B7-h3 antibody-drug conjugate and use thereof

Abstract

The present invention relates to a B7-H3 antibody-drug conjugate and use thereof, and specifically provides an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, a solvate, or a solvate of the salt. The antibody-drug conjugate has a structure represented by formula I, wherein Ab is an anti-B7-H3 antibody. The antibody-drug conjugate of the present invention has good tumor cell growth inhibition activity in vivo and in vitro and has a good application prospect. Ab-(L-D)p Formula I

Description

B7-H3 antibody-drug conjugates and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to CN application No. 202311401463.4, filed on October 25, 2023, and PCT application No. PCT / CN2024 / 126435, filed on October 22, 2024. The contents of the CN application and PCT application are hereby incorporated into this application as a whole. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a B7-H3 antibody-drug conjugate and applications thereof. Background Art

[0004] B7-H3 (also known as CD276) is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. Studies have shown that B7-H3 is overexpressed on the surface of various tumors, including melanoma, leukemia, breast cancer, and prostate cancer, and is closely associated with tumor growth, metastasis, recurrence, and poor prognosis. For example, B7-H3 expression is 33.0% in renal cell carcinoma, 91.8% in hepatocellular carcinoma, 58.0% in gastric cancer, and 88.3% in ovarian cancer. On the other hand, B7-H3 expression is restricted in normal tissues. Immunohistochemical (IHC) analysis of a large number of normal tissues revealed weak staining of B7-H3 only in a few tissues, such as the stomach, gallbladder, prostate, cervix, and basal membrane of the endometrium, with undetectable B7H3 expression in most tissues. This discrepancy in expression levels between tumor and normal tissues makes B7-H3 an attractive target.

[0005] Currently, the development of drugs targeting B7-H3 involves monoclonal antibodies, bispecific antibodies, ADCs, and CAR-T. Several ADCs targeting B7-H3 have entered clinical trials. ABBV-155 (AbbVie) consists of a monoclonal antibody conjugated to a BCL-XL inhibitor via a linker and is currently in Phase I / II trials (NCT03595059). MGC018 (MacroGenics) consists of a humanized B7-H3 antibody conjugated to a duocarmycin payload via a cleavable linker and is currently in Phase I / II trials (NCT03729596). DS7300a (Daiichi Sankyo) consists of a B7-H3-specific monoclonal antibody conjugated to a DNA topoisomerase I inhibitor and is currently in Phase I / II trials (NCT04145622). HS-20093 (Hansoh Pharmaceuticals) consists of a fully human anti-B7-H3 monoclonal antibody linked to a DNA topoisomerase I inhibitor via a cleavable linker and is currently in Phase I / II trials (NCT05276609). BAT8009 (Bio-Thera) consists of a recombinant humanized anti-B7-H3 antibody linked to a DNA topoisomerase I inhibitor via a cleavable linker and is currently in Phase I trials. The development of B7-H3 ADCs carrying different payloads can address diverse clinical needs, such as targeting drug-resistant and sensitive indications.

[0006] Summary of the Invention

[0007] The inventors of the present application prepared a new anti-B7-H3 antibody drug conjugate (ADC) through extensive experiments and creative work, and confirmed that it has good biological activity, thereby completing the present invention.

[0008] To this end, in the first aspect of the present invention, the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody drug conjugate has the structure shown in Formula I,

[0009] Ab-(LD) p

[0010] Formula I

[0011] in:

[0012] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region,

[0013] The heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 1,

[0014] The heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 2,

[0015] The heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 3,

[0016] The light chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 4,

[0017] the light chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 5;

[0018] the light chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 6;

[0019] L is the connector;

[0020] D is cytotoxin;

[0021] p is any number between 1 and 10 (e.g., 1, 1.5, 2, 2.5, 2.7, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, -7, 7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5 or 9.5-10).

[0022] In formula I, LD represents that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule; Ab-(LD) p represents p LD molecules coupled to Ab via covalent bonds.

[0023] In the present invention, the drug antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody drug conjugates described herein can be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody. "p is any value between 1-10", which means that p can be any integer selected from 1-10 (including endpoints 1 and 10) or any decimal selected from 1-10. At the same time, those skilled in the art will understand that even if the same preparation method is used, the DAR values ​​of antibody drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of no more than 0.5. For example, when p is 7.5, it means that its DAR value can fluctuate between 7.0 and 8.0.

[0024] The drug-antibody ratio (DAR) can be determined by conventional means such as mass spectrometry, ELISA assays, hydrophobic interaction chromatography (HIC), and HPLC. The quantitative distribution of ADCs in terms of p can also be determined. In some cases, separation, purification, and validation of homogenous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as HIC, reverse-phase HPLC, or electrophoresis.

[0025] In some embodiments, the linker structure before being connected to Ab is selected from: MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-formyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), MA-PEG4-VC-PAB-DMEA,

[0026] In some embodiments, the linker structure before being connected to Ab is selected from: MCC-AAQ, 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA,

[0027] Among them, the structural formulas of MCC, MCC-AAQ, MC-vc-PAB, and MA-PEG4-VC-PAB-DMEA are as follows:

[0028] In some embodiments, the cytotoxin is selected from the group consisting of: topoisomerase inhibitors, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, gemcitabine, maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, duocarmycin SA,

[0029] I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.

[0030] In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. Exemplary topoisomerase I inhibitors include camptothecin and its derivatives. Exemplary camptothecin derivatives include Exatecan, Belotecan, SN-38, Topotecan, Irinotecan (cpt-11), Deruxtecan, Dxd, etc.

[0031] Among them, the structural formulas of MMAE, duocarmycin SA, Dxd, and ixitecan are as follows:

[0032] In some embodiments, the cytotoxin is selected from the group consisting of Exatecan (abbreviated as Exa), Monomethyl auristatin E (MMAE), Duocarmycin SA,

[0033] In some embodiments, LD before being linked to Ab is selected from the group consisting of: MCC-AAQ-Exa, MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA,

[0034] MC-GGFG-Dxd (i.e.

[0035] The structural formulas of MCC-AAQ-Exa, MC-vc-PAB-MMAE, and MA-PEG4-VC-PAB-DMEA-Duocarmycin SA are as follows:

[0036] When the five LDs are covalently coupled to the Ab, the succinimide at the end of the LDs is coupled to the sulfhydryl groups in the antibody. For example, when MCC-AAQ-Exa is covalently coupled to the Ab, the structure of the resulting ADC is as follows:

[0037] In the ADC formed above, the antibody Ab is linked to the carbon atom of the succinimide at the end of LD via -S-. This -S- is not a thiol group introduced into the Ab, but rather a thiol group contained in the antibody itself after the antibody Ab is reduced to open the disulfide bond.

[0038] In some embodiments, the sequence of the heavy chain variable region CDR1 of the anti-B7-H3 antibody is shown in SEQ ID NO: 1, the sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2, the sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3, the sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4, the sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 5, and the sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 6.

[0039] In some embodiments, the heavy chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 7, and the light chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 8.

[0040] In some embodiments, the sequence of the heavy chain variable region of the anti-B7-H3 antibody is shown in SEQ ID NO: 7, and the sequence of the light chain variable region of the anti-B7-H3 antibody is shown in SEQ ID NO: 8.

[0041] In some embodiments, the heavy chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 9, and the light chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 10.

[0042] In some embodiments, the sequence of the heavy chain variable region of the anti-B7-H3 antibody is shown in SEQ ID NO: 9, and the sequence of the light chain variable region of the anti-B7-H3 antibody is shown in SEQ ID NO: 10.

[0043] In some embodiments, the heavy chain constant region of the anti-B7-H3 antibody is selected from human IgG, IgM, IgA, IgD, IgE constant regions, or mutants of the aforementioned constant regions. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0044] In some embodiments, the light chain constant region of the anti-B7-H3 antibody is selected from a human lambda constant region, a kappa constant region, or a mutant of the aforementioned constant regions.

[0045] In some embodiments, p is any number between 2-8 (eg, 2.7, 3.7, 3.9, 7.8, or 7.9).

[0046] In the second aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.

[0047] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0048] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

[0049] In some embodiments, the chemotherapy drug is, for example, doxorubicin (Adriamycin), cyclophosphamide, taxanes [such as paclitaxel (Taxol), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Felocell, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

[0050] In some embodiments, the immunotherapy drug is, for example, PD-1 monoclonal antibody (e.g., pembrolizumab, nivolumab), PD-L1 monoclonal antibody (e.g., atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclonal antibody (e.g., Ramucirumab, apatinib), HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), etc., or a combination thereof.

[0051] In some embodiments, the immunosuppressant is selected from the group consisting of: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Sule, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

[0052] In the third aspect of the present invention, the present invention provides the use of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating diseases associated with B7-H3.

[0053] In a fourth aspect, the present invention provides the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition, for use in preventing and / or treating diseases associated with B7-H3.

[0054] In a fifth aspect, the present invention provides a method for treating and / or preventing diseases associated with B7-H3, comprising administering to a subject in need thereof a therapeutically and / or preventatively effective amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned pharmaceutical composition.

[0055] In some embodiments, the disease associated with B7-H3 is selected from melanoma, leukemia, breast cancer, prostate cancer, kidney cancer, liver cancer, gastric cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), colon cancer, skin cancer, head and neck cancer, esophageal squamous cell carcinoma, pancreatic cancer, neuroblastoma, and bladder cancer.

[0056] In the sixth aspect of the present invention, the present invention provides an in vitro non-therapeutic method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt, or the aforementioned pharmaceutical composition, wherein the tumor is a tumor expressing B7-H3.

[0057] In some embodiments, the B7-H3 expressing tumor is selected from melanoma, leukemia, breast cancer, prostate cancer, kidney cancer, liver cancer, gastric cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), colon cancer, skin cancer, head and neck cancer, esophageal squamous cell carcinoma, pancreatic cancer, neuroblastoma, and bladder cancer. Beneficial effects

[0058] 1. The newly developed B7-H3-ADC of the present invention exhibits a strong cell-killing effect in various tumor cells (such as melanoma, non-small cell lung cancer, liver cancer, breast cancer, etc.).

[0059] 2. The newly developed B7-H3-ADC of the present invention has shown significant pharmacological effects in inhibiting tumor cell growth in various tumor models (such as melanoma, colon cancer, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 shows the binding activity of B7-H3 antibody to A375 cells;

[0061] FIG2 shows the endocytosis ability of B7-H3 antibody on A375 cells;

[0062] Figure 3 shows the hydrophobic interaction map of 72D1D11-vcMMAE;

[0063] Figure 4 shows the hydrophobic interaction map of 72D1D11-z3-vcMMAE;

[0064] Figure 5 shows the hydrophobic interaction map of hMG.Ab.02-vcMMAE;

[0065] Figure 6 shows the hydrophobic interaction map of 72D1D11-Duocarmycin;

[0066] Figure 7 shows the hydrophobic interaction map of hMG.Ab.02-Duocarmycin;

[0067] FIG8 shows a hydrophobic interaction map of 72D1D11-ZymeLink;

[0068] Figure 9 shows the hydrophobic interaction map of 72D1D11-mcc-AAQ-Exatecan;

[0069] Figure 10 shows the hydrophobic interaction map of 72D1D11-mcGGFG-Dxd;

[0070] Figures 11-17 are representative images of B7-H3 ADCs killing A375.S2, NCI-H1975, Huh-7, and MCF-7 cells;

[0071] FIG18 shows the in vivo anti-tumor activity of different B7-H3-ADCs in the human melanoma nude mouse A375.S2 CDX model;

[0072] FIG19 shows the effects of different B7-H3-ADCs on body weight in nude mice A375.S2 CDX model of human melanoma;

[0073] FIG20 shows the in vivo anti-tumor activity of different B7-H3-ADCs in the nude mouse RKO CDX model of human colon adenoma;

[0074] FIG21 shows the effects of different B7-H3-ADCs on body weight in the nude mouse RKO CDX model of human colon adenoma. DETAILED DESCRIPTION

[0075] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0076] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0077] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0078] In the present invention, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chains of antibodies can be divided into two types: κ and λ. Heavy chains can be divided into five types: μ, δ, γ, α or ε. Depending on the heavy chain, antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE. Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region

[0079] (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C LThe constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system. H and V L The V domain can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antibody binding site. The assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0080] In the present invention, "humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0081] In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, the mutations within the CDRs are typically no more than one or two.

[0082] In the present invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0083] In the present invention, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

[0084] In the present invention, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0085] In the present invention, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0086] In the present invention, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0087] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0088] In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0089] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0090] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0091] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992); Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be directly produced by recombinant host cells (see Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture medium. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0092] In the present invention, algorithms used to determine percentage sequence homology and sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used to determine percentage amino acid sequence homology of the present invention using, for example, parameters described in the literature or using default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).

[0093] In the present invention, the mutant of the amino acid sequence refers to a variant having a homology greater than 70% with the amino acid sequence, for example, greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

[0094] 97%, 98%, 99% of the sequences, for example, sequences with 3, 2, or 1 amino acid substitution, deletion, or addition. Preferably, the number of amino acids substituted, added, or deleted does not exceed 3. More preferably, the number of amino acids substituted, added, or deleted does not exceed 2. Most preferably, the number of amino acids substituted, added, or deleted does not exceed 1.

[0095] "Substitution" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions can be single, in which only one amino acid is substituted in the molecule, or multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Likewise, one amino acid can be substituted by multiple residues, in which case such variants include both substitutions and insertions. "Insertion" (or "addition") variants are variants in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. "Deletion" variants are variants in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0096] In certain embodiments, less than the theoretical maximum number of drug moieties are coupled to the antibody in the coupling reaction. Generally speaking, antibodies do not contain many free and reactive cysteine ​​thiol groups to which drug moieties can be attached; in fact, most cysteine ​​thiol groups in antibodies exist as disulfide bridges. In certain embodiments, antibodies can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups.

[0097] In the present invention, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group present in the conjugate provided by the present invention and a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. and (ii) salts formed between the basic functional groups present in the conjugates provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, and hydroiodides; inorganic acid salts, such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; arylsulfonates, such as benzenesulfonates and p-toluenesulfonates; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, and aspartate.

[0098] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic substance with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic substance with a suitable base to provide a pharmaceutically acceptable cation.

[0099] In the present invention, solvates refer to forms of the antibody-drug conjugates of the present invention that form complexes in solid or liquid form by coordination with solvent molecules. Hydrates are a specific form of solvates that have coordinated water molecules. In the present invention, hydrates are preferred solvates.

[0100] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art based on the present disclosure. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating suitable pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed.

[0101] For the purposes of this invention, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and formulations. These substances, in addition to the active ingredient, have been rationally evaluated for safety and are included in pharmaceutical preparations. In addition to imparting shape, acting as a carrier, and enhancing stability, pharmaceutical excipients also perform important functions such as solubilization, dissolution enhancement, and sustained-release control. They are crucial components that may impact the quality, safety, and efficacy of pharmaceuticals. Based on their source, they can be categorized as natural, semi-synthetic, or fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0102] In the present invention, the pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, and the like. The pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

[0103] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0104] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0105] In the present invention, an "effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect. The "therapeutically effective amount" of the substance / molecule of the present invention may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired preventive effect. Typically, but not necessarily, a prophylactic dose is used in subjects before the onset of disease or in the early stages of the disease, so the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0106] In the present invention, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0107] The present invention will be further explained below with reference to specific examples, but these examples are not intended to limit the scope of the present invention.

[0108] Sequence information:

[0109] Example 1 Preparation, Sequence and Characterization of B7-H3 Antibody

[0110] 1. Preparation of B7-H3 Antibody

[0111] To generate monoclonal antibodies against human B7-H3, Balb / c mice were immunized with human B7-H3 (4Ig) protein. Titered sera from mice were analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) to monitor immune responses. Sera were tested for binding to human B7-H3 protein, a B7-H3-overexpressing CHO-K1 cell line, and a negative control CHO-K1 cell line that did not express B7-H3. Animals with sufficiently high ELISA titers and FACS binding ratios of positive cell lines (B7-H3-overexpressing CHO-K1 cells) to negative cell lines (CHO-K1 expressing the vector alone) were screened for a final boost immunization with B7-H3 protein prior to hybridoma fusion. Spleens were isolated from mice in the final booster immunization and then fused with immortalized mouse myeloma cells via electric field electrofusion to generate hybridoma fusion cells. The fusion cells were cultured in 96-well microplates, and the binding strength and epitope analysis of the hybridoma supernatants were analyzed by ELISA and FACS. To ensure that the subclones were derived from a single parental cell, the primary clones positive for different epitopes in each fusion were subcloned by limiting dilution. The subclones were screened using the same method as the primary clones. Selected subclones were sequenced.

[0112] 2. Sequence of B7-H3 antibody

[0113] The sequence of the obtained B7-H3 antibody 72D1D11 is shown in Table 1 below.

[0114] Table 1: Sequence of B7-H3 antibody 72D1D11 (bold and underlined are antibody CDR sequences)

[0115] 3. Humanization of B7-H3 Antibody

[0116] To engineer the B7-H3 antibody for druggability, humanized antibodies were generated using the variable region sequences of the antibody 72D1D11. The amino acid sequences of 72D1D11-VH and 72D1D11-VL were compared to existing human Ig gene sequence databases to identify the best overall match to human germline Ig sequences. For 72D1D11-VL, IGKV2-28*01 was the most suitable framework sequence, while for 72D1D11-VH, IGHV7-4-1*02 was selected as the humanized framework. Humanized 72D1D11 CDR-grafted antibodies were then designed, with CDRs L1, L2, and L3 grafted onto the IGKV2-28*01 framework sequence and CDRs H1, H2, and H3 grafted onto the IGHV7-4-1*02 framework sequence. A three-dimensional model was then generated to identify amino acids in the native mouse framework regions that are critical for antibody binding and conformation. Based on the analysis results, the grafted antibody was reverse mutated to obtain the humanized antibody 72D1D11-z3. The sequence of the humanized antibody 72D1D11-z3 is shown in Table 2.

[0117] Table 2: Sequence of humanized B7-H3 antibody 72D1D11-z3 (antibody CDR sequences are bold and underlined)

[0118] 4. Characterization of B7-H3 Antibody

[0119] a. Cell binding of B7-H3 antibody

[0120] To evaluate the activity of the antibodies, the binding of the antibodies to A375 cells was tested. A total of 4 × 10 4 A375 cells were incubated with 3-fold serial dilutions of the antibody in FACS buffer at 4°C for 30 minutes. After washing with FACS buffer, a fluorescent dye-linked anti-human IgG antibody was added to each well and incubated at 4°C for 30 minutes. The samples were washed twice with FACS buffer. The geometric mean fluorescence intensity (MFI) was assessed using a MACSQuant Analyzer 16. As shown in Figure 1, 72D1D11 showed stronger cell binding activity than the control antibody hMG.Ab.02. Among them, hMG.Ab.02 is the code name of the B7H3 monoclonal antibody portion of the clinical competitor MGC018, reference (DOI:10.1158 / 1535-7163.MCT-20-0116) and patent WO2017180813A1. The experimental results showed that the humanized antibody 72D1D11-z3 basically maintained the cell binding activity of the mouse antibody 72D1D11. Compared with hMG.Ab.02, both the humanized antibody 72D1D11-z3 and the mouse antibody showed stronger binding ability.

[0121] b. Internalization of B7-H3 Antibody

[0122] To test the internalization ability of the antibody, the endocytic potential of 72D1D11 was tested on A375 cells. pHAb thiol dye is a pH sensor that is sensitive to pH. Its fluorescence is very low at pH>7. When it is internalized into endosomes or lysosomes with a pH of about 6.3 or 4.7, the fluorescence increases significantly. α-hIgG secondary antibody (10nM) labeled with pHAb thiol dye was incubated with 5nM serially diluted antibodies for 30 minutes. The mixture was then added to a 2×10 4 A375 cells were cultured in a 96-well assay plate. After 48 hours of culture, the cells were analyzed using a multimode plate reader ( Fluorescence signals were collected using an ELISA kit (2105) or Operetta. As shown in Figure 2, 72D1D11 exhibited improved endocytosis potential compared to hMG.Ab.02. The EC50 values ​​and maximum endocytosis values ​​(Top) for B7-H3 antibody endocytosis in A375 cells are shown in Table 3.

[0123] Table 3: EC50 and Top values ​​of B7-H3 antibody internalization on A375 cells

[0124] Example 2 Preparation of Linker-Payload

[0125] Preparation of mcc-AAQ-Exatecan

[0126] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0127] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 ml) and DIEA (29 mg 1.5 eq.). TBTU (72.5 mg 1.1 eq.) was added and the reaction was allowed to proceed at room temperature. HPLC monitoring confirmed no residual starting material. After completion of the reaction, the product was purified by medium-pressure column chromatography (DCM / MeOH). The product was collected and concentrated to dryness to obtain Intermediate 1.

[0128] The reaction formula is as follows:

[0129] 2. Synthesis of Intermediate 2 Gln-Exatecan

[0130] Intermediate 1, Fmoc-Gln-Exatecan (130 mg, 1.0 eq.), was added to a reaction flask, followed by DCM (2 ml) and stirred at room temperature. DEA (0.5 ml) was added and the mixture was allowed to react at room temperature. HPLC monitoring confirmed no residual starting material. The reaction was stopped and MTBE (10 ml) was slowly added. A large amount of solid precipitated and the mixture was stirred for 30 min. Filtering and washing with MTBE afforded an off-white solid, which was then dried to yield Intermediate 2.

[0131] The reaction formula is as follows:

[0132] 3. Synthesis of Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan

[0133] Intermediate II, Gln-Exatecan (100 mg, 1.0 eq.), was added to a reaction flask along with DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg, 1.0 eq.), TBTU (68.4 mg, 1.2 eq.), and DIEA (34.4 mg, 1.5 eq.). The reaction was allowed to react at room temperature and monitored by HPLC until completion. The product was purified by column chromatography (DCM / MeOH) at medium pressure to obtain Intermediate III.

[0134] The reaction formula is as follows:

[0135] 4. Synthesis of Intermediate 4: Ala-Ala-Gln-Exatecan

[0136] Intermediate 3, Fmoc-Ala-Ala-Gln-Exatecan (140 mg, 1.0 eq.), was added to a reaction flask, followed by DCM (2 ml) and stirred at room temperature. DEA (0.5 ml) was added and the reaction was allowed to react at room temperature. HPLC monitoring was performed until the reaction was complete. MTBE (10 ml) was slowly added to precipitate a large amount of solid. The mixture was stirred for 30 min. Filtering and washing with MTBE afforded an off-white solid, which was then dried to yield Intermediate 4.

[0137] The reaction formula is as follows:

[0138] 5. Product Synthesis MCC-Ala-Ala-Gln-Exatecan

[0139] Intermediate 4, Ala-Ala-Gln-Exatecan (50 mg, 1.0 eq.), was added to a reaction flask with DMF (1 mL), followed by MCC (18 mg, 1.1 eq.) and DIEA (12.1 mg, 1.5 eq.). The reaction was allowed to proceed at room temperature and monitored by HPLC until no residue of intermediate 4 remained. The product was then collected and concentrated to dryness to yield the product.

[0140] The reaction formula is as follows:

[0141] 1 H NMR (400MHz, DMSO-d6) δ8.17-8.26 (d, J=2.0Hz, 1H), 7.83-7.91 (d, J=2.0Hz, 2H), 7.65-7.74 (d, J=2.0Hz, 2H), 7.21-7.28 (d, J= 8.0Hz,2H), 6.98-7.04(m,2H), 5.43-5.50(m,1H), 5.36-5.43(m,2H), 5.15-5.24(m,1H), 5.01-5.11(m,1H), 4.14-4.24(m,1H), 3.97-4.08(m,1H), 3.47-3.57(m,1H), 3.20-3.27(m,1H), 3.05-3.16(m,2H), 2.30-2.36(m,2H), 2.00-2.15(m,4H), 1.77-1.92(m,3H), 1.65-2.00(m,5H), 1.45-1.64(m,4H), 1.09-1.24(m,5H), 0.95-1.07(m,4H), 0.79-0.94(m,5H).

[0142] Example 3 Preparation of B7-H3-ADC

[0143] 1. Preparation of Antibody Drug Conjugate Anti-B7-H3-vcMMAE

[0144] a. Take 10 mg of B7-H3 antibody (72D1D11, 72D1D11-z3, or hMG.Ab.02) and adjust the pH to 7.5 with 0.7 M Tris-0.5 M EDTA solution. Weigh the antibody, determine the protein concentration, and calculate the total protein. Add 2.8 times the molar ratio of TCEP to the antibody. Incubate on a 3D shaker at room temperature for 105 minutes, mixing continuously.

[0145] b. Add vcMMAE (MC-vc-PAB-MMAE, dissolved in DMSO) (produced by Shanghai Meiyake Biotechnology Co., Ltd. and commissioned by Hubei Huashitong Biopharmaceutical Technology Co., Ltd.) to the reduced antibody at a concentration of 1.6 times the molar number of free thiol groups. Mix thoroughly, then incubate on a 3D shaker at room temperature for 20 minutes with continuous mixing. Add N-acetylcysteine ​​at a concentration of 3 times the molar number of MC-vc-PAB-MMAE added to the reaction mixture, mix thoroughly, and incubate on a 3D shaker at room temperature for 5 minutes with continuous mixing. Use a 15 mL 30KD ultrafiltration device to replace the conjugate storage solution (10 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.6) for a total of 5 replacements, and then filter with a 0.22 μm filter membrane to obtain the antibody drug conjugate product Anti-B7-H3-vcMMAE (collectively referred to as 72D1D11-vcMMAE, 72D1D11-z3-vcMMAE and hMG.Ab.02-vcMMAE) and store at 4°C.

[0146] c. The protein concentration of the antibody-drug conjugate was determined by UV / BCA, the DAR was determined by HIC (as shown in Figures 3-5), and the purity was determined by SEC. The DAR values ​​were 3.9, 3.7, and 4.0, respectively.

[0147] 2. Preparation of Antibody-Drug Conjugate Anti-B7-H3-Duocarmycin

[0148] a. Take 2 mg of B7-H3 antibody (72D1D11 or hMG.Ab.02) and adjust the pH to 7.5 with 0.7 M Tris-0.5 M EDTA solution. Weigh the antibody, determine the protein concentration, and calculate the total protein. Add 2.0 times the molar ratio of TCEP to the antibody. Incubate on a 3D shaker at room temperature for 105 minutes, mixing continuously.

[0149] b. Add duocarmycin (MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, purchased from Lianning (Suzhou) Biopharmaceutical Co., Ltd., dissolved in DMSO) to the reduced antibody at a concentration twice the molar number of free thiol groups. Mix thoroughly, then incubate on a 3D shaker at room temperature for 20 minutes with continuous mixing. Add N-acetylcysteine ​​at a concentration three times the molar number of MA-PEG4-VC-PAB-DMEA-Duocarmycin SA to the reaction mixture, mix thoroughly, and incubate on a 3D shaker at room temperature for 5 minutes with continuous mixing. Use a 0.5 mL 30KD ultrafiltration device to replace the conjugate into the storage solution (10 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.6) for a total of 5 replacements, and then filter with a 0.22 μm filter membrane to obtain the antibody drug conjugate product Anti-B7-H3-Duocarmycin (collectively referred to as 72D1D11-Duocarmycin and hMG.Ab.02-Duocarmycin) and store at 4°C.

[0150] c. The protein concentration of the antibody-drug conjugate was determined by UV / BCA, the DAR was determined by HIC (as shown in Figures 6-7), and the purity was determined by SEC. The DAR values ​​were 2.7 and 1.7, respectively.

[0151] 3. Preparation of Antibody-Drug Conjugate Anti-B7-H3-ZLA

[0152] a. Take 10 mg of B7-H3 antibody (e.g., 72D1D11) and exchange the antibody solution into 40 mM PB + 2 mM EDTA (pH 7.0). Dilute the antibody to 5 mg / mL in 40 mM PB + 2 mM EDTA (pH 6.99). Weigh the sample, measure the protein concentration, and calculate the total protein. Add 2.5 times the molar ratio of TCEP to the antibody, mix the sample, and incubate in an incubator at room temperature for 3 hours with continuous mixing at 200 rpm.

[0153] b. Cool the reduced antibody solution on ice. Add 7 times the molar amount of ZLA (dissolved in DMA, with the total DMA content being 10% of the reaction system) to the cooled antibody solution. Mix rapidly until thoroughly mixed. Incubate the solution in a constant temperature incubator at 4°C for 2 hours with continuous mixing at 200 rpm. Add 6 times the molar amount of N-acetylcysteine ​​to the reaction solution, mix thoroughly, and incubate the solution in a constant temperature incubator at 22°C for 15 minutes with continuous mixing at 200 rpm. Use a 15 mL 50KD ultrafiltration tube to transfer the solution into the conjugate stock solution (20 mM histidine / histidine-HCl, pH 5.41). Sterile filter the solution through a 0.22 μm filter and measure the protein concentration. The antibody-drug conjugate product 72D1D11-ZLA (or 72D1D11-ZymeLink) was obtained and stored at 4°C.

[0154] c. The protein concentration of the antibody-drug conjugate was determined by UV / BCA, the DAR was determined by HIC (as shown in Figure 8), and the purity was determined by SEC. The DAR value was 3.9.

[0155] ZLA structure

[0156] 4. Preparation of Antibody Drug Conjugate Anti-B7-H3-mcc-AAQ-Exatecan

[0157] a. Dilute 2 mg of 72D1D11 antibody with PBS buffer to a concentration of 5 mg / mL. Adjust the antibody solution to the reaction pH with Tris-EDTA solution. Measure the protein concentration using a Nanodrop and weigh the antibody solution to calculate the total protein content. Add TCEP solution to the antibody solution according to the designed TCEP / mAb ratio. Place the solution on a 3D shaker and mix continuously at room temperature until the reduction reaction is complete.

[0158] b. Add an excess of mcc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and continue mixing on a 3D shaker at room temperature until the coupling reaction is complete. Then, add an excess of N-acetylcysteine ​​solution to the reaction mixture and continue mixing on a 3D shaker at room temperature.

[0159] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate 72D1D11-mcc-AAQ-Exatecan, which was stored at 4°C.

[0160] d. The protein concentration of the antibody-drug conjugate was determined by UV / BCA, the DAR was determined by HIC (as shown in Figure 9), and the purity was determined by SEC. The DAR value was 7.8.

[0161] 5. Preparation of Antibody Drug Conjugate Anti-B7-H3-mcGGFG-Dxd

[0162] a. Dilute 2 mg of 72D1D11 antibody with PBS buffer to a concentration of 5 mg / mL. Adjust the antibody solution to the reaction pH with Tris-EDTA solution. Measure the protein concentration using a Nanodrop and weigh the antibody solution to calculate the total protein content. Add TCEP solution to the antibody solution according to the designed TCEP / mAb ratio. Place the solution on a 3D shaker and mix continuously at room temperature until the reduction reaction is complete.

[0163] b. Add an excess of mcGGFG-Dxd solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and continue mixing on a 3D shaker at room temperature until the coupling reaction is complete. Then, add an excess of N-acetylcysteine ​​solution to the reaction mixture and continue mixing on a 3D shaker at room temperature.

[0164] c. Purify the conjugate using a 15 mL 30KD ultrafiltration centrifuge tube and replace the product with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate 72D1D11-mcGGFG-Dxd, which was stored at 4°C.

[0165] d. The protein concentration of the antibody-drug conjugate was determined by UV / BCA, the DAR was determined by HIC (as shown in Figure 10), and the purity was determined by SEC. The DAR was 7.9.

[0166] Example 4 In vitro pharmacodynamic study

[0167] The cell killing activities of ADCs targeting B7-H3 conjugated with various linker-payloads and different DAR values ​​were compared.

[0168] Tumor cells in logarithmic growth phase were collected, centrifuged, resuspended in new culture medium, and counted. The cells were inoculated into 96-well black cell culture plates (Costar) and cultured overnight in a cell culture incubator. The next day, the ADC was diluted 4-fold with culture medium and the dilutions were carefully transferred to the black culture plates. The starting and ending concentrations of the samples were 10,000 ng / mL. After 4 to 6 days of culture in a cell culture incubator, 1 / 10 of the well volume of PrestoBlue reagent (Invitrogen) was added and incubated in a cell culture incubator for 1 hour. The fluorescence signal was read using a SpectraMax M5 plate reader, and the excitation and emission wavelengths of the instrument were set to 560 nm and 590 nm, respectively. The obtained fluorescence signal data were analyzed using SoftMax Pro 6.5 software.

[0169] 1. Experimental reagents and sources:

[0170] Test drug

[0171] Among them, Non-binding means that the antibody is a non-binding human IgG1 isotype control, which does not bind to the target on the surface of the tumor cells used in the experiment and is used as a negative control antibody.

[0172] 2. Experimental results:

[0173] EC cell killing activity of B7-H3 ADCs 50 The average values ​​are shown in Tables 4 to 6. Figures 11 to 17 are representative images of the killing of A375.S2, NCI-H1975, Huh-7, and MCF-7 cells by B7-H3 ADCs.

[0174] Table 4: EC values ​​of B7-H3 ADCs for cytotoxicity in A375.S2 cells 50 value

[0175] Table 5: EC values ​​of B7-H3 ADCs for cytotoxicity in NCI-H1975 cells 50 value

[0176] Table 6: EC values ​​of B7-H3 ADCs for cytotoxicity in Huh-7 and MCF-7 cells 50 value

[0177] The results in Tables 4-6 and Figures 11-17 show that the B7-H3 ADCs of the present invention exhibit specific cell killing activity in A375.S2, NCI-H1975, Huh-7 or MCF-7 tumor cells.

[0178] Example 5 In vivo pharmacodynamic study

[0179] The in vivo antitumor activity of B7-H3-ADC was tested in A375.S2 and RKO CDX mouse models.

[0180] 1. The A375.S2 CDX model was confirmed to be positive for B7-H3 by FACS analysis. The A375.S2 CDX model of human melanoma in nude mice was established by expanding and harvesting A375.S2 cells in the logarithmic growth phase and resuspending them in DMEM basal medium supplemented with 50% Matrigel. Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously on the right side of the back of the mouse. When the average tumor volume reached 120–250 mm, the mouse was inoculated with 0.1 mL of the cell suspension. 3 Around 4:00 p.m., animals were randomly divided into groups according to tumor volume, with the grouping date designated as Day 0. There were 8 mice in each group, with a total of 3 groups, including the Vehicle group (ADC solvent), the Non-binding-vcMMAE administration group (3 mg / kg), and the 72D1D11-vcMMAE administration group (3 mg / kg). The entire experiment was administered twice, once a week, via tail vein injection. The "Non-binding" designation indicates that the antibody is a non-binding human IgG1 isotype control that does not bind to the target on the surface of the tumor cells used in the experiment and serves as a negative control antibody.

[0181] Data analysis: During the experiment, tumor volume was measured twice a week. The calculation formula of tumor volume (TV) is: TV = l × w 2 / 2. Where l and w represent the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. Where V0 is the tumor volume measured at the time of group administration (i.e. Day 0), V f The tumor volume was measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. When T / C (%) ≤ 40% and P < 0.05, the test article was considered to have a significant inhibitory effect on tumor growth.

[0182] The experimental results are shown in Figures 18 and 19. On day 28, the relative tumor proliferation rate (T / C%) of the non-binding-vcMMAE (3 mg / kg) group was 84.38% (P>0.05), and the tumor growth inhibition rate (TGI%) was 15.62%. The relative tumor proliferation rate (T / C%) of the 72D1D11-vcMMAE (3 mg / kg) group was 3.58% (P<0.05), and the tumor growth inhibition rate (TGI%) was 96.42%.

[0183] The results of this experiment showed that for the human melanoma A375.S2 CDX model, 72D1D11-vcMMAE (3 mg / kg) could significantly inhibit tumor growth, while Non-binding-vcMMAE (3 mg / kg) had no significant inhibitory effect on the tumor.

[0184] 2. FACS analysis confirmed the positive expression of B7-H3 in the RKO CDX model. The process of establishing the RKO CDX model of human colon adenoma in nude mice is as follows: RKO cells in the logarithmic growth phase were cultured and collected, and resuspended in DMEM basal medium containing 50% Matrigel. Under sterile conditions, 0.1 mL of cell suspension was inoculated subcutaneously on the right side of the back of the mouse. When the average tumor volume reached 120-250 mm 3 Around 4:00 p.m., animals were randomly divided into groups based on tumor volume, with grouping date designated as Day 0. Three groups consisted of seven mice per group: the Vehicle group (ADC vehicle), the hMG.Ab.02-vcMMAE group (3 mg / kg), and the 72D1D11-vcMMAE group (3 mg / kg). The drug was administered four times, once weekly, via tail vein injection throughout the experiment.

[0185] Data analysis: During the experiment, tumor volume was measured twice a week. The calculation formula of tumor volume (TV) is: TV = l × w 2 / 2. Where l and w represent the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. V0 is the tumor volume measured at the time of group administration (i.e. Day 0), V f The tumor volume was measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. When T / C (%) ≤ 40% and P < 0.05, the test article was considered to have a significant inhibitory effect on tumor growth.

[0186] The experimental results showed that on the 18th day, the relative tumor proliferation rate T / C (%) of the hMG.Ab.02-vcMMAE (3 mg / kg)-treated group was 33.94% (P<0.05), and the tumor growth inhibition rate TGI% was 66.06%; the relative tumor proliferation rate T / C (%) of the 72D1D11-vcMMAE (3 mg / kg)-treated group was 19.21% (P<0.05), and the tumor growth inhibition rate TGI% was 80.79%.

[0187] The results of this experiment are shown in Figures 20 and 21. In the human colon adenoma CDX model RKO, 72D1D11-vcMMAE (3 mg / kg) significantly inhibited tumor growth and was significantly better than hMG.Ab.02-vcMMAE (3 mg / kg).

Claims

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate has a structure as shown in formula I, Ab-(LD) p Formula I in: Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 1, The heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 2, The heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 3, The light chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 4, The light chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 5, The light chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 6; L is the connector; D is cytotoxin; p is any value between 1 and 10.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The linker structure before connection with Ab is selected from: MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), MA-PEG4-VC-PAB-DMEA, Preferably, the linker structure before being connected to Ab is selected from: MCC-AAQ, 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA, 3. The antibody drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The cytotoxin is selected from: Topoisomerase inhibitors, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, Gemcitabine, Maytansine alkaloids (such as Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (such as duocarmycin), doxorubicin, ricin, diphtheria toxin, Duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines, and nanoparticles; Preferably, the cytotoxin is selected from: Exatecan, Monomethyl auristatin E (MMAE), Duocarmycin SA, 4. The antibody drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The LD before being linked to Ab was selected from: MCC-AAQ-Exa, MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, 5. The antibody drug conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The anti-B7-H3 antibody has a technical feature selected from the following (i)-(ii): (i) the heavy chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 7, and the light chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 8; (ii) the heavy chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO: 9, and the light chain variable region of the anti-B7-H3 antibody comprises the sequence shown in SEQ ID NO:

10.

6. The antibody drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The anti-B7-H3 antibody has one or more technical features selected from the following (i)-(ii): (i) the heavy chain constant region of the anti-B7-H3 antibody is selected from human IgG, IgM, IgA, IgD, IgE constant regions or mutants of the above constant regions; Preferably, the IgG is selected from IgG1, IgG2, IgG3 and IgG4; (ii) The light chain constant region of the anti-B7-H3 antibody is selected from a human lambda constant region, a kappa constant region or a mutant of the above constant regions.

7. The antibody drug conjugate according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is any number between 2 and 8 (eg, 2.7, 3.7, 3.9, 7.8 or 7.9).

8. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof; Optionally, the pharmaceutical composition further comprises at least one pharmaceutical excipient; Alternatively, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug and an immunosuppressant for treating tumors.

9. Use of the antibody-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating a disease associated with B7-H3; Preferably, the disease associated with B7-H3 is selected from melanoma, leukemia, breast cancer, prostate cancer, kidney cancer, liver cancer, gastric cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), colon cancer, skin cancer, head and neck cancer, esophageal squamous cell carcinoma, pancreatic cancer, neuroblastoma, and bladder cancer.

10. An in vitro non-therapeutic method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with the antibody-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the pharmaceutical composition according to claim 8, wherein the tumor is a tumor expressing B7-H3; Preferably, the B7-H3 expressing tumor is selected from melanoma, leukemia, breast cancer, prostate cancer, kidney cancer, liver cancer, gastric cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), colon cancer, skin cancer, head and neck cancer, esophageal squamous cell carcinoma, pancreatic cancer, neuroblastoma, and bladder cancer.

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