Anti-B7H3 and PD-L1 bispecific antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention discloses a bispecific antibody drug conjugate as well as a preparation method and application thereof, and the structure of the bispecific antibody drug conjugate comprises the following fragments: an anti-B7H3 and anti-PD-L1 bispecific antibody or an antigen binding fragment of the anti-B7H3 and anti-PD-L1 bispecific antibody, a linker unit L and a cytotoxic drug. The bispecific antibody drug conjugate provided by the invention has good endocytosis effect, proliferation inhibition activity and tumor growth inhibition activity.

Description

Anti-B7H3 and PD-L1 bispecific antibody drug conjugate and its preparation method and use

[0001] This application claims priority to Chinese Patent Application No. 2022116919701 filed on December 27, 2022, and Chinese Patent Application No. 2023116964970 filed on December 11, 2023. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention provides a bispecific antibody-drug conjugate against B7H3 and PD-L1, a preparation method and use thereof, and a pharmaceutical composition comprising the same. Background Art

[0003] The B7-CD28 family, as co-stimulatory signals for T lymphocyte activation, plays a crucial role in T lymphocyte-involved immune responses. Studies have shown that different B7 molecule types have positive or negative regulatory effects on immune cell responses. B7H3 (also known as CD276), a member of the B7 family, is primarily expressed on the surface of tumor cells. Chapoval AI et al. first discovered that it has a co-stimulatory effect on CD4+ and CD8+ T cells. B7H3 signaling induces cellular immunity and selectively enhances interferon-γ (IFN-γ) production under T cell receptor signaling. However, as research on B7H3 continues, inhibitory functions of B7H3 have been gradually discovered. For example, it can inhibit the proliferation of CD4+ and CD8+ T cells. Furthermore, studies have shown that abnormal B7H3 expression is associated with the development, progression, and metastasis of various cancers, and there is substantial evidence that its high expression is associated with a poor prognosis in various malignancies.

[0004] B7H3 is highly expressed in all tested cancer types with limited heterogeneity and is rarely expressed in normal tissues. This suggests that B7H3 can be considered a tumor antigen (TA), providing the possibility of targeted therapy for tumor cells with high B7H3 expression. Currently, B7H3 targeted therapy strategies mainly include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug conjugates (ADCs), monoclonal antibodies that mediate cytotoxicity, and bispecific antibodies (BsAbs).

[0005] Inhibitors targeting the immune checkpoint PD1 / PD-L1 are undoubtedly a focus of tumor immunotherapy. PD-L1 is expressed on the surface of tumor cells, and cytotoxic PD-L1 inhibitory antibodies theoretically have a better anti-tumor effect. However, among the marketed PD-L1 monoclonal antibodies, only avelumab has been reported to mediate ADCC against tumors and exhibit a safety profile comparable to other PD-L1 antibodies. A key reason for this is that the effect of ADCC depends primarily on the abundance of antigen expression, while PD-L1 cannot be considered a typical tumor antigen and exhibits significant heterogeneity within tumor cells. PD1 / PD-L1 pathway-blocking antibodies are often used in combination with cytotoxic antibodies to enhance the efficacy of immunotherapy.

[0006] Antibody drug conjugates (ADCs) consist of three parts: an antibody or its antigen-binding fragment (target), a linker, and a small molecule drug. The antibody or its antigen-binding fragment is conjugated to a small molecule drug with biological activity, such as cytotoxicity, such as a cytotoxin, via a cleavable or non-cleavable linker. This fully utilizes the specificity of the antibody or its antigen-binding fragment targeting the cells of interest (target cells) or the specificity of binding to highly expressed antigens and the high efficiency of small molecule drugs, thereby reducing or avoiding toxic side effects on non-target cells. This means that compared with traditional tumor chemotherapy drugs, antibody drug conjugates for tumors can accurately target tumor cells and reduce the impact on non-tumor cells.

[0007] Among the antibody-drug conjugates targeting B7H3, MacroGenics' MGC018 and Daiichi Sankyo's DS7300 have made the most rapid progress, and both drugs are currently in Phase II clinical trials. MGC018 conjugates the DNA alkylating agent duocarmycin to a humanized B7H3 antibody via a cleavable linker (1inker). Early clinical trials have shown that MGC018 has demonstrated preliminary anti-tumor activity and manageable toxicity in patients with advanced metastatic castration-resistant prostate cancer (mCRPC) and melanoma. DS7300 conjugates an irinotecan derivative (deruxtecan) to a B7H3 antibody via a cleavable linker (1inker). Phase I clinical trials have shown anti-tumor activity in a variety of tumors, including mCRPC, small cell lung cancer, squamous cell lung carcinoma, esophageal squamous cell carcinoma, and endometrial cancer, with a good safety profile.

[0008] Currently, bispecific antibody-drug conjugates (ADCs) are in the early clinical stages, and no drugs have been approved for marketing. There is still a need in this field for bispecific antibody-drug conjugates with synergistic mechanisms, improved tumor selectivity, and excellent stability.

[0009] Summary of the Invention

[0010] The technical problem to be solved by the present invention is to overcome the drawbacks of the prior art of bispecific antibody-drug conjugates, thereby providing a bispecific antibody-drug conjugate, its preparation method, and application. The bispecific antibody-drug conjugate of the present invention has excellent endocytosis effect, proliferation inhibition activity, and tumor growth inhibition activity.

[0011] The present invention mainly solves the above technical problems through the following technical means.

[0012] In one aspect, the present application provides a bispecific antibody-drug conjugate, the structure of which comprises the following fragments: a bispecific antibody against B7H3 and PD-L1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug, wherein the bispecific antibody or antigen-binding fragment thereof comprises:

[0013] A monoclonal antibody unit, which is directed against PD-L1 and includes 2 heavy chains and 2 light chains, a nanobody unit, which is directed against B7H3 and includes 2 identical nanobodies,

[0014] The N-termini of the two nanobodies are respectively connected to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit through connecting peptides.

[0015] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein:

[0016] The light chain variable region of the monoclonal antibody unit includes a CDR1 with an amino acid sequence of SEQ ID NO.: 1, a CDR2 with an amino acid sequence of SEQ ID NO.: 2, and a CDR3 with an amino acid sequence of SEQ ID NO.: 3, the heavy chain variable region of the monoclonal antibody unit includes a CDR1 with an amino acid sequence of SEQ ID NO.: 5, a CDR2 with an amino acid sequence of SEQ ID NO.: 6, and a CDR3 with an amino acid sequence of SEQ ID NO.: 7, and the nanobody includes a CDR1 with an amino acid sequence of SEQ ID NO.: 12, a CDR2 with an amino acid sequence of SEQ ID NO.: 13, and a CDR3 with an amino acid sequence of SEQ ID NO.: 14.

[0017] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein:

[0018] The light chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 4, the heavy chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 8; and the nanobody comprises an amino acid sequence such as SEQ ID NO.: 15.

[0019] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein:

[0020] The monoclonal antibody comprises an immunoglobulin constant region, which is a human IgG constant region, such as a human IgG1 constant region.

[0021] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein:

[0022] The light chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 10, and the Nanobody comprises the amino acid sequence of SEQ ID NO.: 15; or

[0023] The full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO.: 15.

[0024] In some embodiments, in the bispecific antibody-drug conjugate of the present invention, the connecting peptide is a polypeptide comprising glycine and serine and having certain elasticity and protease resistance. Preferably, the amino acid sequence of the connecting peptide is SEQ ID No.: 11.

[0025] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 9.

[0026] In some embodiments, the CDR amino acid sequences may be at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequences. In some embodiments, the variable region amino acid sequences may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequences.

[0027] The Fc regions of the bispecific antibodies of the present invention can be human Fc regions. The Fc regions of the bispecific antibodies of the present invention can be of any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc regions are all of the IgG1 isotype. In some embodiments, the Fc regions are all of the IgG4 isotype.

[0028] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0029] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0030] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the bispecific antibody is DSYE001.

[0031] In some embodiments, in the bispecific drug conjugates, compositions, uses or methods described herein, the bispecific antibody comprises a monoclonal antibody unit and a Nanobody unit, the CDR sequences of the monoclonal antibody unit comprise LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and comprise HCDR 1, HCDR 2 and HCDR 3 with amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; and the CDR sequences of the Nanobody unit comprise HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0032] In some embodiments, in the bispecific drug conjugates, compositions, uses or methods described herein, the light chain variable region of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 4, the heavy chain variable region of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 8; and the Nanobody comprises the amino acid sequence of SEQ ID NO.: 15.

[0033] In some embodiments, in the bispecific drug conjugate, composition, use or method described in the present invention, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO.: 15.

[0034] In some embodiments, in the bispecific drug conjugate, composition or use described in the present invention, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 9.

[0035] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the cytotoxic drug is a structure represented by formula (A-1), its tautomers, enantiomers, diastereomers, or mixtures of isomers, or a pharmaceutically acceptable salt or solvate thereof,

[0036] in,

[0037] M is -L 2 -L 1 -C(O)-;

[0038] L 2 is -O- or -S-, and L 2 Connected to the connector unit L;

[0039] L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a replace;

[0040] m is 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3;

[0041] R 1a and R 1b Each is independently hydrogen, halogen, hydroxy, amino or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens;

[0042] R 2a is halogen, hydroxy, amino or C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted by one or more halogens.

[0043] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 2 It is -O-.

[0044] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is hydrogen, halogen or C1-C6 alkyl; R 1b is hydrogen, halogen or C1-C6 alkyl.

[0045] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R 1b It is hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C3 alkyl.

[0046] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a -CH3; R 1b It is hydrogen or -CH3.

[0047] In some embodiments, in the bispecific antibody drug conjugate of the present invention, m is 1 or 2.

[0048] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 for

[0049] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 is a C3-C6 saturated cycloalkyl or a 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a Replacement, R 2a Each is independently halogen or C1-C6 alkyl.

[0050] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group is optionally substituted by one or more R 2a Replacement, R 2a Each is independently halogen or C1-C6 alkyl.

[0051] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 is optionally replaced by one or more R 2a Substituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, R 2a Each is independently halogen or C1-C6 alkyl.

[0052] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein L 1 for

[0053] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein M is

[0054] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein the structure shown in formula (A-1),

[0055] M is -L 2 -L 1 -C(O)-;

[0056] L 2 is -O-;

[0057] L 1 -(C(R 1a )(R 1b )) m -CH2- or C3-C6 saturated cycloalkylene, the C3-C6 saturated cycloalkylene is optionally substituted by one or more R 2a replace;

[0058] m is selected from 1 or 2;

[0059] R 1a and R 1b are each independently selected from hydrogen, halogen and C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with one or more halogens;

[0060] R 2a is selected from halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0061] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein the cytotoxic drug is selected from any of the following structures:

[0062] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein the linker unit L is -L a -L b -L c -; and said L c connected to the cytotoxic drug;

[0063] -L a -for With the L b connect;

[0064] -L b - Any of the following structures: Preferably The right end of the above structure is preferably with the L c connect;

[0065] -L c -for

[0066] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein the linker unit L is Preferably

[0067] In some embodiments, the bispecific antibody drug conjugate of the present invention has a structure as shown in Formula (A-2):

[0068] in,

[0069] p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95;

[0070] Ab and M are as defined in any embodiment of the present invention; L is the linker unit L described in any embodiment of the present invention.

[0071] In some embodiments, the bispecific antibody drug conjugate of the present invention has a structure as shown in Formula (A-2):

[0072] in,

[0073] p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95;

[0074] M is -L 2 -L 1 -C(O)-;

[0075] L 2 is -O- or -S-, and L 2 Connect with L;

[0076] L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a replace;

[0077] m is 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3;

[0078] R 1a 、R 1b and R 2a Each is independently hydrogen, halogen, hydroxy, amino or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0079] In some embodiments, the bispecific antibody-drug conjugate of the present invention has a structure as shown in Formula (A-2a) or (A-2b):

[0080] in,

[0081] p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95;

[0082] Ab is a bispecific antibody or antigen-binding fragment thereof according to any embodiment of the present invention;

[0083] L 2 is -O- or -S-; preferably -O-;

[0084] X1 is optionally replaced by 1, 2 or 3 R 2a Substituted C3-C6 saturated cycloalkyl;

[0085] X2 is -(C(R 1a )(R 1b ))m -CH2-;

[0086] m is 1 or 2;

[0087] R 1a 、R 1b and R 2a Each is independently hydrogen, halogen or C1-C6 alkyl; the C1-C6 alkyl is optionally substituted by one or more halogens.

[0088] In some embodiments, the bispecific antibody-drug conjugate of the present invention is selected from any one of the following structures:

[0089] in,

[0090] p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95;

[0091] Ab is a bispecific antibody or an antigen-binding fragment thereof according to any embodiment of the present invention.

[0092] In another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate is selected from any one of the following structures:

[0093] in,

[0094] p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; for example, 4.02, 3.95, 6.1 or 7.9;

[0095] DSYE001 is a bispecific antibody against B7H3 and PD-L1. The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.: 9.

[0096] In some embodiments, the bispecific antibody drug conjugates of the present invention (e.g., the bispecific antibody drug conjugates of formula (A-2), (A-2a), or (A-2b) of the present invention), wherein p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; for example, 4.02, 3.95, 6.1, or 7.9.

[0097] In some embodiments, the average connection number p of the present invention can be any integer or decimal from 2 to 8. For example, the average connection number p can be any integer or decimal from 3 to 8. For example, the average connection number p can be any integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0098] In another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate is selected from any one of the following structures (p represents the average number of connections):

[0099] The amino acid sequence of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention is shown in the sequence listing. The CDR numbering method of the antibody of the present invention is: Kabat numbering.

[0100] In another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate is selected from any one of the following structures:

[0101] in,

[0102] t represents the number of connections, and t is any integer from 1 to 10, preferably any integer from 2 to 8, preferably any integer from 4 to 8, for example, 4, 6 or 8.

[0103] The amino acid sequence of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention is shown in the sequence listing. The CDR numbering method of the antibody of the present invention is: Kabat numbering.

[0104] In some embodiments, the connection number t of the present invention is any integer from 1 to 10, preferably any integer from 2 to 8. For example, the connection number t can be any integer from 3 to 8. For example, the connection number t is any integer from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0105] In another aspect, the present invention provides a method for preparing a bispecific antibody-drug conjugate, comprising the following steps: under the action of a reducing agent, mixing the bispecific antibody dissolved in a buffer solution with the linker-cytotoxin dissolved in a solvent to obtain the bispecific antibody-drug conjugate.

[0106] In some embodiments, the reducing agent is a reducing agent conventional in the art for such reactions, such as tris(2-carbonylethyl)phosphine hydrochloride.

[0107] In some embodiments, the buffer is a conventional buffer for such reactions in the art.

[0108] In some embodiments, the solvent is a conventional solvent for such reactions in the art, such as dimethylacetamide.

[0109] In another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody-drug conjugate according to any one of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0110] A further object of the present invention is to provide a method for preparing the pharmaceutical composition of the present invention, comprising combining the bispecific antibody drug conjugate according to any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers or excipients.

[0111] In the present invention, the pharmaceutically acceptable carrier used in the pharmaceutical composition, wherein examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0112] In the present invention, the pharmaceutical composition can be administered in any form, as long as it prevents, alleviates, prevents or cures the symptoms of human or animal patients. For example, it can be prepared into various suitable dosage forms according to the administration route.

[0113] In other embodiments, administration of any of the bispecific antibody drug conjugates or pharmaceutical compositions of the present invention can be combined with another therapeutic approach, which can be selected from, but not limited to, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0114] In another aspect, the present invention provides a pharmaceutical formulation comprising the bispecific antibody drug conjugate of any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof as an active ingredient, or a pharmaceutical composition of any one of the present invention. In some embodiments, the formulation is in the form of a solid formulation, a semisolid formulation, a liquid formulation, or a gaseous formulation.

[0115] In another aspect, the present invention provides use of the bispecific antibody-drug conjugate according to any one of the present invention, or the pharmaceutical composition according to any one of the present invention, in the preparation of a medicament for treating and / or preventing cancer, preferably, the cancer is a cancer that positively expresses B7H3 and / or PD-L1.

[0116] In another aspect, the present invention provides a method for treating and / or preventing cancer, comprising administering to a subject in need thereof the bispecific antibody-drug conjugate according to any one of the present invention, or the pharmaceutical composition according to any one of the present invention, preferably, the cancer is a cancer that positively expresses B7H3 and / or PD-L1.

[0117] In another aspect, the present invention provides a bispecific antibody-drug conjugate as described in any one of the present invention, or a pharmaceutical composition as described in any one of the present invention, for treating and / or preventing cancer. Preferably, the cancer is a cancer that positively expresses B7H3 and / or PD-L1.

[0118] In some embodiments, the cancer described in the present invention is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer and Ewing's sarcoma. Preferably, the cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer or melanoma.

[0119] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0120] In some embodiments, the present invention provides for administering to a subject a therapeutically effective amount of one or more therapies (eg, treatment modalities and / or other therapeutic agents) in combination. In some embodiments, the therapies include surgery and / or radiation therapy.

[0121] In some embodiments, the methods or uses provided herein further comprise administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. The antibody drug conjugates of the present invention or pharmaceutically acceptable salts thereof may be used alone or in combination with other therapeutic agents in the therapy. For example, they may be co-administered with at least one additional therapeutic agent.

[0122] In another aspect, the present invention provides a pharmaceutical combination comprising the anti-B7H3 and PD-L1 bispecific antibody drug conjugate as described herein, or a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0123] In another aspect, the present invention provides a kit comprising the anti-B7H3 and PD-L1 bispecific antibody drug conjugate according to any one of the present invention, or a pharmaceutically acceptable salt thereof or a mixture thereof, or the pharmaceutical composition according to any one of the present invention, preferably further comprising a drug delivery device.

[0124] Definition of terms

[0125] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0126] In order to facilitate understanding of the present invention, certain scientific and technical terms are specifically defined below. Unless otherwise expressly defined elsewhere herein, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. For definitions and terminology in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0127] In the present invention, the term "B7H3", also known as CD276 antigen, refers to a type 1 transmembrane protein belonging to the B7 family, which has an external domain consisting of a single IgV-IgC domain. B7 family proteins contain extracellular IgV-like and IgC-like domains and have a short cytoplasmic tail. B7H3 is an immune checkpoint molecule that is abnormally overexpressed in a variety of cancers. The amino acid sequence of the B7H3 protein includes the full-length B7H3 protein (such as human 4IgB7H3 protein or human 2IgB7H3 protein), or the extracellular domain of B7H3 (B7H3 ECD) or a fragment containing B7H3 ECD; B7H3-ECD fusion protein. Exemplary sequences of B7H3 proteins are shown in Uniprot ID: Q5ZPR3 (human 4IgB7H3), Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse). The amino acid sequence homology of cynomolgus monkey B7H3 to human and mouse B7H3 is approximately 97% and 88%, respectively.

[0128] In the present invention, the term "PD-L1", programmed cell death 1 ligand 1 (PD-L1), also known as surface antigen cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40kDa type I transmembrane protein. PD-L1 is the surface glycoprotein ligand of PD-1, a key immune checkpoint receptor expressed by activated T cells and B cells, and mediates immunosuppression.

[0129] In the present invention, 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 of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1% and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0130] In the present invention, the term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0131] In the present invention, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in the claims, "consisting of..." will refer to only one of the listed numbers or one element of the list.

[0132] Unless the context clearly indicates otherwise, the terms "a" and "an" should be understood as meaning "at least one" in the present invention.

[0133] In the present invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker. In this application, an "antibody-drug conjugate" may be a bispecific antibody-drug conjugate, which may be a bispecific antibody or antigen-binding fragment thereof linked to a biologically active cytotoxic drug fragment via a stable linker.

[0134] In the present invention, the term "cytotoxic drug" generally refers to a toxic drug that has a strong chemical molecule that destroys the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at a sufficiently high concentration. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 or a radioactive isotope of Lu), a toxic drug, a chemotherapeutic drug, an antibiotic or a nucleolytic enzyme, or a derivative thereof, for example, a toxic drug, including but not limited to a camptothecin derivative, for example, the camptothecin derivative exatecan (chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0135] In the present invention, the term "antibody" generally refers to an immunoglobulin that is reactive to a specified protein or peptide or a fragment thereof. The antibody can be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD and IgE, and an antibody from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the present application can be derived from any species. The term "antibody" can include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity.

[0136] In the present invention, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to as an "epitope" as described above. As described above, an antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both. An Fd fragment, for example, has two VH regions and generally retains some of the antigen-binding function of an intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having a VL, VH, constant light chain (CL) and CH1 domains; (2) a F(ab′)2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity determining regions (CDRs); and (7) single-chain Fv (scFv), for example, derived from an scFV library.Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined using recombinant methods by a synthetic linker, which allows them to be prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)); (8) "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J. Biotechnol., 74, 277-302 and reviewed in Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs may also be referred to as nanobodies (Nb).

[0137] In the present invention, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy chain or light chain involved in the binding of the antibody to the antigen. In this application, the term "variable" generally refers to that some parts of the sequence of the variable domain of the antibody vary strongly, forming the binding and specificity of various specific antibodies to their specific antigens. Variability is not evenly distributed in the entire variable region of the antibody. It is concentrated in three segments in the light chain variable region and the heavy chain variable region, known as the complementary determining region (CDR) or hypervariable region (HVR), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved parts in the variable domain are called framework regions (FR). The variable domains of natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a β-pleated sheet configuration and are connected by three CDR structural loop regions. The CDRs in each chain are held together by the FR regions and, together with the CDRs from the other chain, form the antibody antigen-binding site.

[0138] In the present invention, in the art, the variable region of the antibody can be encoded or the CDR of the antibody can be divided by various methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia numbering scheme and definition rules based on the position of the structural loop region (see, Al-Lazikani et al., J Mol Biol 273:927-48, 1997), efranc et al. The IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V genes, as well as Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc., can be seen in Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018.

[0139] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0140] In the present invention, the term "multispecific antibody" refers to an antibody that comprises two or more antigen-binding domains and is capable of binding to two or more different epitopes (e.g., two, three, four or more different epitopes), which may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind to two different antigens or two different epitopes. The bispecific antibodies targeting B7H3 and PD-L1 herein may be referred to as, for example, "anti-B7H3 / PD-L1" or "anti-B7H3 x PD-L1" or "B7H3 x PD-L1" bispecific molecules, or other similar terms.

[0141] In the present invention, the term "nanobody" refers to a heavy chain single-domain antibody (VHH) (variable domain of heavy chain of heavy-chain antibody), which contains only a heavy chain variable region (VHH) and CH2 and CH3 regions. Compared with other antibodies, the light chain is naturally absent. It is composed of the heavy chain variable region of camelids (camels, llamas, alpacas and their close relatives). Nanobody crystals are 2.5nm in diameter and 4nm long, and are the smallest naturally occurring fragments that can bind to antigens.

[0142] In the present invention, the term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0143] In the present invention, the term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0144] In the present invention, an antibody "isotype" refers to the class of antibody provided by the heavy chain constant region gene (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modification has been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0145] In the present invention, the term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0146] In the present invention, the term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Therefore, "cross-reactivity" should be understood as the interspecies reaction of an antigen-binding molecule (e.g., antibody) with a similar molecule (e.g., BDCA2) expressed in a different species. The cross-reactivity specificity of monoclonal antibodies recognizing human BDCA2, monkey, and / or murine BDCA2 (mouse or rat) can be determined by FACS analysis.

[0147] In the present invention, "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on ). Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technology is used to measure affinity, for example, the affinity between the antibody of the present invention and the antigen. In some preferred embodiments of the present invention, a specific method for measuring affinity is the BIAcore method herein.

[0148] In the present invention, the term "not binding" to a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., the K of the binding protein or cell is D 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0149] In the present invention, the term "high affinity" for IgG antibodies refers to the K D 1.0×10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8M or lower.

[0150] In the present invention, the term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0151] In the present invention, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.

[0152] In the present invention, the term "alkyl" generally refers to the residue derived from an alkane by removing a hydrogen atom. Alkyl can be substituted or non-substituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group, which has a residue derived from the removal of a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, and can be a straight or branched group containing 1 to 20 carbon atoms, for example, a chain alkyl containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. Alkyl can be substituted or non-substituted, substituted or non-substituted, for example, when substituted, the substituent can be substituted on any usable point of attachment.

[0153] In the present invention, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group. It can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from a group containing 1 carbon atom by removing two hydrogen atoms. The methylene group can be substituted or unsubstituted, substituted or unsubstituted; for example, an alkylene group containing 1 to 12 carbon atoms, for example, an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), etc. Alkylene groups can be substituted or unsubstituted, substituted or unsubstituted, e.g., when substituted, the substituents can be substituted at any available point of attachment.

[0154] In the present invention, the term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined herein. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0155] In the present invention, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctanyl, etc.; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, for example, when substituted, the substituents may be substituted at any available point of attachment.

[0156] As used herein, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" refers to a moiety having one or more degrees of unsaturation.

[0157] In the present invention, the term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic light substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; more preferably, it contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, it contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls. The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring; the ring connected to the parent structure is the heterocyclyl. A heterocyclyl group may be substituted or unsubstituted, for example, when substituted, the substituents may be substituted at any available point of attachment.

[0158] In the present invention, the term "ring-forming atoms" generally refers to atoms contained in a ring structure. For example, a ring-forming atom may be a carbon atom in a benzene ring or a nitrogen atom in a pyridine ring. When a ring-forming atom is attached to a hydrogen atom, the ring-forming atom may be substituted or unsubstituted. For example, when substituted, the substituent may be substituted at any available attachment point.

[0159] In the present invention, the term "each independently" generally refers to a variable being applicable in any instance, regardless of whether other variables with the same or different definitions exist in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen," both R may be carbon, both R may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0160] In the present invention, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0161] In the present invention, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are independently replaced by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0162] In the present invention, unless otherwise specified, the "connection" of a group to a group can generally be in any orientation; for example, the "connection" of a group X to a group Y can generally be in any orientation, and any orientation generally means that when a group X is used to connect a group Y and a group Z, the two or more connection sites of the group X can be arbitrarily connected to the group Y or the group Z.

[0163] In the present invention, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In the examples, the members of the group may have any number of carbon atoms falling within the range of 1-10.

[0164] In the present invention, the cytotoxic drugs of the present invention can be their tautomers, mesomorphs, racemates, enantiomers, and / or diastereomers. In the present application, the term "diastereomer" generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as, melting points, boiling points, spectral properties and reactivity. In the present application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that can be converted to each other by low energy barriers. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via reorganization of some of the bonding electrons. As used herein, the term "mesomer" generally refers to a molecule containing an asymmetric atom but having factors of symmetry such that the overall intramolecular optical rotation is zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric species.

[0165] In the present invention, the term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end, and can also be connected to a cytotoxic drug after being connected to other linkers. The directly or indirectly connected ligand can refer to the group being directly connected to the ligand through a covalent bond, or it can be connected to the ligand through a linker structure. For example, a chemical structure fragment or bond comprising an acid-labile linker structure (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as the linker structure.

[0166] In the present invention, the term "optionally linked to other molecular moieties" generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures (such as the ligands described herein) that are different from the structure (e.g., linked by a chemical bond or linked by a linker structure).

[0167] In the present invention, the term "drug loading" generally refers to the average number of cytotoxic drugs loaded per ligand, and can also be expressed as the ratio of the cytotoxic drug to the antibody. The cytotoxic drug loading can range from 0 to 12, for example, 1 to 10, cytotoxic drugs per ligand (Ab). In embodiments of the present application, the drug loading is expressed as p, t, or n, and exemplary values ​​may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The drug loading per ADC molecule after the coupling reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0168] In the present invention, certain atoms in the compounds or antibody drug conjugates of the present invention may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125I, or similar isotopes. Thus, the compounds or antibody drug conjugates of the present invention may be enriched in one or more of these isotopes relative to the natural abundance of these isotopes. As is known to those skilled in the art, such isotopically enriched compounds may be used in a variety of applications. For example, heavy isotopes such as deuterium ( 2 Deuterium (H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is one deuterium atom for approximately every 6,500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds or antibody drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as appropriate). Unless otherwise indicated, the structures described herein may also include compounds or antibody drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody drug conjugates that have the same structure as the present invention except for the replacement of hydrogen atoms by deuterium or tritium, or the replacement of carbon atoms by carbon-13 or carbon-14, are within the scope of the present invention.

[0169] In the present invention, the term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical composition preparations can be found in the Chinese Pharmacopoeia. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions prepared in a non-toxic, parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile fixed oils can conveniently be used as solvents or suspending media. For example, any blended fixed oil, including synthetic mono- or diglycerides, can be used. Fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0170] In the present invention, the term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refers to a salt of the compound or antibody-drug conjugate of the present invention, or a salt of the compound or antibody-drug conjugate described in the present invention. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compound or antibody-drug conjugate of the present invention may form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0171] In the present invention, the term "pharmaceutically acceptable carrier" generally refers to a carrier or vehicle for administering therapeutic agents, such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers.

[0172] As used herein, the terms "treatment" and "treating" generally refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic benefit includes, but is not limited to, eradication, inhibition, reduction, or amelioration of the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by eradication, inhibition, reduction, or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, but the patient may still suffer from the underlying disorder.

[0173] As used herein, the terms "prevention" and "preventing" generally refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk for developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0174] In the present invention, the term "subject" or "patient" generally refers to humans (i.e., males or females of any age group, for example, pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults or elderly people) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail and / or turkeys.

[0175] In the present invention, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of a compound or antibody drug conjugate of the present invention, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition when administered to a cell, tissue or subject. A therapeutically effective dose also refers to a dose sufficient to cause amelioration of symptoms, such as an amount that treats, cures, prevents or ameliorate a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0176] As used herein, the term "cancer" refers to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. As used herein, the term "tumor" refers to one or more cells comprising a cancer. As used herein, the term "tumor growth" refers to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0177] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0178] The reagents and raw materials used in the present invention are commercially available.

[0179] The positive progress effect of the present invention is:

[0180] The bispecific antibody-drug conjugate of the present invention, wherein the bispecific antibody has the following advantages:

[0181] 1. It can bind to B7H3 and PD-L1 simultaneously, and can relieve the inhibition of PD-L1 on T cells while targeting tumor cells, and it also shows anti-tumor activity that is superior to the combination of monoclonal antibodies.

[0182] 2. Compared with related monoclonal antibody combination therapies, the bispecific antibodies of this application have the advantages of good compliance and controllable quality.

[0183] 3. The stability characterization of the bispecific antibody in the present invention is mainly reflected in the study of monomer purity and thermal stability. After a single affinity purification, the monomer content of the bispecific antibody can reach 95%, which is even better than the purity after multiple secondary purifications in the industry. The results of the structural and activity analysis of the antibody after heat treatment show that the antibody can still maintain a good molecular conformation and complete biological activity under harsh conditions, which is conducive to the industrial production and packaging and storage of antibodies. In general, the present invention constructs a B7H3 / PD-L1 bispecific antibody in the form of IgG-VHH2; it shows good molecular stability and has in vitro activity (binding molecular level and cellular level) that is significantly better than that of Avelumab and MGA271. In vivo data show that in B7H3+A375 tumor cells, the anti-tumor activity of the bispecific antibody is better than the B7H3 monoclonal antibody combination group. Therefore, the bispecific antibody of the present invention has broad application prospects due to its excellent developability and activity.

[0184] The bispecific antibody drug conjugates of the present invention have one or more of the following advantages:

[0185] 1. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugates of the present invention have significantly enhanced proliferation inhibitory activity against tumor cells in vitro, especially against A375, NCI-H1975, NCI-H441, and NCI-H358 cells that positively express B7H3 and PD-L1.

[0186] 2. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugate of the present invention can more significantly induce downregulation of PD-L1 expression, indicating that the bispecific antibody drug conjugate of the present invention can exert a stronger immunosuppression-relieving effect by reducing the expression level of the target.

[0187] 3. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugates of the present invention have significantly enhanced in vivo tumor growth inhibition activity, for example, in CT26 cells, especially in a syngeneic transplantation mouse model of CT26 cells.

[0188] 3. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugates of the present invention can significantly inhibit the growth of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, especially melanoma A375 cells, esophageal squamous cell carcinoma KYSE-150 cells, small cell lung cancer NCI-H292 cells, liver cancer Huh7 cells, MDA-MB-231 breast cancer cells, or NCI-H1975 non-small cell lung cancer cells.

[0189] 4. Compared with related monoclonal antibody drug conjugates and monoclonal antibody combination therapies, the bispecific antibody drug conjugates of the present invention can significantly inhibit the growth of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, especially MDA-MB-231 breast cancer cells and NCI-H1975 non-small cell lung cancer cells.

[0190] 5. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugates of the present invention have significantly enhanced endocytosis effect.

[0191] 6. The bispecific antibody-drug conjugate of the present invention has good safety.

[0192] The bispecific antibody-drug conjugates of any one of the present invention may have one or more effects selected from the following groups: (1) having inhibitory activity on the proliferation of tumor cells in vitro; (2) having targeted inhibition; (3) having plasma stability; (4) having an in vivo tumor-suppressing effect; (5) having a bystander killing effect; (6) having an anti-transporter transport ability; (7) having an in vivo tumor-targeting ability; (8) having a stronger tumor-suppressing effect in individuals with a healthy immune system; and (9) having good in vivo safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] FIG1 is a diagram showing the affinity screening of the anti-B7H3 VHH humanized antibody prepared by the present invention and B7H3.

[0194] FIG2 is a schematic structural diagram of the bispecific antibody DSYE001 in the bispecific antibody-drug conjugate DSYE001-X1 or DSYE001-X2 of the present invention.

[0195] FIG3 is an SDS-polyacrylamide gel electrophoresis diagram of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0196] FIG4 is the SEC-HPLC purity determination of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0197] FIG5 shows the Tm value determination (DSF) of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0198] FIG6 shows the binding ELISA of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention before and after heat treatment at 60° C., wherein a represents the binding ELISA with PD-L1, and b represents the binding ELISA with B7H3.

[0199] FIG7 shows the binding ELISA of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention, wherein a represents the binding ELISA with PD-L1, and b represents the binding ELISA with B7H3.

[0200] Figure 8 is a BLI affinity test graph of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 B7H3-his or PD-L1-his of the present invention at 5 different concentrations, where a represents the affinity curve with PD-L1, and b represents the affinity curve with B7H3.

[0201] FIG9 is a blocking curve of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 PD-L1 / CHO-PD1 of the present invention.

[0202] FIG10 shows that the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention induces T cells to secrete IFN-γ.

[0203] FIG11 shows that the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention promotes T cell proliferation.

[0204] FIG12 shows the ADCC effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention on cancer cells, wherein a represents the cytotoxic effect on human breast cancer cells MDA-MB-231, and b represents the cytotoxic effect on human ovarian clear cell carcinoma cells ES-2.

[0205] FIG13 shows the in vivo tumor inhibition effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0206] FIG14 shows the effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention on the body weight of mice.

[0207] FIG15 shows the endocytic activity test results of the bispecific antibody-drug conjugate DSYE001-X2 of the present invention.

[0208] FIG16 is a test showing the inhibition of tumor cell proliferation in vitro by the bispecific antibody-drug conjugate DSYE001-X1 (DAR8) of the present invention.

[0209] FIG17 shows the effect of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention on PD-L1 expression in NCI-H1975 cells.

[0210] FIG18 is an evaluation of the efficacy of the bispecific antibody-drug conjugate DSYE001-X2 of the present invention in a syngeneic transplant mouse model.

[0211] FIG19 is a diagram showing the efficacy evaluation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR4) of the present invention in human non-small cell lung cancer cell NCI-H1975 tumor-bearing mice.

[0212] FIG20 is an evaluation of the efficacy of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention in mice bearing human breast cancer cells MDA-MB-231.

[0213] FIG21 is a diagram showing the efficacy evaluation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention in mice bearing human non-small cell lung cancer cells NCI-H1975. DETAILED DESCRIPTION

[0214] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0215] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0216] Sample testing

[0217] 1. ADC DAR value analysis method - HIC-HPLC (hydrophobic chromatography)

[0218] High performance liquid chromatography: Waters e2965 high performance liquid chromatography system.

[0219] Column: MabPac TM HIC-Butyl 5μm 4.6×100mm (Manufacturer: Thermo);

[0220] Mobile phase A: 1.5 M (NH 4 ) 2 SO 4 + 50 mM K 2 HPO 4 (pH 7.0);

[0221] Mobile phase B: 50 mM K2HPO4 (pH 7.0) / isopropanol (75:25 V / V);

[0222] Elution was performed according to the following elution procedure:

[0223] Detection conditions: Set the mobile phase flow rate to 1 mL / min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0224] 2.SEC purity analysis - SEC-HPLC (size exclusion chromatography)

[0225] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0226] Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0227] Mobile phase: 50 mM NaH2PO4 + 200 mM arginine (pH 6.80) + 10% isopropanol

[0228] Detection conditions: Set the mobile phase flow rate to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0229] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0230] Example

[0231] The following examples are provided to demonstrate and further explain some preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

[0232] Example 1: Preparation and testing of bispecific antibody DSYE001

[0233] In the anti-B7H3 and PD-L1 bispecific antibody drug conjugate of the present invention, the anti-B7H3 and PD-L1 bispecific antibody or antigen-binding fragment thereof is prepared with reference to the method of PCT / CN2022 / 125089, as shown below (the CDR regions of the bispecific antibody are determined according to the Kabat numbering convention):

[0234] 1. Humanization of camel-derived anti-B7H3

[0235] Using human antibody germline genes as templates, a framework shuffling approach was used to humanize camel-derived anti-B7H3 nanobodies. The corresponding VHH framework-shuffled libraries were generated by full in vitro synthesis using overlapping PCR. The VHH phage library was then cloned, screened, and characterized.

[0236] More specifically, a one-step strategy was used to humanize a camel-derived anti-B7H3 nanobody. Approximately 1,000 clones were screened from this sublibrary, and selected positive clones were screened for phage-level thermostability using ELISA. Highly absorbable 96-well ELISA plates were coated with 5 μg / mL huB7H3 antigen, and the supernatant from overnight amplification of the screened phage was reacted, selecting clones that displayed high OD450 readings.

[0237] The above-mentioned phage clone was sequenced to obtain the B7H3 VHH gene sequence, and its C-terminus was fused with the human Fc protein gene to construct and express B7H3-Fc. Using biofilm interferometry, 100 nM of B7H3 VHH-Fc was captured using a Protein A probe and bound to a 200 nM starting concentration of 2-fold diluted B7H3 antigen. The KD value of the antibody binding to the antigen was calculated. The results showed that 75-16 (whose amino acid sequence is SEQ ID NO.: 15, CDR1 sequence is SEQ ID NO.: 12, CDR2 sequence is SEQ ID NO.: 13, and CDR3 sequence is SEQ ID NO.: 14) had the highest affinity for B7H3, with a KD of 3.24×10 -9 M (see Figure 1), so it was selected for the next step of bispecific antibody construction.

[0238] 2. Construction and Expression of Bispecific Antibodies Against B7H3 and PD-L1

[0239] The amino acid sequences of the PD-L1 monoclonal antibody light chain (amino acid sequence of SEQ ID NO.: 9, variable region sequence of SEQ ID NO.: 4, CDR1 sequence of SEQ ID NO.: 1, CDR2 sequence of SEQ ID NO.: 2, and CDR3 sequence of SEQ ID NO.: 3) and heavy chain (amino acid sequence of SEQ ID NO.: 10, variable region sequence of SEQ ID NO.: 8, CDR1 sequence of SEQ ID NO.: 5, CDR2 sequence of SEQ ID NO.: 6, and CDR3 sequence of SEQ ID NO.: 7) are derived from the existing PD-L1hIgG1 humanized monoclonal antibody, and the N-terminus of the anti-B7H3 VHH (75-16 described above) is connected to the C-terminus of the Fc fragment via a connecting peptide (SEQ ID NO.: 11) (the structure is shown in Figure 2).

[0240] A DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in Escherichia coli. The purified plasmid was transfected into HEK293 cells using PEI. The cells were then suspended in OPM-CD05 expression medium and cultured. After 6 days of culture, the cell culture supernatant was collected and the antibody was purified using a protein A column. The purified IgG1 was dialyzed against phosphate-buffered saline (PBS), snap-frozen, and stored at -80°C.

[0241] The purified anti-B7H3 and PD-L1 bispecific antibody DSYE001 has a heavy chain amino acid sequence of SEQ ID NO.: 16, and a light chain amino acid sequence of SEQ ID NO.: 9.

[0242] Test section

[0243] Hereinafter, unless otherwise specified, the term "bispecific antibody" refers to the bispecific antibody against B7H3 and PD-L1 in the present invention, which is also referred to as "B7H3 / PD-L1 bispecific antibody", "B7H3 / PD-L1 bispecific antibody" or "bispecific antibody".

[0244] Test method:

[0245] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0246] 5 μg of the bispecific antibody was mixed with protein reducing and non-reducing buffers, and the mixture was made up to 10 μL with PBS. After heating at 100°C for 10 minutes to fully denature the protein, 9 μL was added to the wells of a precast polyacrylamide gel (Bio-Rad). Separation was performed at 80 V for 30 minutes and then at 120 V for 60 minutes. The gel was then stained with Coomassie Brilliant Blue for 30 minutes and destained with a destaining solution (acetic acid: ethanol: water = 1:3:6) for 15 minutes. This destaining was repeated three times to fade the background, and images were acquired using a gel imager. Results showed that the B7H3 / PD-L1 bispecific antibody exhibited good monomeric purity under non-reducing conditions. Under reducing conditions, two bands, one for the heavy and one for the light chains, were formed due to the disruption of the disulfide bonds between the heavy and light chains, with no contaminants (Figure 3).

[0247] (2) Size Exclusion Chromatography (SEC-HPLC)

[0248] SEC-HPLC analysis was used to assess the monomeric purity of the bispecific antibodies. The B7H3 / PD-L1 bispecific antibody was analyzed using a 1260 HPLC system (Agilent, Santa Clara, CA) on a ThermoMAbPac SEC-1, 5 μm, (7.8 × 300 mm) P / N 088460 and compared with the PD-L1 monoclonal antibody and B7H3 VHH-Fc. The mobile phase used was phosphate-buffered saline (PBS). The flow rate was set to 0.7 mL / min; the injection volume was 15 μL. SEC chromatograms were recorded by monitoring the absorbance at 280 nm using a UV detector at a constant temperature of 25°C. As shown in Figure 4, all antibodies tested had a very high proportion of monomeric peaks, with the peak area ratio of the monomeric peak exceeding 95%. This indicates that the bispecific antibody has good monomeric purity and a low number of aggregates under PBS buffer conditions.

[0249] (3) Differential scanning fluorimetry (DSF) to detect the Tm value of the antibody

[0250] DSF was detected using a real-time PCR instrument (Biorad cfx96, USA). The B7H3 / PD-L1 bispecific antibody, B7H3 VHH-Fc, and PD-L1 monoclonal antibody were diluted to 1 mg / mL in PBS. SYPRO Orange was diluted 1000-fold from a 5000-fold concentrated stock solution with ddH2O. A 20 μL sample was placed in a PCR tube, and the SYPRO Orange working solution was added to the reaction to prevent photobleaching. The reaction was centrifuged briefly to pool the liquid at the bottom of the PCR tube. The qPCR instrument was turned on and programmed to heat from 25°C to 95°C at a rate of 0.3°C per second. Data were collected, temperature and signal values ​​were plotted, and the melting temperature (Tm) was calculated. The data showed that the Tm of the Fc and Fab of the B7H3 / PD-L1 bispecific antibody were 69°C and 90°C, respectively, demonstrating excellent high-temperature resistance and similar to the Tm of the B7H3 VHH-Fc and PD-L1 monoclonal antibodies (Figure 5).

[0251] (4) Verify the thermal stability and binding activity of the antibody by combining ELISA

[0252] A 96-well ELISA plate was coated with 2 μg / ml his-tagged PD-L1 or B7H3 antigen protein overnight at 4°C. The next day, 100 μL of Casein blocking solution was added to each well and blocked at 37°C for 1 hour. Three-fold serial dilutions of the B7H3 / PD-L1 bispecific antibody (for thermal stability testing, the bispecific antibody was treated in a 60°C water bath for 1 hour) and monoclonal antibodies (for ELISA testing of bispecific antibody binding to B7H3, the control monoclonal antibodies used were MGA271, isotype hIgG1, and B7H3 VHH-Fc; for PD-L1 binding testing, the control monoclonal antibodies used were PD-L1 monoclonal antibody, avelumab, and isotype hIgG1) were added to the plate. After incubation at 37°C for 1 hour, unbound antibodies were washed away with 0.1% PBST, and bound antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, USA). 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (TMB) was used for color development. After standing for 5 minutes, 50 μL of 2 M sulfuric acid was added to stop the color development. The absorbance was measured at OD450 nm using a SpectraMax M5e (Molecular Devices) microplate reader, and the antibody concentration was plotted against the OD450 reading using a four-parameter fitting method to calculate the EC 50Figures 6a and 6b show the binding curves of the bispecific antibody to PD-L1 and B7H3 before and after treatment at 60°C for 1 hour. As can be seen from the figure, the binding curves of the bispecific antibody before and after heating are basically overlapping, regardless of whether it is for PD-L1 or B7H3, which indicates that the bispecific antibody can withstand the high temperature of 60°C and maintain strong binding activity to the dual targets of PD-L1 and B7H3. On the other hand, the bispecific antibody has similar binding ability to the positive antibody, PD-L1 monoclonal antibody, and B7H3 VHH-Fc for both targets. The EC values ​​of the bispecific antibody, PD-L1 monoclonal antibody, and control Avelumab for binding to human PD-L1 are 0. 50 The binding activity of bispecific antibody, B7H3 VHH-Fc and MGA271 to human B7H3 was similar, and the EC 50 0.04105 nM, 0.02515 nM and 0.05476 nM respectively ( FIG. 7 b ).

[0253] (5) BLI method to determine the affinity of bispecific antibodies binding to antigens

[0254] B7H3 / PD-L1 bispecific antibody, PD-L1 monoclonal antibody, and B7H3 VHH-Fc were diluted to 100 nM in sample buffer (0.02% tween20 and 0.1% BSA in PBS). The affinity of the bispecific antibody to specific human B7H3 and human PD-L1 antigens was analyzed by OCTET 96. The probe used was made of Protein A material to fix the antibody; B7H3-his and PD-L1-his antigens were diluted to an initial concentration of 200 nM with sample buffer, and multiple antigen gradients were set up according to a 2-fold dilution for binding to the antibody to obtain rate constants and affinity. The Kon and Koff values ​​were calculated using the software provided by the supplier, and the KD value of the antibody was obtained. As can be seen from the figure, the bispecific antibody has a high affinity for PD-L1 and B7H3, with KD values ​​reaching 5.85×10 -10 M and 8.11×10 -9 M (Figure 8).

[0255] (6) Flow cytometry to detect the ability of bispecific antibodies to block the PD1 / PD-L1 pathway

[0256] Flow cytometry was used to evaluate the ability of B7H3 / PD-L1 bispecific antibody to block the binding of human PD-L1 and human PD1-CHO cells, and compared with Avelumab and PD-L1 monoclonal antibody. 5Individual PD1-CHO cells were evenly plated in a 96-well culture plate and incubated with a mixture of 400 nM starting, serially diluted antibodies (B7H3 / PD-L1 bispecific antibody, Avelumab, PD-L1 monoclonal antibody, IsotypehIgG1, B7H3 VHH-Fc) and biotinylated PD-L1 antigen (50 nM) at room temperature for 30 minutes. The mixed solution was then incubated with the cells at 4 ° C for 45 minutes, and the unbound antigen was washed away with PBS. The cells were then fluorescently stained with PE-streptavidin. Finally, the mean fluorescence intensity (MFI) of the PE channel in the flow cytometer was read, the antibody concentration and MFI were plotted, and the IC of the antibody was calculated using four-parameter fitting. 50 Flow cytometry analysis results showed that the bispecific antibody was able to block the binding of PD-L1 to CHO-PD1 cells, and its IC 50 The blocking activity was 106.4 nM and was comparable to that of PD-L1 monoclonal antibody (IC 50 is 94.20nM), Avelumab (IC 50 was 115.0 nM) ( FIG9 ).

[0257] (7) Mixed lymphocyte reaction (MLR) detection of the ability of bispecific antibodies to activate T cells

[0258] Dendritic cells (DCs) were induced by culturing monocytes isolated from peripheral blood mononuclear cells (PBMCs) in vitro for 7 days with 500 U / mL interleukin-4 (IL-4) and 250 U / mL GM-CSF using a monocyte purification kit (Miltenyi Biotec, Germany). CD4 + T cells (1 × 10 5 ) and allogeneic DCs (1.25×10 4 ) were co-cultured in RPMI 1640 complete medium supplemented with 10% FBS at 5% CO2 and a constant temperature of 37°C. Groups were set up with no antibody or with varying concentrations of B7H3 / PD-L1 bispecific antibody, PD-L1 monoclonal antibody, avelumab, B7H3 VHH-Fc, MGA271, and hIgG1 isotype. After 5 days, IFN-γ concentrations in the culture supernatants were analyzed using an IFN-γ ELISA kit. MLR results showed that the bispecific antibody stimulated CD4+ T cells to secrete IFN-γ, and its T cell activation ability was superior to that of PD-L1 monoclonal antibody and avelumab at both low and high concentrations. Furthermore, when only B7H3 antibody was present, T cells were partially activated due to the bispecific antibody blocking B7H3 inhibitory signaling, despite the lack of significant PD-L1 signaling blocking antibodies (Figure 10).

[0259] (8) T cell proliferation assay

[0260] 1 μg / mL CD3 antibody (Clone HIT3a), 1 μg / mL CD28 antibody (Clone CD28.2) and 5 μg / mL human PD-L1 were coated on a 96-well cell plate (Corning, USA) at 4°C for 1 hour. Control wells were coated with mouse IgG2a isotype control alone or with CD3 and CD28 antibodies using the same method. Dynabeads were used. TM CD4+ T cells were isolated using the CD4 Positive Isolation Kit. CD4+ T cells were cultured in pre-coated 96-well plates with different concentrations of B7H3 / PD-L1 bispecific antibodies, Avelumab, and PD-L1 monoclonal antibodies at 37°C in RPMI1640 medium containing 10% FBS (Gibco) for 4 days. After 4 days, the CCK8 kit was used to detect changes in T cell numbers. The data showed that freshly isolated human CD4+ T cells cultured on plates coated with anti-CD3 and anti-CD28 antibodies showed increased proliferation. When PD-L1 was added to the plates, the proliferation capacity was significantly reduced, confirming that PD-L1 provides an inhibitory signal to T cells. Avelumab, PD-L1 monoclonal antibodies, and bispecific antibodies significantly promoted T cell proliferation at concentrations of 100nM and 500nM (Figure 11).

[0261] (9) Antibody-dependent cellular cytotoxicity (ADCC)

[0262] The primary antitumor effects of MGA271 and avelumab are derived from the ADCC function of the antibodies, which is related to their IgG1 subtype. Bispecific antibodies also have IgG1 functional regions. Antibody ADCC was measured using an LDH cytotoxicity assay kit. Human PBMCs were purified from leukocytes using Ficoll gradient centrifugation and NK cells were isolated from human PBMCs using negative selection magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10 6 ) and MDA-MB-231 and ES-2 cells (3×10 5 ) At the beginning of the assay, cells were co-cultured with or without the addition of different concentrations of bispecific antibody and Avelumab. After 18 hours, the lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. + PD-L1 + When MDA-MB-231 was used as the target cell, both the bispecific antibody and Avelumab showed ADCC activity, but the activity of the bispecific antibody was stronger at low concentrations; using PD-L1 +Using ES-2 cells as target cells, it was observed that the activity of the bispecific antibody at a dose of 100 nM was comparable to that of Avelumab, and it also exhibited strong ADCC activity at low concentrations ( FIG12 ).

[0263] (10) Study on the anti-tumor activity of bispecific antibodies in vivo

[0264] Human PBMC (6.67×10 6 ) were injected into 41 NPSG mice via tail vein one day before inoculation of A375 tumor cells, and 5×10 6 A375 tumor cells were injected five days after tumor inoculation, and subcutaneous tumor formation was observed. Ten animals were enrolled in each group, with groups divided into isotype control, monoclonal antibody combination therapy (Pembrolizumab + MGA271 and Avelumab + MGA271), and monoclonal and bispecific antibody groups. Dosing began on day 5 after model establishment and continued twice weekly until the end of the experiment. Tumor volume and animal body weight were measured and recorded on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39, and 42 of the experiment. Efficacy and safety were evaluated based on tumor growth inhibition values ​​based on relative tumor volume (TGIRTV) and changes in animal body weight. The bispecific antibody maintained significantly stronger activity throughout the experiment than the PD-1 monoclonal antibody + MGA271 combination group. Over time, the bispecific antibody gradually demonstrated superior efficacy to the avelumab + MGA271 group, with TGIs of 39.29% and 26.45%, respectively, at the end of the experiment (Figure 13). Furthermore, no mice experienced significant weight loss during the experiment, demonstrating the safety of the bispecific antibody treatment (Figure 14).

[0265] From the test results of (1) to (10) above, it can be seen that the bispecific antibody constructed by the present invention can bind to B7H3 and PD-L1 simultaneously, can relieve the inhibition of PD-L1 on T cells while targeting tumor cells, and at the same time shows anti-tumor activity that is superior to the combination of monoclonal antibodies.

[0266] The anti-B7H3 antibody DSYE002 is the reference antibody for the bispecific antibody DSYE001 without the PD-L1 antibody; the anti-B7H3 antibody DSYE003 is the reference antibody for the bispecific antibody DSYE001 with the PD-L1 antibody replaced (the PD-L1 antibody variable regions in the bispecific antibody are replaced with the Human Anti-HIV-1 gp120 clone b12 VH and VL); and the anti-PD-L1 antibody DSYE004 is the reference antibody for the PD-L1 antibody in the bispecific antibody DSYE001.

[0267] Antibodies DSYE002, DSYE003 and DSYE004 are prepared according to conventional methods, for example, vector construction is performed, followed by transfection into eukaryotic cells such as HEK293 cells and CHO cells for purification and expression.

[0268] The amino acid sequences of the anti-B7H3 and PD-L1 bispecific antibody DSYE001, and the anti-B7H3 antibodies DSYE002, DSYE003, and DSYE004 are shown in the sequence listing.

[0269] Example 2: Preparation of bispecific antibody drug conjugates (ADCs)

[0270] 2.1. Preparation of linker-payload

[0271] Linker-Cytotoxin X1:

[0272] Synthesis route:

[0273] first step:

[0274] To a solution of 27a (5.00 g, 43.0 mmol) and NaHCO₃ (10.9 g, 129 mmol) in DMF (50 mL) was added benzyl bromide (11.0 g, 64.6 mmol) dropwise under nitrogen. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 2 / 1) indicated completion of the reaction. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). The mixture was separated, washed with saturated aqueous sodium chloride solution (500 mL), dried over anhydrous Na₂SO₄, and concentrated through a column chromatography (PE:EA = 3:2) to afford 5.1 g of a colorless liquid (yield: 57.1%).

[0275] Step 2:

[0276] Under nitrogen, a solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0°C. The mixture was allowed to react at 25°C for 2 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was added to 200 mL of water and extracted twice with EA (200 mL). The mixture was dried over anhydrous Na2SO4 and concentrated through a column chromatography (PE / EA = 3 / 2) to afford 1.56 g of a white solid (yield: 26%).

[0277] Step 3:

[0278] To a solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) was added Pd / C (80 mg) under hydrogen atmosphere at 0°C and stirred at 0°C for 2.5 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered through celite, washed with EA (200 mL), concentrated, and dissolved in THF (20 mL) and dried to afford 600 mg of a white solid (yield: 91%).

[0279] Step 4:

[0280] Under nitrogen, DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol) in DMF (6 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to aqueous citric acid (pH = 4) (150 mL), filtered, and the filter cake was washed with 175 mL of water, filtered, and pulled dry using an oil pump to afford 260 mg of a brown solid (yield: 66%).

[0281] Step 5:

[0282] Under nitrogen, diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 mL) at 0°C. The mixture was allowed to react for 3 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to 600 mL of petroleum ether at 0°C, resulting in the precipitation of a solid. After the solid was allowed to adsorb to the bottom of the flask, the solution was decanted and pumped dry with an oil pump to afford 90 mg of a brown solid (yield: 47.1%).

[0283] Step 6:

[0284] Under nitrogen, HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) at 0°C and allowed to react for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added to a pH 4 aqueous citric acid solution (30 mL) at 0°C. A flocculent solid precipitated, which was filtered and purified by preparative chromatography (DCM / MecOH = 10 / 1) to afford 9.2 mg of a light yellow solid, X1, in a 6% yield.

[0285] MS m / z (ESI): 1074 [M+1].

[0286] H-NMR (400MHz, MeOD): 7.65 (d, 1H), 7.62 (s, 1H), 7.30-7.21 (m, 5H), 6.79 (s, 2H), 5.69-5.65 (m, 1H), 5.57(d, 1H), 5.43-5.10(m, 3H), 4.70(d, 2H), 4.48-4.39(m, 2H), 4.10-4.05(m, 1H), 4.01-3 .75(m, 5H), 3.46(t, 2H), 3.22-3.15(m, 2H), 3.07-3.00(m, 1H), 2.75(m, 1H), 2.62(m, 1H), 2.45( s, 3H), 2.37-2.20 (m, 6H), 2.10-2.02 (m, 2H), 2.00-1.92 (m, 2H) 1.68-1.57 (m, 6H), 1.01 (t, 3H).

[0287] Linker-Cytotoxin X2:

[0288] Synthesis route:

[0289] first step

[0290] 34a (5 g, 48.0 mmol) and K2CO3 (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 3 / 1) confirmed the complete reaction. The reaction solution was added to water (200 mL) and extracted with EA (250 mL). The mixture was washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated to give 8.7 g of 34b as a colorless liquid (93% yield). MS-ESI: m / z 195.1 [M+H]+.

[0291] Step 2

[0292] 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL) under nitrogen and cooled to 0°C. A solution of 43b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise. After complete addition, the mixture was allowed to react at 0°C for 2 hours. TLC (PE / EA = 2 / 1) showed that most of the starting material had reacted. The reaction solution was poured into 100 mL of water and extracted with DCM (100 mL). The mixture was separated and washed with saturated NaCl, dried over anhydrous Na2SO4, and filtered through a column (PE / EA = 1 / 1) to give 3.9 g of a colorless viscous product 34d (yield: 39%). MS-ESI: m / z 503.3 [M+H]+.

[0293] Step 3

[0294] To a solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL) was added Pd / C (1 g, 10 wt.%) under hydrogen at 0°C for 3 hours. TLC (PE / EA = 2 / 1) indicated the reaction was complete. The reaction mixture was filtered through celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL x 3). The filtrate was concentrated and dissolved in THF (50 mL x 3) and the mixture was dried by spin drying. This was repeated three times to afford 1 g of a gray solid 34e in a 64% yield. MS-ESI: m / z 435.2 [M+Na]+.

[0295] Step 4

[0296] Under nitrogen, DIEA (303 mg, 2.35 mmol) was added dropwise to a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) in DMF (20 mL) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, allowed to stand for 5 minutes, and filtered. The filter cake was dissolved in DCM / MeOH (10:1, 100 mL), dried, and then spin-dried. The sample was purified by column chromatography (EA:MeOH = 30:1) to afford 600 mg of 34f as a yellow solid, in a 77% yield. MS-ESI: m / z 830.3 [M+H]+.

[0297] Step 5

[0298] Under nitrogen, diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0°C. The mixture was allowed to react for 2 hours. LCMS indicated the reaction was complete. Petroleum ether (100 mL x 6) was added to the reaction mixture, causing solid precipitation. After allowing the solid to settle, the solution was decanted and pumped dry using an oil pump to yield 120 mg (34 g) of a white powder. LCMS indicated a 70% product content, for a yield of 76%. MS-ESI: m / z 608.3 [M+H]+.

[0299] Step 6

[0300] Under nitrogen, a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated complete reaction of the starting materials. The reaction solution was directly purified by a reverse phase column using an eluent (MeCN / MeOH = 1 / 1): HO = 60%: 40%) to afford 14.8 mg of a yellow solid, X2, in a 14% yield.

[0301] MS-ESI: m / z 1062.4[M+H]+.

[0302] 1H NMR (400MHz, Methanol-d4) δ7.69-7.61 (m, 2H), 7.22-7.16 (m, 2H), 7.16-7.09 (m, 3H), 6.76 (s, 2H), 5.70-5.64 (m, 1H), 5.6 0 (d, J=16.4Hz, 1H), 5.40-5.31 (m, 2H), 5.26 (d, J=19.0Hz, 1H), 4.65-4.50 (m, 7H), 4.25-4.16 (m, 1H), 3.87 (d, J=16.7Hz, 1 H), 3.83-3.76 (m, 3H), 3.72 (d, J=17.0Hz, 2H), 3.44 (t, J=7.1Hz, 2H), 3.25-3.17 (m, 2H), 3.10-3.02 (m, 1H), 2.92-2.83 (m, 1H), 2.45-2.39 (m, 5H), 2.32-2.20 (m, 5H), 1.97-1.89 (m, 2H), 1.63-1.50 (m, 4H), 1.34-1.20 (m, 6H), 0.99 (t, J=7.3Hz, 3H).

[0303] Linker-Cytotoxin X3:

[0304] Synthesis route:

[0305] first step:

[0306] To a solution of 32a (2.00 g, 6.6 mmol) and KCO (1.82 g, 13.2 mmol) in MeCN (20 mL) was added allyl bromide (960 mg, 7.92 mmol), and the mixture was stirred at 20°C for 5 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (PE / EA = 2 / 1) to afford 1.83 g of 32b as a white solid (yield: 81%).

[0307] Step 2:

[0308] To a solution of 32b (1.38 g, 4.02 mmol) in DCM (10 mL) was added TFA (10 mL) and stirred at 25°C for 17 hours. TLC (PE / EA = 1 / 3) indicated the reaction was complete. The reaction solution was dried to afford 0.91 g of a yellow viscous product 32c. The yield was negligible.

[0309] Step 3:

[0310] To a solution of 32c (910 mg, 4.87 mmol) and NaHCO₃ (613 mg, 7.3 mmol) in DME / H₂O (20 mL / 10 mL) was added 41d (1.92 g, 4.87 mmol) and stirred at 25°C for 3 hours. TLC (DCM / MeOH = 1 / 1) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5 with aq. HCl (1 N), and the mixture was extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (DCM / MeOH = 20 / 1) to afford 1.53 g of 32e as a white solid in a 67% yield. MS-ESI: m / z 467.4 [M+H]⁺.

[0311] Step 4:

[0312] To a solution of 32f (3 g, 5.83 mmol) in MeOH (50 mL) was added Pd / C (600 mg) and stirred at 25°C under a hydrogen balloon for 5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.9 g of a white solid (32g, yield: 77%).

[0313] Step 5:

[0314] HATU (707 mg, 1.86 mmol) was added to 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL). The mixture was stirred at 0°C for 3.5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into H2O (80 mL), extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (EA) to afford 1.186 g of a white solid (32 h yield: 83%). MS-ESI: m / z 842.3 [M+H]+.

[0315] Step 6:

[0316] A solution of 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was poured into petroleum ether (200 mL) and filtered to afford 768 mg of 32i as a white solid in an 88% yield. MS-ESI: m / z 620.3 [M+H]+.

[0317] Step 7:

[0318] To a solution of 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol) in DMF (10 mL) was added HATU (414 mg, 1.09 mmol), and the mixture was stirred at 20°C for 17 hours. TLC (PE / EA = 1 / 5) indicated the reaction was complete. The reaction solution was poured into water (30 mL) and filtered. The filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to afford 511 mg of 32j as a white solid in a 44% yield. MS-ESI: m / z 1068.3 [M+H]+.

[0319] Step 8:

[0320] A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred at 10°C for 17 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to afford 32k as a white solid (301 mg). The yield was negligible.

[0321] Step 9:

[0322] To a solution of 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL) was added morpholine (93 mg, 1.07 mmol) and stirred at 25°C for 5 hours. LCMS indicated the reaction was complete. The reaction mixture afforded 108 mg of 32l as a white solid, yield: 38%. MS-ESI: m / z 806.3 [M+H]+.

[0323] Step 10:

[0324] To a solution of 321 (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL) was added bromoacetyl bromide (27 mg, 0.134 mmol), and the mixture was stirred at 0°C for 1 hour. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was directly used to obtain 15 mg of a white solid, X3, in a 12% yield.

[0325] MS-ESI: m / z 926.3[M+H]+.

[0326] 1H NMR (400MHz, DMSO-d6) δ12.11 (s, 1H), 8.54-8.42 (m, 3H), 8.27-8.16 (m, 2H), 7.78 (d, J=11.0Hz, 1H), 7.3 0(s, 1H), 6.53(s, 1H), 5.61-5.51(m, 1H), 5.42(s, 2H), 5.20-5.05(m, 2H), 4.56-4.42(m, 2H), 4.32-4.22 (m, 1H), 3.96-3.87 (m, 3H), 3.79 (d, J=5.6Hz, 2H), 3.70 (d, J=5.9Hz, 2H), 3.253.08 (m, 2H), 2.61-2.53 (m , 2H), 2.45-2.36 (m, 4H), 2.36-2.22 (m, 3H), 2.20-2.03 (m, 4H), 1.99-1.68 (m, 4H), 0.87 (t, J=7.3Hz, 3H).

[0327] Linker-Cytotoxin X4:

[0328] Synthesis route:

[0329] first step:

[0330] To a solution of 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) was added Pd / C (400 mg, 10 wt.%) and stirred at 20°C for 5 h. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.3 g of 33b as a white solid (yield: 74%).

[0331] Step 2:

[0332] To a solution of 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL) was added HATU (305 mg, 0.802 mmol), and the mixture was stirred at 0°C for 2 hours. TLC (DCM / MeOH = 1 / 10) indicated the reaction was complete. The reaction solution was poured into water (40 mL) and filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to afford 33c as a yellow solid (360 mg, 41% yield).

[0333] Step 3:

[0334] To a solution of 33c (360 mg, 0.326 mmol) in DCM (10 mL) was added diethylamine (2 mL). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) indicated the reaction was complete. The reaction solution was poured into PE (100 mL) and filtered to afford 205 mg of 33d as a white solid in a 71% yield. MS-ESI: m / z 881.3 [M+H]+.

[0335] Step 4:

[0336] To a solution of 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL) was added a solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL), and the mixture was stirred at 0°C for 1 hour. The reaction solution directly produced 15 mg of a white solid X4, with a yield of 6%.

[0337] MS-ESI: m / z 1001.2[M+H]+.

[0338] 1H NMR (400MHz, DMSO-d6) δ 8.57-8.50 (m, 1H), 8.50-8.43 (m, 2H), 8.35-8.29 (m, 1H), 8.198.12 (m, 2H), 7. 80(d, J=10.8Hz, 1H), 7.27-7.14(m, 7H), 6.53(s, 1H), 5.59-5.51(m, 1H), 5.44-5.39(m, 2H), 5.20-5.07 (m, 2H), 4.56-4.44 (m, 3H), 3.92 (s, 3H), 3.80-3.68 (m, 5H), 3.41 (s, 1H), 3.21-3.12 (m, 2H), 2.83-2.7 4 (m, 1H), 2.58-2.55 (m, 3H), 2.39 (s, 4H), 2.18-2.03 (m, 4H), 1.93-1.78 (m, 2H), 0.87 (t, J=7.3Hz, 3H).

[0339] 2.2 Preparation of Bispecific Antibody-Drug Conjugates

[0340] Preparation of bispecific antibody-drug conjugate DSYE001-X1 (DAR4):

[0341] At 37°C, the prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0 mM, 0.215 mL, 1.503 μmol) was added to the buffer of the bispecific antibody DSYE001 (30 mM histidine-acetate + 20 mM EDTA pH 5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), placed in a constant temperature water bath shaker, and oscillated at 37°C for 2 hours to stop the reaction.

[0342] The linker-cytotoxin X1 (3.10 mg, 2.89 μmol) was dissolved in 1.0 mL DMA (N, N-Dimethylacetamide) and added to the above antibody solution. The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid pH 5.5) and concentrated by ultrafiltration to obtain a solution of the bispecific antibody drug conjugate ADC DSYE001-X1 (DAR4) (30 mM histidine-hydrochloric acid pH 5.5; 93.8 mg, 12.7 mg / mL, yield: 93.8%), which was stored at 4°C in the dark.

[0343] The HIC DAR analysis detected and calculated the DAR value p=4.02.

[0344] Preparation of bispecific antibody-drug conjugate DSYE001-X1 (DAR6):

[0345] A solution of antibody DSYE001 (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10,000 mg, 20.52 mg / mL, 57.80 μmol) was added to a reaction vessel. Reducing buffer (20 mM PB, pH 6.7, 93.59 mL) was added, followed by 10 mM DTPA solution (66.67 mL). The pH of the reaction solution was adjusted to 6.72 using 0.4 M Na2HPO4 solution (7.0 mL). The prepared tris(2-carbonylethyl)phosphine hydrochloride solution (10 mM, 19.08 mL, 190.74 μmol) was then added to the reaction vessel. The reaction was stirred at 50-100 rpm and the reaction temperature was controlled at 25°C for 3.5 h. The pH of the reaction solution was adjusted to 5.5 using 0.5 M NaH2PO4 solution (200 mL). The reaction solution was then cooled to 20°C. DMSO (32.06 mL) was then added to the reaction system. Linker-cytotoxin X1 (465.7 mg, 433.56 μmol) was dissolved in 43.35 mL of DMSO and added to the solution. The reaction was stirred at 50-100 rpm and maintained at 22°C for 1.25 hours. The prepared NAC solution (50 mM, 43.35 mL, 2.17 mM) was then added to the reaction solution. The reaction was stirred at 50-100 rpm and maintained at 22°C for 0.5 hours. The feed solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm2), and then ultrafiltration was performed using an ultrafiltration membrane package (30KD, 0.11 m2) to obtain the exemplary product DSYE001-X1 (DAR6) (10 mM histidine-succinate, pH 5.0, 14.3 mg / ml), which was stored at -80°C.

[0346] The DAR value detected and calculated by HIC DAR analysis was n=6.1.

[0347] Preparation of bispecific antibody-drug conjugate DSYE001-X1 (DAR8):

[0348] A solution of antibody DSYE001 (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10,000 mg, 20.52 mg / mL, 57.80 μmol) was added to a reaction vessel. Reducing buffer (45 mM PB, pH 7.35, 72.2 mL) was added, followed by 10 mM DTPA solution (66.67 mL). Finally, a prepared tris(2-carbonylethyl)phosphine hydrochloride solution (10 mM, 40.46 mL, 404.6 μmol) was added to the reaction vessel. The mixture was stirred at 50-100 rpm and the reaction temperature was controlled at 25°C for 4.5 hours. The pH of the reaction solution was adjusted to 5.5 using 0.5 M NaH2PO4 solution (200 mL). The reaction solution was then cooled to 20°C. DMSO (63.40 mL) was then added to the reaction system. Linker-cytotoxin X1 (651.9 mg, 606.91 μmol) was dissolved in 60.69 mL of DMSO and added to the solution. The reaction was stirred at 50-100 rpm and maintained at 22°C for 1.25 hours. The prepared NAC solution (50 mM, 60.69 mL, 3.03 mM) was then added to the reaction solution. The reaction was stirred at 50-100 rpm and maintained at 22°C for 0.5 hours. The feed solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm2), and then ultrafiltration was performed using an ultrafiltration membrane package (30KD, 0.11 m2) to obtain the exemplary product DSYE001-X1 (DAR8) (10 mM histidine-succinate, pH 5.0, 29.5 mg / ml), which was stored at -80°C.

[0349] The DAR value detected and calculated by HIC DAR analysis was n=7.9.

[0350] Preparation of anti-B7H3 antibody-drug conjugate DSYE003-X1 (DAR6) (refer to ADC-1)

[0351] To the buffer solution of antibody DSYE003 (PBS pH 7.4; 40 mg, 5.36 mg / mL, 0.23 μmol), 30 mM His-HAc pH 5.5 buffer (1.845 mL) was added, followed by 100 mM EDTA solution (0.5 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (6.977 mM, 0.208 mL, 1.45 μmol). The mixture was placed in a thermostatic stirrer at 500 rpm for 2 hours at 37°C, and the reaction was stopped. 0.750 mL of DMA was added to the above solution, and then linker-cytotoxin X1 (2.5 mg, 2.32 μmol) was dissolved in 0.25 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 500 rpm for oscillation at 4°C for 1 hour, and the reaction was stopped. A 300 mg / mL activated carbon suspension (Charcoal, Dextran Coated, Sigma-Aldrich) was added to 10% of the total volume of the reaction solution, mixed by vortexing, and then placed on a rotary mixer at 4°C for 1 hour. After 1 hour, the supernatant was centrifuged and then added with 10% of the total volume of the activated carbon suspension. The mixture was then mixed by vortexing and then placed on a rotary mixer at 4°C for 1 hour. After centrifugation at 4300 rcf for 10 minutes, the supernatant was filtered through a 0.22 μm syringe filter (Merck Millipore) and concentrated by four DV ultrafiltration using 30 mM His-HAc pH 5.5 buffer in a 30 kDa ultrafiltration tube to obtain a solution of the exemplary product DSYE003-X1DAR6 (30 mM His-HAc pH 5.5; 35.89 mg, 6.14 mg / mL, yield: 89.74%), which was stored at -80°C.

[0352] The DAR value detected and calculated by HIC DAR analysis was n=6.32.

[0353] Preparation of anti-B7H3 antibody-drug conjugate DSYE002-X1 (DAR6) (refer to ADC-2)

[0354] Using linker-cytotoxin X1 and anti-B7H3 antibody DSYE002, the preparation method was referenced to the bispecific antibody drug conjugate ADC DSYE001-X1 to prepare DSYE002-X1 (DAR6).

[0355] Preparation of anti-PD-L1 antibody-drug conjugate DSYE004-X1 (DAR6) (referring to ADC-3)

[0356] Using linker-cytotoxin X1 and anti-PD-L1 antibody DSYE004, the preparation method was based on the bispecific antibody drug conjugate ADC DSYE001-X1 to prepare DSYE004-X1 (DAR6).

[0357] Preparation of bispecific antibody-drug conjugate DSYE001-X2:

[0358] At 37°C, the prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0 mM, 0.207 mL, 1.449 μmol) was added to the buffer of the bispecific antibody DSYE001 (30 mM histidine-acetate + 20 mM EDTA pH 5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), placed in a constant temperature water bath shaker, and oscillated at 37°C for 2 hours to stop the reaction.

[0359] The linker-cytotoxin X2 (3.07 mg, 2.89 μmol) was dissolved in 1.0 mL DMA (N, N-Dimethylacetamide, dimethylacetamide), added to the above antibody solution, placed in a water bath shaker, and shaken at 22°C for 2 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration were performed to obtain a solution of the bispecific antibody drug conjugate ADCDSYE001-X2 (30 mM histidine-hydrochloric acid pH 5.5; 96.5 mg, 15.8 mg / mL, yield: 96.5%), which was stored at 4°C in the dark.

[0360] The HIC DAR analysis detected and calculated the DAR value p=3.95.

[0361] Preparation of anti-B7H3 antibody-drug conjugate DSYE002-X2 (referring to ADC-4)

[0362] Antibody DSYE002 serves as a reference for the bispecific antibody DSYE001, which removes PD-L1. The amino acid sequence of DSYE002 is shown in the sequence listing.

[0363] At 37°C, prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0 mM, 1.08 mL, 7.55 μmol) was added to the buffer of anti-B7H3 antibody DSYE002 (30 mM histidine-acetate + 20 mM EDTA pH 5.5; 60 mg, 5.5 mg / mL, 0.755 μmol), and the mixture was placed in a constant temperature water bath shaker and shaken at 37°C for 2 hours to stop the reaction.

[0364] Linker-cytotoxin X2 (10.8 mg, 9.82 μmol) was dissolved in 1.2 mL of DMA and added to the above solution. The mixture was placed in a water bath shaker and shaken at 4°C for 1 hour to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid pH 5.5) and concentrated by ultrafiltration to obtain a solution of the exemplary product DSYE002-X2 (30 mM histidine-hydrochloric acid pH 5.5; 30.5 mg, 7.5 mg / mL, yield: 50.8%), which was stored at 4°C in the dark.

[0365] The HIC DAR analysis detected and calculated the DAR value p=3.98.

[0366] Preparation of isotype control ADC

[0367] Using isotype control antibodies and linker-cytotoxin X1, referring to the preparation method of the bispecific antibody drug conjugate ADC DSYE001-X1, isotype control ADC (DAR4), isotype control ADC (DAR6) and isotype control ADC (DAR8) were prepared respectively.

[0368] Example 3: Endocytic activity of antibody drug conjugates

[0369] Purpose of the test

[0370] The endocytosis of the anti-B7H3 and PD-L1 bispecific antibody-drug conjugates of the present invention was tested in A375 and NCI-H1975 cells that co-express B7H3 and PD-L1. The cells were incubated with a fixed concentration of the test drug and the endocytosis indicator reagent pHrodo. The endocytosis ability of the test drug was evaluated by observing the fluorescence signal produced by pHrodo upon entry of the antibody drug into the cells at different time points.

[0371] Experimental methods:

[0372] 1. Add A375 and NCI-H1975 cells at a density of 1.2E4 / well into a 95-well black culture plate that had been pretreated with 8 μg / ml of 5% CO2 and culture overnight at 37°C in 5% CO2.

[0373] 2. The drug to be tested was mixed with Fab-pHrodo at a ratio of 1:1.2 and incubated at 37°C, 5% CO2 for 0.5 h. The mixture was diluted 4-fold starting from 100 nM to 0.0061 nM.

[0374] 3. Add the diluted sample to the culture plate at 50 μL / well and incubate at 37°C, 5% CO2 for 24 hours.

[0375] 4. After incubation, remove the supernatant and rinse the cell culture plate once with 1% BSA.

[0376] 5. Add 1 μg / mL Hoechst 33342 and 0.5 μg / mL Calcein AM in DPBS to the culture plate at 100 μL / well for staining and incubate at room temperature for 15 minutes.

[0377] 6. After incubation, remove the supernatant and rinse the cell culture plate once with 1% BSA.

[0378] 7. The fluorescent spots formed by internalized antibodies in each cell were counted using the Perkin Elmer Operetta CLS High-Content Analysis System.

[0379] The results are shown in Table 1 and Figure 15.

[0380] Table 1. Endocytic activity of DSYE001-X2 cells

[0381] The experimental results showed that compared with the anti-B7H3 antibody-drug conjugate DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of the present application had a better endocytosis effect in the test cells, for example, a higher maximum mean fluorescence point number or a lower EC50 value.

[0382] Example 4: Antibody-drug conjugates inhibit tumor cell proliferation in vitro

[0383] use The chemiluminescence cell viability assay (i.e., CTG method) was used to evaluate the inhibitory effect of the anti-B7H3 / PD-L1 bispecific antibody drug conjugate DSYE001-X2 and the anti-B7H3 antibody drug conjugate DSYE002-X2 on cell proliferation after incubation for 6 days in A375 and NCI-H1975 cells expressing B7H3 and PD-L1.

[0384] Logarithmically growing cells were harvested and plated at 400 cells / well for A375 and 1000 cells / well for NCI-H1975, respectively. 50 μL of the cell plates were plated per well and incubated overnight at 37°C, 5% CO2. On the second day of the experiment, DSYE001-X2 and DSYE002-X2 were diluted three-fold in complete medium to create an eight-dose concentration gradient (starting at 1000 nM). 50 μL of the drug was then added to the cell culture plates, with complete medium serving as a blank control. Two replicates were set up. Incubation was continued at 37°C, 5% CO2 for 6 days. After incubation, the plates were removed and equilibrated to room temperature. 50 μL of CTG detection reagent (Promega, Cat# G7573) was added to each well. The mixture was mixed by vortexing and allowed to stand in the dark for 10 minutes before the signal was measured using a microplate reader. GraphPad Prism software was used to plot sigmoidal dose-response curves using a nonlinear regression model and calculate the IC 50 Cell viability calculation formula = (Lum 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 )×100%.

[0385] The experimental results showed that compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of the present application had comparable proliferation inhibitory activity against A375 and NCI-H1975 cells that positively expressed B7H3 and PD-L1.

[0386] Example 5: Antibody-drug conjugates inhibit tumor cell proliferation in vitro

[0387] use The chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of anti-DSYE001-X1 (DAR8) and isotype control ADC (DAR8) on cell proliferation after incubation with human tumor cells positive for B7H3 and PD-L1 for 7 days.

[0388] Cells in the logarithmic growth phase were collected and plated at a density of 1000-3000 cells / well. The cell plates were placed in a 37°C, 5% CO2 incubator for overnight culture. On the second day of the experiment, DSYE001-X1 (DAR8) was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 1000nM). After the drug was added, 50μL / well was added to the cell culture plate. Complete culture medium was used as a blank control, and 3 replicates were set up; the plate was continued to be incubated in a 37°C, 5% CO2 incubator for 6 days. After the incubation was completed, the cell culture plate was removed and equilibrated to room temperature. 50μL of CTG detection reagent was added to each well. After shaking and mixing, it was placed in the dark for 10 minutes, and the signal value was read using an enzyme reader. GraphPad Prism software was used to draw an S-type dose-response curve using a nonlinear regression model and calculate the IC 50 Cell viability calculation formula = (Lum 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 )×100%.

[0389] The results are shown in Table 2 and Figure 16.

[0390] Table 2. Inhibitory effect of DSYE001-X1 (DAR8) on tumor cell proliferation in vitro

[0391] The experimental results showed that on various tumor cells, the bispecific antibody-drug conjugate DSYE001-X1 of the present application had significant proliferation inhibitory activity against A375, NCI-H1975, NCI-H441, and NCI-H358 cells that positively expressed B7H3 and PD-L1.

[0392] Example 6: Effect of Antibody Drug Conjugates on PD-L1 Expression in NCI-H1975 Cells

[0393] The purpose of this study was to investigate the differences in the effects of bispecific antibody-drug conjugates compared with their parental monoclonal antibody conjugates on PD-L1 expression in tumor cells.

[0394] Experimental methods

[0395] 1) NCI-H1975 tumor cells were cultured in RPMI1640+10% FBS medium in a 37°C 5% CO2 constant temperature incubator.

[0396] 2) Treat the cells with trypsin and count them to ensure that the cell viability is above 90.0%. Add 2×10 6 cells / 2 mL to each well of a 6-well plate.

[0397] 3) On the second day, culture medium (blank control), isotype control antibody, isotype control antibody ADC, DSYE001-X1 (DAR6), DSYE003-X1 (DAR6) and Anti-PDL1-X1 (final concentration of 100 nM) were added and incubated at 37°C for 72 hours.

[0398] 4) After incubation, wash the cells twice with PBS, add 500 μL of ice-cold RIPA lysis buffer (with 1% protease inhibitor and 1% phosphatase inhibitor) to each culture plate (six-well plate), and incubate on ice for 30 minutes, mixing several times.

[0399] 5) Centrifuge at approximately 14,000 rpm for 10 minutes at 4°C to remove cell debris. Transfer the supernatant to a fresh tube for protein concentration determination and subsequent experiments.

[0400] 6) Quantify protein using a BCA quantification kit. Based on the results, prepare the protein sample for loading to a uniform protein concentration of 1-2 μg / μL. Add LDS loading buffer (4X) and sample reducing agent (10X). Heat the sample at 100°C for 10 minutes.

[0401] 7) Alternatively, store the denatured samples in a -80°C refrigerator.

[0402] 8) Thaw the loaded sample.

[0403] 9) Western Blot: Load 10 μL of sample into each well of an SDS-PAGE gel (the amount of sample loaded depends on the antibody titer); electrophoresis at 80 V for 30 minutes, followed by 120 V for 90 minutes; transfer to the membrane using the iBlot2 transfer kit and a transfer apparatus, using the P3 program for 7 minutes.

[0404] 10) After transfer, cut the membrane according to the molecular weight of the protein to be detected and wash the membrane with 1x TBST three times, 5 minutes each time, at room temperature with shaking.

[0405] 11) Blocking: The membrane was placed in blocking solution (5% skim milk prepared with 1xTBST) for blocking at room temperature with shaking for 1 hour.

[0406] 12) Wash the membrane with 1x TBST three times, 5 minutes each time, at room temperature with shaking.

[0407] 13) Incubation with primary antibody: Add primary antibody of appropriate dilution (diluted with 5% bovine serum albumin in 1xTBST) and incubate at 4°C overnight with slow shaking.

[0408] 14) Wash the membrane with 1x TBST three times, 10 minutes each time, at room temperature with shaking.

[0409] 15) Incubation with secondary antibody: Add secondary antibody of appropriate dilution and incubate at room temperature with slow shaking for 1 hour.

[0410] 16) Wash the membrane with 1x TBST three times, 10 minutes each time, at room temperature with shaking.

[0411] 17) Chemiluminescence: HRP substrate from the West Femto Ultra-Sensitive Chemiluminescence Kit was added to the membrane.

[0412] 18) Detect chemiluminescence and take photos using a Bio-Rad ChemiDoc™ xrs+ or Tanon 5200 Multi instrument.

[0413] 29) Detection indicators: PD-L1, β-actin.

[0414] The results are shown in Table 3 and Figure 17.

[0415] Table 3. Effect of DSYE001-X1 (DAR6) on PD-L1 expression in NCI-H1975 cells

[0416] The results showed that the bispecific antibody-drug conjugate DSYE001-X1 could more significantly induce downregulation of PD-L1 expression than the parent B7H3 single-arm antibody conjugate DSYE003-X1 and the parent PD-L1 antibody conjugate anti-PD-L1-X1 (DSYE004-X1), suggesting that the bispecific antibody-drug conjugate DSYE001-X1, by jointly binding to two targets, not only exerts an immunomodulatory effect by inhibiting binding to the corresponding receptors, but also can exert a stronger immunosuppression-relieving effect by reducing target expression.

[0417] Example 7: Evaluation of the efficacy of antibody drug conjugates in a syngeneic transplant mouse model

[0418] To investigate the inhibitory effect of the anti-B7H3 and PD-L1 bispecific antibody drug conjugate of the present invention on tumor growth, a syngeneic transplant mouse model was established in B7H3 and PD-L1 humanized BALB / c mice (BALB / c-hPD-L1 / hB7H3) using CT26 stably expressing human B7H3 and PD-L1 (CT26-hPD-L1 / hB7H3) to evaluate the anti-tumor effect of the bispecific antibody drug conjugate DSYE001-X2.

[0419] 1. Test drugs and materials

[0420] Isotype control ADC: 10 mg / kg

[0421] G1: blank control group (control group): normal saline

[0422] G2: DSYE002-X2 (refer to ADC-4, treatment group): 12 mg / kg

[0423] G3: DSYE001-X2 (treatment group): 5.5 mg / kg

[0424] Note: Each test substance was administered in equimolar amounts.

[0425] 2. Preparation method: All samples were diluted with physiological saline.

[0426] 3. Experimental animals: BALB / c-hPD-L1 / hB7-H3 female mice, 6-8 weeks old, weighing approximately 18-22 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0427] 4. Test method:

[0428] CT26-hPD-L1 / hB7H3 cells were revived and cultured, and the cell freezing batch was recorded. CT26-hPD-L1 / hB7H3 cells in the logarithmic growth phase (passage 3-4 after resuscitation) were collected, the culture medium was removed, and the cells were washed twice with DPBS before inoculation (cell survival rate was tested before and after tumor loading). The inoculation volume was 1×10 6 cells / 100μL / mouse (without adding matrix gel) were inoculated subcutaneously on the right side of the mouse. Grouping was carried out when the tumor volume reached 80-120mm3. The day of grouping was defined as D0, and drug administration began on D0. The coefficient of variation CV of tumor volume did not exceed 1 / 3. Isotype control ADC, DSYE001-X2 and DSYE002-X2 were injected intravenously (iv) once a week for a total of 2 injections, with doses of 10mg / kg, 12mg / kg and 5.5mg / kg respectively. The experiment was terminated after 19 days of administration. Tumor volume and body weight were measured twice a week and the data were recorded. The tumor inhibition rate was calculated by measuring the tumor volume.

[0429] At the end of the experiment, the mice were euthanized and the tumor inhibition rate TGI was calculated (TGI (%) = [1-(T i -T0) / (V i -V0)]×100). T i : mean tumor volume of the treatment group and the positive control group on the i-th day of administration; T0: mean tumor volume of the treatment group and the positive control group on the 0th day of administration; V i : the mean tumor volume of the negative control group on the i-th day of administration; V0: the mean tumor volume of the negative control group on the 0th day of administration.

[0430] The experimental results are shown in Figure 18 and Tables 4-6.

[0431] Table 4. Tumor volume of each group at different time points 1 (mm3 )

[0432] Note:

[0433] 1. Tumor volume is expressed as mean ± standard error;

[0434] 2. Days after starting medication.

[0435] Table 5. Changes in tumor volume inhibition rate (TGI) of the test substance in the CT26 (CT26-hPD-L1 / hB7H3) homologous transplant mouse model TV )

[0436] Note: TGI TV All calculations were compared with G1.

[0437] Table 6. Statistical analysis of the tumor volume in different groups of the CT26 (CT26-hPDL1 / hB7H3) homologous transplant mouse model

[0438] Note: Independent sample T test was used, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0439] The experimental results showed that compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of the present invention exhibited significantly enhanced tumor growth inhibitory activity after administration.

[0440] Example 8: Evaluation of the efficacy of antibody drug conjugates in human non-small cell lung cancer cell NCI-H1975 tumor-bearing mice

[0441] To investigate the inhibitory effect of DSYE001-X1(DAR4) on tumor formation in vivo, a subcutaneous xenograft tumor model was constructed in mice using human lung cancer NCI-H1975 cells mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1(DAR4).

[0442] NCG mice, female, 6-8 weeks old, weighing approximately 18-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. NCI-H1975 was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. NCI-H1975 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. Frozen PBMCs were purchased, resuscitated and counted, and the resuspended PBMCs were added to NCI-H1975 cells. PBMCs were co-cultured with NCI-H1975 cells for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0443] After PBMCs were co-cultured with NCI-H1975 cells for 5 days, PBMCs were collected and freshly digested NCI-H1975 cells were used as the culture medium. 5 NCI-H1975 cells 2×10 6 0.2 mL / mouse (containing 50% matrix gel) was inoculated subcutaneously on the right side of NCG mice. When the tumor volume reached about 100-200 mm3, 20 mice were selected according to the tumor volume and randomly divided into 4 groups with 5 mice in each group. On the day of grouping (day 0), isotype control ADC, DSYE001-X1 (DAR4), reference ADC-DSYE002-X1 (DAR4) and DSYE002-X1 (DAR4) in combination with PDL1 monoclonal antibody were injected intravenously (iv.) twice a week for 2 weeks, for a total of 4 times. The dosage was 18 mg / kg for dual-antibody ADC and 15 mg / kg for isotype control ADC and PDL1 monoclonal antibody (equimolar dose). The experiment was terminated on the 28th day. The experimental grouping and dosing are as follows. The tumor volume and mouse body weight were measured twice a week and the data were recorded.

[0444] Isotype control ADC: 15 mg / kg

[0445] DSYE001-X1(DAR4) (treatment group): 18 mg / kg

[0446] DSYE002-X1(DAR4) (treatment group): 8 mg / kg

[0447] DSYE002-X1 (refer to ADC-2, DAR4) + anti-PD-L1 monoclonal antibody DSYE004 (treatment group): 8 mg / kg + 15 mg / kg

[0448] All samples were diluted with PBS.

[0449] Relative tumor growth rate (T / C) (%): Calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). Relative tumor volume (RTV) was calculated based on tumor measurement results using the formula: RTV = Vt - V0, where V0 is the average tumor volume measured at the time of group dosing (i.e., D0), and Vt is the average tumor volume at a single measurement. TRTV and CRTV data were collected on the same day.

[0450] Tumor Growth Inhibition (TGI) (%) = (1 - T / C) x 100%. T / C% represents the relative tumor growth rate, i.e., the percentage of tumor volume in the treatment group relative to the control group at a specific time point. T and C represent the tumor volume (TV) in the treatment and control groups, respectively, at a specific time point.

[0451] At the end of the experiment, the mice were euthanized and the tumor weights were measured.

[0452] The experimental results are shown in Figure 19 and Tables 7-8.

[0453] Table 7. Average tumor volume of mice in each group (Mean ± SEM)

[0454] Note: Compared with the isotype control ADC, 15 mg / kg control group, **p < 0.01, *p < 0.05 is considered to be significantly different

[0455] Table 8. Average tumor weight of mice in each group (Mean ± SEM)

[0456] Note: Compared with the isotype control ADC, 15 mg / kg control group, **p < 0.01, *p < 0.05 is considered to be significantly different

[0457] The results showed that DSYE001-X1 could significantly inhibit tumor growth, and its anti-tumor activity was stronger than that of B7H3 monoclonal antibody ADC (DSYE002-X1). It had a better tumor inhibition effect than the combination of B7H3 monoclonal antibody ADC and PDL1 monoclonal antibody.

[0458] Example 9: Evaluation of the efficacy of antibody drug conjugates in mice bearing human breast cancer cells MDA-MB-231

[0459] To investigate the inhibitory effect of DSYE001-X1(DAR6) on tumor formation in vivo, a subcutaneous xenograft tumor model was constructed in mice using human breast cancer MDA-MB-231 cells mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1(DAR6).

[0460] Female NCG mice aged 6-8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used as experimental animals. MDA-MB-231 cells (provided by Jicui Yaokang, mycoplasma test results were negative) were revived and subcultured, and the revived generation was N+13. MDA-MB-231 cells in the logarithmic growth phase were collected (inoculation generation was N+17, the culture medium was removed and washed twice with DPBS before inoculation (cell survival rates before and after tumor loading were: 99.27% ​​and 97.00%, respectively), and the inoculation size was 5×106 / 100μL / mouse (1:1 Matrigel inoculation). One week after tumor cell inoculation, human PBMC were inoculated by tail vein injection. The inoculation dose was 7.5×10 6 / 100μL / mouse. On day 9 after inoculation, when the average tumor volume reached 85.44mm³, 12 mice were randomly divided into two groups of 6 mice each based on tumor volume. The day of grouping was defined as D0, and dosing began on D0 and continued on D7. The experiment ended on day 41. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0461] At the end of the experiment, the mice were euthanized and TGI was calculated. TV (Relative tumor inhibition rate). TGI TV The calculation formula for (relative tumor inhibition rate) is:

[0462] Among them, mean RTV treat : Mean RTV value of the drug-treated group;

[0463] mean RTV vehicle : Average RTV value of the Vehicle group (vehicle group, in this example, the group only administered with normal saline);

[0464] The calculation formula of the RTV is:

[0465] Among them, V nt : Tumor volume of mouse numbered n on day t;

[0466] V n0 : Tumor volume of mice numbered n on day 0;

[0467] RTV n : The relative tumor volume of the mouse numbered n on day t.

[0468] The experimental results are shown in Figure 20, Table 9 and Table 10.

[0469] Table 9. Changes in tumor volume in different groups

[0470] Table 10. Relative tumor inhibition rate (TGI) of different groups TV )

[0471] The results showed that DSYE001-X1 could significantly inhibit the growth of breast cancer cells.

[0472] Example 10: Evaluation of the efficacy of antibody drug conjugates in human non-small cell lung cancer cell NCI-H1975 tumor-bearing mice

[0473] To investigate the inhibitory effect of DSYE001-X1(DAR6) on tumor formation in vivo, a subcutaneous xenograft tumor model was constructed in mice using human lung cancer NCI-H1975 cells mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1(DAR6).

[0474] NCG mice, female, 6-8 weeks old, weighing approximately 18-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. NCI-H1975 was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. NCI-H1975 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. Frozen PBMCs were purchased, resuscitated and counted, and the resuspended PBMCs were added to NCI-H1975 cells. PBMCs were co-cultured with NCI-H1975 cells for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0475] After PBMCs were co-cultured with NCI-H1975 cells for 5 days, PBMCs were collected and freshly digested NCI-H1975 cells were used as the culture medium. 5 NCI-H1975 cells 2×10 6 0.2 mL / mouse (containing 50% matrix gel) was inoculated subcutaneously on the right side of NCG mice. When the tumor volume reached about 100-200 mm3, 15 mice were selected according to the tumor volume and randomly divided into 3 groups according to the tumor volume, with 5 mice in each group. On the day of grouping (day 0), DSYE001-X1 (DAR6) and DSYE003-X1 (DAR6) were injected intravenously (iv.) in combination with pembrolizumab twice a week for 2 weeks, for a total of 4 times. The dosages were 18 mg / kg for dual-antibody ADC and 15 mg / kg for isotype control ADC and pembrolizumab (equimolar doses). The experiment was terminated on the 21st day. The experimental groups and dosing were as follows. The tumor volume and mouse body weight were measured twice a week and the data were recorded.

[0476] Isotype control ADC: 15 mg / kg

[0477] DSYE001-X1(DAR6) (treatment group): 18 mg / kg

[0478] DSYE003-X1 (refer to ADC-1, DAR6) + pembrolizumab (treatment group): 18 mg / kg + 15 mg / kg

[0479] All samples were diluted with PBS. Pablolide beads were prepared using conventional methods, and the sequence was from Recommended INN list R72 (2014).

[0480] Relative tumor growth rate (T / C) (%): Calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). Relative tumor volume (RTV) was calculated based on tumor measurement results using the formula RTV = Vt - V0, where V0 is the average tumor volume measured at the time of group dosing (i.e., d0), and Vt is the average tumor volume at a single measurement. TRTV and CRTV data were collected on the same day.

[0481] Tumor Growth Inhibition (TGI) (%) = (1 - T / C) x 100%. T / C% represents the relative tumor growth rate, i.e., the percentage of tumor volume in the treatment group relative to the control group at a specific time point. T and C represent the tumor volume (TV) in the treatment and control groups, respectively, at a specific time point.

[0482] At the end of the experiment, the mice were euthanized and the tumor weights were measured.

[0483] The experimental results are shown in Figure 21 and Tables 11-12.

[0484] Table 11. Average tumor volume of mice in each group (Mean ± SEM)

[0485] Note: Compared with the isotype control ADC, 15 mg / kg control group, ****p < 0.0001 is considered to be significantly different.

[0486] Table 12. Average tumor weight of mice in each group (Mean ± SEM)

[0487] Note: Compared with the isotype control ADC, 15 mg / kg control group, ****p < 0.0001 is considered to be significantly different.

[0488] The results showed that DSYE001-X1 could significantly inhibit tumor growth and had a better tumor inhibition effect than the combination of B7H3 monoclonal antibody ADC and pembrolizumab.

[0489] Example 11: Pharmacokinetic and toxicity studies of antibody drug conjugates after multiple administration

[0490] Purpose of the test

[0491] DSYE001-X1(DAR6) was administered intravenously to cynomolgus monkeys once every three weeks for a total of two doses. The purpose was to observe the nature, extent, dose-effect, and time-effect relationships of potential toxic reactions induced by the antibody-drug conjugate and to determine the target organs or tissues of toxicity to provide a reference for subsequent studies.

[0492] Experimental methods

[0493] Four cynomolgus macaques (two per sex) were randomly divided into three groups based on body weight, with one macaque per sex per group. Groups 1 and 2 received the test article (DSYE001-X1(DAR6)) at 30 and 120 mg / kg, respectively, representing the low and high doses. The animals were administered via intravenous infusion at a volume of 10 mL / kg over approximately 30 minutes, once every three weeks for a total of two doses.

[0494] During the trial, the animals were observed clinically, and their body weight, food intake, body temperature, electrocardiogram, and clinical pathology (blood cell count, coagulation function, and blood biochemistry) were measured. One week after the last dose (D29), the animals were euthanized and subjected to gross anatomy and histopathology (gross abnormalities, bone and bone marrow) examinations. At the same time, the total antibody protein and DSYE001-X1 (DAR6) concentrations in the serum and the small molecule concentrations in the plasma of the animals dosed on D1 and D22 were measured, and toxicokinetic analysis was performed.

[0495] Table 13. Toxicokinetic parameters

[0496] Experimental Conclusion

[0497] During the trial, no animals in any dose group died or were near death. No abnormal changes related to the test article were observed in body weight, food intake, body temperature, electrocardiogram parameters and waveforms, clinical pathology (blood cell count, coagulation function, and blood biochemistry), gross anatomy, and histopathology (bone and bone marrow). This indicates that the antibody-drug conjugate has good safety.

[0498] Example 12: Evaluation of the efficacy of the antibody drug conjugate DSYE001-X1 (DAR8) in a syngeneic transplant mouse model

[0499] To study the inhibitory effect of DSYE001-X1 (DAR8) on tumor growth, a pharmacodynamic evaluation was performed in BALB / c-hPD-L1 / hB7H3 mice. For specific methods, refer to Example 7.

[0500] The antibody-drug conjugate DSYE001-X1 of the present application has better anti-tumor effect than B7H3 monoclonal antibody ADC or its combination with PD-L1 antibody.

[0501] Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention as claimed should not be limited to such specific embodiments. In fact, various modifications of the described modes for implementing the present invention will be apparent to those skilled in the art of molecular biology, immunology, or related fields and are intended to be within the scope of the appended claims.

[0502] Sequence Listing

[0503] DSYE001 amino acid sequence (the underlined part is CDR)

[0504] Light chain variable region of monoclonal antibody:

[0505] Heavy chain variable region of monoclonal antibody:

[0506] Heavy chain variable region of nanobody:

[0507] Connector peptide between monoclonal antibody and nanobody:

[0508] Heavy chain of monoclonal antibody:

[0509] Light chain of monoclonal antibody:

[0510] Heavy chain of bispecific antibody:

[0511] Light chain of bispecific antibody:

[0512] DSYE002 amino acid sequence (reference antibody)

[0513] DSYE003 heavy chain

[0514] DSYE003 light chain (HumanAnti-HIV-1gp120clone b12 light chain)

[0515] PD-L1 monoclonal antibody amino acid sequence (DSYE004)

[0516] PD-L1 monoclonal antibody heavy chain

[0517] PD-L1 monoclonal antibody light chain: the same as the light chain of the bispecific antibody

[0518] Pembrolizumab amino acid sequence

[0519] heavy chain

[0520] light chain

Claims

1. A bispecific antibody-drug conjugate, the structure of which comprises the following fragments: a bispecific antibody against B7H3 and PD-L1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug, wherein: The bispecific antibody or antigen-binding fragment thereof comprises: A monoclonal antibody unit, which is directed against PD-L1 and comprises 2 heavy chains and 2 light chains, A Nanobody unit, which is directed against B7H3 and comprises 2 identical Nanobodies, The N-termini of the two nanobodies are respectively connected to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit through connecting peptides.

2. The bispecific antibody drug conjugate according to claim 1, wherein: The light chain variable region of the monoclonal antibody unit includes a CDR1 with an amino acid sequence of SEQ ID NO.: 1, a CDR2 with an amino acid sequence of SEQ ID NO.: 2, and a CDR3 with an amino acid sequence of SEQ ID NO.: 3, the heavy chain variable region of the monoclonal antibody unit includes a CDR1 with an amino acid sequence of SEQ ID NO.: 5, a CDR2 with an amino acid sequence of SEQ ID NO.: 6, and a CDR3 with an amino acid sequence of SEQ ID NO.: 7, and the nanobody includes a CDR1 with an amino acid sequence of SEQ ID NO.: 12, a CDR2 with an amino acid sequence of SEQ ID NO.: 13, and a CDR3 with an amino acid sequence of SEQ ID NO.:

14.

3. The bispecific antibody-drug conjugate according to claim 1 or 2, wherein: The light chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 4, the heavy chain variable region of the monoclonal antibody unit comprises an amino acid sequence such as SEQ ID NO.: 8; and the nanobody comprises an amino acid sequence such as SEQ ID NO.:

15.

4. The bispecific antibody drug conjugate according to any one of claims 1 to 3, wherein: The monoclonal antibody comprises an immunoglobulin constant region, which is a human IgG constant region, such as a human IgG1 constant region.

5. The bispecific antibody drug conjugate according to any one of claims 1 to 4, wherein: The light chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO.: 10, and the Nanobody comprises the amino acid sequence of SEQ ID NO.: 15; or The full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO.:

15.

6. The bispecific antibody drug conjugate according to any one of claims 1 to 5, wherein: The connecting peptide is a polypeptide containing glycine and serine and having certain elasticity and protease resistance. Preferably, the amino acid sequence of the connecting peptide is SEQ ID No.:

11.

7. The bispecific antibody drug conjugate according to any one of claims 1 to 6, wherein: The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.:

9.

8. The bispecific antibody-drug conjugate according to any one of claims 1 to 7, wherein the cytotoxic drug is a structure represented by formula (A-1), a tautomer, an enantiomer, a diastereomer, or a mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof, in, M is -L 2 -L 1 -C(O)-; L 2 is -O- or -S-, preferably -O-, and L 2 Connected to the connector unit L; L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a replace; m is 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3; R 1a and R 1b Each is independently hydrogen, halogen, hydroxyl, amino or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a is halogen, hydroxy, amino or C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted by one or more halogens.

9. The bispecific antibody drug conjugate according to claim 8, wherein: L 1 -(C(R 1a )(R 1b )) m -CH2-; each R 1a Each R is independently hydrogen, halogen or C1-C6 alkyl; 1b Each is independently hydrogen, halogen or C1-C6 alkyl; Preferably, L 1 -(C(R 1a )(R 1b )) m -CH2-; each R 1a Each R is independently a C1-C6 alkyl group, preferably a C1-C3 alkyl group; 1b Each is independently hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C3 alkyl; Preferably, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is -CH3; each R 1b Each is independently hydrogen or -CH3; Preferably, L 1 for and / or, the cycloalkyl, heterocyclyl and alkyl are each independently unsubstituted; And / or, the atoms in the bispecific antibody drug conjugate are atoms of natural abundance.

10. The bispecific antibody drug conjugate according to claim 8, wherein: L 1 is a C3-C6 saturated cycloalkyl or a 3-6 membered saturated heterocyclic group, preferably, L 1 is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group and the 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a Replacement, R 2a Each is independently halogen or C1-C6 alkyl; Preferably, L 1 is optionally replaced by one or more R 2a Substituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, R 2a Each is independently halogen or C1-C6 alkyl; Preferably, L 1 for 11. The bispecific antibody drug conjugate according to claim 8, wherein: In the structure shown in formula (A-1), M is -L 2 -L 1 -C(O)-; L 2 for -O-; L 1 -(C(R 1a )(R 1b )) m -CH2- or C3-C6 saturated cycloalkyl, the C3-C6 saturated cycloalkyl is optionally substituted by one or more R 2a replace; m is selected from 1 or 2; R 1a and R 1b Each is independently selected from hydrogen, halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a Each is independently selected from halogen and C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with one or more halogens.

12. The bispecific antibody drug conjugate of claim 8, wherein the cytotoxic drug is selected from any of the following structures:

13. The bispecific antibody drug conjugate according to any one of claims 1 to 12, wherein: The linker unit L is -L a -L b -L c -; and said L c connected to the cytotoxic drug; -L a -for -L b -Selected from any of the following structures: Preferably -L c -for 14. The bispecific antibody drug conjugate of claim 13, wherein the linker unit L is Preferably 15. The bispecific antibody drug conjugate according to any one of claims 1 to 14, wherein: The structure of the bispecific antibody drug conjugate is shown in formula (A-2): Wherein, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 2 to 8; Ab is a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7; M is defined as in any one of claims 8 to 12; L is the connector unit L according to claim 13 or 14.

16. The bispecific antibody drug conjugate according to claim 15, wherein: The structure of the bispecific antibody-drug conjugate is shown in formula (A-2a) or (A-2b): in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8; Ab is a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7; L 2 is -O- or -S-; preferably -O-; X1 is optionally replaced by 1, 2 or 3 R 2a Substituted C3-C6 saturated cycloalkyl; X2 is -(C(R 1a )(R 1b )) m -CH2-; m is 1 or 2; R 1a , R 1b and R 2a Each is independently hydrogen, halogen or C1-C6 alkyl; the C1-C6 alkyl is optionally substituted by one or more halogens.

17. The bispecific antibody drug conjugate according to any one of claims 1 to 16, wherein the bispecific antibody drug conjugate is selected from any of the following structures: in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; Ab is a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

18. A bispecific antibody-drug conjugate, wherein: The bispecific antibody drug conjugate is selected from any of the following structures: Wherein, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; DSYE001 is a bispecific antibody against B7H3 and PD-L1, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.:

9.

19. A pharmaceutical composition comprising the bispecific antibody drug conjugate according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier or excipient.

20. Use of the bispecific antibody drug conjugate according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating and / or preventing cancer, preferably, the cancer is a cancer that positively expresses B7H3 and / or PD-L1.

21. The use according to claim 20, wherein: The cancer is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer and Ewing's sarcoma. Preferably, the cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer or melanoma.

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