Antibody-drug conjugates targeting B7-H3 and uses thereof

Abstract

The present invention relates to antibody-drug conjugates targeting B7-H3, and to compositions containing said antibody-drug conjugates. The invention also relates to therapeutic and diagnostic uses of these antibody-drug conjugates.

Description

Technical Field

[0001] The present invention relates to antibody-drug conjugates (ADCs) targeting B7-H3 and compositions containing the antibody-drug conjugates. The present invention also relates to therapeutic and diagnostic uses of these ADCs. Background of the Invention

[0003] Therapy using cytotoxic agents is an important therapy for treating cancer, but low-selectivity cytotoxic agents often kill normal cells and cause serious toxic side effects. Although biological macromolecule drugs, such as antibodies or antibody fragments, have high targeting, their therapeutic effects on solid tumors are limited, which limits their application. Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, combining the targeting effect of antibodies with the high activity of small molecule drugs (such as cytotoxic agents), and have advantages such as high efficacy and high safety.

[0004] B7-H3 is a type I transmembrane protein of the B7 family. It is not constitutively expressed in many immune cells (e.g., natural killer (NK) cells, T cells, and antigen presenting cells (APC)); however, its expression can be induced. In addition, the expression of B7-H3 is not limited to immune cells. B7-H3 transcripts are expressed in a variety of human tissues (including colon, heart, liver, placenta, prostate, small intestine, testis, and uterus) as well as osteoblasts, fibroblasts, epithelial cells, and other non-lymphoid lineage cells that may indicate immune and non-immune functions (Nygren et al. Front Biosci. 3: 989-93 (2011)). However, protein expression in normal tissues is usually maintained at a low level and may therefore be subject to post-transcriptional regulation. B7-H3 is also expressed in a variety of human cancers, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, breast cancer, and the like.

[0005] At present, research has been conducted on ADCs targeting B7-H3 (see, for example, US20200338209A1; Scribner et al., Mol Cancer Ther (2020) 19(11):2235–2244; Yamato et al., Mol Cancer Ther. 2022Apr1; 21(4):635–646). Among them, DS-7300a developed by Daiichi Sankyo Co., Ltd. is one of the most typical blockbuster new drugs in this field, which has high efficacy against various cancers (Yamato et al., Mol Cancer Ther. 2022Apr 1; ​​21(4):635–646). However, existing ADCs including DS-7300a still have some defects, such as the efficacy needs to be further improved, the endocytosis efficiency is not high, there are side effects that need to be improved, the uneven distribution of product DAR values ​​affects the efficacy, the DAR value is low and / or the half-life is short.

[0006] Therefore, there is still an urgent need to develop new B7-H3 antibody molecules and ADC molecules containing the same. Preferably, the molecules have advantages such as high efficacy, high safety (including low side effects) and / or high product uniformity. SUMMARY OF THE INVENTION

[0008] The present invention provides an immunoconjugate comprising an antibody targeting B7-H3 (eg, an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 of the present invention) and other payloads.

[0009] In some aspects, the immunoconjugate is an antibody-drug conjugate.

[0010] Therefore, the present invention provides an antibody targeting B7-H3 and an antibody-drug conjugate (ADC) targeting B7-H3. The antibody of the present invention has advantages such as higher endocytosis efficiency and higher affinity.

[0011] In some embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof suitable for constructing the immunoconjugates of the invention bind to B7-H3 (e.g., human B7-H3) with sufficient affinity, for example, with an equilibrium dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.5 nM, or ≤0.4 nM, or ≤0.35 nM, for example, less than 10-7 M, for example, 10-7 M to 10-10 M, for example, between about 0.1 nM-1 nM, between greater than 0.2-0.5 nM, between about 0.3-0.4 nM, for example, as detected by ForteBio. In some embodiments, B7-H3 is human or cynomolgus monkey B7-H3. In some embodiments, the antibody binding affinity is determined using bio-optical interferometry.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to B7-H3 expressed on the surface of a cell.

[0013] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can induce ADCC effects. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit and / or reduce the growth and / or volume of tumors in vivo.

[0014] In one aspect, the present invention provides an antibody-drug conjugate having formula (I):

[0015] Ab-(LD) p (I)

[0016] or a pharmaceutically acceptable salt or solvate thereof,

[0017] in:

[0018] Ab is an antibody or a fragment thereof that binds to B7-H3 (eg, human B7-H3);

[0019] L is a linker;

[0020] D is a drug, preferably an anti-tumor compound; and

[0021] p is the average drug-to-antibody ratio DAR, which is a value from 1 to 16, for example, 4-10,

[0022] Wherein Ab in formula (I) comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 7 or 15, 8 and 9, respectively.

[0023] In some embodiments, Ab in formula (I) comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or 13; or

[0024] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4 or 13; or

[0025] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4 or 13, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions; and / or

[0026] Light chain variable region

[0027] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or 16; or

[0028] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 16; or

[0029] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10 or 16, consisting of said amino acid sequence, and preferably, said amino acid changes do not occur in the CDR regions.

[0030] In some embodiments, Ab in formula (I) comprises VH and VL, wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO:4, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:10; or wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO:13, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:16.

[0031] In some embodiments, Ab in Formula (I) further comprises a heavy chain constant region and / or a light chain constant region.

[0032] In some embodiments, Ab in Formula (I) is an IgG antibody.

[0033] In some embodiments, the heavy chain constant region of the Ab in Formula (I) is from IgG1 or IgG2 or IgG3 or IgG4, such as IgG1.

[0034] In some embodiments, Ab in Formula (I) comprises a heavy chain and a light chain, wherein the heavy chain

[0035] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 14;

[0036] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 14; or

[0037] (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 6 or 14; and / or

[0038] Light chain

[0039] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12 or 17;

[0040] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 17; or

[0041] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or 17.

[0042] In some embodiments, Ab in formula (I) comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:12; or wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:14, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:17.

[0043] In some embodiments, Ab in Formula (I) is a chimeric antibody or a humanized antibody.

[0044] In some embodiments, Ab in formula (I) is an antigen-binding fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibody; single-chain antibody (such as scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0045] In some embodiments, the anti-tumor compound is a cytotoxic agent, such as a camptothecin, an auristatin, or a maytansine.

[0046] In some embodiments, D has the structure shown in formula (D-1):

[0047]

[0048] Where R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0049] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0050] R 4 Selected from H or C1-C4 alkyl.

[0051] In some embodiments, R 1 For H, R 2 is C1-C6 alkyl, R 3 is -F.

[0052] In some embodiments, D has a structure shown in formula (D-2):

[0053]

[0054] Where R 1 , R 2 and R 3 As defined above.

[0055] In some embodiments, D has a structure shown in formula (D-3) or formula (D-4):

[0056]

[0057] In some embodiments, -L- has the structure:

[0058] -ZE-NH-CH2-QL 2 -L 1 -

[0059] Where Z is connected to Ab, L 1 Connect with D;

[0060] Z is selected from wherein m is an integer of 1 to 10; the carbonyl group at the right end of Z is covalently linked to E;

[0061] E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z;

[0062] Q is -O- or -S-;

[0063] L 1 is not present or -(C1-C 10 Alkylene)-;

[0064] L 2 Does not exist, -N(R 5 )C(O)-(C1-C 10 Alkylene)-* or -C(O)N(R 5 )-(C1-C 10 Alkylene)-*; wherein * indicates that the terminus is covalently linked to Q; and

[0065] R 5 It is H or C1-C6 alkyl.

[0066] In some embodiments, E is a peptide residue of 2, 3 or 4 amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group; and

[0067] -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10Alkylene)-*, wherein * indicates that the terminus is covalently linked to Q;

[0068] R 5 It is H or C1-C6 alkyl.

[0069] In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, or Where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N-terminus; and

[0070] -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0071] In some embodiments, -ZE-NH-CH2-QL 2 -L 1 - has the following structure

[0072]

[0073] Its right end is connected to D.

[0074] In some embodiments, the antibody-drug conjugate of formula (I) can be represented by the following formula:

[0075] Ab'-(SLD)p(I')

[0076] wherein Ab' is as defined above for Ab, and L, D and p are as defined above.

[0077] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR value of 5-11, such as 7.5-8.5.

[0078] In some embodiments, the antibody-drug conjugate is selected from

[0079]

[0080] wherein Ab is antibody HZ5C2.9; q represents the average DAR, for example, 5-11 or 7.5-8.5.

[0081] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

[0082] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents, such as a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug or an immunomodulator.

[0083] In another aspect, the present invention provides a method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition as described above, or the pharmaceutical combination as described above.

[0084] In some embodiments, the tumor is a cancer, and preferably, the cancer has elevated levels (eg, nucleic acid or protein levels) of B7-H3, eg, compared to healthy individuals or healthy tissue adjacent to the patient's cancer tissue.

[0085] In some embodiments, the cancer is selected from lung cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, pancreatic cancer, or breast cancer.

[0086] In some embodiments, the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modality comprises radiation therapy or surgery, or the therapeutic agent comprises a chemotherapeutic agent, angiogenesis inhibitor, cytokine, cytotoxic agent, other antibodies, small molecule drugs or immunomodulators.

[0087] The antibody-drug conjugate of the present invention has the following advantages:

[0088] (1) Bind to target cells expressing human B7-H3 with high affinity;

[0089] (2) Ability to enter cells through endocytosis and kill target cells; the ADC of the present invention has high endocytosis efficiency;

[0090] (3) There is a significant bystander effect;

[0091] (4) It has high anti-tumor efficacy, has a stronger killing effect on tumor cells, and has a stronger inhibitory effect on tumor growth, especially tumors with high expression of B7-H3; it has significantly improved or even unexpected anti-tumor activity compared to DS-7300a;

[0092] (5) With a DAR value as high as 8, the product is more uniform; while the antibody-drug ratio (DAR value) of DS-7300a is 4, the distribution of DAR value is uneven and affects the efficacy.

[0093] (6) Low toxicity;

[0094] (7) having good stability, and the stability of the linker-payload used in the present invention is high;

[0095] (8) It has good drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 Binding of anti-B7-H3 monoclonal antibody HZ5C2.9 and control antibody M30 to the human lung cancer cell line Calu-6 is shown.

[0097] Figure 2 The results of endocytosis experiments of HZ5C2.9 and control antibody M30 are shown.

[0098] Figure 3A The results of the cellular binding assay of ADC-01 molecule and HZ5C2.9 to Calu-6 are shown.

[0099] Figure 3B The results of the cell binding assay of ADC-01 molecule and HZ5C2.9 to MDA-MB-453 are shown.

[0100] Figure 4 The results of the endocytosis experiments of ADC-01 molecules and HZ5C2.9 are shown.

[0101] Figure 5A The in vitro cell killing effect of ADC-01 molecule in the MDA-MB-453 cell line is shown.

[0102] Figure 5B The in vitro cell killing effect of ADC-01 molecule in the MDA-MB-453-B7-H3 cell line is shown.

[0103] Figure 5C The in vitro cell killing effect of ADC-01 molecule in Calu-6 cell line is shown.

[0104] Figure 5D The in vitro cell killing effect of ADC-01 molecule in A375 cell line is shown.

[0105] Figure 6 The bystander killing effect of the ADC-01 molecule is shown.

[0106] Fig. 7A The tumor inhibitory effect of ADC-01 molecule on Calu-6 mouse transplant tumor model was shown.

[0107] Figure 7B Shown are the changes in mouse body weight in a tumor inhibition experiment using the Calu-6 mouse xenograft tumor model.

[0108] Fig. 8A The tumor inhibitory effect of ADC-01 molecule on A375 mouse transplant tumor model was shown.

[0109] Figure 8B Shown are the changes in mouse body weight in a tumor inhibition experiment using the A375 mouse xenograft tumor model.

[0110] Fig.9A The tumor inhibitory effect of ADC-01 molecule on the BXPC3 mouse transplant tumor model was shown.

[0111] Fig. 9B Shown are the changes in mouse body weight in a tumor inhibition experiment using the BXPC3 mouse xenograft tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0113] I. Definitions

[0114] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.

[0115] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0116] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0117] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0118] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0119] Unless otherwise specified, the terms "B7-H3", "B7H3" and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein that is a member of the B7 / CD28 superfamily and is similar in sequence to the extracellular domain of PD-L1. B7-H3 has 316 amino acids, including a putative signal peptide consisting of 28 amino acids, an extracellular region consisting of 217 amino acids, a transmembrane region and a cytoplasmic domain consisting of 45 amino acids, with a molecular weight of approximately 45-66 kDa. In humans, due to exon duplication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3), or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 has approximately 90% homology with its human counterpart. In some embodiments of the invention, B7-H3 is human B7-H3. In some embodiments, B7-H3 is a protein under UniProt database accession number Q5ZPR3.

[0120] The terms "anti-B7-H3 antibody", "anti-B7-H3", "B7-H3 antibody" or "anti-B7-H3 antibody" as used herein refer to an antibody that can bind to the B7-H3 protein with sufficient affinity. The antibody can be used as a diagnostic and / or therapeutic agent targeting B7-H3, or used to construct an immunoconjugate, such as an antibody drug conjugate.

[0121] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, full-length antibodies comprise two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, full-length antibodies comprise two heavy chains (H) interconnected by disulfide bonds.

[0122] For conventional four-chain IgG antibodies, the full-length antibody heavy chain is usually composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0123] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0124] "Antigen-binding fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0125] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., B7-H3) that specifically interacts with an antibody molecule.

[0126] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0127] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0128]

[0129]

[0130] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0131] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0132] Unless otherwise indicated, in the present invention, when referring to the residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0133] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules:

[0134] VH CDR1 was determined according to the AbM rule; and VH CDR2 and 3 were both determined according to the Kabat rule.

[0135] In one embodiment, the light chain variable region CDRs of the antibodies of the invention are determined according to the Kabat rules.

[0136] In one embodiment, the heavy chain variable region CDRs of the antibodies of the invention are determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule; and the light chain variable region CDRs are determined according to the Kabat rule.

[0137] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0138] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A natural immunoglobulin "Fc domain" comprises two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a natural antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or comprises the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0139] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0140] "Immunoconjugate" refers herein to a payload connected to an antibody or its antigen binding fragment by a linker, so that the antibody or its antigen binding fragment can be used as a carrier to target and transport the payload to a target site. The term "payload" refers to the active portion of the antibody or antibody fragment of the present invention that is conjugated, and may include any portion for attaching the antibody or antibody fragment. In some embodiments, the payload may be a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide. In some embodiments, immunoconjugates include antibody drug conjugates (ADC), antibody immunostimulatory drug conjugates (ISAC), antibody oligonucleotide conjugates (AOC), antibody polypeptide drug conjugates (APC), antibody nuclide drug conjugates (RDC), or antibody degradation drug conjugates (ADeC), etc. The payload or active part suitable for being linked to the antibody includes, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonist (TLR) ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drug XMT-2056, Treg cell regulation ISAC drug ADCT-301, etc.), immunomodulators, therapeutic oligonucleotides (siRNA, PMO, etc.) or radionuclides, etc. In some embodiments, the immunoconjugate of the present invention is an antibody drug conjugate, i.e., ADC.

[0141] As used herein, "antibody-drug conjugate (ADC)" refers to a structure / molecule obtained by linking an antibody and a (small molecule) drug.

[0142] The term "linker" refers to a structural fragment that connects a drug (e.g., a small molecule drug) to an antibody portion. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being attached to the antibody or its antigen-binding fragment.

[0143] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (eg, a small molecule drug), to a linker.

[0144] The term "site-specific conjugation" as described herein refers to conjugation in which a drug is specifically linked to a specific site of an antibody via a linker.

[0145] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0146] The term "alkylene" refers to an alkyl group as defined above, but it is divalent, i.e., has two single bonds connected to the other two groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0147] The term "alkenyl" refers to a straight or branched hydrocarbon group containing 2-16 carbon atoms and containing at least one double bond and no triple bonds. The alkenyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms or 2-4 carbon atoms. Representative examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0148] The term "alkynyl" refers to a straight or branched hydrocarbon group containing 2-16 carbon atoms and comprising at least one triple bond. Alkynyl groups preferably contain 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.

[0149] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br) and iodine (-I).

[0150] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted by one or more halogen groups as defined herein. Halogenated alkyl may preferably be monohalogenated alkyl, dihalogenated alkyl or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halogenated groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10 or 8 or 6 or 4 or 3 or 2 halogen groups. Non-limiting examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. Perhalogenated alkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0151] The term "haloalkenyl" refers to an alkenyl as defined herein substituted with one or more halogen groups as defined herein. The term "haloalkynyl" refers to an alkynyl as defined herein substituted with one or more halogen groups as defined herein. The meaning of "halo" defined for "haloalkyl" is applicable to "haloalkenyl" and "haloalkynyl".

[0152] The term "polyol group" refers to an alkyl group as defined above containing a plurality (e.g. 2-10, e.g. 3, 4, 5, 6, 7 or 8) of hydroxyl groups, which optionally contains 1 or more (e.g. 2, 3 or 4) other groups (e.g. amino, carbonyl). Non-limiting examples of "polyol groups" include, for example Among them, the chiral center without indicating the stereo configuration can be R or S configuration, preferably

[0153] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. The compilation of conventional amino acids involved herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in the present disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, glycine can be represented by Gly, alanine can be represented by Ala, valine can be represented by Val, glutamine can be represented by Gln, glutamic acid can be represented by Glu, phenylalanine can be represented by Phe, and leucine can be represented by Leu.

[0154] The term "optional" or "optionally": means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted", the group or structure may be substituted or may not be substituted.

[0155] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention, and the salt is not biologically or otherwise undesirable. The ADC conjugate of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the present invention and an organic or inorganic acid, and the organic or inorganic acid includes but is not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention and organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts formed by organic bases containing N groups.

[0156] The term "solvate" refers to an association formed by one or more solvent molecules and the ADC antibody-drug conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0157] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0158] The term "drug: antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be determined by the number of -LD in Formula I, for example, the DAR can be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The DAR can also be calculated as the average DAR of the population of molecules in the product, that is, the overall ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to herein as the average DAR or the average DAR of the drug to antibody ratio. It should be understood that the average DAR can be any value within the range, including decimals and integers. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 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, 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 or 10.0, and ranges with two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules, which comprises ADC molecules with the same and / or different DARs.

[0159] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0160] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0161] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0162] The term "drug" refers to an organic compound capable of modulating biological processes, particularly altering or preventing pathological processes.

[0163] The term "prodrug" refers to a chemically modified active or inactive compound that, after administration to an individual, becomes an active drug through physiological actions in the body (e.g., hydrolysis, neogenesis, etc.). The techniques for making and using prodrugs are well known to those skilled in the art.

[0164] The term "small molecule drug" refers to low molecular weight organic compounds that can regulate biological processes, especially change or prevent pathological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 10 kD, more preferably less than 500 D. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting immune responses than macromolecules.

[0165] "Anti-tumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as cytotoxic agents disclosed in WO2021 / 173773, camptothecin compounds Exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE) or maytansine compounds such as small molecule microtubule inhibitor DM1. It should be understood that anti-tumor compounds can be substituted by isotopes including but not limited to deuterium, tritium, etc. For example, after substitution with deuterium, after the carbon-deuterium bond replaces the carbon-hydrogen bond, since the former is more stable than the latter, the substitution can directly affect the absorption, distribution, metabolism and excretion of certain drugs, thereby improving the efficacy, safety and tolerability of the drug. Therefore, the "anti-tumor compound" of the present invention can cover compounds substituted with deuterium.

[0166] "Deuterium substituted" means that a hydrogen in the molecule is replaced with deuterium, for example, 1 or more hydrogens, for example 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) hydrogens are replaced with deuterium.

[0167] "Camptothecin compounds" refers to compounds having a camptothecin core structure (e.g., a pentacyclic ring of camptothecin or a tetracyclic ring structure thereof) and having anti-tumor activity. "Camptothecin compounds" is a term conventionally used in pharmaceutical chemistry. Based on the structure of the compound, a person skilled in the art can easily determine whether the compound belongs to the camptothecin compounds.

[0168] "Auristatin compounds" refer to compounds that have the core structure of auristatin and have anti-tumor activity.

[0169] “Maytansine compounds” refer to compounds that have a maytansine core structure and have anti-tumor activity.

[0170] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0171] The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention, which produces a desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses. Depending on the desired effect, "therapeutically effective amount" and "prophylactically effective amount" may be included.

[0172] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0173] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0174] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as in the initially transformed cell are included herein.

[0175] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.

[0176] "Individuals" or "subjects" include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0177] An "isolated" antibody or other molecule (e.g., ADC molecule) is an antibody or molecule that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0178] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0179] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can be in early, intermediate or advanced stages or be a metastatic cancer. Cancers suitable for treatment by the molecules of the invention include, but are not limited to, lung cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, pancreatic cancer or breast cancer, including metastatic forms of those cancers.

[0180] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues."Tumor" encompasses solid tumors and hematological tumors as well as metastatic lesions. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

[0181] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0182] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0183] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the ADC molecules of the present invention, and (ii) other therapeutic agents) are administered to the patient in sequence as separate entities, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the ADC molecules of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those when they are used alone. The term "fixed combination" means that two or more active agents are administered to the patient simultaneously in the form of a single entity. It is preferred that the dosage and / or time interval of the two or more active agents be selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0184] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to a desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.

[0185] As used herein, "treat," ...

[0186] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0187] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0188] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0189] II. Antibody-drug conjugates

[0190] The present invention provides an antibody-drug conjugate having formula (I):

[0191] Ab-(LD) p (I)

[0192] or a pharmaceutically acceptable salt or solvate thereof,

[0193] in:

[0194] Ab is an antibody or a fragment thereof that binds to B7-H3 (eg, human B7-H3);

[0195] L is a linker;

[0196] D is a drug, preferably an anti-tumor compound; and

[0197] p is the average drug to antibody ratio, DAR, of 1 to 16, such as 1-10, 1-9, 2-8, 4-10, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0198] In some embodiments, Ab in formula (I) of the present invention is a chimeric antibody or a humanized antibody, preferably a humanized antibody. In some embodiments, Ab in formula (I) of the present invention is an antibody fragment, such as an antigen-binding fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibody; single-chain antibody (such as scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0199] In some embodiments, Ab in formula (I) of the present invention is a bispecific antibody or a multispecific antibody.

[0200] In a preferred embodiment of the invention, the Ab comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, from the heavy chain variable region (VH).

[0201] In a preferred embodiment of the invention, the Ab comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region (VL).

[0202] In some embodiments, the Ab comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0203] In some aspects, Ab comprises a heavy chain variable region (VH). In some aspects, Ab comprises a light chain variable region (VL). In some aspects, Ab comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

[0204] In some embodiments, the heavy chain variable region VH

[0205] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or 13; or

[0206] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4 or 13; or

[0207] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4 or 13, consisting of said amino acid sequence, and preferably, said amino acid changes do not occur in the CDR regions.

[0208] In some embodiments, the light chain variable region VL

[0209] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or 16; or

[0210] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 16; or

[0211] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10 or 16, consisting of said amino acid sequence, and preferably, said amino acid changes do not occur in the CDR regions.

[0212] In some embodiments, the three complementary determining regions (HCDRs) from VH of the present invention, HCDR1, HCDR2 and HCDR3 are

[0213] (i) three complementary determining regions HCDR1, HCDR2 and -HCDR3 contained in VH as shown in SEQ ID NO: 4 or 13, or

[0214] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions relative to the sequence of (i).

[0215] In some embodiments, the three complementarity determining regions (LCDRs) from VL of the present invention, LCDR1, LCDR2 and LCDR3 are

[0216] (i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 10 or 16,

[0217] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three LCDR regions relative to the sequence of (i).

[0218] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0219] In some embodiments, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence, or HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2.

[0220] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, or HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0221] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 7 or 15, or consists of the amino acid sequence, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7 or 15.

[0222] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO:8, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0223] In some embodiments, LCDR3 comprises the amino acid sequence of SEQ ID NO:9, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

[0224] In some embodiments, the Ab in formula (I) of the present invention further comprises a heavy chain constant region. In some embodiments, the Ab in formula (I) of the present invention further comprises a light chain constant region. In some embodiments, the Ab in formula (I) of the present invention further comprises a heavy chain constant region and a light chain constant region.

[0225] In some embodiments, the heavy chain constant region HC of the present invention is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, such as a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a lambda or kappa light chain constant region.

[0226] In some preferred embodiments, the heavy chain constant region HC of the present invention

[0227] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5;

[0228] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 5; or

[0229] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0230] In some embodiments, the antibody light chain constant region LC of the present invention is

[0231] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11;

[0232] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or

[0233] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 11.

[0234] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a light chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain and a light chain.

[0235] In some specific embodiments of the present invention, the heavy chain of the present invention comprises a heavy chain variable region and a heavy chain constant region, or consists of the heavy chain variable region and the heavy chain constant region. In some specific embodiments of the present invention, the light chain of the present invention comprises a light chain variable region and a light chain constant region, or consists of the light chain variable region and the light chain constant region.

[0236] In some embodiments, the heavy chain

[0237] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 14;

[0238] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 14; or

[0239] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 6 or 14, or consists of said amino acid sequence.

[0240] In some embodiments, the light chain

[0241] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12 or 17;

[0242] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 17; or

[0243] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or 17.

[0244] In some specific embodiments, the Ab in formula (I) of the present invention specifically binds to B7H3 and comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:4 or 13, and / or the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:10 or 16.

[0245] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and / or LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 7, 8 and 9, respectively.

[0246] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and / or LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 15, 8 and 9, respectively.

[0247] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises VH and VL, wherein

[0248] (i) VH comprises or consists of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0249] VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 90% identity thereto; or

[0250] (ii) VH comprises or consists of the amino acid sequence of SEQ ID NO:13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0251] VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90% identity thereto.

[0252] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO:4, and VL comprises the amino acid sequence shown in SEQ ID NO:10; or wherein VH comprises the amino acid sequence shown by SEQ ID NO:13, and VL comprises the amino acid sequence shown by SEQ ID NO:16.

[0253] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises VH and VL, wherein the amino acid sequence of VH is shown by SEQ ID NO:4, and the amino acid sequence of VL is shown by SEQ ID NO:10; or wherein the amino acid sequence of VH is shown by SEQ ID NO:13, and the amino acid sequence of VL is shown by SEQ ID NO:16.

[0254] In some specific embodiments of the present invention, the Ab in formula (I) of the present invention is an IgG antibody, i.e., it comprises a heavy chain and / or a light chain, such as a heavy chain and a light chain, that binds to B7H3. In some embodiments, the Ab in formula (I) of the present invention is a complete antibody.

[0255] In some embodiments, the heavy chain of Ab in Formula (I)

[0256] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 14;

[0257] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 14; or

[0258] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 6 or 14, or consists of said amino acid sequence.

[0259] In some embodiments, the light chain of Ab in Formula (I)

[0260] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12 or 17;

[0261] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 17; or

[0262] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or 17.

[0263] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown by SEQ ID NO:6, and the amino acid sequence of the light chain is the amino acid sequence shown by SEQ ID NO:12; or wherein the heavy chain comprises the amino acid sequence shown by SEQ ID NO:14, and the light chain comprises the amino acid sequence shown by SEQ ID NO:17.

[0264] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown by SEQ ID NO:6, and the amino acid sequence of the light chain is shown by SEQ ID NO:12; or wherein the amino acid sequence of the heavy chain is shown by SEQ ID NO:14, and the amino acid sequence of the light chain is shown by SEQ ID NO:17.

[0265] In one embodiment of the invention, the amino acid changes described herein include replacement, insertion or deletion of amino acids. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the heavy chain constant region of an antibody.

[0266] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to an amino acid substitution by another amino acid within the same class, such as an acidic amino acid substitution by another acidic amino acid substitution, a basic amino acid substitution by another basic amino acid substitution, or a neutral amino acid substitution by another neutral amino acid substitution. In certain embodiments, the substitution occurs in the CDR region of an antibody. Typically, the variant obtained has modifications (e.g., improvements) and / or will have certain biological properties that are substantially retained relative to the parent antibody in terms of certain biological properties (e.g., increased affinity). An exemplary substitution variant is an affinity matured antibody.

[0267] In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has an antitumor effect and has a substituent or a partial structure that can be connected to the linker structure, without any particular limitation. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, part or all of the linker can preferably be cut off in tumor cells to free the antitumor compound portion, thereby showing an antitumor effect. When the linker is cut off from the connecting portion of the drug, the antitumor compound is freed with an unmodified structure, and its original antitumor effect can be exerted.

[0268] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent or a chemotherapeutic agent, such as a camptothecin compound Exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), an auristatin compound such as monomethyl auristatin E (MMAE), or a maytansine compound such as a small molecule microtubule inhibitor DM1.

[0269] In some embodiments, D has the structure shown in formula (D-1):

[0270]

[0271] Where R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0272] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0273] R 4 Selected from H or C1-C4 alkyl.

[0274] In some embodiments, R 1 For H, R 2 is C1-C6 alkyl, R 3 is -F.

[0275] In some embodiments, D has a structure shown in formula (D-2):

[0276]

[0277] Where R 1 , R 2 and R 3 As defined above.

[0278] In some embodiments, D has a structure shown in formula (D-3):

[0279]

[0280] In some embodiments, D has a structure shown in formula (D-4):

[0281]

[0282] In some embodiments, -L- has the following structure: -ZE-NH-CH2-QL 2 -L 1 -

[0283] Where Z is connected to Ab, L 1 Connect with D;

[0284] Z is selected from

[0285] Where m a1 and m a2 An integer independently selected from 0-20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0286] m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; the carbonyl group at the right end of Z is covalently linked to E;

[0287] E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted by 1 or more (e.g. 2, 3 or 4) groups selected from C 1-6 an alkyl group and a polyol group, wherein the N-terminus of the peptide residue is covalently linked to Z;

[0288] Q is -O- or -S-;

[0289] L 1 is not present or -(C1-C 10 Alkylene)-;

[0290] L 2 Does not exist, -N(R 5 )C(O)-(C1-C10 Alkylene)-* or -C(O)N(R 5 )-(C1-C 10 Alkylene)-*; wherein * indicates that the terminus is covalently linked to Q; and

[0291] R 5 It is H or C1-C6 alkyl.

[0292] In some embodiments, Z is selected from wherein m is an integer of 1-10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5; the carbonyl group at the right end is covalently linked to E;

[0293] E is a peptide comprising 2-10 amino acids, wherein the peptide is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide is covalently linked to Z;

[0294] Q is -O- or -S-;

[0295] L 1 is not present or -(C1-C 10 Alkylene)-;

[0296] L 2 Does not exist, -N(R 5 )C(O)-(C1-C 10 Alkylene)-* or -C(O)N(R 5 )-(C1-C 10 Alkylene)-*; wherein * indicates that the terminus is covalently linked to Q; and

[0297] R 5 It is H or C1-C6 alkyl.

[0298] In some embodiments, E is a peptide residue consisting of 2-8, 2-6, 2-5, or 2-4 amino acids.

[0299] In some embodiments, the amino acid is selected from, for example, glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally with 1 C 1-6 In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally with 1 C 1-6 Alkyl substitution.

[0300] In some embodiments, E is a peptide residue consisting of 2, 3 or 4 amino acids.

[0301] In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group.

[0302] In some embodiments, the substituted glutamine or glutamic acid has the structure shown below:

[0303] Where R 6 is H or C1-C6 alkyl;

[0304] Preferably

[0305] Where R 6 It is H or C1-C6 alkyl.

[0306] In some embodiments, E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu-Cit-, -Lys-Cit -, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-Glu-, -Val-L ys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-L ys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys-Leu-, -A la-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with 1 polyol group and optionally with 1 C 1-6 Alkyl substitution,

[0307] Preferably, the substituted Gln or Glu has a structure as shown in Formula (G-1a), Formula (G-1a) or Formula (G-1a) as described above.

[0308] In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, or Where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N terminus.

[0309] In some embodiments, -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10 Alkylene)-*, wherein * indicates that the terminus is covalently linked to Q;

[0310] R 5 It is H or C1-C6 alkyl.

[0311] In some embodiments, -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0312] In some embodiments, -ZE-NH-CH2-QL 2 -L 1 - has the following structure

[0313]

[0314] It should be understood that the right end of the structure is connected to the D portion.

[0315] In some embodiments, the payload (e.g., drug) of the immunoconjugates of the invention is attached to the modified antibody or antibody fragment via a thiol group of a free cysteine ​​of the antibody or antigen-binding fragment thereof (optionally via a linker). In some embodiments, the payload (e.g., drug) of the immunoconjugates of the invention is linked to the thiol group of the cysteine ​​via a cleavable or non-cleavable linker.

[0316] In some embodiments, Ab is linked to L via the sulfur atom on its sulfhydryl group, in which case the antibody-drug conjugate of formula (I) can be represented by the following formula:

[0317] Ab'-(SLD) p (I')

[0318] wherein Ab' is as defined above for Ab, and L, D and p are as defined above.

[0319] In some embodiments, the antibody-drug conjugate has an average DAR value of 5-11 or 7.5-8.5.

[0320] In some embodiments, the antibody-drug conjugate is selected from

[0321]

[0322] wherein Ab is an antibody or fragment thereof that binds to B7-H3 as defined herein, for example antibody HZ5C2.9; q represents the average DAR, for example as defined above for p or as defined herein for the average DAR, for example 5-11, 6-10, 7-9 or 7.5-8.5, preferably 8.0.

[0323] It should be understood that the S atom connected to Ab in the above ADC comes from the antibody Ab. Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a thiol -SH, which is then connected to the maleimide part of the linker.

[0324] In another aspect, the present invention provides an antibody-drug conjugate having formula (II):

[0325] Ab-(LD)n(II)

[0326] or a pharmaceutically acceptable salt or solvate thereof,

[0327] in:

[0328] Ab, L and D are as defined herein for formula (I);

[0329] n represents the number of -LDs attached to Ab, and is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0330] III. Preparation of ADC molecules of the present invention

[0331] Another aspect of the present invention provides a method for preparing ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to an active substance (D) having biological and / or pharmaceutical activity through a linker (L). The method comprises coupling the antibody (Ab) of the present invention to one or more active substances D through one or more linkers (L) defined in the present invention. Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0332] In some embodiments, the method includes preparing an Ab for ADC, which comprises culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for expression of the Ab or its chains, as provided above, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0333] Ab-encoding nucleic acids suitable for use in the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 6, 10, 12, 13, 14, 16 or 17, or nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 6, 10, 12, 13, 14, 16 or 17 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0334] The polynucleotide encoding the polypeptide chain of Ab of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0335] Expression vectors suitable for the present invention include, but are not limited to, viruses, plasmids, cosmids, lambda phages or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0336] Abs prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0337] In some embodiments, the method comprises the steps of:

[0338] (a) adding antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0339] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0340] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0341] wherein Ab is as defined above.

[0342] It will be appreciated that the linker-payload reacts with Ab to provide the -LD moiety in the compound of Formula I, and where -LD is clearly defined, the structure of the linker-payload can be determined.

[0343] In some embodiments, the buffer solution of step a) is a PBS buffer, preferably, the pH thereof is 5.0-9.0, such as 6.0-8.0.

[0344] In some embodiments, the reducing agent of step a) is TCEP.

[0345] In some embodiments, the linker-payload has the following structure: Z'-E-NH-CH2-QL 2 -L 1 -D, where E, Q, L 2 , L 1 , D is as defined above, Z' is m is as defined above.

[0346] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0347] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0348] IV. Pharmaceutical Compositions

[0349] In some embodiments, the present invention provides a composition comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof, preferably a composition is a pharmaceutical composition or a pharmaceutical preparation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an ADC molecule of the present invention, and a combination of one or more other therapeutic agents.

[0350] The present invention also includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the present invention or pharmaceutically acceptable salts thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0351] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0352] For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Seskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0353] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0354] A medicament comprising the ADC described herein can be prepared by mixing the ADC molecule of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0355] The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), etc. The active ingredient is suitably combined in an amount effective for the intended use.

[0356] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0357] VII. Drug Combinations and Kits

[0358] In some embodiments, the present invention also provides a drug combination or a drug combination product comprising an ADC molecule of the present invention and one or more other therapeutic agents (e.g., therapeutic agents including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc.).

[0359] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, so that the dosage unit can be provided according to the dosage regimen or the interval of drug administration.

[0360] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0361] - a first container containing a pharmaceutical composition comprising the ADC molecule of the present invention;

[0362] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0363] VIII. Use and Methods

[0364] In one aspect, the present invention provides a method for preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of the ADC molecule, pharmaceutical composition, pharmaceutical combination or kit of the present invention.

[0365] In some embodiments, the tumor, such as cancer, is B7-H3 positive, for example, it comprises tumor cells expressing B7-H3. In some embodiments, the patient's tumor comprises tumor cells expressing B7-H3. In some embodiments, the patient's tumor cells express B7-H3, for example, moderate expression of B7-H3, preferably high expression of B7-H3. In some embodiments, the tumor tissue of the tumor (e.g., cancer) patient has (e.g., elevated levels, such as nucleic acid or protein levels) B7-H3, for example, compared with the same tissue of a healthy individual or with the level of B7-H3 in a healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels, such as nucleic acid or protein levels) B7-H3, for example, compared with the same cells of a healthy individual or with the level of B7-H3 in a healthy cell adjacent to the patient's tumor cell.

[0366] In some embodiments, the tumor, such as cancer, includes solid tumors and blood tumors and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in early, middle or late stages or be metastatic cancer.

[0367] In a specific embodiment, the ADC molecule of the present invention is capable of killing tumor cells, and / or inhibiting the proliferation of tumor cells, such as tumor cells expressing B7-H3, such as lung cancer cells, head and neck tumor cells, prostate cancer cells, melanoma cells, pancreatic cancer cells, breast cancer cells, esophageal cancer cells, cervical cancer cells, renal cancer cells, bladder cancer cells or ovarian cancer cells.

[0368] In some embodiments, the tumor is an immune evasive tumor.

[0369] In some embodiments, the tumor is a cancer, such as lung cancer, head and neck cancer, prostate cancer, melanoma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, or ovarian cancer.

[0370] The subject can be a mammal, e.g., a primate, preferably, a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or at risk of suffering from a disease described herein. In some embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0371] In other aspects, the present invention provides the use of the above-mentioned ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits in the production or preparation of drugs for the purposes described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0372] In some embodiments, the ADC molecule or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of a disorder and / or symptoms associated with a disorder.

[0373] In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for preventing and / or treating the relevant diseases or disorders mentioned herein.

[0374] In some embodiments, the treatment modality includes surgery; radiation therapy, localized or focused irradiation, and the like.

[0375] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0376] Exemplary additional antibodies include antibodies that specifically bind to immune checkpoints.

[0377] The combination therapies of the invention encompass combined administration (e.g., two or more therapeutic agents are contained in the same formulation or in separate formulations), and separate administration, in which case administration of the ADC molecules of the invention may occur prior to, concurrently with, and / or after administration of the other therapeutic agents and / or agents.

[0378] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional route; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0379] The composition can also be administered locally via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ and can deliver the desired molecule via diffusion, a timed-release bolus, or continuous administration.

[0380] These and other aspects and embodiments of the present invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout the present invention may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0381] Example 1.1 Preparation of recombinant B7-H3 monoclonal antibody

[0382] According to the antibody preparation method of WO2022135467A1 (anti-B7-H3 antibodies and their uses), the chimeric antibody CH5C2 was obtained, and the humanized antibody HZ5C2.9 was obtained after humanization. Its sequence is as follows:

[0383]

[0384] Antibody expression and purification

[0385] First, the expression vector was constructed, and the heavy chain variable region and light chain variable region (see sequence table information) of the chimeric antibody ch5C2, humanized antibody HZ5C2.9 and control antibody (positive control antibody M30, negative control antibody IgG) were placed at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and the light chain kappa constant region (SEQ ID NO: 11). Then, it was constructed into the pcDNA3.1 expression vector with an N-terminal signal peptide to obtain the light and heavy chain expression vectors. HEK293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5×10 6 cells / ml. The cell density on the day of transfection was about 3×10 6cells / ml. Take Opti-MEM medium (Gibco catalog number: 31985-070) with a final volume of 1 / 10 (v / v) as transfection buffer, add the constructed expression vector, mix well, and filter with a 0.22μm filter for later use. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid in the previous step (the mass ratio of plasmid to PEI is 1:3), mix well, and incubate at room temperature for 10 minutes to obtain a DNA / PEI mixture. Gently pour the DNA / PEI mixture into HEK293 cells and mix well. After culturing for 24 hours at 37°C and 8% CO 2, add VPA (Sigma, catalog number: P4543-100G) with a final concentration of 2mM and 2% (v / v) Feed solution (1g / L Phytone Peptone + 1g / L Difco SelectPhytone), and continue culturing for 6 days. After cell culture, the cell culture fluid was centrifuged at 13000 rpm for 20 min, and the supernatant was collected. According to the manufacturer's instructions, the supernatant was purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95), and the concentration was determined. 100 μg of purified protein was taken, the concentration was adjusted to 1 mg / mL, and the protein purity was determined using a gel filtration column SW3000 (TOSOH Product No.: 18675).

[0386] Example 1.2 Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen using biofilm thin layer interferometry

[0387] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human B7-H3 was determined using the biofilm thin layer interferometry technique (ForteBio). The ForteBio affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitopebinning. MAbs, 2013. 5 (2): p. 270-8).

[0388] Briefly, the sensor was equilibrated offline in the assay buffer for 30 minutes, then online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to human B7-H3 protein, after which the sensor was transferred to the assay buffer for dissociation rate measurement. KD values ​​were analyzed using the ForteBio analysis software.

[0389] Table 1. Affinity constants (equilibrium dissociation constants) of B7-H3 antibodies detected by ForteBio

[0390]

[0391] Example 1.3 Binding of B7-H3 Antibody to Tumor Cell Lines

[0392] The binding of the anti-B7-H3 monoclonal antibody HZ5C2.9 and the control antibody M30 to the human lung cancer cell line Calu-6 (ATCC, HTB-56) was determined by flow cytometry (FACS). Specifically, the cells to be tested were counted and diluted to 2×10 6 cells / ml, add 50μl / well to a U-bottom 96-well plate. Centrifuge at 500g for 5min and remove the cell culture medium. Add the anti-B7-H3 monoclonal antibody HZ5C2.9 and the control antibody M30 to the U-shaped plate and resuspend the cells. Add 50μl of the antibody to each well at a starting concentration of 140nM and then dilute it 3 times in sequence, for a total of 8 concentration points. Let stand on ice for 30min. Remove the supernatant at 500g for 5min and wash the cells once with PBS. Add 100μl of goat anti-human IgG PE-labeled secondary antibody (SouthernBiotech, 51380) to each well. Incubate on ice for 30min in the dark. Remove the supernatant at 500g for 5min and wash the cells once with PBS. Resuspend the cells with 50μl 1×PBS and detect on the FACS machine. Analyze the experimental data using GraphPad Prism software to obtain Figure 1 .like Figure 1 The fully human antibody HZ5C2.9 showed good tumor cell-specific binding, which was better than the control antibody M30.

[0393] Example 1.4 Endocytosis experiment of B7-H3 antibody

[0394] After digestion of Calu-6 cells, the cell density was adjusted to 1*10 5Cells / well were plated in 96-well plates. Centrifuge at 500g for 3min and discard the supernatant. Take 50μl of the molecule to be detected with a final concentration of 10nM and incubate with PE-labeled anti-human IgG Fc (SEQ IDNID NO:32) at 4℃ for 30min, then add the primary antibody and secondary antibody complex to the cells. Each sample was repeated 2 times (the endocytosis time of each group was set to 0h, 30min, 1h, 2h, 3h, and 4h respectively). Incubate at 37℃. According to the experimental design, the corresponding cells were taken out from the incubator at different time points and added to the 96-well plate on ice for use; after the cells at the last time point were collected, centrifuged and discarded the supernatant, washed once with pre-cooled 1XPBS, added 200μl / well with a final concentration of 0.5mg / ml Papain (diluted with 1XPBS), pipetted and mixed, and incubated at room temperature for 20min; after incubation, centrifuged and discarded the supernatant, and washed twice with FACS buffer. After the second wash, discard the supernatant, add FACS buffer at 100 μl / well to resuspend the cells and test on the machine. GraphPadPrism software was used to analyze the experimental data. Figure 2 .like Figure 2 The results showed that HZ5C2.9 had a faster endocytosis rate, which was better than the control antibody M30.

[0395] Example 2.1 Synthesis of ADC molecule (ADC-01)

[0396] Based on HZ5C2.9, ADCs coupled with small molecule compounds were further designed and synthesized.

[0397] Example 2.1.1 Synthesis of Compound NT3

[0398]

[0399] According to the method disclosed in Example 4 of WO2021173773A1, compound NT3 was prepared. Its physicochemical data include 1 The HNMR and mass spectrometry data are consistent with those disclosed in WO2021173773A1.

[0400] Example 2.1.2 Synthesis of ADC-01

[0401]

[0402] wherein Ab is antibody HZ5C2.9; q represents the average DAR, for example 5-11, 6-10, 7-9 or 7.5-8.5, such as 7.98 determined in the experiments described below.

[0403] The specific process is as follows:

[0404] Preparation of ADC-01 (HZ5C2.9-NT3)

[0405] (a) The antibody HZ5C2.9 prepared according to the method of Example 1.1 was dissolved in PBS buffer (thermofisher, 10010023). A reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) was added, and the reaction mixture was placed at room temperature for 2-4 hours.

[0406] (i) The optimal concentration of HZ5C2.9 is 2-10 mg / mL,

[0407] (ii) The optimal molar ratio of TCEP / mAb is 10.0-20.0,

[0408] (iii) The optimal reaction temperature is 20-37°C,

[0409] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0410] (b) adding an excess of linker-toxin (NT3, dissolved in DMSO) to react with the antibody reduced in step (a), and the reaction mixture is placed at room temperature for 1-2 hours, wherein

[0411] (i) The optimal molar ratio of NT3 / mAb is 10.0-16.0,

[0412] (ii) The optimal reaction temperature is 20-37°C,

[0413] The crude ADC product was obtained.

[0414] (c) The obtained ADC crude product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product ADC-01.

[0415] (d) Using RP-HPLC, the DAR value was calculated based on the peak area of ​​each peak at UV280nm. The DAR of HZ5C2.9-NT3 was calculated to be 7.98.

[0416] Example 2.1.3 Synthesis of M30-DXd

[0417]

[0418] wherein Ab is antibody M30; q represents the average DAR, for example, 5-11, 6-10, 7-9 or 7.5-8.5, such as 3.95 determined in the experiments described below.

[0419] First, the proportion of the reducing agent TCEP (Aldrich, Lot #SLBZ2552) was tested. Different proportions of TCEP were added to the M30 antibody solution, and after reduction for 6 hours in an ice-water bath, 8 times the molar ratio of mc-GGFG-DXd was added, and DMSO was added to make the final system contain 10% by volume, and the reaction was continued in an ice-water bath for 1 hour. Then, Zeba TM The desalting centrifugal column removes unreacted small molecule drugs and other impurities and replaces the ADC in a 20mM histidine solution at pH 5.5 for storage. The DAR is calculated using RP-HPLC, and after linear regression of different TCEP ratios, the TCEP ratio required when the DAR is 4 is calculated. According to the obtained TCEP ratio, the ADC molecule is prepared according to the above experimental method. The DAR value of the ADC is determined using RP-HPLC. A small amount of M30-DXd-DAR4 ADC is reduced by adding a reducing agent, and then analyzed using the same analytical conditions as ADC-01. The DAR value is calculated based on the peak area of ​​each peak at UV280nm, and the DAR of M30-DXd ADC is 3.95. The purity analysis was performed using the same analytical method as ADC-01, and its SEC purity was 99.55%.

[0420] Example 2.1.4 Synthesis of Control IgG1-NT3

[0421] The preparation process was similar to that of ADC-01, except that the HZ5C2.9 monoclonal antibody was replaced with the control antibody IgG. The same analytical method as ADC-01 was used to measure the DAR to be 7.94 and the purity to be 99.35%.

[0422] Example 2.1.5 Synthesis of Control IgG1-DXd

[0423] The preparation process is similar to that of M30-DXd, except that the M30 monoclonal antibody is replaced with the control antibody IgG. The same analytical method as ADC-01 was used to measure the DAR to be 4.1 and the purity to be 99.43.

[0424] Example 2.2 Cell Binding Experiment of ADC-01 Molecule

[0425] In order to detect whether small molecule conjugation would change the binding properties of ADC-01 monoclonal antibody to target cells, we used human lung cancer cell lines Calu-6 (ATCC, HTB-56) and MDA-MB-453 (Cobioer, CBP60386 expression negative) to detect the affinity of ADC-01 to the target by flow cytometry. The experimental method was the same as that in Example 1.2. Figure 3A and Figure 3BThe results showed that both HZ5C2.9 and ADC-01 did not bind to the non-target cell MDA-MB-453, but had a strong affinity to Calu-6, indicating that the binding of the antibody depends on the expression specificity of the target, and the coupling of small molecules (i.e., the payload part) will not affect the binding of the antibody.

[0426] Example 2.3: Endocytosis experiment of ADC-01 molecule

[0427] Strong endocytosis is one of the important characteristics of ADC drugs. When ADC binds to the antigen on the cell membrane surface, the ADC-antigen complex enters the cell through endocytosis and kills the target cell. Therefore, the endocytosis efficiency of ADC is one of the important indicators that determine the tumor suppression effect.

[0428] In order to detect whether the coupling of small molecule compounds affects the internalization efficiency of antibodies, we detected the internalization of ADC-01 molecules on Calu-6 by flow cytometry. The experimental method is the same as that of Example 1.4. Figure 4 The results showed that ADC-01 and HZ5C2.9 have similar endocytosis efficiency.

[0429] Example 2.4: Construction of stable expression cell lines

[0430] Preparation of human B7-H3 overexpressing cell lines

[0431] First, the full-length coding gene of human B7-H3 (UniProt ID: Q5ZPR3) was constructed into the vector pcDNA3.1 to construct an expression plasmid and transfected with a reagent The constructed expression plasmid was transferred into MDA-MB-453 cells using 3000 (Invitrogen, L3000008), and the transfected cells were screened by Geneticin (Gibco, 10131-027) to obtain a pool of cells expressing B7-H3. Then, a flow cytometer (MoFlo XDP, Beckman Coulter) was used to sort out MDA-MB-453-B7-H3 cells with high expression of B7-H3.

[0432] Preparation of luciferase-expressing cell lines

[0433] First, the full-length coding gene of Luciferase (UniProt ID: V9VFW4) was constructed into the vector pcDNA3.1 to construct a plasmid and use a transfection reagent The constructed plasmid was transferred into MDA-MB-453 cells using 3000 (Invitrogen, L3000008). The transfected cells were screened by Geneticin (Gibco, 10131-027) to obtain the luciferase-expressing cells MDA-MB-453-Luc.

[0434] Example 2.5: In vitro cell killing effect of ADC-01 molecule

[0435] The Cell Titer-Glo (Promega, G9242) detection kit was used to detect the effect of ADC on cell viability in various cell lines expressing B7-H3 as shown in Table 2.

[0436] After the cells were digested with Trypsin, the density was adjusted and evenly plated in a 96-well plate (Table 2) and diluted sample ADC-01 molecules and control molecules M30-DXd (Daiichi Sankyo targeting B7-H3 ADC, see, for example, Mol Cancer Ther. 2022Apr 1; ​​21(4): 635–646) were added with a specific concentration of dilution (the initial dilution concentration was 20 nM and the dilution factor was 3), and incubated in a 37°C incubator for 5 days. After 5 days, 100 μl of Cell Titer Glo detection reagent was added to each well, incubated at room temperature for 30 minutes, and detected with an enzyme reader. The relative cell viability was calculated (relative cell viability = experimental well / negative control well * 100%), and the curve was fitted using GraphPad Prism 8.0.

[0437] like Figure 5A-5D As shown in the figure, the killing effect of ADC molecules on cell lines depends on the expression level of B7-H3 on the cell surface. In B7-H3-negative MDA-MB-453 cells, ADC-01 molecules had no significant effect on cell viability; on the constructed MDA-MB-453-B7-H3 and medium- and high-expressing cell lines Calu-6 and A375, ADC-01 molecules had significant killing effects. This shows that ADC-01 molecules have good selectivity and effectiveness.

[0438] Table 2 Cell seeding density

[0439] Cell lines Number of cells per well (cell killing assay) MDA-MB-453 2000 MDA-MB-453-B7-H3 2000 A375 1000 Calu-6 1000

[0440] Example 2.5: Bystander Killing Effect

[0441] During the synthesis of ADC drugs, small molecule compounds can be connected to antibodies through cleavable linkers. After being internalized into the cell membrane, the linkers are cleaved and the small molecules are released to kill the target cells. After the target cells die, small molecule compounds are released from the target cells into the intercellular space, further killing non-target cells within a certain range. This effect is called the bystander effect. Since cells inside the tumor can have great differences in target expression levels (tumor heterogeneity), the bystander effect is very important for effectively killing tumor cells and inhibiting tumor growth.

[0442] The present invention utilizes non-target cells MDA-MB-453-Luc and target cells MDA-MB-453-B7-H3 to detect the bystander effect of ADC-01 molecules. After the cells are digested with Trypsin, the density is adjusted, a 96-well cell culture plate is prepared, 1000 / well of MDA-MB-453-Luc and 4000 / well of MDA-MB-453-B7-H3 are added, and the two types of cells are co-cultured for 24 hours, the supernatant is discarded, and 100 μl of the test samples ADC-01 and M30-DXd molecules are added to each well, with a final concentration of 2 nM, and 3 replicates are set for each sample. The cell culture plate is placed in a 37°C incubator and cultured for 5 days. After 5 days, 100 μl of One-Glo ((Promega, E6120) detection reagent is added to each well, incubated at room temperature for 5 minutes, the luminescence value is detected with an enzyme reader, and a bar graph is drawn with Graph Pad Prism 8.0. The results are shown in FIG. Figure 6 shown.

[0443] Example 2.7: Antitumor efficacy of ADC-01 in the Calu-6 mouse transplant tumor model

[0444] In order to demonstrate the in vivo efficacy of ADC-01, we used Calu-6 cells to inoculate CB17-SCID mice to determine the anti-tumor efficacy of the molecule of the present invention. The experiment used SPF grade female mice (14-17g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).

[0445] Calu-6 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1X) to prepare a cell concentration of 2.5x10 7 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB17-SCID mice to establish a Calu-6 tumor-bearing mouse model. On the 13th day after tumor cell inoculation, all mice were randomly divided into 5 groups (5 mice in each group). The dosage and method of administration are shown in Table 3. The mice were given drugs on the 13th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Fig. 7A ,7B As shown, monitoring ended after 38 days.

[0446] The relative tumor inhibition rate (TGI%) was calculated on the 38th day after inoculation, and the calculation formula was as follows:

[0447] TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before administration).

[0448] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula:

[0449] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0450] Table 3. Experimental design table

[0451] Group Dosage Dosing frequency Dosage Vehicle control N / A 1 vein Control IgG1-DXd 3mg / kg 1 vein Control IgG1-NT3 3mg / kg 1 vein M30-DXd 3mg / kg 1 vein ADC-01 3mg / kg 1 vein

[0452] The tumor inhibition rate results are shown in Table 4 and Fig. 7A As shown in Figure 2, the tumor inhibition rate of ADC-01 after a single administration reached 88.44% on the 38th day after inoculation, which was significantly better than the tumor inhibition rate of M30-DXd (69.49%). At the same time, we tested the weight of the mice, and the results were as follows: Figure 7B There were no significant differences in body weights of the mice shown.

[0453] Table 4. Tumor inhibition rate on day 38

[0454]

[0455] Example 2.8: Antitumor efficacy of ADC-01 in A375 mouse transplant tumor model

[0456] In order to demonstrate the in vivo efficacy of ADC-01, we used A375 cells (ATCC, CRL-1619) to inoculate CB17-SCID mice to determine the anti-tumor efficacy of the antibody molecule of the present invention. The experiment used SPF grade female mice (14-17g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).

[0457] A375 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1X) to prepare a cell concentration of 2.5x10 7On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB17-SCID mice to establish an A375 tumor-bearing mouse model. On day 19 after tumor cell inoculation, all mice were randomly divided into 5 groups (5 mice in each group). The dosage and method of administration are shown in Table 5. The mice were administered on day 19 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Fig. 8A , 8B As shown, monitoring ended after 44 days.

[0458] The relative tumor inhibition rate (TGI%) was calculated on the 44th day after inoculation, and the calculation formula was as follows:

[0459] TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before administration).

[0460] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula:

[0461] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0462] Table 5. Experimental design table

[0463] Group Dosage Dosing frequency Dosage Vehicle control N / A 1 vein Control IgG1-DXd 1mg / kg 1 vein Control IgG1-NT3 1mg / kg 1 vein M30-DXd 1mg / kg 1 vein ADC-01 1mg / kg 1 vein

[0464] The tumor inhibition rate results are shown in Table 6 and Fig. 8A As shown: The tumor inhibition rate of ADC-01 single administration reached 44 days after vaccination.

[0465] 103.53%, which is significantly better than the tumor inhibition rate of M30-DXd of 44.17%. At the same time, we tested the weight of mice. The results are as follows Figure 8B There were no significant differences in body weights of the mice shown.

[0466] Table 6 Tumor inhibition rate on day 44

[0467]

[0468]

[0469] Example 2.8: Antitumor efficacy of ADC-01 in the BXPC3 mouse transplant tumor model

[0470] In order to demonstrate the in vivo efficacy of ADC-01, we used BXPC3 cells (Cobioer, CBP60542) to inoculate CB17-SCID mice to determine the anti-tumor efficacy of the antibody molecule of the present invention. The experiment used SPF grade female mice (14-17g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).

[0471] BXPC3 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1X) to prepare a cell concentration of 2.5x10 7 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB17-SCID mice to establish a BXPC3 tumor-bearing mouse model. On the 13th day after tumor cell inoculation, all mice were randomly divided into 4 groups (5 mice in each group). The dosage and method of administration are shown in Table 7. The mice were given drugs on the 13th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Fig.9A , 9B As shown, monitoring ended after 45 days.

[0472] The relative tumor inhibition rate (TGI%) was calculated on the 45th day after inoculation, and the calculation formula was as follows:

[0473] TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before administration).

[0474] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula:

[0475] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0476] Table 9. Experimental design table (Note: administration once a week, 3 times in total)

[0477] Group Dosage Dosing frequency Dosage Vehicle control N / A 3 Abdominal cavity Control IgG1-DXd 10mg / kg 3 Abdominal cavity Control IgG1-NT3 10mg / kg 3 Abdominal cavity M30-DXd 10mg / kg 3 Abdominal cavity ADC-01 10mg / kg 3 Abdominal cavity

[0478] Table 8. Tumor inhibition rate on day 45

[0479]

[0480] The tumor inhibition rate results are shown in Table 8 and Fig.9A As shown in Figure 2, the tumor inhibition rate of ADC-01 reached 82.5% on the 45th day after inoculation, which was significantly better than the tumor inhibition rate of M30-DXd (30.98%). At the same time, we tested the weight of the mice. The results are as follows: Fig. 9B There were no significant differences in the body weights of the mice shown.

[0481]

[0482]

[0483]

[0484]

Claims

1. An antibody-drug conjugate having formula (I): Ab-(L-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, in: Ab is an antibody or a fragment thereof that binds to B7-H3 (eg, human B7-H3); L is a linker; D is a drug, preferably an anti-tumor compound; and p is the average drug-to-antibody ratio DAR, which is 1 to 16, such as 4-10, Wherein Ab in formula (I) comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 7 or 15, 8 and 9, respectively.

2. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein Ab in formula (I) comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or 13; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4 or 13; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4 or 13, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions; and / or Light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or 16; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 16; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10 or 16, consisting of said amino acid sequence, and preferably, said amino acid changes do not occur in the CDR regions.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 2, wherein Ab in formula (I) comprises VH and VL, wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; or wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

16.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein Ab in formula (I) further comprises a heavy chain constant region and / or a light chain constant region.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein Ab in formula (I) is an IgG antibody.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the heavy chain constant region of Ab in formula (I) is from IgG1 or IgG2 or IgG3 or IgG4, such as IgG1.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein Ab in formula (I) comprises a heavy chain and a light chain, wherein the heavy chain (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 14; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 14; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 6 or 14; and / or Light chain (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12 or 17; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 17; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or 17.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein Ab in formula (I) comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; or wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

17.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 8, wherein Ab in formula (I) is a chimeric antibody or a humanized antibody.

10. The antibody-drug conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab in formula (I) is an antigen-binding fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); a linear antibody; a single-chain antibody (such as scFv); a single-domain antibody such as VHH; a bivalent antibody or a fragment thereof; or a camelid antibody.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, wherein the anti-tumor compound is a cytotoxic agent, such as a camptothecin compound, an auristatin compound or a maytansine compound.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 11, wherein D has a structure represented by formula (D-1): Where R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2; R 4 Selected from H or C1-C4 alkyl.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein R 1 For H, R 2 is C1-C6 alkyl, R 3 is -F.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein D has a structure represented by formula (D-2): Where R 1 , R 2 and R 3 As defined in claim 12 or 13.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein D has a structure represented by formula (D-3) or formula (D-4):

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 15, wherein -L- has the following structure: -Z-E-NH-CH2-Q-L 2 -L 1 - Where Z is connected to Ab, L 1 Connect with D; Z is selected from Where m a1 and m a2 independently selected from integers from 0 to 20; m is an integer selected from 1 to 10, Preferably, Z is selected from Wherein m is an integer from 1 to 10; The carbonyl group at the right end of Z is covalently linked to E; E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted by one or more groups selected from C 1-6 an alkyl group and a polyol group, wherein the N-terminus of the peptide residue is covalently linked to Z; Q is -O- or -S-; L 1 is not present or -(C1-C 10 Alkylene)-; L 2 Does not exist, -N(R 5 )C(O)-(C1-C 10 Alkylene)-* or -C(O)N(R 5 )-(C1-C 10 Alkylene)-*; wherein * indicates that the terminus is covalently linked to Q; and R 5 It is H or C1-C6 alkyl.

17. The antibody-drug conjugate according to claim 16 or a pharmaceutically acceptable salt or solvate thereof, wherein E is a peptide residue consisting of 2, 3 or 4 amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine and proline, and wherein the glutamine or glutamic acid is optionally substituted by 1 polyol group and optionally by 1 C 1-6 alkyl substitution; preferably, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group; and -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10 Alkylene)-*, wherein * indicates that the terminus is covalently linked to Q; R 5 It is H or C1-C6 alkyl.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 17, wherein the substituted glutamine or glutamic acid has the structure shown below: Where R 6 is H or C1-C6 alkyl; Preferably Where R 6 It is H or C1-C6 alkyl.

19. The antibody-drug conjugate according to claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Al a-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val- , -Leu-Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, - Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala- Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys- -Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with 1 polyol group and optionally with 1 C 1-6 Alkyl substitution.

20. The antibody-drug conjugate according to claim 17 or a pharmaceutically acceptable salt or solvate thereof, in, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala- or Where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N-terminus; and -L 2 -L 1 - is -(C1-C6 alkylene)-.

21. The antibody-drug conjugate according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, Among them -ZE-NH-CH2-QL 2 -L 1 - has the following structure Its right end is connected to D.

22. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 21, wherein the antibody-drug conjugate of formula (I) has the following structure: Ab’-(S-L-D) p (I’) wherein Ab' is as defined in any one of claims 1-10 for Ab, and L, D and p are as defined in any one of claims 1-21.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 22, having an average DAR value of 5-11, such as 7.5-8.

5.

24. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from wherein Ab is as defined in any one of claims 1-10; q represents the average DAR, for example, 5-11 or 7.5-8.

5.

25. Antibody-drug conjugate of formula (II): Ab-(LD) n (II) or a pharmaceutically acceptable salt or solvate thereof, in: Ab, L and D are as defined in any one of claims 1 to 24; n represents the number of -LDs linked to Ab, and is an integer selected from 1 to 16, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

26. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 25 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

27. A pharmaceutical combination comprising the antibody-drug conjugate of any one of claims 1 to 25 or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

28. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, or the pharmaceutical composition of claim 26, or the pharmaceutical combination of claim 27.

29. The method of claim 28, wherein the tumor is a cancer, preferably the cancer has elevated levels (eg, nucleic acid or protein levels) of B7-H3, eg, compared to healthy individuals or healthy tissue adjacent to the patient's cancer tissue.

30. The method of claim 29, wherein the cancer is selected from lung cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, pancreatic cancer, or breast cancer.

31. The method of any one of claims 29-30, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modalities comprise radiation therapy or surgery, or the therapeutic agents comprise chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

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