dose) of the active compound.

[0216] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable, within the scope of sound medical judgment, for use in contact with the tissues of the subject in question (e.g., a human) without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0217] The formulations can be prepared by any method well known in the pharmaceutical art. Such methods include the step of associating the active compound with a carrier that constitutes one or more auxiliary ingredients. Typically, the formulation is prepared by uniformly and intimately associating the active compound with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.), and then shaping the product as desired.

[0218] The formulations may be prepared to provide fast or slow release; immediate, delayed, timed or sustained release; or a combination thereof.

[0219] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other particulate matter). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes, which can make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng / ml to about 10 μg / ml, for example, from about 10 ng / ml to about 1 μg / ml. The formulation can be present in unit dose or multi-dose sealed containers (e.g., ampoules and vials) and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., water) for injection just before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0220] 5. Products and Kits

[0221] In another aspect, a product or kit is provided, comprising an anti-PDL1 antibody-drug conjugate as described herein. The product or kit may also include instructions for use of the anti-PDL1 antibody-drug conjugate as described herein in the method of the present invention. Therefore, in certain embodiments, the product or kit includes instructions for use of the anti-PDL1 antibody-drug conjugate as described herein in the method for treating a subject's cancer (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, prostate cancer, pancreatic cancer, and glioma), the method comprising administering an effective amount of an anti-PDL1 antibody-drug conjugate as described herein to the subject. In some embodiments, the subject is a person.

[0222] The article or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single-chamber or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container can be made of various materials (e.g., glass or plastic). The container holds the preparation.

[0223] The product or kit may also include a label or package insert on or associated with the container that may indicate instructions for reconstructing and / or using the preparation. The label or package insert may further indicate that the preparation may be used for or intended for subcutaneous, intravenous (e.g., intravenous infusion) or other modes of administration to treat cancer (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, breast cancer (e.g., triple negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, prostate cancer, pancreatic cancer and glioma) in the subject. The container for holding the preparation may be a disposable bottle or a reusable bottle that allows repeated administration of the reconstructed preparation. The product or kit may also include a second container containing a suitable diluent. The product or kit may also include other materials desired from a commercial, therapeutic and user perspective, including other buffers, diluents, filters, needles, syringes and a package insert printed with instructions for use.

[0224] The article of manufacture or kit herein optionally further comprises a container containing a second agent, wherein the anti-PDL1 antibody-drug conjugate is a first agent, and the article of manufacture or kit further comprises instructions on a label or package insert for treating a subject with an effective amount of the second agent. In some embodiments, the second agent is used to eliminate or reduce the severity of one or more adverse events.

[0225] In some embodiments, the anti-PDL1 antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in an airtight container (such as a vial, ampoule or pouch), and the container indicates the amount of the active agent. In the case of administering the drug by injection, an ampoule of sterile water for injection or normal saline can, for example, be optionally provided as a part of the kit so that the ingredients can be mixed before administration. If necessary, such kits can also include one or more various conventional pharmaceutical components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., which are clear to those skilled in the art. The kit can also include printed instructions as an insert or as a label, which indicate the amount of the component to be administered, the guidance of the administration guide and / or the mixing of the components.

[0226] Sequence Listing Example

[0227] The present invention is further described in detail below by way of examples, which should not be construed as limiting the present invention.

[0228] Reagents and materials:

[0229] Organic solvents such as dimethyl sulfoxide were purchased from Spectrum Chemical Mfg Corp, and TCEP-HCl reagent was purchased from Thermo Fisher.

[0230] IgG1 is the isotype control and was purchased from Beijing Sino Biological Technology Co., Ltd.

[0231] pH Sensitive Zenon TM pHrodo TM iFL IgG labeling reagent was purchased from Thermo Fisher.

[0232] CellTiter-Glo solution was purchased from Promega.

[0233] Cell lines NCI-H292, BXPC-3 (for in vivo models), and MDA-MB-231 were purchased from the Cell Bank of the Chinese Academy of Sciences, NCI-H441 was purchased from ATCC, and BXPC-3 (for in vitro cell killing) was purchased from Pronose Biotechnology Co., Ltd. Various PDX xenografts were established and maintained by Shanghai Lidi Biotechnology Co., Ltd.

[0234] Preparation plan:

[0235] The structures of the compounds described in the following examples were determined by H NMR spectroscopy ( 1 HNMR) or liquid chromatography-mass spectrometry (LC-MS).

[0236] H NMR spectroscopy ( 1 HNMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard substance was tetramethylsilane (TMS).

[0237] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0238] s: singlet; d: doublet; t: triplet; q: quartet; dd: double doublet; qd: quarter doublet; ddd: double double doublet; ddt: double double triplet; dddd: double double double doublet; m: multiplet; br: broad; J: coupling constant; Hz: hertz; DMSO-d6: deuterated dimethyl sulfoxide. Δ values ​​are expressed in ppm.

[0239] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0240] Example 1. Preparation of Linker-Toxin (LP62)

[0241] Example 1.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4',6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3)

[0242] The synthetic route of compound A1.3 is shown in Scheme 1 below:

[0243] Scheme 1: Preparation of active compound A1.3

[0244] The specific steps are as follows:

[0245] Step 1: To a 75% sulfuric acid solution (5 mL) of (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4',6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.1, 500 mg) was added ferrous sulfate heptahydrate (570 mg dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) under ice-cooling conditions. The reaction mixture was stirred for three minutes, followed by the dropwise addition of hydrogen peroxide (29%, 2.5 mL) under ice-cooling conditions. After the addition was complete, the reaction mixture was stirred at 0°C for 5 minutes, then allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (80 mL x 2). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was evaporated under reduced pressure and concentrated to obtain a crude product. The crude product was purified by preparative liquid chromatography equipped with a C18 column (acetonitrile / 0.05% formic acid in water; 5%-60%) to obtain the target compound (A1.2, yellow solid, 400 mg, yield: 67%).

[0246] LC-MS (ESI) [M+H] + :468.9:

[0247] 1 HNMR(400Hz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.42(s, 2H), 5.26 (s, 2H), 3.81 (d, J = 5.9Hz, 2H), 3.22 (s, 2H), 1.98 (d, J = 6.7Hz, 4H), 0.88 (t, J = 7.2Hz, 3H).

[0248] Step 2: (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4',6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2, 100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL). The reaction mixture was reacted at 110°C for 48 hours. Saturated sodium bicarbonate solution (30 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10 mL x 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC (acetonitrile / water containing 0.05% formic acid) to give (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4',6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3, 1.78 mg).

[0249] LC-MS (ESI) [M+H] + :451.0:

[0250] 1HNMR(400Hz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.47-5.37( m,2H),5.32-5.19(m,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.92-1.76(m,4H),0.90-0.84(m,3H).

[0251] Example 1.2: Synthesis of Compound A2.3

[0252] The synthetic route of compound A2.3 is shown in Scheme 2 below:

[0253] Scheme 2: Synthesis route of compound A2.3

[0254] The specific steps for synthesizing compound A2.3 are as follows:

[0255] Step 1: Compound A2.1 (368 mg), compound A1.3 (440 mg), and pyridinium p-toluenesulfonate (PPTS, 25 mg) were refluxed in dichloromethane (20 mL) at 40°C for 20 hours. The mixture was then washed with aqueous sodium bicarbonate and aqueous hydrochloric acid, respectively. The organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the target compound A2.2 (240 mg).

[0256] LC-MS (ESI) [M+H] + :747:

[0257] Step 2: Dissolve A2.2 (240 mg) in DMF (5 mL), add piperidine (1 mL), and stir at room temperature for 20 minutes. Remove low-boiling components under pressure, and use the residue directly in the next step. A small amount of the crude product is purified by reverse-phase chromatography (acetonitrile / 0.05% FA in water: 5%-50%) to yield the target compound A2.3.

[0258] LC-MS (ESI) [M+H] + :525:

[0259] 1 H NMR(400Hz,DMSO-d6)δ9.13(t,1H),8.04(br,2H),7.58(s,1H),7.51(s,1H),7.25(s,1H),6.29(s,2H),5 .43(s,2H),5.21(s,2H),4.65(d,2H),3.63(m,2H),3.53(m,2H),3.11(m,2H),1.87(m,4H),0.88(t,3H).

[0260] Example 1.3: Synthesis of Compound A3.5

[0261] The synthetic route of compound A3.5 is shown in Scheme 3 below:

[0262] Scheme 3: Synthesis route of compound A3.5

[0263] The specific synthesis steps of compound A3.5 are as follows:

[0264] Step 1: Add a solution of hydrogen chloride-dioxone (C4H8O2) (100 mL) to compound A3.1 (10.0 g) and allow to react at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the target compound A3.2 (8.0 g) as a white solid.

[0265] LC-MS (ESI) [M+H] + :380.1

[0266] 1H NMR (400MHz, DMSO-d6) δ8.18(d,J=7.4Hz,1H),7.40-7.30(m,5H),7.26(d,J=8.8Hz,1H),5.08-4.99(m,2H),4.23-4.11(m,1H) ,3.94-3.88(m,1H),2.78-2.73(m,2H),2.03-1.94(m,1H),1.77-1.51(m,4H),1.44-1.31(m,2H),0.87(dd,J=17.3,6.6Hz,6H).

[0267] Step 2: Dissolve compound A3.2 (5.0 g, 12 mmol) in dichloromethane (100 mL). Add n-propionaldehyde (4.2 g, 72.3 mmol) to the reaction mixture and stir at room temperature for 10 minutes. Then, add sodium triacetoxyborohydride (12.8 g, 60.25 mmol) and stir at room temperature for 1 hour. LC-MS indicated completion of the reaction. Add saturated aqueous ammonium chloride to the reaction mixture and stir for 1 hour. Rotary dry, filter, and purify the filtrate using a C18 reverse-phase column to obtain the target compound A3.3 (4.57 g, 82.0% yield) as a white solid.

[0268] LC-MS (ESI) [M+H] + :464.0:

[0269] 1 H NMR (400MHz, DMSO-d6) δ7.81(d,J=7.3Hz,IH),7.38-7.28(m,5H),5.04(d,J=1.7Hz,2H),4.12-4.02(m,IH),3.94-3.82(m,1H),2.6 5-2.52(m,6H),2.06-1.94(m,1H),1.76-1.64(m,1H),1.64-1.53(m,1H),1.52-1.40(m,6H),1.34-1.18(m,2H),0.92-0.80(m,12H).

[0270] Step 3: Compound 3.3 (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL) at room temperature, and then Pd / C (0.16 g) was added to the reaction solution. The reaction was stirred at room temperature under hydrogen for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was filtered and concentrated under reduced pressure to obtain the target compound A3.4 (1.2 g, yield 85.5%) as a white solid.

[0271] Step 4: 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in DMF (1 mL), followed by the addition of HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol). The system was stirred for 30 minutes, followed by the addition of compound A3.4 (122 mg, 0.371 mmol), and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound N. 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (Compound A3.5, 50 mg, yield 28%) was a light yellow solid.

[0272] LC-MS (ESI) [M+H] + :580.0:

[0273] 1HNMR(400MHz,DMSO-d6)δ8.24(s,2H),7.98-7.93(m,2H),4.24-4.16(m,1H) ,4.10(d,J=5.2Hz,1H),3.41(s,3H),2.79-2.64(m,6H),2.55(t,J=7.1Hz,2H) ,2.45-2.26(m,2H),2.06-1.91(m,1H),1.89-1.78(m,2H),1.76-1.66(m,1H), 1.64-1.57(m,1H),1.57-1.42(m,6H).1.37-1.24(m,2H),0.93-0.78(m,12H).

[0274] Example 1.4: Synthesis of Linker-Toxin (LP62)

[0275] Step 1: Dissolve compound A3.5 (43 mg, 0.074 mmol) and compound A2.3 (40 mg, 0.074 mmol) in 1 mL of DMF. HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were then added sequentially. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, as determined by LC-MS, the reaction mixture was directly purified by preparative chromatography (0.01% trifluoroacetic acid in water, acetonitrile) to afford the trifluoroacetate salt of the target compound LP62 (8.5 mg, 10% yield) as a yellow solid. LC-MS (ESI) [M+H] + :1098.6:

[0276] 1 H NMR (400MHz, DMSO-d6): δ9.10 (s, 2H), 9.03 (s, 1H), 8.64 (t, J = 6.4Hz, 1H), 8.19 (m, J = 5.9Hz, 1H), 8.08 (d, J = 7.4Hz, 1H),7.92(d,J=8.5Hz,1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),6.29(s,2H),5.42(s,2H),5.24(s,2 H),4.66-4.52(m,2H),4.31-4.21(m,1H),4.19-4.10(m,1H),3.74(d,J=5.5Hz,2H),3.49-3.48(m,2H),3.40(s,3H) ,3.15-3.06(m,2H),3.02-2.95(m,6H),2.59-2.52(m,3H),2.41-2.29(m,2H),2.50-1.90(m,2H),1.91-1.77(m,6H), 1.63-1.57(m,6H),1.31-1.29(m,2H),0.92-0.80(m,15H).

[0277] Example 2. Preparation of antibodies

[0278] Unless otherwise indicated, the heavy and light chain amino acid sequences of the anti-PDL1 antibodies used in the present invention are described in the sequence listing.

[0279] Generally, the antibodies are prepared as follows: a stable CHO cell line secreting and expressing an anti-PDL1 antibody is constructed. The stable CHO cell line is cultured in a suitable culture medium. The culture supernatant is harvested and subjected to a series of processes, including depth filtration, protein A affinity chromatography, viral inactivation, filtration, anion chromatography, cation chromatography, and nanofiltration / ultrafiltration. The target antibody is ultimately obtained.

[0280] Example 3. Preparation and detection of antibody-drug conjugates

[0281] Example 3.1. Preparation of MAB20-LP62

[0282] Where n=8 represents the molar ratio of linker-drug payload to antibody.

[0283] Place 2-5 mL of anti-PDL1 antibody MAB20 (Anti-PDL1_mAb, 10-30 mg / mL) in a dedicated antibody coupling reactor. Add EDTA to a final concentration of 10-20 mM, then adjust the pH to 5.5-7.5 with 0.5-1 M sodium dihydrogen phosphate solution. Slowly add 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 4-10 equivalents, 0.1-0.8 mL), mix thoroughly, and react at room temperature for 50-80 minutes. After the anti-PDL1 antibody reduction reaction is complete, add the linker-toxin LP62 solution (8-15 equivalents, 2-10 μmol) previously dissolved in dimethyl sulfoxide (DMSO) or DMAC solvent to the above solution system, stir to mix, and react at room temperature for 100-150 minutes. After purification by cationic chromatography, the buffer was exchanged to 20-50 mM PB, pH 5.5-7.5, using an Amicon Ultra-15. The conjugate product, MAB20-YL62, consisting of the anti-PDL1 antibody MAB20 and the linker-toxin LP62, was obtained. Its DAR value and monomer content were confirmed to meet standards by LC-MS, HIC, and HPLC-SEC analysis.

[0284] Example 3.2. Preparation of reference ADC MAB20-VC-MMAE

[0285] Structure of MAB20-VC-MMAE (here Ab is MAB20 and k is the DAR value)

[0286] MAB20-VC-MMAE was prepared according to conventional methods. Specifically, 2-5 mL of the PDL1 antibody MAB20 (Anti-PDL1_mAb, 10-30 mg / mL) was placed in a dedicated antibody coupling reactor. EDTA was added to a final concentration of 10-20 mM, followed by adjusting the pH to 5.5-7.5 with 0.5-1 M sodium dihydrogen phosphate solution. 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 2-4 equivalents, 0.1-0.8 mL) was slowly added, mixed, and allowed to react at room temperature for 50-80 minutes. After the MAB20 reduction reaction was complete, a solution of the linker-toxin MC-VC-PAB-MMAE (structure shown below), pre-dissolved in dimethyl sulfoxide (DMSO) or DMAC (4-15 equivalents, 1-10 μmol), was added to the solution, mixed, and allowed to react at room temperature for 100-150 minutes. After purification by cationic chromatography, the buffer was exchanged with 20-50 mM PB (pH 5.5-7.5) using an Amicon Ultra-15 to obtain the conjugate product, MAB20-VC-MMAE, derived from the anti-PDL1 antibody MAB20 and MC-VC-PAB-MMAE. Its DAR value and monomer content were confirmed to meet standards by LC-MS, HIC, and HPLC-SEC analysis.

[0287] MC-VC-PABC-MMAE

[0288] DAR values ​​of anti-PDL1 antibody-drug conjugate samples

[0289] Example 3.3. Preparation of reference ADC SGN-PDL1V

[0290] Structure of SGN-PDL1V (here Ab is the antibody portion of SGN-PDL1V, and k is the DAR value)

[0291] SGN-PDL1V can be prepared with reference to the method described in WO2021067776A1. Alternatively, it can be prepared as follows: take 2-5 mL of the antibody portion of SGN-PDL1V (10-30 mg / mL) and place it in a dedicated antibody coupling reactor. Add EDTA to a final concentration of 10-20 mM, and then adjust the pH to 5.5-7.5 with 0.5-1 M disodium hydrogen phosphate solution. Slowly add 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 2-4 equivalents, 0.1-0.8 mL), mix well and react at room temperature for 50-80 minutes. After the antibody reduction reaction is complete, add a linker-toxin MC-VC-PAB-MMAE (its structure is shown below) solution (4-15 times equivalent, 1-10 μmol) dissolved in dimethyl sulfoxide DMSO or DMAC solvent in advance to the above solution system, stir and mix well and react at room temperature for 100-150 minutes. After purification by cationic chromatography, the buffer was exchanged with 20-50 mM PB, pH 5.5-7.5, using an Amicon Ultra-15. The conjugate product, SGN-PDL1V, consisting of the SGN-PDL1V antibody moiety and MC-VC-PAB-MMAE, was obtained. Its DAR value (DAR4) and monomer content were confirmed to meet standards by LC-MS, HIC, and HPLC-SEC analysis.

[0292] MC-VC-PABC-MMAE

[0293] Example 3.4. Preparation of reference ADC MAB20-GGFG-DXD

[0294] Structure of MAB20-GGFG-DXD (here Ab is MAB20, and k is the DAR value)

[0295] MAB20-GGFG-DXD was prepared according to a general method in the prior art. Specifically, 2-5 mL of the anti-PDL1 antibody MAB20 (Anti-PDL1_mAb, 10-25 mg / mL) was placed in a reaction tube. EDTA was added to a final concentration of 10-20 mM, followed by adjusting the pH to 6.0-7.5 with 0.5-1 M sodium dihydrogen phosphate solution. 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 4-10 equivalents, 0.1-0.4 mL) was slowly added, mixed, and allowed to react at room temperature for 50-80 minutes. After the MAB20 reduction reaction was completed, a solution of the linker-toxin MC-GGFG-DXD (its structure is shown below) pre-dissolved in dimethyl sulfoxide (DMSO) or DMAC solvent (8-12 equivalents, 2-10 μmol) was added to the above solution system, mixed, and allowed to react at room temperature for 100-150 minutes. An Amicon Ultra-15 was used to exchange the system buffer with 10-50 mM PB or histidine-histidine hydrochloride, pH 5.5-7.5, to obtain the conjugated product, MAB20-GGFG-DXD, consisting of MAB20 and the linker toxin MC-GGFG-DXD. The DAR value and monomer content were confirmed to be within the specified range by LC-MS, HIC, and HPLC-SEC analysis.

[0296] MC-GGFG-DXD

[0297] Example 3.5: Analysis of Drug-to-Antibody Ratio (DAR) of Antibody-Drug Conjugates by LC-MS

[0298] The antibody-drug conjugate sample was reduced with DTT at 37°C for 10 minutes, and then the DAR value of the conjugate was analyzed using LC-MS. Specifically, the reduced sample was injected into an Acquity UPLC BEH C4 reverse phase column (Waters Corporation, 2.1 mm × 50 mm, 1.7 μm, The drug:antibody ratio (DAR) was calculated using the deconvoluted LC-MS results provided.

[0299] Example 4. Determination of the affinity of anti-PDL1 antibody-drug conjugates for PDL1-positive cells

[0300] Normally grown NCI-H292 and NCI-H441 tumor cells were collected, washed with PBS, and resuspended at 1×10 6Cells / well / 50μl were inoculated into a 96-well plate. The test sample was then diluted in an isogradient manner with FACS buffer (PBS+2% FBS), and the diluted test samples and control samples were added sequentially to the 96-well plate inoculated with cells, wherein the control sample IgG1 was human IgG1, and the κ isotype control was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The sample plate was incubated at 4°C for 0.5h, centrifuged, the supernatant was removed, and then washed with 300μl / well of FACS buffer. Subsequently, the fluorescently labeled goat anti-human IgG antibody detection solution was added to the sample plate (100μl / well) and incubated with the cells. The sample plate was incubated at 4°C for 0.5h, washed with 300μl / well of FACS buffer, and finally the cells were resuspended. The fluorescence values ​​of each sample were detected by flow cytometry. The data were analyzed with Graphpad prism 8.1 and fitted with a four-parameter equation.

[0301] As shown in Figure 1A, MAB20-LP62 (DAR8) exhibited good affinity for the PDL1-positive cell line NCI-H292, with an affinity comparable to that of the unconjugated antibody MAB20. Similarly, as shown in Figure 1B, MAB20-LP62 (DAR8) exhibited good affinity for the PDL1-positive cell line NCI-H441.

[0302] Example 5. Determination of internalization of anti-PDL1 antibody-drug conjugates into PDL1-positive cells

[0303] Take a certain volume of NCI-H292 cells that have been grown and passaged normally, centrifuge, remove the supernatant, and resuspend in cell culture medium (RPMI-1640 medium + 10% FBS). Inoculate 50 μl of cell suspension into a 96-well plate for use (1×10 5 cells / well). Use cell culture medium to transfer pH-sensitive Zenon TM pHrodo TM iFL IgG labeling reagent (purchased from ThermoFisher) was prepared into 4x working solution 1 (480nM). This pH-sensitive dye conjugate does not fluoresce outside the cell, but emits bright fluorescence in an acidic environment (including lysosomes). The test substance was prepared into 4x working solution 2 (160nM) using cell culture medium. 25μl of working solution 1 and working solution 2 were taken into a 96-well plate, mixed and incubated. The mixed liquid was aspirated into 50μl of cell suspension, mixed, and placed in an incubator for culture. The test substance was taken at 0, 2, 16, and 24h time points, centrifuged, and the supernatant was removed. Then, the cells were washed with 300μl / well FACS buffer, resuspended, and the fluorescence value of each sample was measured by flow cytometry.

[0304] As shown in Figure 2 , compared with the IgG1 isotype control data, the ratio of MAB20-LP62 (DAR8) and MAB20 internalization increased with increasing incubation time, reaching a peak at 4 h, indicating that the anti-MAB20 antibody-drug conjugate MAB20-LP62 (DAR8) can effectively bind to the PDL1 antigen on the cell surface and internalize into the lysosomal pathway in the cell.

[0305] Example 6. Determination of the cell-killing activity of anti-PDL1 antibody-drug conjugates

[0306] Cells in the exponential growth phase were collected and live cells were counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to an appropriate concentration using culture medium. 80 μl of cell suspension was added to each well of the cell culture plate. The test sample was gradiently diluted (5-fold dilution), and 20 μl of the diluted test sample was added to the cell suspension in the cell culture plate. The cell culture plate was placed in an incubator and cultured for 72 hours. After 72 hours, according to the CellTiter-Glo operating instructions, 50 μl of CellTiter-Glo solution (purchased from Promega) that had been pre-melted and equilibrated to room temperature was added to each well, mixed for 2 minutes using a microplate shaker, placed at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision2104 plate reader. The raw data was analyzed using Graphpad prism 8.1 and fitted with a four-parameter equation.

[0307] As shown in FIG3 , MAB20-LP62 (DAR8) showed a strong cell-killing effect against MAB20-insensitive PDL1-positive tumor cells NCI-H292, BXPC-3, and NCI-H441.

[0308] Example 7. Cross-binding activity of anti-PDL1 antibody-drug conjugates against PDL1 from different species

[0309] The extracellular domain (ECD) of PDL1 derived from humans, crab-eating macaques, rats and mice was diluted with 1×PBS to a working concentration of 2μg / mL as ELISA coating solution. The test plate was washed 3 times with PBST (PBS containing 0.05% Tween20). The plate was incubated at room temperature with blocking solution SuperBlock T20 (PBS) Blocking Buffer (60μL / well), and then the test samples were diluted in equal gradients and added to the test plate respectively, and incubated at room temperature. The test plate was washed, and HRP enzyme-labeled goat anti-human IgG was added as a secondary antibody and incubated. The test plate was washed, the stop solution (30μL / well) was added, centrifuged, and then the light absorption at a wavelength of 450nm was read. The raw data was analyzed using Graphpad prism 8.1 and fitted with a four-parameter equation.

[0310] As shown in Figure 4, MAB20-LP62 binds to cynomolgus monkey and mouse PDL1 ECD with similar affinities to human PDL1 ECD, with EC50 values ​​of 0.039 nM, 0.094 nM, and 0.054 nM, respectively. MAB20-LP62 (DAR8) exhibits weak specific binding to rat PDL1 ECD.

[0311] Example 8. Bystander Effect Assay of Anti-PDL1 Antibody-Drug Conjugates

[0312] PDL1-negative tumor cells MDA-MB-468 cells in the exponential growth phase were counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to an appropriate concentration using culture medium. 80 μl of cell suspension was added to each well of the cell culture plate. The test drug was diluted stepwise (5-fold dilution), and 20 μl of the diluted test article was added to the cell suspension in the cell culture plate. The cell culture plate was placed in an incubator and cultured for 96 hours. According to the CellTiter-Glo operating instructions, 50 μl of CellTiter-Glo solution (purchased from Promega) that had been pre-melted and equilibrated to room temperature was added to each well, mixed for 2 minutes using a microplate shaker, placed at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision2104 plate reader.

[0313] HEK293 cells overexpressing PDL1 in exponential growth were cultured and viable cells were counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to the appropriate concentration using culture medium. 80 μl of the cell suspension was added to each well of the cell culture plate. A serial dilution of the test drug (5-fold dilution) was performed, and 20 μl of each diluted test article was added to the cell suspension in the cell culture plate. The cell culture plate was placed in an incubator and incubated for 96 hours. After 96 hours, the supernatant of the conditioned culture medium from the PDL1-overexpressing HEK293 cells was collected and an equal volume was transferred to culture MDA-MB-468 cells. The cells were cultured for an additional 96 hours. Following the CellTiter-Glo instructions, 50 μl of pre-thawed and room temperature CellTiter-Glo solution (purchased from Promega) was added to each well. The cells were mixed using a microplate shaker for 2 minutes, incubated at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision 2104 plate reader.

[0314] As shown in Figures 5A and 5B, MAB20-LP62 (DAR8) had a significant bystander effect.

[0315] Example 9. Immunotoxicity Assay of Anti-PDL1 Antibody-Drug Conjugates

[0316] Because some human immune cells, such as antigen-presenting cells (macrophages and dendritic cells), express PDL1, immunotoxicity of PDL1-targeting antibody-drug conjugates is an unavoidable concern. Human dendritic cells and macrophages were induced to differentiate and viable cells were counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to an appropriate concentration using culture medium. 80 μl of the cell suspension was added to each well of a cell culture plate, supplemented with 500 IU / mL IFN-γ. A serial dilution of the test drug (5-fold dilutions) was performed, and 20 μl of each diluted test article was added to the cell suspension in the cell culture plate. The cell culture plate was then placed in an incubator and incubated for 96 hours. Following the CellTiter-Glo operating instructions, 50 μl of pre-thawed and room temperature CellTiter-Glo solution (purchased from Promega) was added to each well. The mixture was mixed using a microplate shaker for 2 minutes, incubated at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision 2104 plate reader. SGN-PDL1V was used as one of the control molecules, which was disclosed in U.S. Patent No. US20210101982A1.

[0317] As shown in Figures 6 and 7, the control molecule SGN-PDL1V exhibited similar immunotoxicity to the isotype control ADC (IgG1-MMAE) in macrophages and dendritic cells in vitro. However, when the concentration was higher than 20nM, SGN-PDL1V caused non-specific killing of both immune cells. In contrast, MAB20-LP62 (DAR8) had no significant cytotoxicity against macrophages and dendritic cells even at a high concentration (500nM). Therefore, compared with SGN-PDL1V, MAB20-LP62 has negligible immunotoxicity and a higher safety profile.

[0318] Example 10. Antitumor activity of anti-PDL1 antibody-drug conjugate in NCG mouse model inoculated subcutaneously with human breast cancer MDA-MB-231 cells

[0319] MDA-MB-231 cells were cultured in growth medium (RPMI 1640 + 10% FBS + IL15). Cells in the logarithmic growth phase were collected, washed twice with DPBS, and then used for inoculation. The cells were resuspended in PBS and Matrixgel matrix at a ratio of 1:1 and MDA-MB-231 cells (5x10 6 / 100μl / mouse). When the tumor grows to an average volume of 204.54mm 3The mice were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes of the different groups were similar. The day of grouping was defined as day 0. After the start of drug administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0320] As shown in Figure 8, on day 35 after administration, MAB20-LP62 (DAR4) (3 mg / kg and 8 mg / kg) and MAB20-LP62 (DAR8) (3 mg / kg and 8 mg / kg) both showed significant tumor growth inhibition compared to the control group (MAB20, 8 mg / kg, tumor growth inhibition rate (TGI) 21.26%), with TGIs of 46.89% (p<0.001), 74.50% (p<0.001), 80.33% (p<0.001), and 92.91% (p<0.001), respectively. MAB20-LP62 (DAR8) exhibited significant anti-tumor effects even at a low dose. MAB20-LP62 (DAR8) demonstrated significantly superior anti-tumor activity compared to the antibody-drug conjugates MAB20-VC-MMAE (DAR4) (3 mg / kg TGI 38.79%) and MAB20-GGFG-DXD (DAR8) (3 mg / kg TGI 44.60%, 8 mg / kg TGI 72.98%), both constructed using existing conjugation technologies. Throughout the treatment period, no animals experienced significant weight loss.

[0321] Example 11. Antitumor activity of anti-PDL1 antibody-drug conjugates in a subcutaneous inoculation of PDL1 low-expressing human pancreatic cancer cell BxPC3 cells in NCG mouse model

[0322] BXPC3 cells were cultured in growth medium (RPMI 1640 + 10% FBS). Cells in the logarithmic growth phase were collected and washed twice with DPBS before inoculation. The cells were resuspended in a 1:1 ratio of PBS to Matrixgel matrix. BXPC3 cells (5x10 6 / 100μl / mouse). When the tumor grows to an average volume of 210.90mm 3The mice were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes of the different groups were similar. The day of grouping was defined as day 0. After the start of drug administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: tumor volume (mm 3 )=1 / 2×(a×b2)(where a represents the major diameter and b represents the minor diameter).

[0323] As shown in Figure 9, on day 35 after administration, MAB20-LP62(DAR4) (8 mg / kg) and MAB20-LP62(DAR8) (8 mg / kg) both exhibited tumor inhibition, with TGIs of 14% and 40.9%, respectively, compared to the control group (MAB20) (TGI -28.31%). Compared to the other test drugs, MAB20-VC-MMAE(DAR4) (3 mg / kg TGI -6.79%) and MAB20-GGFG-DXD(DAR8) (3 mg / kg TGI -18.00%, 8 mg / kg TGI -0.12%), only MAB20-LP62 achieved tumor growth inhibition in the PDL1 low-expressing model. Throughout the treatment period, none of the animals experienced significant weight loss.

[0324] Example 12. Antitumor Effect of Anti-PDL1 Antibody-Drug Conjugate in BALB / c Nude Mice Bearing Human Non-Small Cell Lung Cancer LU6437 PDX Tumors

[0325] from Tumor tissues were collected from lung cancer xenograft model LU6437 tumor-bearing mice after recovery, cut into 2-3 mm diameter tumor masses and inoculated subcutaneously at the right anterior scapula of BALB / c nude mice. The tumors were grown to a volume of approximately 200 mm. 3 The patients were randomly divided into groups according to tumor size to ensure that the tumor volume in each group was similar. The actual average volume of the groups was 253.70 mm 3 The day of grouping was defined as day 0. After the start of dosing, dosing was continued once a week for three times, and the body weight and tumor size of the mice were measured twice a week. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2 × (a × b2) (where a represents the major diameter and b represents the minor diameter).

[0326] It should be noted that preliminary studies have shown that SGN-PDL1V at a dose of 8 mg / kg is severely lethal to mice. For ethical reasons, this experimental group was not included in the experimental design.

[0327] As shown in Figure 10 , in the anti-PD1 monoclonal antibody-resistant human non-small cell lung cancer PDX model LU6437, MAB20-LP62 at 1 mg / kg (day 21, TGI -0.16%), 3 mg / kg (day 21, TGI 43.23%), and 8 mg / kg (day 21, TGI 106.85%) demonstrated superior performance to the control molecule SGN-PDL1V (1 mg / kg, day 21, TGI -15.17%).

[0328] MAB20-LP62 at 8 mg / kg showed the strongest tumor suppression effect. No animals experienced severe weight loss during the entire treatment period.

[0329] Example 13. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PDL1-positive liver cancer LD1-0011-200617 PDX xenografts

[0330] The humanized liver cancer LD1-0011-200617 model was revived in NCG mice and the tumors were grown to 500-800 mm. 3 When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (approximately 30-60mg) and inoculated subcutaneously on the right side of NU / NU mice. When the average tumor volume of grouped mice reached 155.24mm3, they were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0331] As shown in Figure 11, in the PDX model LD1-0011-200617 for PDL1-positive liver cancer, MAB20 at 10 mg / kg achieved a TGI of 30.3% on day 21 after treatment. In contrast, MAB20-LP62 (DAR8) exhibited dose-dependent tumor suppression at both 3 mg / kg (TGI 75.24% on day 21) and 10 mg / kg (TGI 89.51% on day 21). The highest dose of MAB20-LP62 (DAR8) demonstrated the strongest tumor suppression effect. Throughout the treatment period, no animals experienced significant weight loss.

[0332] Example 14. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in NCG mice bearing human PDL1-2023-411020 head and neck squamous cell carcinoma PDX xenografts

[0333] The human PDL1-positive head and neck squamous cell carcinoma LD1-2023-411020 model was revived in NCG mice until the tumor grew to 500-800 mm. 3 When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (about 20-30mg) and inoculated subcutaneously on the right side of NCG mice. When the average tumor volume of grouped mice reached 138.88mm3, they were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drugs were administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0334] As shown in Figure 12, in the anti-PD1 monoclonal antibody-resistant human PDL1-positive head and neck squamous cell carcinoma PDX model LD1-2023-411020, MAB20 at 10 mg / kg did not exhibit a tumor inhibitory effect (day 28, TGI -34.46%) and free payload drug A1.3 did not exhibit a tumor inhibitory effect (day 28, TGI -8.74%), but MAB20-LP62 (DAR8) at the same dose (day 28, TGI 102.31%) exhibited a strong tumor inhibitory effect. Throughout the treatment period, no animals experienced severe weight loss.

[0335] Example 15. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in Balb / c nude mice bearing human PDL1-positive cervical cancer LD1-0010-200614 PDX xenografts

[0336] The human PDL1-positive cervical cancer LD1-0010-200614 model was revived in Balb / c nude mice and the tumor was grown to 500-800 mm. 3When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (about 25-35mg) and inoculated subcutaneously on the right side of Balb / c nude mice. When the average tumor volume of grouped mice reached 146.74mm3, they were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0337] As shown in Figure 13, in the human PDL1-positive cervical cancer PDX model LD1-0010-200614, MAB20 exhibited moderate tumor inhibition at 10 mg / kg (day 35, TGI 67.38%), free payload drug A1.3 exhibited moderate tumor inhibition (day 35, TGI 56.5%), but MAB20-LP62 (DAR8) at the same dose (day 35, TGI 109.33%) exhibited a strong tumor inhibition effect. Throughout the treatment period, no animals experienced significant weight loss.

[0338] Example 16. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in Balb / c nude mice bearing human PDL1-positive esophageal squamous cell carcinoma LD1-0015-362448 PDX xenografts

[0339] The human PDL1-positive esophageal squamous cell carcinoma LD1-0015-362448 model was revived in Balb / c nude mice and the tumor was grown to 500-800 mm. 3 When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (about 25-35mg) and inoculated subcutaneously on the right side of Balb / c nude mice. When the average tumor volume of grouped mice reached 140.46mm3, they were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0340] As shown in Figure 14, in the human PDL1-positive esophageal squamous cell carcinoma PDX model LD1-0015-362448, 10 mg / kg of MAB20-LP62 (DAR8) exhibited a strong tumor inhibitory effect (TGI 92.02% on day 45). During the entire treatment period, all animals did not experience severe weight loss.

[0341] Example 17. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PDL1-positive gastric cancer LD1-0017-200636 PDX xenografts

[0342] The human PDL1-positive gastric cancer LD1-0017-200636 model was revived in NU / NU mice and the tumors were grown to 500-800 mm. 3 When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (approximately 30-60mg) and inoculated subcutaneously on the right side of NU / NU mice. When the average tumor volume of grouped mice reached 138.70mm3, they were randomly divided into groups according to the tumor volume to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0343] As shown in Figure 15 , in the human PDL1-positive gastric cancer PDX model LD1-0017-200636, at day 21 after treatment, the TGI of 10 mg / kg MAB20 was 14.89% and the TGI of the free drug A1.3 was 34.12%, but 10 mg / kg MAB20-LP62 (DAR8) (TGI 108.18% on day 21) exhibited a strong tumor inhibitory effect. Throughout the treatment period, no animal experienced significant weight loss.

[0344] Example 18. Anti-tumor effect of anti-PDL1 antibody-drug conjugate in NU / NU mice bearing human PDL1-2013-362125 colorectal cancer PDX xenografts

[0345] The human PDL1-positive colorectal cancer LD1-2013-362125 model was revived in NU / NU mice until the tumor grew to 500-800 mm. 3When the tumor tissue was removed surgically and sterile, non-tumor tissue and necrotic tissue were removed. The tumor tissue was peeled off and evenly cut into tumor blocks of approximately 3mm×3mm×3mm (approximately 25-35mg) and inoculated subcutaneously on the right side of NU / NU mice. When the average tumor volume of grouped mice reached 156.99mm3, they were randomly divided into groups according to the size of the tumor to ensure that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated according to the following formula: Tumor volume (mm3) = 1 / 2×(a×b2) (where a represents the long diameter and b represents the short diameter).

[0346] As shown in Figure 16, in the human PDL1-positive colorectal cancer PDX model LD1-2013-362125, at day 31 after treatment, the TGI of 10 mg / kg MAB20 was 50.27% and the TGI of the free drug A1.3 was 37.16%. However, the same dose of MAB20-LP62 (DAR8) (TGI 101.59%) exhibited a strong tumor inhibitory effect. Throughout the treatment period, no animals experienced significant weight loss.

[0347] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, so that the disclosed subject matter includes any suitable combination of features disclosed herein. The above description of specific embodiments of the disclosed subject matter has been presented for the purposes of illustration and description. The above description is not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0348] It will be apparent to those skilled in the art that various modifications and variations can be made to the composition and method of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter include modifications and variations within the scope of the appended claims and their equivalents.

[0349] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entirety.

Claims

1. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by formula (I): TP-[L1-L2-L3-D] k (I) in: TP is a targeting moiety that selectively binds to or recognizes PDL1; L1 is an extension unit, which connects TP and L2; L2 is an optional amino acid residue or a short peptide consisting of 2-10 amino acid residues; L3 is a spacer element; D is a bioactive molecule; k represents any value between 0.1 and 10.

0.

2. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to claim 1, wherein the TP is an antibody or an antigen-binding fragment thereof.

3. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to claim 2, wherein the antigen-binding fragment is selected from the group consisting of a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab') 2 , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabodies, triabodies, tetrabodies or any combination thereof.

4. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the TP is a humanized antibody or an antigen-binding fragment thereof.

5. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt according to any one of the preceding claims, wherein the TP comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3 contained in SEQ ID NO: 7, and the light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 contained in SEQ ID NO:

8.

6. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to claim 5, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, and the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5 and the amino acid sequence of LCDR3 is SEQ ID NO:

6.

7. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the TP comprises the Fc sequence of human IgG.

8. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the IgG is selected from IgG1, IgG2, IgG3 and IgG4.

9. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any of the preceding claims, wherein the TP comprises a heavy chain variable region (VH), and the amino acid sequence of the heavy chain variable region (VH) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:

7.

10. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any of the preceding claims, wherein the TP comprises a light chain variable region (VL), and the amino acid sequence of the light chain variable region (VL) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:

8.

11. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any of the preceding claims, wherein the TP comprises a heavy chain (HC) and the amino acid sequence of the heavy chain (HC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:

9.

12. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any of the preceding claims, wherein the TP comprises a light chain (LC) and the amino acid sequence of the light chain (LC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:

10.

13. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the TP is a multispecific antibody.

14. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the TP is an afucosylated antibody.

15. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the Fc region comprises a C-terminal lysine.

16. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the Fc region comprises a deletion of the C-terminal lysine.

17. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L1 is selected from wherein L1 is connected to TP via an S atom at position a and is connected to L2 at position b, m is any integer between 1 and 10, n is any integer between 1 and 10, and X and Y are each independently C, O, S or N.

18. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L1 is in, m is selected from 1, 2, 3, 4, 5, Among them, L1 is connected to TP through the S atom at the a position and is connected to L2 at the b position.

19. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L1 is Among them, L1 is connected to TP through the S atom at the a position and is connected to L2 at the b position.

20. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L2 is selected from Wherein L2 is connected to L1 at the c position and to L3 at the d position.

21. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L2 is Wherein L2 is connected to L1 at the c position and to L3 at the d position.

22. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein L3 is a single bond or a group selected from the following: Where L3 is connected to L2 at the e position and to D at the f position. Z is selected from hydrogen, halogen, C 1 -C 20 Alkyl, C 1 -C 20 Haloalkyl, cyano, sulfonyl, carboxyl, C 1 -C 20 Alkoxy, C 2 -C 10 Unsaturated alkyl groups, natural amino acids, unnatural amino acids, and short peptides composed of the above amino acids.

23. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein Z is selected from: wherein Z is connected to the rest of L3 at position g, z1-z9 are each independently an integer between 0 and 10, and T is NH 2 or OH.

24. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein D is a cytotoxic agent.

25. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein D is selected from Wherein D is connected to L3 at the h position.

26. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein the antibody-drug conjugate is selected from: wherein Ab represents the antibody or an antigen-binding fragment thereof.

27. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein k is about 4.0 to about 8.0, preferably k is about 4.0, about 6.0 or about 8.

0.

28. A pharmaceutical composition comprising the antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to any one of claims 1 to 27, and optionally, a pharmaceutically acceptable carrier, diluent or excipient.

29. The antibody-drug conjugate, the prodrug, the pharmaceutically acceptable salt, the solvate or the solvate of the pharmaceutically acceptable salt thereof according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 28 for use as a medicament.

30. The antibody-drug conjugate, the prodrug, the pharmaceutically acceptable salt, the solvate or the solvate of the pharmaceutically acceptable salt thereof according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 28, for use in treating or preventing tumors or cancers.

31. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing tumors or cancers according to claim 30, wherein the tumor or cancer is a tumor or cancer associated with PDL1 expression.

32. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate or a pharmaceutical composition thereof for treating or preventing a tumor or cancer according to claim 30, wherein the tumor or cancer is selected from melanoma, lung cancer, head and neck cancer, breast cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, bladder cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer and brain glioma.

33. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing tumors or cancer according to claim 32, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

34. The antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing tumors or cancer according to claim 32, wherein the breast cancer is triple-negative breast cancer (TNBC).

35. An article of manufacture or kit comprising the antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 28 and instructions for use.

36. A method for preventing or treating tumors or cancers, comprising administering to a subject in need thereof a preventively or therapeutically effective amount of an antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt according to any one of claims 1 to 27, or a pharmaceutical composition according to claim 28.

37. The method of claim 36, wherein the tumor or cancer is a tumor or cancer associated with PDL1 expression.

38. The method of claim 36, wherein the tumor or cancer associated with PDL1 expression is selected from melanoma, lung cancer, head and neck cancer, breast cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, bladder cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer and brain glioma.

39. The method of claim 38, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

40. The method of claim 38, wherein the breast cancer is triple negative breast cancer (TNBC).

41. The method of any one of claims 36-40, wherein the method comprises administering to the subject an additional therapeutic agent selected from the group consisting of an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent, and a cytotoxic agent.

42. The method of any one of claims 36-41, wherein the method comprises further administering radiation therapy to the subject.

43. Use of the antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to any one of claims 1 to 27, or the pharmaceutical composition according to claim 28 in the preparation of a medicament for preventing or treating tumors or cancer.

44. Use of the antibody-drug conjugate, its prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt according to any one of claims 1 to 27, or the pharmaceutical composition according to claim 28 in combination with an additional therapeutic agent in the preparation of a medicament for preventing or treating tumors or cancer. The use according to claim 43 or 44, wherein the tumor or cancer is a tumor or cancer associated with PDL1 expression.

46. ​​The use according to claim 45, wherein the tumor or cancer associated with PDL1 expression is selected from melanoma, lung cancer, head and neck cancer, breast cancer, ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, bladder cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer and brain glioma.

47. The use according to claim 46, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

48. The use according to claim 46, wherein the breast cancer is triple negative breast cancer (TNBC).

49. The use according to any one of claims 44-48, wherein the additional therapeutic agent is selected from an anti-tumor agent, a chemotherapeutic agent, a growth inhibitory agent and a cytotoxic agent.