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

Abstract

The present invention discloses a bispecific antibody-drug conjugate and its preparation and use, wherein the bispecific antibody-drug conjugate structure comprises an anti-B7H3 and PD-L1 bispecific antibody or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug. The bispecific antibody-drug conjugate of the present invention has excellent endocytosis effect, proliferation inhibitory activity, and tumor growth inhibitory activity.

Description

[Technical Field]

[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 cites the above Chinese patent applications in their entirety.

[0002] The present invention provides anti-B7H3 and PD-L1 bispecific antibody-drug conjugates, methods for their preparation, use, and pharmaceutical compositions containing them. [Background technology]

[0003] As a costimulatory signal for T lymphocyte activation, the B7-CD28 family plays an important role in T lymphocyte-mediated immune responses. Research has shown that various types of B7 molecules have positive or negative regulatory effects on immune cell responses. B7H3 (also known as CD276) is a member of the B7 family and is primarily expressed on the surface of tumor cells. Chapoval AI et al. first discovered that it has a costimulatory effect on CD4+ and CD8+ T cells. B7H3 signaling induces cell-mediated immunity and selectively promotes interferon-γ (IFN-γ) production under T cell receptor signaling. However, as research on B7H3 deepens, its inhibitory functions have gradually been discovered, such as inhibiting 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 growing evidence that its high expression is associated with poor prognosis in various malignant tumors.

[0004] B7H3 is highly expressed in all types of cancer tested, with limited heterogeneity, but is rarely expressed in normal tissues. This suggests that B7H3 may be considered a tumor antigen (TA) and offers the potential for targeted therapy against tumor cells with high B7H3 expression. Currently, therapeutic strategies targeting B7H3 primarily include inhibitory monoclonal antibodies, radioimmunotherapy, antibody-drug conjugates (ADCs), cytotoxicity-mediated monoclonal antibodies, 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-blocking antibodies are theoretically considered to have superior antitumor efficacy. However, among commercially available PD-L1 monoclonal antibodies, only Avelumab has been reported to mediate ADCC effects against tumors and exhibit safety profiles comparable to other PD-L1 antibodies. One important reason is that the effectiveness of ADCC is primarily dependent on antigen abundance, whereas PD-L1 is not 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 components: an antibody or its antigen-binding fragment (target), a linker, and a small molecule drug. An antibody or its antigen-binding fragment is linked to a biologically active small molecule drug, such as a cytotoxic or cytotoxin, via a cleavable or non-cleavable linker. This takes full advantage of the specificity of the antibody or its antigen-binding fragment in targeting cells of interest (target cells) or binding to highly expressed antigens, and the high efficacy of the small molecule drug, reducing or avoiding toxic side effects on non-target cells. This means that, compared with conventional cancer chemotherapy drugs, antibody-drug conjugates for cancer can precisely target tumor cells and reduce the impact on non-tumor cells.

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

[0008] Currently, bispecific ADCs are in the early stages of clinical trials, and no drugs have been approved for sale. There remains a need in the art for bispecific antibody-drug conjugates with synergistic mechanisms, better tumor selectivity, and superior stability. Summary of the Invention [Problem to be solved by the invention]

[0009] The technical problem to be solved by the present invention is to provide a bispecific antibody-drug conjugate, a method for producing the same, and uses thereof, which overcomes the drawback of the limited number of bispecific antibody-drug conjugates available in the prior art. The bispecific antibody-drug conjugate of the present invention has excellent endocytosis effect, growth inhibitory activity, and tumor growth inhibitory activity. [Means for solving the problem]

[0010] The present invention mainly solves the above technical problems through the following technical solutions.

[0011] In one aspect, the present disclosure provides a bispecific antibody-drug conjugate comprising an anti-B7H3 and PD-L1 bispecific antibody or antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug, wherein the bispecific antibody or antigen-binding fragment thereof is: a monoclonal antibody unit targeting PD-L1 and comprising two heavy chains and two light chains; a nanobody unit targeting B7H3 and comprising two identical nanobodies; Here, the N-termini of the two nanobodies are linked to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit via linker peptides, respectively.

[0012] In some embodiments, the bispecific antibody drug conjugate according to the present invention comprises: The light chain variable region of the monoclonal antibody unit comprises a CDR1 whose amino acid sequence is SEQ ID NO: 1, a CDR2 whose amino acid sequence is SEQ ID NO: 2, and a CDR3 whose amino acid sequence is SEQ ID NO: 3; the heavy chain variable region of the monoclonal antibody unit comprises a CDR1 whose amino acid sequence is SEQ ID NO: 5, a CDR2 whose amino acid sequence is SEQ ID NO: 6, and a CDR3 whose amino acid sequence is SEQ ID NO: 7; and the Nanobody comprises a CDR1 whose amino acid sequence is SEQ ID NO: 12, a CDR2 whose amino acid sequence is SEQ ID NO: 13, and a CDR3 whose amino acid sequence is SEQ ID NO: 14.

[0013] In some embodiments, the bispecific antibody drug conjugate according to the present invention comprises: 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.

[0014] In some embodiments, the bispecific antibody drug conjugate according to the present invention comprises: The monoclonal antibody comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, for example, a human IgG1 constant region.

[0015] In some embodiments, the bispecific antibody drug conjugate according to the present invention comprises: the light chain of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 9, the heavy chain of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 10, and said 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 as set forth in SEQ ID NO: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as set forth in SEQ ID NO: 10, and the amino acid sequence of the Nanobody is as set forth in SEQ ID NO: 15.

[0016] In some embodiments, in the bispecific antibody-drug conjugate described in the present invention, the linker peptide is a polypeptide containing glycine and serine and having predetermined elasticity and protease resistance, and preferably, the amino acid sequence of the linker peptide is SEQ ID NO: 11.

[0017] In some embodiments, in a bispecific antibody drug conjugate described herein, the heavy chain amino acid sequence of the bispecific antibody is as set forth in SEQ ID NO: 16 and the light chain amino acid sequence is as set forth in SEQ ID NO: 9.

[0018] In some embodiments, the CDR amino acid sequences are at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each of the above sequences, hi some embodiments, the variable region amino acid sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each of the above sequences.

[0019] The Fc region of the bispecific antibody of the present invention may be a human Fc region. The Fc region of the bispecific antibody of the present invention may 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.

[0020] 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 may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0021] In some embodiments, the antibodies provided herein can be further modified to contain other nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0022] In some embodiments, in the bispecific antibody drug conjugate described herein, the bispecific antibody is DSYE001.

[0023] In some embodiments, in a bispecific drug conjugate, composition, use, or method 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 having amino acid sequences as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and comprise HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth 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 having amino acid sequences as set forth in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively.

[0024] In some embodiments, in the bispecific drug conjugate, composition, use or method described herein, the light chain variable region of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 4, the heavy chain variable region of said monoclonal antibody unit comprises the amino acid sequence of SEQ ID NO: 8, and said Nanobody comprises the amino acid sequence of SEQ ID NO: 15.

[0025] In some embodiments, in the bispecific drug conjugates, compositions, uses or methods described herein, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is as set forth in SEQ ID NO: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as set forth in SEQ ID NO: 10, and the amino acid sequence of the Nanobody is as set forth in SEQ ID NO: 15.

[0026] In some embodiments, in a bispecific drug conjugate, composition or use according to the invention, the heavy chain amino acid sequence of said bispecific antibody is as set forth in SEQ ID NO: 16 and the light chain amino acid sequence is as set forth in SEQ ID NO: 9.

[0027] In some embodiments, in the bispecific antibody drug conjugate described herein, the cytotoxic drug has a structure represented by formula (A-1), a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0028] [ka]

[0029] however, M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, and L 2 is linked to said linker unit L,

[0030] L 1 is -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl, or 3- to 6-membered saturated heterocyclyl, and the C3-C6 saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl are each independently one or more R 2a is optionally replaced by

[0031] M is 1, 2, 3, or 4, and the heteroatoms in the 3- to 6-membered saturated heterocyclyl are each independently N, O, or S, and the number of heteroatoms is 1, 2, or 3;

[0032] R 1a and R 1b are each independently hydrogen, halogen, hydroxy, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens;

[0033] R 2a is halogen, hydroxy, amino or C1-C6 alkyl, said C1-C6 alkyl being optionally substituted with one or more halogens.

[0034] In some embodiments, in the bispecific antibody drug conjugates described herein, L 2 is -O-.

[0035] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is -(C(R 1a )(R 1b )) m -CH2- and R 1a is hydrogen, halogen, or C1-C6 alkyl, and R 1b is hydrogen, halogen or C1-C6 alkyl.

[0036] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is -(C(R 1a )(R 1b )) m -CH2- and R 1a is C1 to C6 alkyl, preferably C1 to C3 alkyl, and R 1b is hydrogen or C1 to C6 alkyl, preferably hydrogen or C1 to C3 alkyl.

[0037] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is -(C(R 1a )(R 1b )) m -CH2- and R 1a is -CH3 and R 1b is hydrogen or -CH3.

[0038] In some embodiments, M is 1 or 2 in the bispecific antibody drug conjugate described herein.

[0039] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 teeth, [ka] is.

[0040] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is a C3-C6 saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, and the C3-C6 saturated cycloalkyl and the 3- to 6-membered saturated heterocyclyl each independently represent one or more R 2a optionally replaced by R 2a are each independently halogen or C1 to C6 alkyl.

[0041] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is a C3-C6 saturated cycloalkyl, and the C3-C6 saturated cycloalkyl is one or more R 2a optionally replaced by R 2a are each independently halogen or C1 to C6 alkyl.

[0042] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 is one or more R 2a and R is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl optionally substituted by 2a are each independently halogen or C1 to C6 alkyl.

[0043] In some embodiments, in the bispecific antibody drug conjugates described herein, L 1 teeth, [ka] is.

[0044] In some embodiments, in the bispecific antibody drug conjugate described herein, M is [ka] is.

[0045] In some embodiments, in the bispecific antibody-drug conjugate described in the present invention, in the structure represented by formula (A-1), M is -L 2 -L 1 -C(O)-, L 2 is -O-,

[0046] L 1 is -(C(R 1a )(R 1b )) m -CH2- or C3 to C6 saturated cycloalkylene, and the C3 to C6 saturated cycloalkylene is one or more R 2a is optionally replaced by m is selected from 1 or 2;

[0047] R 1a and R 1b are each independently selected from hydrogen, halogen, and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens;

[0048] R 2a is selected from halogen and C1-C6 alkyl, wherein said C1-C6 alkyl is optionally substituted with one or more halogens.

[0049] In some embodiments, in the bispecific antibody drug conjugate described in the present invention, the cytotoxic drug is selected from any one of the following structures:

[0050] [ka]

[0051] In some embodiments, in the bispecific antibody drug conjugate described herein, the linker unit L is -L a -L b -Lc - and said L c is linked to the cytotoxic drug,

[0052] -L a -teeth, [ka] and L b is connected to

[0053] -L b -teeth, [ka] and preferably,

[0054] [ka] and the right end of the structure is preferably the L c is connected to

[0055] -L c -teeth, [ka] is.

[0056] In some embodiments, in the bispecific antibody drug conjugate described herein, the linker unit L is [ka] and preferably

[0057] [ka] is.

[0058] In some embodiments, the bispecific antibody-drug conjugate according to the present invention has a structure represented by formula (A-2).

[0059] [ka]

[0060] however, p represents the average number of connections, and is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, for example, 4.02 or 3.95.

[0061] Ab and M are each as defined in any one embodiment of the present invention, and L is a linker unit L as described in any one embodiment of the present invention.

[0062] In some embodiments, the bispecific antibody-drug conjugate according to the present invention has a structure represented by formula (A-2).

[0063] [ka]

[0064] however, p represents the average number of connections, and is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, for example, 4.02 or 3.95.

[0065] M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, and L 2 is connected to L,

[0066] L 1 is -(C(R 1a )(R 1b )) m-CH2-, C3-C6 saturated cycloalkyl, or 3- to 6-membered saturated heterocyclyl, and the C3-C6 saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl are each independently one or more R 2a is optionally replaced by

[0067] M is 1, 2, 3, or 4, and the heteroatoms in the 3- to 6-membered saturated heterocyclyl are each independently N, O, or S, and the number of heteroatoms is 1, 2, or 3;

[0068] R 1a , R 1b and R 2a are each independently hydrogen, halogen, hydroxy, amino, or C1-C6 alkyl, wherein said C1-C6 alkyl is optionally substituted with one or more halogens.

[0069] In some embodiments, the bispecific antibody drug conjugate described in the present invention has a structure as represented by formula (A-2a) or (A-2b).

[0070] [ka]

[0071] however, p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, for example, 4.02 or 3.95;

[0072] Ab is a bispecific antibody or antigen-binding fragment thereof according to any one of the embodiments of the invention, L 2 is —O— or —S—, preferably —O—;

[0073] X1 is 1, 2 or 3 R 2a is a C3-C6 saturated cycloalkyl optionally substituted by X2 is -(C(R1a )(R 1b )) m -CH2-,

[0074] m is 1 or 2; R 1a , R 1b and R 2a are each independently hydrogen, halogen, or C1-C6 alkyl, wherein said C1-C6 alkyl is optionally substituted with one or more halogens.

[0075] In some embodiments, in the bispecific antibody drug conjugate described herein, the bispecific antibody drug conjugate is selected from any one of the following structures:

[0076] [ka]

[0077] [ka]

[0078] [ka]

[0079] [ka]

[0080] however, p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, for example, 4.02 or 3.95; The Ab is a bispecific antibody or antigen-binding fragment thereof according to any one embodiment of the invention.

[0081] In yet another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate has any one of the following structures:

[0082] [ka]

[0083] [ka]

[0084] however, p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 3 to 8, preferably any one integer or decimal number from 4 to 8, preferably any one integer or decimal number from 6 to 8, for example, 4.02, 3.95, 6.1, or 7.9; DSYE001 is an anti-B7H3 and anti-PD-L1 bispecific antibody, the heavy chain amino acid sequence of which is set forth in SEQ ID NO: 16, and the light chain amino acid sequence of which is set forth in SEQ ID NO: 9.

[0085] In some embodiments, in the bispecific antibody-drug conjugates described in the present invention (e.g., bispecific antibody-drug conjugates represented by Formula (A-2), Formula (A-2a), or (A-2b) described in the present invention), p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, preferably any one integer or decimal number from 4 to 8, preferably any one integer or decimal number from 6 to 8, for example, 4.02, 3.95, 6.1, or 7.9.

[0086] In some embodiments, the average number of connections p described in the present invention may be any one of an integer or decimal number from 2 to 8. For example, the average number of connections p may be any one of an integer or decimal number from 3 to 8. For example, the average number of connections p may be any one of an integer or decimal number 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, and 9 to 10.

[0087] In yet 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):

[0088] [ka]

[0089] [ka]

[0090] The amino acid sequence of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention is as shown in the Sequence Listing. The numbering system for the CDRs of the antibodies of the present invention is Kabat numbering.

[0091] In yet another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate has any one of the following structures:

[0092] [ka]

[0093] however, t represents the number of connections, and is any one integer from 1 to 10, preferably any one integer from 2 to 8, and preferably any one integer from 4 to 8, for example, 4, 6, or 8.

[0094] The amino acid sequence of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention is as shown in the Sequence Listing. The numbering system for the CDRs of the antibodies of the present invention is Kabat numbering.

[0095] In some embodiments, the number of connections t described in the present invention is any one integer from 1 to 10, and preferably any one integer from 2 to 8. For example, the number of connections t may be any one integer from 3 to 8. For example, the number of connections t is any one integer from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0096] In yet another aspect, the present invention provides a method for preparing a bispecific antibody-drug conjugate, comprising the step of mixing the bispecific antibody dissolved in a buffer solution with the linker-cytotoxin dissolved in a solvent under the action of a reducing agent.

[0097] In some embodiments, the reducing agent is one conventional in the art for such reactions, for example, tris(2-carboxyethyl)phosphine hydrochloride.

[0098] In some embodiments, the buffer is a buffer conventional in the art for such reactions.

[0099] In some embodiments, the solvent is a solvent conventional in the art for such reactions, for example, dimethylacetamide.

[0100] In yet another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody drug conjugate according to any one of the present invention and a pharmaceutically acceptable carrier or excipient.

[0101] A further object of the present invention is to provide a process for preparing the pharmaceutical composition of the present invention, which comprises combining a 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.

[0102] In the present invention, examples of suitable pharmaceutically acceptable carriers used in the pharmaceutical composition are those described in Remington's Pharmaceutical Sciences (2005).

[0103] In the present invention, the pharmaceutical composition can be administered in any form as long as it prevents, alleviates, prevents, or cures symptoms in human or animal patients. For example, it can be prepared into various appropriate dosage forms depending on the administration route.

[0104] In another embodiment, the administration of the bispecific antibody drug conjugate or said pharmaceutical composition according to any one of the present invention can be combined with another therapeutic method, which may be selected from, but is not limited to, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0105] In yet another aspect, the present invention provides a pharmaceutical formulation comprising a bispecific antibody-drug conjugate according to any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof as an active ingredient, or a pharmaceutical composition according to any one of the present invention, in some embodiments, the formulation is in the form of a solid, semi-solid, liquid, or gaseous formulation.

[0106] In yet another aspect, the present invention provides use of a bispecific antibody drug conjugate according to any one of the present invention or a pharmaceutical composition according to any one of the present invention in the manufacture of a medicament for treating and / or preventing cancer, preferably wherein said cancer is a B7H3 and / or PD-L1 positive expressing cancer.

[0107] In yet another aspect, the present invention provides a method for treating and / or preventing cancer, comprising administering to a subject in need thereof a bispecific antibody drug conjugate described in any one of the present invention or a pharmaceutical composition described in any one of the present invention, preferably wherein said cancer is a B7H3 and / or PD-L1 positive expressing cancer.

[0108] In yet another aspect, the present invention provides a bispecific antibody-drug conjugate according to any one of the present invention or a pharmaceutical composition according to any one of the present invention for treating and / or preventing cancer, preferably wherein the cancer is a B7H3 and / or PD-L1 positive-expressing cancer.

[0109] In some embodiments, the cancer according to the present invention is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney cancer, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain cancer, cervical cancer, blood cancer, bone cancer, lymphoma, pancreatic cancer and Ewing's sarcoma, preferably said cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer or melanoma.

[0110] In some embodiments, methods of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., intraarticular in arthritic joints), inhalation, aerosol delivery, or intratumoral administration.

[0111] In some embodiments, the present invention provides for administering to a subject a therapeutically effective amount of one or more concomitant treatments (e.g., therapeutic modalities and / or other therapeutic agents), which in some embodiments include surgical treatment and / or radiation therapy.

[0112] In some embodiments, the methods or uses provided by the present invention further comprise administering to the individual one or more therapeutic methods (e.g., therapeutic modalities and / or other therapeutic agents). The antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt thereof, can be used alone or in combination with other therapeutic agents in the therapeutic method. For example, it can be used in combination with at least one additional therapeutic agent.

[0113] In yet another aspect, the present invention provides a pharmaceutical combination comprising an 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.

[0114] In yet another aspect, the present invention provides a kit comprising an anti-B7H3 and PD-L1 bispecific antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, or a mixture thereof, of any one of the pharmaceutical compositions of the present invention, preferably further comprising a drug administration device.

[0115] Definition of Terms Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, within the skill of the art.

[0116] In order to make the present invention more readily understandable, certain technical terms are specifically defined below. Unless expressly defined elsewhere herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. For definitions and terminology in this field, experts can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0117] In the present invention, the term "B7H3," also referred to as CD276 antigen, refers to a type 1 transmembrane protein belonging to the B7 family, which has an ectodomain consisting of a single IgV-IgC domain. B7 family proteins contain extracellular IgV-like and IgC-like domains and a short cytoplasmic tail. B7H3 is an immune checkpoint molecule that is abnormally overexpressed in many cancers. The amino acid sequence of the B7H3 protein includes full-length B7H3 proteins (such as human 4IgB7H3 protein or human 2IgB7H3 protein), the extracellular domain of B7H3 (B7H3 ECD), or fragments containing the B7H3 ECD, such as B7H3-ECD fusion proteins. An exemplary sequence of the B7H3 protein is set forth 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 between cynomolgus monkey B7H3 and human and mouse B7H3 is approximately 97% and 88%, respectively.

[0118] As used herein, the term "PD-L1" refers to programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), a 40 kDa type I transmembrane protein. PD-L1 is expressed by activated T cells and B cells and is the surface glycoprotein ligand for PD-1, a key immune checkpoint receptor that mediates immune inhibition.

[0119] In the present invention, the term "about" when used in conjunction with a numerical value encompasses numerical values ​​within a range having a lower limit of 5% below the specified numerical value and an upper limit of 5% above the specified numerical value, including, but not limited to, ±5%, ±2%, ±1%, and ±0.1%, as these variations are appropriate for carrying out the disclosed methods.

[0120] In the present invention, the term "and / or" should be understood to mean any one of any items or any two or more combinations of any items.

[0121] For purposes of 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, i.e., including at least one of the listed quantities or elements, and also including a plurality, and optionally additional unlisted items. Unless terms such as "only one" or "exactly one" expressly state the contrary, or as used in the claims, "consisting of" refers to only one listed number or one element of the list.

[0122] Unless the context clearly dictates otherwise, in the present invention the terms "one" and "one" should be understood to mean "at least one."

[0123] In the present invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linking unit. In the present application, "antibody-drug conjugate" may also be a bispecific antibody-drug conjugate, which may refer to a bispecific antibody or antigen-binding fragment thereof linked to a biologically active cytotoxic drug fragment via a stable linking unit.

[0124] In the present invention, the term "cytotoxic drug" generally refers to a toxic drug having a chemical molecule that potently disrupts the normal growth of tumor cells within the tumor cells. A cytotoxic drug can kill tumor cells at a sufficient concentration. The "cytotoxic drug" may include small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals, radioactive isotopes (e.g., 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 nuclease, or a derivative thereof, such as a toxic drug including, but not limited to, a camptothecin derivative, for example, 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).

[0125] In the present invention, the term "antibody" generally refers to an immunoglobulin that reacts with a specific protein or peptide, or a fragment thereof. The antibody may be of any class, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may further have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibody of the present application may be derived from any species. The term "antibody" includes complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and other modified immunoglobulin molecules, so long as these antibodies exhibit the desired biological activity.

[0126] 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 an antibody is referred to as an "epitope," as described above. As described above, an antigen-binding domain can typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily include both. For example, an Fd fragment has two VH regions and typically retains some of the antigen-binding function of the complete antigen-binding domain. Examples of antigen-binding fragments of antibodies include the following: (1) Fab fragment, a monovalent fragment having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab')2 fragment, a bivalent fragment of two Fab fragments linked by disulfide bonds in the hinge region; (3) Fd fragment, having two VH and CH1 domains; (4) Fv fragment, having the VL and VH domains of a single antibody arm; (5) dAb fragment, having a VH domain (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), the entire contents of which are incorporated herein by reference); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv) derived from an scFV library.The two domains of an Fv fragment, VL and VH, are encoded by independent genes, but can be linked using recombinant methods via a synthetic linker, and can be produced as a single protein chain (referred to as single-chain Fv (scFv)) in which the VL and VH domains pair to form a monovalent molecule (see 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 camelids (camel, dromedary, llama, alpaca, etc.) (Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J. Biotechnol., 74, 277-302 and Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs are also called nanobodies (Nbs).

[0127] In the present invention, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. In this application, the term "variable" generally refers to the fact that certain portions of the antibody variable domain sequence vary significantly, resulting in the binding and specificity of each particular antibody to a particular antigen. Variation is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments of the light and heavy chain variable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs): LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively. The more highly conserved portions of the variable domain are called framework regions (FRs). Naturally occurring heavy and light chain variable domains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, and L-FR4), most of which adopt a β-sheet structure and are connected by three CDR structural loop regions. The CDRs of each chain are adjacent to each other via the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody.

[0128] In the present invention, antibody variable regions or antibody CDRs are encoded by various methods in the art, such as the Kabat numbering scheme and definition rules based on sequence variability (see Kabat et al., Immunological Protein Sequences, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia numbering scheme and definition rules based on structural loop region positions (see A1-Lazikani et al., J Mol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules based on amino acid sequence alignment of germline V genes by efranc et al., Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc.; Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2014; 2018. can be referred to.

[0129] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific because they target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain 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 substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0130] As used herein, the term "multispecific antibody" refers to an antibody that contains two or more antigen-binding domains (e.g., two, three, four, or more different epitopes) capable of binding to two or more different epitopes, which may be on the same antigen or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind to two different antigens or two different epitopes. As used herein, bispecific antibodies targeting B7H3 and PD-L1 may be referred to, for example, as "anti-B7H3 / PD-L1" or "anti-B7H3xPD-L1" or "B7H3xPD-L1" bispecific molecules, or other similar terms.

[0131] In the present invention, the term "nanobody" refers to a heavy chain single domain antibody (VHH) that contains only one heavy chain variable region (VHH) and CH2 and CH3 regions, and is naturally devoid of light chains compared to other antibodies. It is composed of the heavy chain variable region of camelids (camels, llamas, alpacas, and related species). Nanobody crystals are 2.5 nm in diameter and 4 nm in length, making them the smallest naturally occurring fragments capable of binding to antigens.

[0132] In the present invention, the term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region. Optionally, 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 antigen or different antigens.

[0133] In the present invention, the term "humanized antibody" refers to antibody forms comprising human and non-human (e.g., murine, rat) antibody sequences. Generally, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which 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 those of a human immunoglobulin sequence. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0134] As used herein, the "isotype" of an antibody refers to the antibody class (e.g., IgM, IgE, IgG such as IgG1, IgG2, and IgG4) provided by the heavy chain constant region genes. Isotypes also include modified forms of any of these species, which may be performed to alter Fc function, for example, to enhance or reduce effector function or binding to Fc receptors.

[0135] In the present invention, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. In some embodiments, a 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 numbers in this paragraph are based on the EU numbering system, referred to as the EU index; see, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.)

[0136] In the present invention, the term "cross-reactivity" refers to binding to antigen fragments of the same target molecule of human, monkey, and / or mouse (mouse or rat) origin. Therefore, "cross-reactivity" should be understood as an interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a cognate molecule (e.g., BDCA2) expressed in a different species. The cross-reactivity specificity of a monoclonal antibody that recognizes human BDCA2, monkey, and / or mouse BDCA2 (mouse or rat) can be determined by FACS analysis.

[0137] 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 between a molecule X and its partner Y is generally determined by the dissociation rate constant and the association rate constant (k dis and k on ) and the equilibrium dissociation constant (K D ) Affinity can be measured by common methods known in the art. In some embodiments of the invention, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between an antibody of the invention and an antigen. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0138] In the present invention, "not binding" to a protein or cell means not binding to a protein or cell, or not binding to a protein or cell with high affinity, i.e., the K D is 1.0 x 10 -6 M or more, more preferably 1.0 × 10 -5 M or more, more preferably 1.0 × 10 -4 M or more, 1.0 x 10 -3 M or more, more preferably 1.0 × 10 -2 M or above.

[0139] In the present invention, "high affinity" for an IgG antibody means a K D is 1.0×10 -6 M or less, preferably 5.0 × 10-8 M or less, more preferably 1.0×10 -8 M or less, 5.0 x 10 -9 M or less, more preferably 1.0×10 -9 For other antibody subtypes, "high affinity" binding may be different. For example, "high affinity" binding for IgM subtypes is defined as K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.

[0140] 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 those in a reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0141] In the present invention, "halogen" generally refers to fluorine, chlorine, bromine, and iodine, and may be, for example, fluorine or chlorine.

[0142] In the present invention, the term "alkyl" generally refers to a residue obtained by removing a hydrogen atom from an alkane. The alkyl may be substituted or unsubstituted, and may be substituted or unsubstituted. The term "alkyl" generally refers to a saturated straight-chain or branched-chain aliphatic hydrocarbon group having a residue derived by removing hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, and may be a straight-chain or branched-chain group containing 1 to 20 carbon atoms, for example, 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, etc. The alkyl may be substituted or unsubstituted, and may be substituted or unsubstituted; for example, if substituted, the substituent may be substituted at any available point of attachment.

[0143] In the present invention, the term "alkylene" generally refers to a saturated straight-chain or branched-chain aliphatic hydrocarbon group having a residue derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of two parent alkanes, and may refer to a straight-chain or branched-chain group containing 1 to 20 carbon atoms, for example, the term "methylene" may refer to a residue derived from a group of one carbon atom by removing two hydrogen atoms. The methylene may be substituted or unsubstituted, substituted or unsubstituted, for example, an alkylene containing 1 to 12 carbon atoms, for example, an alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH-), 1,1-ethylene (-CH(CH)-), 1,2-ethylene (-CHCH)-, 1,1-propylene (-CH(CHCH)-), 1,2-propylene (-CHCH(CH)-), 1,3-propylene (-CHCHCH-), 1,4-butylene (-CHCHCHCHCH-), and 1,5-butylene (-CHCHCHCHCHCH-), etc. Alkylene can be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent can be substituted at any available point of attachment.

[0144] As used herein, the term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl are as defined herein. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.

[0145] As used herein, 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, cyclooctane, etc., and polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted; for example, if substituted, the substituent may be substituted at any available point of attachment.

[0146] In the present invention, "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between ring molecules. The term "partially unsaturated" includes cyclic structures with multiple sites of unsaturation, but does not include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" indicates that the moiety has one or more degrees of unsaturation.

[0147] As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more ring atoms are 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 3 to 8 ring atoms, of which 1 to 3 are heteroatoms, more preferably 3 to 6 ring atoms, of which 1 to 3 are heteroatoms, and most preferably 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused-ring, and bridged-ring heterocyclyls. The heterocyclyl ring can be fused to an aryl, heteroaryl, or cycloalkyl ring, and the ring connected to the core structure is a heterocyclyl. The heterocyclyl can be substituted or unsubstituted, for example, if substituted, the substituents can be substituted at any available point of attachment.

[0148] In the present invention, the term "ring atom" generally refers to an atom contained in a ring structure. For example, a ring atom may be a carbon atom in a benzene ring or a nitrogen atom in a pyridine ring. When a ring atom is connected to a hydrogen atom, the ring atom may be substituted or unsubstituted; for example, when substituted, the substituent may be substituted at any available connection point.

[0149] In the present invention, the term "independently" generally refers to the variable being applied in all instances, regardless of whether the variable is present in the same compound with the same or different definitions. For example, the variable may refer to the type or number of substituents on the compound or the type of atom in the compound. For example, if R appears twice in a compound and R is defined as "independently carbon or nitrogen," then both Rs may be carbons, both Rs may be nitrogens, or one R may be carbon and the other R may be nitrogen.

[0150] In the present invention, "optional" or "optionally" generally means that the following item or circumstance may occur but does not necessarily occur, and the description includes cases where the described item or circumstance occurs and cases where the item or circumstance does not occur. For example, "a multi-ring group optionally substituted with alkyl" means that alkyl may be present but does not necessarily have to be present, and the description may include a situation where the multi-ring group is substituted with alkyl and a situation where the multi-ring group is not substituted with alkyl.

[0151] In the present invention, "substituted" generally refers to one or more hydrogen atoms of a group, for example, up to 5, e.g., 1 to 3 hydrogen atoms, being independently replaced with the corresponding number of substituents. Substituents are only available at their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions (through experiment and theory) without much effort. For example, when an amino or hydroxyl containing free hydrogen is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond, it may be unstable.

[0152] In the present invention, unless otherwise specified, the "connection" between groups may generally be in any direction. The "connection" between group X and group Y can generally be set in any direction, and "any direction" generally means that when group X is used to connect group Y and group Z, two or more bonding sites of group X can be arbitrarily connected to group Y or group Z.

[0153] In the present invention, as known to those skilled in the art, the terms "alkyl", "alkenyl", "cycloalkyl", etc. may in certain cases be preceded by an identifier indicating the number of atoms present in the group, such as C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript number following the "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl having three carbon atoms (e.g., n-propyl, isopropyl), C 1-10 In the formula, the group members can have any number of carbon atoms in the range of 1 to 10.

[0154] In the present invention, the cytotoxic drug of the present invention may be a tautomer, mesomers, racemates, enantiomers, and / or diastereomers thereof. In this application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror-images of each other. Diastereomers may have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. In this application, the terms "tautomer" and "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via rearrangement of some bond electrons. In this application, the term "racemate" generally refers to a molecule that contains an asymmetric atom but has a symmetry factor such that the total optical rotation of the molecule is zero. The terms "racemate" or "racemic mixture" refer to a composition consisting of equimolar amounts of two enantiomeric substances.

[0155] In the present invention, the term "linker unit" or "linker structure" generally refers to a chemical fragment or bond that is linked at one end to a ligand and at the other end to a cytotoxic drug, or to another linker and then to a cytotoxic drug. The term "directly or indirectly linked ligand" refers to the group being directly connected to the ligand via a covalent bond or being connected to the ligand via a linker structure. For example, a chemical fragment or bond containing an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., 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.

[0156] As used herein, the term "optionally linked to other molecular moieties" of a particular structure generally refers to either the structure not being linked to other chemical structures, or the structure being linked (e.g., linked by a chemical bond or linked by a linker structure) to one or more other chemical structures that are different from the structure (e.g., a ligand described herein).

[0157] In the present invention, the term "drug loading capacity" generally refers to the average amount of cytotoxic drug loaded on each ligand and can be expressed as the ratio of the amount of cytotoxic drug to the amount of antibody, with the cytotoxic drug loading range being 0 to 12, e.g., 1 to 10, cytotoxic drugs per ligand (Ab). In embodiments of the present application, the drug loading capacity is represented by p, t, or n, and may illustratively be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The drug loading capacity of each ADC molecule after the coupling reaction can be determined by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA testing, and HPLC characterization.

[0158] In the present invention, certain atoms of the compounds or antibody-drug conjugates of the present invention may exist in one or more isotopic forms. For example, hydrogen may be a hydrogen atom ( 1 H), deuterium ( 2 H) and tritium ( 3 H), and carbon exists as three different isotopes ( 12 C. 13 C. 14 Examples of isotopes that can be incorporated into compounds of the present application include: 15 N, 18 O. 17 O. 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125These include, but are not limited to, I or similar isotopes. Therefore, the compounds or antibody-drug conjugates of the present invention may be enriched in one or more of these isotopes compared to the natural abundance of these isotopes. As known to those skilled in the art, such isotope-enriched compounds can be used for various purposes. For example, deuterium ( 2 Substitution with heavier isotopes, such as deuterium (H), may offer certain therapeutic advantages due to increased metabolic stability. 2 The natural abundance of hydrogen (H) is approximately 0.015%. Thus, in nature, there is approximately 1 deuterium atom for every 6,500 hydrogen atoms. Thus, deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance of greater than 0.015% at one or more positions (as the case may be). Unless otherwise defined, structures depicted 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 having the same structure as those of the present invention except that a hydrogen atom is replaced with deuterium or tritium, or a carbon atom is replaced with carbon-13 or carbon-14, are all within the scope of the present invention.

[0159] In the present invention, the term "pharmaceutical composition" generally refers to a mixture of one or more compounds described herein or their physiologically / pharmacologically 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 and promote absorption of the active ingredients, thereby exerting their biological activity. The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia. Pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. These suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents as described above. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Furthermore, sterile fixed oils are conveniently used as solvents or suspending media. For example, any mixed fixed oil, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids such as oleic acid can also be used in the preparation of injectable preparations.

[0160] In the present invention, the term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refers to a salt of a compound or antibody-drug conjugate of the present invention, or a salt of a 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.

[0161] 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 undue toxicity. Suitable carriers include large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactivated virus particles. Those skilled in the art are familiar with such carriers. Pharmaceutically acceptable carriers in therapeutic compositions include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like.

[0162] As used herein, "treatment" and "treating" generally refer to an approach for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit. A therapeutic benefit includes, but is not limited to, eradicating, inhibiting, reducing, or ameliorating the underlying disease being treated. Furthermore, a therapeutic benefit is achieved through eradicating, reducing, or ameliorating one or more physiological symptoms associated with the underlying disease, such that the patient experiences improvement, but may still be suffering from the underlying disease.

[0163] In the present invention, "prevention" and "preventing" generally refer to a method for obtaining a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, pharmaceutical compositions can be administered to patients who are at risk of developing a particular disease or who report one or more physiological symptoms of the disease, even if the disease has not yet been diagnosed.

[0164] In the present invention, the term "subject" or "patient" generally refers to humans (i.e., males or females of any age, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or the elderly) 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).

[0165] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a compound or antibody-drug conjugate of the invention, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate one or more symptoms of a disease or condition, or the occurrence of a disease or condition. A therapeutically effective amount also refers to an amount sufficient to cause improvement of symptoms, such as an amount that treats, cures, prevents, or ameliorates the associated pathology, or an amount that increases the rate of treatment, cure, prevention, or amelioration of such pathology. When an active ingredient is administered to a subject alone, a therapeutically effective dose refers to that ingredient alone. When administered in combination, a therapeutically effective amount refers to the combined amounts of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent results in an improvement in a diagnostic criterion or parameter by at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0166] As used herein, the term "cancer" refers to a group of cells that exhibits abnormally high proliferation and growth. Cancers may be benign (also called benign tumors), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemic cancer cells. As used herein, the term "tumor" refers to one or more cells that comprise a cancer. As used herein, the term "tumor growth" refers to the proliferation or growth of one or more cells that make up a cancer, resulting in an increase proportional to the size or extent of the cancer.

[0167] Unless contrary to common knowledge in the art, the above-mentioned preferable conditions can be arbitrarily combined to obtain each preferable embodiment of the present invention.

[0168] The reagents and raw materials used in the present invention are commercially available. [Effects of the Invention]

[0169] The positive advances of the present invention are: In the bispecific antibody-drug conjugate of the present invention, the bispecific antibody has the following advantages:

[0170] 1. It simultaneously binds to B7H3 and PD-L1, targeting tumor cells while relieving the inhibition of PD-L1 on T cells, demonstrating superior antitumor activity compared to the combined administration of monoclonal antibodies.

[0171] 2. Compared with related monoclonal antibody combination therapy, the bispecific antibody of the present application has the advantages of good compliance and controllable quality.

[0172] 3. The stability characteristics of the bispecific antibody in this invention are mainly reflected in the monomer purity and thermal stability studies. After a single affinity purification, the bispecific antibody monomer content reached 95%, which is superior to the purity achieved in the industry after multiple secondary purification rounds. Analysis of the antibody structure and activity after heat treatment demonstrated that the antibody maintained good molecular structure and full biological activity even under harsh conditions, which is beneficial for the industrial production, packaging, and storage of antibodies. Overall, this invention constructed a B7H3 / PD-L1 bispecific antibody in the IgG-VHH2 format, which exhibited good molecular stability and significantly better in vitro activity (at the binding molecule and cellular levels) than Avelumab and MGA271. In vivo data showed that B7H3 + The antitumor activity of the bispecific antibody was demonstrated to be superior to that of the B7H3 monoclonal antibody in A375 tumor cells. Therefore, the bispecific antibody of the present invention has a wide range of potential applications due to its excellent development potential and activity.

[0173] The bispecific antibody-drug conjugates of the present invention have one or more of the following advantages:

[0174] 1. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugates of the present invention have significantly improved in vitro growth inhibitory activity against tumor cells, particularly A375, NCI-H1975, NCI-H441, and NCI-H358 cells that positively express B7H3 and PD-L1.

[0175] 2. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugates of the present invention can more significantly induce downregulation of PD-L1 expression, indicating that the bispecific antibody-drug conjugates of the present invention can exert a more potent immune suppression alleviation effect by reducing target expression levels.

[0176] 3. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugates of the present invention have significantly improved tumor growth inhibitory activity against CT26 cells in vivo, particularly in the establishment of a syngeneic mouse model of CT26 cells.

[0177] 3. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugates of the present invention can significantly inhibit the proliferation of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, particularly 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, and NCI-H1975 non-small cell lung cancer cells.

[0178] 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 proliferation of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, particularly MDA-MB-231 breast cancer cells and NCI-H1975 non-small cell lung cancer cells.

[0179] 5. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugates of the present invention have significantly improved endocytosis efficiency.

[0180] 6. The bispecific antibody-drug conjugate of the present invention has excellent safety.

[0181] The bispecific antibody-drug conjugate according to any one of the present invention may have one or more effects selected from the following: (1) inhibitory activity against tumor cell growth in vitro, (2) target inhibition, (3) plasma stability, (4) tumor-inhibitory effect in vivo, (5) bystander effect, (6) anti-transporter trafficking ability, (7) tumor-targeting ability in vivo, (8) stronger tumor-inhibitory effect in individuals with healthy immune systems, and (9) good in vivo safety. [Brief explanation of the drawings]

[0182] [Figure 1] 1 is a graph showing affinity screening of anti-B7H3 VHH humanized antibodies produced according to the present invention with B7H3. [Figure 2] FIG. 1 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. [Figure 3] Figure 1 shows an SDS-polyacrylamide gel electrophoresis image of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention. [Figure 4] Figure 1 shows SEC-HPLC purity measurement of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention. [Figure 5] Figure 1 shows the Tm value (DSF) of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention. [Figure 6]Figure 1 shows the binding ELISA of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the invention before and after heat treatment at 60°C, where a shows the binding ELISA for PD-L1 and b shows the binding ELISA for B7H3. [Figure 7] Figure 1 shows the binding ELISA of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention, where a shows the binding ELISA for PD-L1 and b shows the binding ELISA for B7H3. [Figure 8] Figure 1 shows graphs of BLI affinity detection of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention at five different concentrations of B7H3-his or PD-L1-his. (a) shows the affinity curve with PD-L1, and (b) shows the affinity curve with B7H3. [Figure 9] Figure 1 shows the blocking curve of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 PD-L1 / CHO-PD1 of the invention. [Figure 10] 1 shows that the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention induces IFN-γ secretion from T cells. [Figure 11] Figure 1 shows that the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention promotes T cell proliferation. [Figure 12] 1 shows the ADCC effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention on cancer cells, where (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. [Figure 13] 1 shows the in vivo tumor-inhibiting effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention. [Figure 14] 1 shows the effect of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the invention on mouse body weight. [Figure 15] 1 shows the test results of the endocytosis activity of the bispecific antibody-drug conjugate DSYE001-X2 of the present invention. [Figure 16]1 shows an in vitro tumor cell growth inhibition test by the bispecific antibody-drug conjugate DSYE001-X1 (DAR8) of the present invention. [Figure 17] Figure 1 shows the effect of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention on PD-L1 expression in NCI-H1975 cells. [Figure 18] 1 shows the efficacy evaluation of the bispecific antibody drug conjugate DSYE001-X2 of the present invention in a syngeneic transplant mouse model. [Figure 19] 1 shows an evaluation of the efficacy of the bispecific antibody-drug conjugate DSYE001-X1 (DAR4) of the present invention in mice bearing human non-small cell lung cancer cells NCI-H1975. [Figure 20] 1 is a graph showing the efficacy evaluation of the bispecific antibody-drug conjugate DSYE001-X1(DAR6) of the present invention in mice bearing human breast cancer cell MDA-MB-231. [Figure 21] 1 is a graph 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 OF THE INVENTION

[0183] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples. In the following examples, experimental methods for which specific conditions are not described are selected according to conventional methods and conditions or product instructions.

[0184] In the following examples, experimental methods for which no specific conditions are described were selected according to conventional methods and conditions or product instructions.

[0185] Sample detection 1. ADC DAR value analysis method - HIC-HPLC (hydrophobic chromatography) High-performance liquid chromatograph: Waters e2965 high-performance liquid chromatograph system. Chromatography column: MabPac™ HIC-Butyl 5 μm 4.6 × 100 mm (manufacturer: Thermo); Mobile phase A: 1.5 M (NH4)2SO4 + 50 mM K2HPO4 (pH 7.0), Mobile phase B: 50 mM K2HPO4 (pH 7.0) / isopropanol (75:25 V / V),

[0186] Elution was carried out according to the following elution program. [Table 1]

[0187] 2. SEC Purity Analysis - SEC-HPLC (Size Exclusion Chromatography) High-performance liquid chromatograph: 1260Agilent liquid chromatograph. Chromatography column: Waters Xbridge BEH200 SEC (7.8 x 300 mm, 3.5 μm) Mobile phase: 50 mM NaH2PO4 + 200 mM arginine (pH 6.80) + 10% isopropanol

[0188] [Table 2]

[0189] The present invention includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of example only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including those cited by reference, are hereby incorporated by reference in their entirety. [Example]

[0190] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope of the invention.

[0191] Example 1: Production and testing of bispecific antibody DSYE001 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 according to the preparation method described in PCT / CN2022 / 125089, and is specifically as shown below (the CDR regions of the bispecific antibody are determined according to the Kabat numbering convention):

[0192] 1. Humanization of camel-derived anti-B7H3 A camel-derived anti-B7H3 nanobody was humanized using framework shuffling, using germline human antibody genes as a template. The corresponding VHH framework-shuffled library was generated by in vitro overlap PCR synthesis. The VHH phage library was then cloned, screened, and identified.

[0193] More specifically, a one-step strategy was used to humanize camel-derived anti-B7H3 nanobodies. Approximately 1,000 clones from the sublibrary were screened, and selected positive clones were screened for phage-level thermostability using ELISA. 5 μg / mL of huB7H3 antigen was coated onto a high-adsorption 96-well ELISA plate, and the supernatant after overnight amplification of the screened phage was then reacted to select clones with higher OD450 values.

[0194] The above phage clones were sequenced to obtain the B7H3 VHH gene sequence, and the C-terminus was fused to a human Fc protein gene to construct and express B7H3-Fc. Biomembrane interferometry was then performed to capture 100 nM of B7H3 VHH-Fc using a Protein A probe, which bound to the B7H3 antigen at a starting concentration of 200 nM and diluted two-fold. The KD values ​​of the antibodies binding to the antigen were calculated, and the result was that antibody 75-16 (its 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 Since the clone reached M (see Figure 1), it was selected and used in the next step, construction of a bispecific antibody.

[0195] 2. Construction and Expression of Anti-B7H3 and Anti-PD-L1 Bispecific Antibody The amino acid sequences of the PD-L1 monoclonal antibody light chain (amino acid sequence SEQ ID NO: 9, variable region sequence SEQ ID NO: 4, CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 2, CDR3 sequence SEQ ID NO: 3) and heavy chain (amino acid sequence SEQ ID NO: 10, variable region sequence SEQ ID NO: 8, CDR1 sequence SEQ ID NO: 5, CDR2 sequence SEQ ID NO: 6, CDR3 sequence SEQ ID NO: 7) were derived from an existing PD-L1 hIgG1 humanized monoclonal antibody, with the N-terminus of the anti-B7H3 VHH (75-16 above) linked to the C-terminus of the Fc fragment (the structure is shown in Figure 2 ) via a linker peptide (SEQ ID NO: 11).

[0196] The DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in E. coli. The purified plasmid was transfected into HEK293 cells using PEI. The cells were then cultured in suspension in OPM-CD05 expression medium. After 6 days of culture, the cell culture supernatant was collected and the antibody was purified through a protein A column. The purified IgG1 was dialyzed against phosphate-buffered saline (PBS), flash-frozen, and stored at -80°C.

[0197] The purified anti-B7H3 and PD-L1 bispecific antibody DSYE001 had a heavy chain amino acid sequence of SEQ ID NO: 16 and a light chain amino acid sequence of SEQ ID NO: 9.

[0198] Testing section Hereinafter, unless otherwise specified, the term "bispecific antibody" refers to the anti-B7H3 and anti-PD-L1 bispecific antibody of the invention, which may also be abbreviated as "B7H3 / PD-L1 bispecific antibody," "B7H3 / PD-L1 bispecific antibody," or "bispecific antibody."

[0199] Test Method: (1) Sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Five micrograms of bispecific antibody was mixed with protein reducing and non-reducing buffers, the reaction mixture was supplemented with PBS to a total volume of 10 μL, and heated at 100°C for 10 minutes to fully denature the protein. 9 μL was then loaded onto a precast polyacrylamide gel well (Bio-Rad). The voltage was set at 80 V for 30 minutes, followed by 120 V for 60 minutes. The gel was then stained with Coomassie Brilliant Blue stain for 30 minutes and destained with a destaining solution (acetic acid:ethanol:water = 1:3:6) for 15 minutes. The destaining was repeated three times to lighten the background, and images were captured using a gel imager. Results showed that under non-reducing conditions, the B7H3 / PD-L1 bispecific antibody exhibited good monomer purity. Under reducing conditions, the disulfide bond between the light and heavy chains was opened, resulting in the appearance of two bands, one for the heavy chain and one for the light chain, with no impurity bands (Figure 3).

[0200] (2) Size Exclusion Chromatography (SEC-HPLC) The monomer purity of the bispecific antibody was assessed using SEC-HPLC analysis. The B7H3 / PD-L1 bispecific antibody was analyzed using a ThermoMAbPac SEC-1, 5 μm, (7.8 × 300 mm) column (Product No. 088460) on a 1260 HPLC system (Agilent, Santa Clara, CA) and simultaneously compared with the PD-L1 monoclonal antibody, B7H3 VHH-Fc. The mobile phase used was phosphate buffer solution (PBS). The flow rate was set at 0.7 mL / min, with an injection volume of 15 μL. As shown in Figure 4, the SEC chromatograms were recorded using a UV detector at 280 nm at a constant temperature of 25°C. All detected antibodies had a very high proportion of monomer peaks, with the peak area ratio of the monomer peak exceeding 95%, indicating good monomer purity and low levels of aggregates in PBS buffer conditions.

[0201] (3) Detection of antibody Tm values ​​by differential scanning fluorescence (DSF) DSF was detected using a real-time PCR system (Biorad cfx96, USA). 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 5000x concentrated stock solution with ddH2O. 20μL of the sample was transferred to a PCR tube, and SYPRO Orange working solution was added to the reaction to prevent bleaching. The liquid was collected at the bottom of the PCR tube by instantaneous centrifugation. The qPCR instrument was then turned on and programmed to increase the temperature from 25°C to 95°C at a rate of 0.3°C per second. Data was collected, and the temperature and signal values ​​were plotted to calculate the melting temperature (Tm). The data showed that the Tm of the Fc and Fab of the B7H3 / PD-L1 bispecific antibody was 69°C and 90°C, respectively, demonstrating good high-temperature resistance and close to the Tm of the B7H3 VHH-Fc and PD-L1 monoclonal antibody (Figure 5).

[0202] (4) Validate the thermostability and binding activity of the antibody by combining with ELISA. A 96-well enzyme plate was coated with 2μg / ml of his-tagged PD-L1 or B7H3 antigen protein overnight at 4℃. The next day, 100μL of casein blocking solution was added to each well and blocked for 1 hour at 37℃. Three-fold dilutions of B7H3 / PD-L1 bispecific antibody (when testing thermal stability, the bispecific antibody was treated in a 60℃ water bath for 1 hour) and monoclonal antibody (when testing bispecific antibody binding to B7H3 by ELISA, the control monoclonal antibody used was MGA271, isotype hIgG1, B7H3 VHH-Fc; when testing binding to PD-L1, the control monoclonal antibody used was PD-L1 monoclonal antibody, avelumab, isotype hIgG1) were added to the wells. After incubation at 37°C for 1 hour, unbound antibody was washed away with 0.1% PBST. Bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, USA). Color development was performed using 50 μL of 3,3',5,5'-tetramethylbenzidine substrate (TMB). After incubation for 5 minutes, color development was stopped by adding 50 μL of 2 M sulfuric acid. Absorbance was detected at OD450 nm using a SpectraMax M5e (Molecular Devices) microplate reader. The antibody concentration was plotted against the OD450 reading using a four-parameter fitting method, and the EC 50 was calculated. Figures 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. The figures show that the binding curves of the bispecific antibody before and after heating are essentially overlapping, whether it is PD-L1 or B7H3, indicating that the bispecific antibody can withstand the high temperature of 60°C and maintain strong binding activity to its dual targets of PD-L1 and B7H3. In other words, the bispecific antibody exhibited similar binding ability to both targets as the positive antibody, PD-L1 monoclonal antibody, and B7H3 VHH-Fc, and the EC of the bispecific antibody, PD-L1 monoclonal antibody, and control avelumab for binding to human PD-L1. 50The EC values ​​were 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which are of the same order of magnitude (Figure 7a). The bispecific antibodies, B7H3 VHH-Fc and MGA271, also showed similar binding activity to human B7H3, and the EC values ​​were 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which are of the same order of magnitude (Figure 7a). 50 were 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively (Fig. 7b).

[0203] (5) Measurement of binding affinity between bispecific antibodies and antigens using BLI The B7H3 / PD-L1 bispecific antibody, PD-L1 monoantibody, and B7H3 VHH-Fc were diluted to 100 nM in sample buffer (PBS containing 0.02% Tween 20 and 0.1% BSA) and analyzed for affinity of the bispecific antibody to specific human B7H3 and human PD-L1 antigens using OCTET 96. Protein A material was used as the probe for antibody immobilization. B7H3-his and PD-L1-his antigens were diluted to an initial concentration of 200 nM in sample buffer. Multiple antigen concentration gradients were set up using two-fold dilutions to measure antibody binding. Rate constants and affinities were obtained, and the K on and K off values ​​were calculated using software provided by the supplier to obtain the antibody K values. As can be seen from the figures, the bispecific antibody exhibited high affinity for PD-L1 and B7H3, with K values ​​of 5.85 x 10, respectively. -10 M and 8.11 x 10 -9 M was reached (Fig. 8).

[0204] (6) Measurement of the ability of bispecific antibodies to block the PD1 / PD-L1 pathway by flow cytometry Flow cytometry was used to assess the ability of the B7H3 / PD-L1 bispecific antibody to inhibit binding of human PD-L1 to human PD1-CHO cells and compare it with avelumab, a PD-L1 monoclonal antibody. 5Human PD1-CHO cells were uniformly seeded in a 96-well culture plate and incubated with a mixture of serially diluted antibodies (B7H3 / PD-L1 bispecific antibody, avelumab, PD-L1 monoclonal antibody, isotype hIgG1, B7H3 VHH-Fc) starting at 400 nM and biotinylated PD-L1 antigen (50 nM) at room temperature for 30 minutes. The mixture was then further incubated with the cells for 45 minutes at 4°C, and unbound antigen was washed away with PBS. Next, the cells were fluorescently stained using PE-streptavidin. Finally, the mean fluorescence intensity (MFI) in the PE channel of the flow cytometer was read, and the antibody concentration vs. MFI was plotted. The IC value of the antibody was calculated using a four-parameter fitting. 50 Flow cytometry analysis showed that the bispecific antibody inhibited the binding of PD-L1 to CHO-PD1 cells, and its IC 50 The inhibitory activity was 106.4 nM, and the inhibitory activity was 106.4 nM. 50 94.20 nM), avelumab (IC 50 The ATP concentration was shown to be equivalent to that of the ATP-dependent agonist (115.0 nM) (Figure 9).

[0205] (7) Detection of T cell activation ability of bispecific antibodies by mixed lymphocyte reaction (MLR) Dendritic cells (DCs) were induced by in vitro culture of mononucleated monocytes from peripheral blood mononuclear cells (PBMCs) for 7 days using a mononuclear cell purification kit (Miltenyi Biotec, Germany) with 500 U / mL interleukin-4 (IL-4) and 250 U / mL GM-CSF. CD4+ T cells (1 × 10 5 ) and allogeneic DCs (1.25 × 10 4) were co-cultured in RPMI 1640 complete medium containing 10% FBS under 5% CO2 at 37°C. The culture conditions included a no-antibody group and groups containing different concentrations of the B7H3 / PD-L1 bispecific antibody, PD-L1 monoclonal antibody, avelumab, B7H3 VHH-Fc, MGA271, and isotype hIgG1. After 5 days, IFN-γ levels in the culture supernatants were analyzed using an IFN-γ ELISA kit. MLR results demonstrated that the bispecific antibody stimulated CD4+ T cells to secrete IFN-γ, and its T cell activation potency was superior to that of the PD-L1 monoclonal antibody and avelumab, even at low and high concentrations. Furthermore, the presence of the B7H3 antibody alone partially activated T cells by inhibiting B7H3 inhibitory signaling, although this effect was less pronounced than with the PD-L1 signal-blocking antibody (Figure 10).

[0206] (8) T cell proliferation experiment 96-well cell culture plates (Corning, USA) were coated with 1 μg / mL of CD3 antibody (Clone HIT3a), 1 μg / mL of CD28 antibody (Clone CD28.2), and 5 μg / mL of human PD-L1 at 4°C for 1 hour. Control wells were coated with mouse IgG2a isotype control alone or with CD3 and CD28 antibodies in the same manner. CD4+ T cells were isolated using the Dynabeads™ CD4 Positive Isolation Kit. CD4+ T cells were cultured in pre-coated 96-well plates with different concentrations of B7H3 / PD-L1 bispecific antibody, avelumab, and PD-L1 monoclonal antibody in RPMI1640 medium containing 10% FBS (Gibco) at 37°C for 4 days. After 4 days, changes in T cell numbers were measured using the CCK8 kit. Freshly isolated human CD4 T cells cultured on culture plates coated with anti-CD3 and anti-CD28 antibodies were +T cell proliferation increased, but the addition of PD-L1 significantly reduced the proliferation ability, confirming that PD-L1 transmits an inhibitory signal to T cells. Avelumab, a PD-L1 monoclonal antibody, and a bispecific antibody significantly promoted T cell proliferation at concentrations of 100 nM and 500 nM (Figure 11).

[0207] (9) Antibody-dependent cytotoxicity (ADCC) The primary antitumor activity of MGA271 and avelumab is due to the ADCC function of the antibodies, which is related to their IgG1 subtype; bispecific antibodies also contain IgG1 functional domains. The ADCC of the antibodies was measured using an LDH cytotoxicity assay kit. Human PBMCs were purified from white blood cell bags by Ficoll gradient centrifugation, and NK cells were isolated from the human PBMCs using negative selection magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10 6 ) and MDA-MB-231, ES-2 cells (3 × 10 5 ) were co-cultured with or without the bispecific antibody avelumab at different concentrations at the start of the assay. After 18 hours, lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. + PD-L1 + When MDA-MB-231 cells were used as target cells, both the bispecific antibody and avelumab demonstrated ADCC activity, but at lower concentrations, the bispecific antibody activity was stronger and PD-L1 activity was higher. + When ES-2 cells were used as target cells, the bispecific antibody exhibited activity equivalent to that of avelumab at a dose of 100 nM, and strong ADCC activity was observed even at lower concentrations (Figure 12).

[0208] (10) Study of in vivo antitumor activity of bispecific antibodies Human PBMCs (6.67 × 10 6 ) was administered intravenously to 41 NPSG mice via the tail vein on the day before A375 tumor cell inoculation, and 5 × 10 6A375 tumor cells were injected subcutaneously. Five days after tumor inoculation, subcutaneous tumor formation was observed. Each group consisted of 10 animals. The groups were divided into a homogenous control group, a monoclonal antibody group, a combination treatment group (pembrolizumab + MGA271 and avelumab + MGA271), a monoclonal antibody group, and a bispecific antibody group. Treatment began on day 5 of the model and continued twice weekly until the end of the experiment. Tumor volume and animal 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 using tumor growth inhibition scores based on relative tumor volume (TGIRTV) and animal weight change. The bispecific antibody maintained significantly stronger activity than the PD1 monoclonal antibody and MGA271 combination group throughout the experimental period. As the experimental time progressed, the bispecific antibody gradually demonstrated superior efficacy over the avelumab + MGA271 group, with TGIs of 39.29% and 26.45%, respectively, at the end of the experiment (Figure 13). Furthermore, the mice did not experience significant weight loss during the experimental period, confirming the safety of this bispecific antibody treatment (Figure 14).

[0209] The test results (1) to (10) above demonstrate that the bispecific antibody constructed according to the present invention can simultaneously bind to B7H3 and PD-L1, target tumor cells, and release the inhibition of PD-L1 on T cells, and at the same time, exhibit superior antitumor activity compared to the combined administration of monoclonal antibodies. The anti-B7H3 antibody DSYE002 is a reference antibody in which the PD-L1 antibody has been removed from the bispecific antibody DSYE001. The anti-B7H3 antibody DSYE003 is a reference antibody in which the PD-L1 antibody in the bispecific antibody DSYE001 has been replaced (the PD-L1 antibody variable regions in the bispecific antibody have been replaced with Human Anti-HIV-1 gp120 clone b12 VH and VL). The anti-PD-L1 antibody DSYE004 is a reference antibody for the PD-L1 antibody in the bispecific antibody DSYE001.

[0210] The antibodies DSYE002, DSYE003 and DSYE004 were produced according to conventional methods, for example, by constructing vectors and then transfecting them into eukaryotic cells such as HEK293 cells, CHO cells, etc. for purification and expression.

[0211] 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.

[0212] Example 2: Preparation of bispecific antibody drug conjugates (ADCs) 2.1. Linker-payload production

[0213] Linker-cytotoxin X1: [ka]

[0214] Synthetic Route: [ka]

[0215] Step 1: Under nitrogen gas protection, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol) in DMF (50 mL), and the mixture was allowed to react at 25 °C for 17 h. After confirming the completion of the reaction by TLC (PE / EA = 2 / 1), the reaction mixture was added to 500 mL of water, extracted twice with EA (250 mL), separated, washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE:EA = 3:2) to give 5.1 g of a colorless liquid in 57.1% yield.

[0216] Step 2: Under nitrogen gas protection, a solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0 °C, and the mixture was allowed to react for 2 h at 25 °C. After confirming the completion of the reaction by TLC (PE / EA = 1 / 2), the reaction solution was added to 200 mL of water, extracted twice with EA (200 mL), separated, dried over anhydrous NaSO, concentrated, and then purified by column chromatography (PE / EA = 3 / 2) to give 1.56 g of a white solid in 26% yield.

[0217] Step 3: 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 a hydrogen atmosphere at 0°C, and the mixture was stirred at 0°C for 2.5 h. LCMS showed the reaction was complete. The reaction mixture was filtered through diatomaceous earth, and the cake was washed with EA (200 mL). The mixture was then concentrated, dissolved in THF (20 mL), and spin-dried to give 600 mg of a white solid (91% yield).

[0218] Step 4: Under nitrogen gas protection, 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 for 2 h. LCMS showed the reaction was complete. The reaction mixture was added to an aqueous citric acid solution (pH = 4) (150 mL), filtered, and the cake was washed with 175 mL of water. The mixture was then dried and dried under oil pump suction to give 260 mg of a brown solid (yield: 66%).

[0219] Step 5: Under nitrogen gas protection, diethylamine (8 ml) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 ml) at 0°C, and the mixture was allowed to react for 3 hours. LCMS showed that the reaction was complete. The reaction mixture was added to a petroleum ether solution (600 ml) at 0°C, and a solid precipitated. After standing, the solid adsorbed to the bottom of the bottle. The solution was discarded, and the mixture was dried by suction with an oil pump to give 90 mg of a brown solid in a 47.1% yield.

[0220] Step 6: Under nitrogen gas protection, 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 the mixture was allowed to react for 2 h. LCMS showed that the reaction was essentially complete. At 0 °C, the reaction mixture was added to a pH 4 aqueous citric acid solution (30 mL). A fluffy solid precipitated. After filtration, the solid was treated with a purification plate (DCM / MeOH = 10 / 1) to give 9.2 mg of a pale yellow solid, X1, in a 6% yield.

[0221] MS m / z (ESI): 1074 [M+1]. H-NMR (400 MHz,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).

[0222] Linker-cytotoxin X2: [ka]

[0223] Synthetic Route: [ka]

[0224] Step 1 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 h. TLC (PE / EA = 3:1) confirmed the completion of the reaction. The reaction mixture was added to water (200 mL), extracted with EA (250 mL), washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated. Column chromatography (PE:EA = 2:1) afforded 8.7 g of colorless liquid 34b in a 93% yield. MS-ESI: m / z 195.1 [M+H]+.

[0225] Step 2 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL) and cooled to 0 °C under nitrogen gas protection. A solution of 43b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise. After the addition was complete, the mixture was allowed to react at 0 °C for 2 h. TLC (PE / EA = 2 / 1) showed that most of the starting material had reacted. The reaction mixture was poured into 100 mL of water, extracted with DCM (100 mL), separated, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by column chromatography (PE / EA = 1 / 1) to give 3.9 g of colorless viscous product 34d (39% yield). MS-ESI: m / z 503.3 [M+H]+.

[0226] Step 3 Pd / C (1 g, 10 wt.%) was added to a solution of 34d (1.9 g, 3.78 mmol) in 100 mL of EtOH and 100 mL of EA under a hydrogen atmosphere at 0 °C for 3 h. TLC (PE / EA = 2:1) showed the reaction was complete. The reaction mixture was filtered through diatomaceous earth, and the cake was washed with EA / EtOH (1:1, 100 mL x 3). The filtrate was concentrated, dissolved in THF (50 mL x 3), and spin-dried. This procedure was repeated three times to give 1 g of a gray solid, 34e, in a 64% yield. MS-ESI: m / z 435.2 [M+Na]+.

[0227] Step 4 Under nitrogen gas protection, 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. After the addition was complete, the mixture was allowed to react at 0 °C for 2 h. LCMS showed the reaction was complete. The reaction mixture was added dropwise to 300 mL of water, stirred, and then allowed to stand for 5 min. After filtration, the cake was dissolved in DCM / MeOH (10:1, 100 mL), dried, and spun. The mixture was mixed and purified by column chromatography (EA:MeOH = 30:1) to give 600 mg of a yellow solid, 34f, in a 77% yield. MS-ESI: m / z 830.3 [M+H]+

[0228] Step 5 Under nitrogen gas protection, diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0 °C, and the reaction was allowed to proceed for 2 h. LCMS analysis showed the reaction was complete. Petroleum ether solution (100 mL × 6) was added to the reaction mixture, and a solid precipitated. After standing until the solid precipitated, the solution was removed and the mixture was further dried with an oil pump to give 120 mg of a white powder, 34g. LCMS analysis revealed a product content of 70% and a yield of 76%. MS-ESI: m / z 608.3 [M+H]+.

[0229] Step 6 Under nitrogen gas protection, a solution of 34g (60 mg, 0.099 mmol), 43h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) in DMF (1 mL) was added to a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) at 0 °C, and the mixture was allowed to react at 0 °C for 2 h. LCMS showed that the starting material had reacted completely. The reaction mixture was directly passed through a reverse-phase column and purified with an eluent (MeCN / MeOH = 1 / 1):HO = 60%:40%) to give 14.8 mg of a yellow solid, X2, in a 14% yield.

[0230] MS-ESI: m / z 1062.4 [M+H]+. 1H NMR (400 MHz, Methanol-d4) δ 7.69 - 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), 5.60 (d, J = 16.4 Hz, 1H), 5.40 - 5.31 (m, 2H), 5.26 (d, J = 19.0 Hz, 1H), 4.65 - 4.50 (m, 7H), 4.25 - 4.16 (m, 1H), 3.87 (d, J = 16.7 Hz, 1H), 3.83 - 3.76 (m, 3H), 3.72 (d, J = 17.0 Hz, 2H), 3.44 (t, J = 7.1 Hz, 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 = 7.3 Hz, 3H).

[0231] Linker-cytotoxin X3: [ka]

[0232] Synthetic Route: [ka]

[0233] Step 1: Allyl bromide (960 mg, 7.92 mmol) was added to 32a (2.00 g, 6.6 mmol), KCO (1.82 g, 13.2 mmol), and MeCN (20 mL) and stirred at 20 °C for 5 h. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL). The mixture was dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography (PE / EA = 2 / 1) to give 1.83 g of a white solid, 32b, in an 81% yield.

[0234] Step 2: To 32b (1.38 g, 4.02 mmol) in DCM (10 mL) was added TFA (10 mL) and the mixture was stirred at 25 °C for 17 h. TLC (PE / EA = 1 / 3) showed the reaction was complete. The reaction solution was spun dry to give 0.91 g of a yellow viscous product 32c; the yield was not calculated.

[0235] Step 3: To a solution of 32c (910 mg, 4.87 mmol), NaHCO3 (613 mg, 7.3 mmol) in DME / HO (20 mL / 10 mL) was added 41d (1.92 g, 4.87 mmol), and the mixture was stirred at 25 °C for 3 h. TLC (DCM / MeOH = 1 / 1) showed the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH adjusted to 5 with 1 N aqueous HCl, extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography (DCM / MeOH = 20 / 1) to give 1.53 g of white solid 32e in 67% yield. MS-ESI: m / z 467.4 [M+H]+.

[0236] Step 4: A solution of 32f (3 g, 5.83 mmol) in MeOH (50 mL) was added to Pd / C (600 mg) and stirred at 25 °C under a hydrogen balloon for 5 h. TLC (EA) showed the reaction was complete. The reaction solution was filtered and spin-dried to give 1.9 g of a white solid, 32f, in a 77% yield.

[0237] Step 5: HATU (707 mg, 1.86 mmol) was added to 32g (789 mg, 1.86 mmol), KI (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL) and stirred at 0 °C for 3.5 h. TLC (EA) showed the reaction was complete. The reaction mixture was poured into HO (80 mL), extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography (EA) to give 1.186 g of white solid 32h in an 83% yield. MS-ESI: m / z 842.3 [M+H]+.

[0238] Step 6: A solution of 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25 °C for 17 h. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. The reaction mixture was poured into petroleum ether (200 mL) and filtered to give 768 mg of a white solid, 32i, in an 88% yield. MS-ESI: m / z 620.3 [M+H]+.

[0239] Step 7: 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 with HATU (414 mg, 1.09 mmol) and stirred at 20 °C for 17 h. TLC (PE / EA = 1 / 5) showed the reaction was complete. The reaction mixture was poured into water (30 mL), filtered, and the cake was purified by column chromatography (DCM / MeOH = 50 / 1) to give 511 mg of white solid 32j in a 44% yield. MS-ESI: m / z 1068.3 [M+H]+.

[0240] Step 8: A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred at 10 °C for 17 h. TLC (EA) showed the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to give 301 mg of a white solid, 32k; the yield was not calculated.

[0241] Step 9: Morpholine (93 mg, 1.07 mmol) was added to 32k (301 mg, 0.356 mmol), Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL) and stirred at 25 °C for 5 h. LCMS showed the reaction was complete. The reaction mixture was purified to give 108 mg of a white solid, 32l, for a yield of 38%. MS-ESI: m / z 806.3 [M+H]+.

[0242] Step 10: Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32l (108 mg, 0.134 mmol), triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL) and stirred at 0°C for 1 hour. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. The reaction solution was directly purified to give 15 mg of a white solid, X3, with a yield of 12%.

[0243] MS-ESI: m / z 926.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 - 8.42 (m, 3H), 8.27 - 8.16 (m, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (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 = 5.6 Hz, 2H), 3.70 (d, J = 5.9 Hz, 2H), 3.25 - 3.08 (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 = 7.3 Hz, 3H).

[0244] Linker-cytotoxin X4: [ka]

[0245] Synthetic Route: [ka]

[0246] Step 1: Pd / C (400 mg, 10 wt.%) was added to 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) and stirred at 20 °C for 5 h. TLC (EA) showed the reaction was complete. The reaction mixture was filtered and spin-dried to give 1.3 g of white solid 33b, a yield of 74%.

[0247] Step 2: 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 stirred at 0 °C for 2 h. TLC (DCM / MeOH = 1 / 10) showed the reaction was complete. The reaction mixture was poured into water (40 mL) and filtered to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 20 / 1) to give 360 ​​mg of a yellow solid, 33c, in a yield of 41%.

[0248] Step 3: To 33c (360 mg, 0.326 mmol) in DCM (10 mL) was added diethylamine (2 mL). The mixture was stirred at 25 °C for 17 h. TLC (DCM / MeOH = 5 / 1) showed the reaction was complete. The reaction solution was poured into PE (100 mL) and filtered to give 205 mg of white solid 33d, a yield of 71%. MS-ESI: m / z 881.3 [M+H]+.

[0249] Step 4: 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 with a solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) and stirred at 0°C for 1 hour. The reaction mixture was directly purified to give 15 mg of a white solid, X4, in a 6% yield.

[0250] MS-ESI: m / z 1001.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.57 - 8.50 (m, 1H), 8.50 - 8.43 (m, 2H), 8.35 - 8.29 (m, 1H), 8.19 - 8.12 (m, 2H), 7.80 (d, J = 10.8 Hz, 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 - 2.74 (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 = 7.3 Hz, 3H).

[0251] 2.2. Preparation of bispecific antibody-drug conjugates Preparation of bispecific antibody drug conjugate DSYE001-X1 (DAR4):

[0252] [ka]

[0253] At 37°C, prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0 mM, 0.215 mL, 1.503 μmol) was added to a buffer solution of bispecific antibody DSYE001 (30 mM histidine-acetic acid + 20 mM EDTA pH 5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), and the mixture was placed in a thermostatic water bath shaker and reacted with shaking at 37°C for 2 hours, after which the reaction was terminated.

[0254] Linker-cytotoxin X1 (3.10 mg, 2.89 μmol) was dissolved in 1.0 mL of DMA (N,N-dimethylacetamide) and added to the antibody solution. The reaction was stopped by shaking in a water bath shaker at 22°C for 2 hours. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-HCl, pH 5.5). After concentration by ultrafiltration, a solution of bispecific antibody-drug conjugate ADC DSYE001-X1(DAR4) (30 mM histidine-HCl, pH 5.5; 93.8 mg, 12.7 mg / mL, yield: 93.8%) was obtained and stored at 4°C, protected from light.

[0255] A DAR value of p=4.02 was detected and calculated by HIC DAR analysis.

[0256] Preparation of bispecific antibody drug conjugate DSYE001-X1 (DAR6): 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 kettle, followed by the addition of a reducing buffer (20 mM PB, pH 6.7, 93.59 mL), followed by the addition of a 10 mM DTPA solution (66.67 mL), and the pH of the reaction solution was adjusted to 6.72 using 0.4 M NaHPO solution (7.0 mL). The prepared tris(2-carbonylethyl)phosphine hydrochloride solution (10 mM, 19.08 mL, 190.74 μmol) was added to the reaction kettle, and the reaction was carried out at a stirring speed of 50-100 rpm and a reaction temperature of 25 °C for 3.5 hours while controlling the temperature. 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, and DMSO (32.06 mL) was added to the reaction system. Linker-cytotoxin X1 (465.7 mg, 433.56 μmol) was then dissolved in 43.35 mL of DMSO and added to the solution. The reaction was carried out at a temperature of 22 °C for 1.25 hours while stirring at a rotation speed of 50-100 rpm and controlled under temperature control. The prepared NAC solution (50 mM, 43.35 mL, 2.17 mM) was added to the reaction solution, and the reaction was carried out at a rotation speed of 50-100 rpm and controlled under temperature control for 0.5 hours while stirring at a rotation speed of 50-100 rpm and controlled under temperature control. The raw solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm), followed by ultrafiltration solution exchange with an ultrafiltration membrane (30 KD, 0.11 m), to obtain the exemplary product DSYE001-X1 (DAR6) (10 mM histidine-succinic acid, pH 5.0, 14.3 mg / ml), which was stored at -80°C.

[0257] By HIC DAR analysis, a DAR value of n=6.1 was detected and calculated.

[0258] Preparation of bispecific antibody drug conjugate DSYE001-X1 (DAR8): 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 kettle, followed by the addition of a reduction buffer (45 mM PB, pH 7.35, 72.2 mL), followed by the addition of a 10 mM DTPA solution (66.67 mL), and the addition of the prepared tris(2-carbonylethyl)phosphine hydrochloride solution (10 mM, 40.46 mL, 404.6 μmol) to the reaction kettle. The reaction was carried out at a stirring speed of 50-100 rpm and a reaction temperature of 25 °C for 4.5 hours while controlling the temperature. 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, and DMSO (63.40 mL) was added to the reaction system. Linker-cytotoxin X1 (651.9 mg, 606.91 μmol) was then dissolved in 60.69 mL of DMSO and added to the solution. The reaction was carried out at a temperature of 22°C for 1.25 hours while stirring at a rotation speed of 50-100 rpm. The prepared NAC solution (50 mM, 60.69 mL, 3.03 mM) was added to the reaction solution, and the reaction was carried out at a rotation speed of 50-100 rpm for 0.5 hours while stirring at a temperature of 22°C. The raw solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm), followed by ultrafiltration solution exchange with an ultrafiltration membrane (30 KD, 0.11 m), to obtain the exemplary product DSYE001-X1 (DAR8) (10 mM histidine-succinic acid, pH 5.0, 29.5 mg / ml), which was stored at -80°C.

[0259] By HIC DAR analysis, a DAR value of n=7.9 was detected and calculated.

[0260] Preparation of anti-B7H3 antibody-drug conjugate DSYE003-X1 (DAR6) (see ADC-1)

[0261] [ka]

[0262] To a buffer solution of antibody DSYE003 (PBS pH 7.4, 40 mg, 5.36 mg / mL, 0.23 μmol) was added 30 mM His-HAc pH 5.5 buffer solution (1.845 mL), 100 mM EDTA solution (0.5 mL), and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (6.977 mM, 0.208 mL, 1.45 μmol) were added. The mixture was placed in an incubator and incubated at 37°C for 2 hours at 500 rpm. After quenching, 0.750 mL of DMA was added to the solution. Linker-cytotoxin X1 (2.5 mg, 2.32 μmol) was dissolved in 0.25 mL of DMA and added to the solution. The mixture was then placed in an incubator and incubated at 4°C for 1 hour with shaking at 500 rpm. After quenching, the reaction was terminated. A 300 mg / mL suspension of activated charcoal (Charcoal, Dextran Coated, manufacturer: Sigma Aldrich) equivalent to 10% of the total volume of the reaction solution was added, and the mixture was shaken to mix uniformly. The mixture was then mixed uniformly for 1 hour using a rotary mixer at 4°C. After 1 hour, the mixture was centrifuged to recover the supernatant, and a further 10% suspension of activated charcoal was added to the supernatant. The mixture was shaken to mix uniformly, and the mixture was then mixed uniformly for 1 hour using a rotary mixer at 4°C. After completion, the mixture was centrifuged at 4300 rcf for 10 minutes, and the supernatant was filtered through a 0.22 μm syringe filter (Merck Millipore), followed by four rounds of DV ultrafiltration concentration in a 30KD ultrafiltration tube using 30 mM His-HAc pH 5.5 buffer to obtain a solution of exemplary product DSYE003-X1 DAR6 (30 mM His-HAc pH 5.5, 35.89 mg, 6.14 mg / mL, yield: 89.74%), which was stored at -80°C.

[0263] A DAR value of n=6.32 was detected and calculated by HIC DAR analysis.

[0264] Preparation of anti-B7H3 antibody-drug conjugate DSYE002-X1 (DAR6) (see ADC-2) Using linker-cytotoxin X1 and anti-B7H3 antibody DSYE002, DSYE002-X1 (DAR6) was prepared by following the preparation method of bispecific antibody-drug conjugate ADC DSYE001-X1.

[0265] Manufacturing of anti-PD-L1 antibody-drug conjugate DSYE004-X1 (DAR6) (see ADC-3) Using linker-cytotoxin X1 and anti-PD-L1 antibody DSYE004, DSYE004-X1 (DAR6) was prepared by following the manufacturing method of bispecific antibody-drug conjugate ADC DSYE001-X1.

[0266] Preparation of bispecific antibody drug conjugate DSYE001-X2:

[0267] [ka]

[0268] At 37°C, prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0 mM, 0.207 mL, 1.449 μmol) was added to a buffer solution of bispecific antibody DSYE001 (30 mM histidine-acetic acid + 20 mM EDTA pH 5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), and the mixture was placed in a thermostatic water bath shaker and reacted with shaking at 37°C for 2 hours, after which the reaction was terminated.

[0269] Linker-cytotoxin X2 (3.07 mg, 2.89 μmol) was dissolved in 1.0 mL of N,N-dimethylacetamide (DMA) and added to the antibody solution. The reaction was stopped by shaking in a water bath shaker at 22°C for 2 hours. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-HCl, pH 5.5). After ultrafiltration and concentration, the bispecific antibody-drug conjugate ADCDSYE001-X2 solution (30 mM histidine-HCl, pH 5.5, 96.5 mg, 15.8 mg / mL, yield: 96.5%) was obtained and stored at 4°C, protected from light.

[0270] A DAR value of p=3.95 was detected and calculated by HIC DAR analysis.

[0271] Manufacturing of anti-B7H3 antibody drug conjugate DSYE002-X2 (see ADC-4)

[0272] [ka]

[0273] Antibody DSYE002 is a reference antibody in which the PD-L1 antibody has been removed from the bispecific antibody DSYE001. The amino acid sequence of DSYE002 is as shown in the Sequence Listing.

[0274] At 37°C, prepared tris(2-carbonylethyl)phosphine hydrochloride (7.0mM, 1.08mL, 7.55µmol) was added to a buffer solution of anti-B7H3 antibody DSYE002 (30mM histidine-acetic acid + 20mM EDTA pH 5.5, 60mg, 5.5mg / mL, 0.755µmol), and the mixture was placed in a thermostatic water bath shaker and shaken at 37°C for 2 hours, after which the reaction was stopped.

[0275] Linker-cytotoxin X2 (10.8 mg, 9.82 μmol) was dissolved in 1.2 mL of DMA and added to the above solution. The reaction was stopped by shaking in a water bath shaker at 4° C. for 1 hour. The reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 30 mM histidine-HCl pH 5.5). After concentration by ultrafiltration, an exemplary product DSYE002-X2 solution (30 mM histidine-HCl pH 5.5, 30.5 mg, 7.5 mg / mL, yield: 50.8%) was obtained and stored at 4° C. in the dark.

[0276] A DAR value of p=3.98 was detected and calculated by HIC DAR analysis.

[0277] Production of isotype control ADC Using the isotype control antibody and linker-cytotoxin X1, isotype control ADC (DAR4), isotype control ADC (DAR6), and isotype control ADC (DAR8) were prepared, respectively, following the manufacturing method of the bispecific antibody-drug conjugate ADC DSYE001-X1.

[0278] Example 3: Endocytic activity of antibody-drug conjugates Test Purpose The endocytosis effect of the anti-B7H3 and anti-PD-L1 bispecific antibody-drug conjugate of the present invention in A375 and NCI-H1975 cells, which co-express B7H3 and PD-L1, was assessed. Cells were co-incubated with a fixed concentration of test drug and the endocytosis indicator pHrodo, and the endocytosis ability of the test drug was assessed by observing the fluorescent signal emitted by pHrodo, which entered the cells together with the antibody drug, at different time points.

[0279] Experimental Method: 1. A375 and NCI-H1975 cells were added at a density of 1.2 E4 / well to a 96-well black culture plate pretreated with 8 μg / ml, and cultured overnight at 37°C, 5% CO2.

[0280] 2. The test drug and Fab-pHrodo were mixed at a ratio of 1:1.2 and incubated at 37°C and 5% CO2 for 0.5 hours. The mixture was diluted 4-fold from 100 nM to 0.0061 nM.

[0281] 3. The diluted sample was added to the culture plate at 50 μL / well and cultured at 37°C and 5% CO 2 for 24 hours.

[0282] 4. After the culture was completed, the supernatant was removed and the cell culture plate was washed once with 1% BSA.

[0283] 5. DPBS containing 1 μg / mL Hoechst 33342 and 0.5 μg / mL Calcein AM was added to the culture plate at 100 μL / well, and staining was carried out at room temperature for 15 minutes.

[0284] 6. After the culture was completed, the supernatant was removed and the cell culture plate was washed once with 1% BSA.

[0285] 7. Fluorescent puncta formed by internalized antibodies within each cell were counted using a Perkin Elmer Operetta CLS High-Content Analysis System.

[0286] The results are shown in Table 1 and FIG.

[0287] [Table 3]

[0288] Example 4: In vitro tumor cell growth inhibition test using antibody-drug conjugates The CellTiter-Glo® chemiluminescence detection cell viability assay (i.e., CTG method) was used to assess the inhibitory effects of the anti-B7H3 / PD-L1 bispecific antibody-drug conjugates DSYE001-X2 and the anti-B7H3 antibody-drug conjugates DSYE002-X2 on cell proliferation in B7H3- and PD-L1-positive A375 and NCI-H1975 cells after 6 days of culture treatment.

[0289] Logarithmic growth phase cells were harvested and seeded at densities of 400 and 1000 cells / well, respectively, for A375 and NCI-H1975. Cell plates were cultured overnight in a 37°C, 5% CO2 incubator with 50 μL of each solution. On the second day of the experiment, DSYE001-X2 and DSYE002-X2 were diluted 3-fold with complete medium to obtain eight drug concentrations (starting with the highest concentration of 1000 nM). 50 μL of each solution was added to the cell culture plate in duplicate. Complete medium served as a blank control. The culture was continued for 6 days in a 37°C, 5% CO2 incubator. After incubation, the cell culture plate was removed and allowed to equilibrate to room temperature. 50 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, shaken to mix evenly, and then left in the dark for 10 minutes. Signal values ​​were then detected and read using a microplate reader. Using GraphPad Prism software, an S-shaped dose-response curve was constructed using a nonlinear regression model, and the IC was calculated. 50 The value was calculated. Cell viability calculation formula = (Lum 試験薬 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0290] Experimental results showed that compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of the present application exhibited comparable growth inhibitory activity against B7H3 and PD-L1 positively expressing A375 and NCI-H1975 cells.

[0291] Example 5: In vitro tumor cell growth inhibition test using antibody-drug conjugates The CellTiter-Glo® chemiluminescent cell viability assay (i.e., CTG method) was used to evaluate the inhibitory effects of anti-DSYE001-X1 (DAR8) and isotype control ADC (DAR8) on cell proliferation after 7 days of culture treatment with B7H3- and PD-L1-positive expressing human tumor cells.

[0292] Logarithmic growth phase cells were harvested and seeded at densities of 1,000–3,000 cells / well. The cell plates were cultured overnight in an incubator at 37°C with 5% CO2. On the second day of the experiment, DSYE001-X1 (DAR8) was diluted 3-fold with complete medium to obtain nine drug concentrations (starting with the highest concentration of 1,000 nM). 50 μL of each was added to the cell culture plate. Complete medium served as a blank control, and three replicates were set up. The culture was continued for 6 days in an incubator at 37°C with 5% CO2. After the culture was completed, the cell culture plate was removed and equilibrated to room temperature. 50 μL of CTG detection reagent was added to each well, shaken to mix evenly, and then incubated in the dark for 10 minutes. Signal values ​​were detected and read using a microplate reader. GraphPad Prism software was used to construct an S-shaped dose-response curve using a nonlinear regression model, and the IC was calculated. 50 The value was calculated. Cell viability calculation formula = (Lum 試験薬 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0293] The results are shown in Table 2 and FIG.

[0294] [Table 4]

[0295] Example 6: Effect of antibody-drug conjugates on NCI-H1975 cell PD-L1 expression The purpose of this study was to investigate the differential effects of bispecific antibody-drug conjugates on PD-L1 expression in tumor cells compared to their parent monoclonal antibody conjugates.

[0296] Experimental Method 1) NCI-H1975 tumor cells were cultured in RPMI1640 + 10% FBS medium in a constant temperature incubator at 37°C and 5% CO2.

[0297] 2) The cells were trypsinized, counted, and confirmed to have a cell viability of 90.0% or higher. Then, 2 x 106 cells / 2 mL were added to each well of a 6-well plate.

[0298] 3) The next day, medium (blank control), isotype control antibody, isotype control antibody ADC, DSYE001-X1(DAR6), DSYE003-X1(DAR6), and Anti-PDL1-X1 (final concentration 100 nM) were added, and the cells were cultured at 37°C for 72 hours.

[0299] 4) After incubation, the cells were washed twice with PBS, and 500 μL of ice-cold RIPA lysis solution (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added to each 6-well plate. The plate was incubated on ice for 30 minutes, with mixing several times during this time.

[0300] 5) The mixture was centrifuged at approximately 14,000 rpm for 10 minutes at 4°C to remove cell debris, and the supernatant was transferred to a new tube for protein concentration measurement and subsequent experiments.

[0301] 6) Protein was quantified using a BCA quantification kit. Based on the quantification results, the protein samples to be loaded were determined, and the protein concentrations of the samples were standardized to 1-2 μg / μL. LDS spiking buffer (4X) and sample reducing agent (10X) were added, and the samples were heated at 100°C for 10 minutes.

[0302] 7) Or the denatured samples were stored in a refrigerator at -80°C.

[0303] 8) The load sample was thawed.

[0304] 9) Western blotting: Loading: 10 μL of SDS-PAGE gel was loaded into each well (the loading amount depended on the titer of the antibody). Electrophoresis was performed at 80 V for 30 minutes, followed by 120 V for 90 minutes. Transfer was performed using the iBlot2 transfer kit and transfer device with the P3 program for 7 minutes.

[0305] 10) After the transfer was completed, the membrane was cut to match the molecular weight of the protein to be detected and washed with 1x TBST three times, for 5 minutes each time, at room temperature with shaking.

[0306] 11) Sealing: The membrane was sealed with blocking solution (5% skim milk in 1xTBST) for 1 hour at room temperature with shaking.

[0307] 12) The membrane was washed with 1x TBST three times for 5 minutes each and shaken at room temperature.

[0308] 13) Primary antibody incubation: Add appropriate dilution of primary antibody (diluted with 5% bovine serum albumin prepared in 1xTBST) and shake gently overnight at 4°C.

[0309] 14) The membrane was washed with 1x TBST three times for 10 minutes each and shaken at room temperature.

[0310] 15) Secondary antibody incubation: Add appropriate dilution of secondary antibody and shake gently at room temperature for 1 hour.

[0311] 16) The membrane was washed with 1x TBST three times for 10 minutes each and shaken at room temperature.

[0312] 17) Chemiluminescence: HRP substrate from West Femto Ultrasensitive Chemiluminescence Kit was added to the membrane.

[0313] 18) Chemiluminescence was detected and photographed using a Bio-Rad ChemiDoc™ xrs+ or Tanon 5200 Multi machine.

[0314] 29) Detection indicators: PD-L1, β-actin.

[0315] The results are shown in Table 3 and FIG.

[0316] [Table 5]

[0317] Example 7: Evaluation of the efficacy of antibody drug conjugates in a syngeneic transplant mouse model In this study, to investigate the inhibitory effect of the anti-B7H3 and PD-L1 bispecific antibody-drug conjugate of the present invention on tumor growth, an allograft mouse model was established using CT26 (CT26-hPD-L1 / hB7H3) mice stably expressing human B7H3 and PD-L1 in B7H3- and PD-L1-humanized BALB / c mice (BALB / c-hPD-L1 / hB7H3), and the antitumor effect of the bispecific antibody-drug conjugate DSYE001-X2 was evaluated.

[0318] 1. Test Drugs and Materials Isotype control ADC: 10 mg / kg G1: Blank control group (control group): Physiological saline G2:DSYE002-X2 (see ADC-4, treatment group): 12mg / kg G3:DSYE001-X2 (treatment group): 5.5mg / kg Note: Each test substance was administered at equimolar doses.

[0319] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0320] 3. Test animals: BALB / c-hPD-L1 / hB7-H3 mice, female, 6-8 weeks old, weighing approximately 18-22 g. Purchased from Gempharmatech Co., Ltd.

[0321] 4. Testing method: CT26-hPD-L1 / hB7H3 cells were resuscitated and cultured, and a cell freezing batch was recorded. CT26-hPD-L1 / hB7H3 cells in the logarithmic growth phase (3rd to 4th generation after resuscitation) were harvested, the medium was removed, and the cells were washed twice with DPBS. Then, cells were inoculated (cell viability was measured before and after tumor formation). The inoculation dose was 1 x 10 6 The cells were inoculated subcutaneously into the right side of the mouse at 100 μL per mouse (without Matrigel). 3 Grouping was performed when the tumor volume reached 10 mg / kg, and the day of grouping was defined as D0, with treatment starting on D0. The coefficient of variation (CV) of tumor volume did not exceed 1 / 3. The isotype control ADCs, DSYE001-X2, and DSYE002-X2 were injected intravenously (iv) once a week for a total of two doses at 10 mg / kg, 12 mg / kg, and 5.5 mg / kg, respectively. The experiment was terminated 19 days after treatment. Tumor volume and body weight were measured twice a week and the data were recorded. Tumor volume was measured and tumor inhibition rate was calculated.

[0322] 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 on day i of administration in the treatment group and the positive control group; TO: Mean tumor volume on day 0 of administration in the treatment group and the positive control group; V i : Mean tumor volume on day i of administration in the negative control group; V0: Mean tumor volume on day 0 of administration in the negative control group.

[0323] The experimental results are shown in FIG. 18 and Tables 4 to 6.

[0324] [Table 6]

[0325] [Table 7]

[0326] [Table 8]

[0327] 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.

[0328] Example 8: Efficacy evaluation of antibody-drug conjugates in mice bearing human non-small cell lung cancer cells NCI-H1975 To investigate the in vivo tumorigenesis inhibitory effect of DSYE001-X1(DAR4), a subcutaneous tumor model was established in mice by mixing human PBMCs with human lung cancer NCI-H1975 cells, and the in vivo antitumor effect of DSYE001-X1(DAR4) was evaluated.

[0329] NCG mice, female, 6-8 weeks old, weighing 18-22 g, were purchased from Gempharmatech Co., Ltd. NCI-H1975 were continuously cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. Logarithmically growing NCI-H1975 cells were harvested, resuspended in HBSS to the appropriate concentration, and used for subcutaneous tumor inoculation in NCG mice. Cryopreserved PBMCs were purchased, resuspended, counted, and added to NCI-H1975 cells. PBMCs and NCI-H1975 cells were cocultured for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0330] After co-culture of PBMCs and NCI-H1975 cells for 5 days, PBMCs and freshly digested NCI-H1975 cells were collected, and PBMCs 3 × 10 5 pcs, NCI-H1975 cells 2 x 10 6The cells were inoculated subcutaneously into the right side of NCG mice at 0.2 mL / mouse (containing 50% Matrigel). 3 At the time of tumor volume arrival, 20 mice were selected based on tumor volume and randomly divided into four groups, each with five mice. On the day of grouping (day 0), mice were intravenously administered with the isotype control ADC, DSYE001-X1(DAR4), the reference ADC-DSYE002-X1(DAR4), and the combination of DSYE002-X1(DAR4) and PDL1 monoclonal antibody twice weekly for two weeks, for a total of four doses. The doses were 18 mg / kg for the dual antibody ADC and 15 mg / kg (equimolar) for the isotype control ADC and PDL1 monoclonal antibody, respectively. The experiment was terminated on day 28. The experimental grouping and administration are shown below. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0331] Isotype control ADC: 15 mg / kg DSYE001-X1(DAR4) (treatment group): 18mg / kg DSYE002-X1(DAR4) (treatment group): 8mg / kg DSYE002-X1 (reference ADC-2, DAR4) + anti-PD-L1 monoclonal antibody DSYE004 (treatment group): 8 mg / kg + 15 mg / kg All samples were prepared by dilution with PBS.

[0332] Relative tumor growth rate (T / C) (%) was calculated using the following formula: T / C% = TRTV / CRTV × 100% (TRTV: treatment group RTV; CRTV: negative control group RTV). Relative tumor volume (RTV) was calculated from tumor measurement results using the formula: RTV = Vt - V0, where V0 is the mean tumor volume measured at the time of administration (i.e., D0) and Vt is the mean tumor volume at a particular measurement. TRTV and CRTV were obtained on the same day.

[0333] Tumor growth inhibition rate (TGI) (%) = (1-T / C) x 100%. T / C% represents the relative tumor growth rate, and indicates the percentage of tumor volume in the treatment group compared to the control group at a specific time point. T and C represent the tumor volume (TV) at a specific time point in the treatment group and control group, respectively. At the end of the experiment, the mice were euthanized and tumor weights were measured. The experimental results are shown in FIG. 19 and Tables 7-8.

[0334] [Table 9]

[0335] [Table 10]

[0336] The results showed that DSYE001-X1 significantly inhibited tumor growth, its antitumor activity was stronger than that of the B7H3 monoclonal antibody ADC (DSYE002-X1), and it showed superior tumor inhibitory effects compared to the combination group of B7H3 monoclonal antibody ADC and PDL1 monoclonal antibody.

[0337] Example 9: Efficacy evaluation of antibody-drug conjugates in mice bearing human breast cancer cell MDA-MB-231 To investigate the in vivo tumorigenesis inhibitory effect of DSYE001-X1(DAR6), a subcutaneous tumor model was established in mice by mixing human PBMCs with human breast cancer cells MDA-MB-231 cells, and the in vivo antitumor effect of DSYE001-X1(DAR6) was evaluated.

[0338] The experimental animals were 6-8 week-old female NCG mice (purchased from Gempharmatech Co., Ltd.). MDA-MB-231 cells (provided by Gempharmatech Co., Ltd., mycoplasma test results were negative) were revived and subcultured. The revived passage number was N+13. MDA-MB-231 cells in the logarithmic growth phase were collected (seeding passage N+17), the medium was removed, and the cells were washed twice with DPBS before inoculation (cell viability before and after tumor inoculation was 99.27% ​​and 97.00%, respectively). The inoculation dose was 5 × 10 6 One week after tumor cell inoculation, human PBMCs were inoculated via tail vein injection at a dose of 7.5 × 10 6 On the 9th day after inoculation, the average tumor volume was 85.44 mm 3 At the time of tumor volume reaching 1000 mg / kg, 12 mice were selected and randomly divided into two groups, each with six mice, according to tumor volume. The day of grouping was defined as D0, and treatment began on D0. Treatment days were D0 and D7. The experiment was terminated on day 41. Tumor volume and mouse weight were measured twice weekly and recorded.

[0339] At the end of the experiment, mice were euthanized and TGI TV The relative tumor inhibition rate (TGI) was calculated. TV (Relative tumor inhibition rate) calculation formula:

number

[0340] where mean RTV treat : Mean RTV of the treatment group, mean RTV vehicle : Mean RTV value of the vehicle group (solvent group, in this example, the group administered only physiological saline),

[0341] The formula for calculating RTV is:

number

[0342] where V nt : tumor volume of mouse number n on day t, V n0 : tumor volume of mouse no. n on day 0, RTV n : Relative tumor volume of mouse number n on day t.

[0343] The experimental results are shown in FIG. 20 and Tables 9 and 10.

[0344] [Table 11]

[0345] [Table 12]

[0346] Example 10: Efficacy evaluation of antibody-drug conjugates in mice bearing human non-small cell lung cancer cells NCI-H1975 To investigate the in vivo tumorigenesis inhibitory effect of DSYE001-X1(DAR6), a subcutaneous tumor model was established in mice by mixing human PBMCs with human lung cancer NCI-H1975 cells, and the in vivo antitumor effect of DSYE001-X1(DAR6) was evaluated.

[0347] NCG mice, female, 6-8 weeks old, weighing 18-22 g, were purchased from Gempharmatech Co., Ltd. NCI-H1975 were continuously cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. Logarithmically growing NCI-H1975 cells were harvested, resuspended in HBSS to the appropriate concentration, and used for subcutaneous tumor inoculation in NCG mice. Cryopreserved PBMCs were purchased, resuspended, counted, and added to NCI-H1975 cells. PBMCs and NCI-H1975 cells were cocultured for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0348] After co-culture of PBMCs and NCI-H1975 cells for 5 days, PBMCs and freshly digested NCI-H1975 cells were collected, and PBMCs 3 × 10 5 pcs, NCI-H1975 cells 2 x 10 6 The cells were inoculated subcutaneously into the right side of NCG mice at 0.2 mL / mouse (containing 50% Matrigel). 3 When tumor volume reached approximately 1000 mcg, 15 mice were selected based on tumor volume and randomly divided into three groups, each with five mice. On the day of grouping (day 0), DSYE001-X1(DAR6) and DSYE003-X1(DAR6) were administered intravenously (iv) in combination with pembrolizumab twice weekly for two weeks for a total of four doses. The doses were 18 mg / kg for the dual antibody ADC and 15 mg / kg (equimolar) for the isotype control ADC and pembrolizumab, respectively. The experiment was terminated on day 21. The experimental grouping and administration are shown below. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0349] Isotype control ADC: 15 mg / kg DSYE001-X1(DAR6) (treatment group): 18mg / kg DSYE003-X1 (reference ADC-1, DAR6) + pembrolizumab (treatment group): 18 mg / kg + 15 mg / kg

[0350] All samples were prepared by dilution in PBS. Pabrolizumab was prepared by standard methods, and the sequence was derived from the Recommended INN list R72 (2014).

[0351] Relative tumor growth rate (T / C) (%) was calculated using the following formula: T / C% = TRTV / CRTV × 100% (TRTV: treatment group RTV, CRTV: negative control group RTV). Relative tumor volume (RTV) was calculated from tumor measurement results using the formula: RTV = Vt - V0, where V0 is the mean tumor volume measured at the time of administration (i.e., d0), Vt is the mean tumor volume at a particular measurement, and TRTV and CRTV were taken on the same day.

[0352] Tumor growth inhibition rate (TGI) (%) = (1-T / C) x 100%. T / C% represents the relative tumor growth rate, and indicates the percentage of tumor volume in the treatment group compared to the control group at a specific time point. T and C represent the tumor volume (TV) at a specific time point in the treatment group and control group, respectively.

[0353] At the end of the experiment, the mice were euthanized and tumor weights were measured.

[0354] The experimental results are shown in FIG. 21 and Tables 11 and 12.

[0355] [Table 13]

[0356] [Table 14]

[0357] The results showed that DSYE001-X1 significantly inhibited tumor growth and demonstrated superior tumor inhibition effects compared to the combination of B7H3 monoclonal antibody ADC and pembrolizumab.

[0358] Example 11: Pharmacokinetic and Toxicity Studies of Repeated Administration of Antibody-Drug Conjugates Test Purpose DSYE001-X1 (DAR6) was administered intravenously to cynomolgus monkeys twice, once every three weeks. The nature, severity, dose-effect relationship, and time-dependent relationship of toxic reactions that may be caused by the antibody-drug conjugate were observed, and the target organs or tissues of toxicity were determined, providing a basis for future studies.

[0359] Experimental Method A total of four cynomolgus monkeys (two per sex) were used in the study, randomly divided into three groups based on body weight, with one monkey per sex per group. Animals in Groups 1 and 2 were administered 30 and 120 mg / kg of the test substance (DSYE001-X1(DAR6)), respectively, and these were designated the low-dose and high-dose groups of the test substance. Animals were intravenously administered 10 mL / kg over approximately 30 minutes, once every three weeks for three consecutive weeks (a total of two doses).

[0360] During the study period, clinical observations, body weight, food consumption, body temperature, electrocardiogram, and clinical pathology (blood cell count, coagulation function, blood biochemistry) were performed on the animals. One week after the final administration (D29), the animals were euthanized and macroscopic dissection and histopathological examination (gross abnormalities, bone and bone marrow) were performed. At the same time, for the animals administered on D1 and D22, the total antibody protein and DSYE001-X1(DAR6) concentrations in serum, as well as the small molecule concentrations in plasma, were measured, and toxicokinetic analysis was performed.

[0361] [Table 15]

[0362] Experimental Conclusion During the study period, no deaths or moribundity were observed in any of the animals in any of the dose groups, and no abnormal findings related to the test substance 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 histopathological examination (bone and bone marrow), indicating that the antibody-drug conjugate had a good safety profile.

[0363] Example 12: Efficacy evaluation of antibody drug conjugate DSYE001-X1 (DAR8) in a syngeneic transplant mouse model To investigate the inhibitory effect of DSYE001-X1(DAR8) on tumor growth, efficacy evaluation was carried out in BALB / c-hPD-L1 / hB7H3 mice. The specific method was as described in Example 7.

[0364] The antibody-drug conjugate DSYE001-X1 of the present application showed superior antitumor effects compared to combination with B7H3 monoclonal antibody ADC or PD-L1 antibody.

[0365] Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific preferred embodiments of the invention have been described, it should be understood that the invention as claimed is not limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the art of molecular biology, immunology, or other fields are intended to be within the scope of the appended claims.

[0366] Sequence Listing DSYE001 amino acid sequence (underlined CDRs)

[0367] Monoclonal antibody light chain variable region: [ka]

[0368] Monoclonal antibody heavy chain variable region: [ka]

[0369] Nanobody heavy chain variable region: [ka]

[0370] Linker peptide between monoclonal antibody and nanobody: GGGGSGGGGTGGGGS (SEQ ID NO: 11)

[0371] Monoclonal antibody heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVTITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10)

[0372] Monoclonal antibody light chain: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9)

[0373] Bispecific antibody heavy chain: (SEQ ID NO: 16)

[0374] Bispecific antibody light chain: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9)

[0375] DSYE002 amino acid sequence (reference antibody) [ka]

[0376] DSYE003 heavy chain (SEQ ID NO: 18)

[0377] DSYE003 light chain (Human Anti-HIV-1 gp120 clone b12 light chain) EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19)

[0378] PD-L1 monoclonal antibody amino acid sequence (DSYE004)

[0379] PD-L1 monoclonal antibody heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVTITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20)

[0380] PD-L1 monoclonal antibody light chain: similar to bispecific antibody light chain

[0381] Pembrolizumab amino acid sequence

[0382] heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 21)

[0383] Light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22)

Claims

1. A bispecific antibody-drug conjugate comprising an anti-B7H3 and PD-L1 bispecific antibody or antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug, However, the bispecific antibody or antigen-binding fragment thereof is a monoclonal antibody unit that targets PD-L1 and comprises two heavy chains and two light chains; a nanobody unit that targets B7H3 and comprises two identical nanobodies, wherein the N-termini of the two nanobodies are linked to the C-termini of the Fc fragments of the two heavy chains of the monoclonal antibody unit via linker peptides, respectively.

2. The bispecific antibody-drug conjugate of claim 1, wherein the light chain variable region of the monoclonal antibody unit comprises CDR1 whose amino acid sequence is SEQ ID NO: 1, CDR2 whose amino acid sequence is SEQ ID NO: 2, and CDR3 whose amino acid sequence is SEQ ID NO: 3; the heavy chain variable region of the monoclonal antibody unit comprises CDR1 whose amino acid sequence is SEQ ID NO: 5, CDR2 whose amino acid sequence is SEQ ID NO: 6, and CDR3 whose amino acid sequence is SEQ ID NO: 7; and the nanobody comprises CDR1 whose amino acid sequence is SEQ ID NO: 12, CDR2 whose amino acid sequence is SEQ ID NO: 13, and CDR3 whose amino acid sequence is SEQ ID NO:

14.

3. 3. The bispecific antibody-drug conjugate of claim 1, wherein 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.

4. The bispecific antibody-drug conjugate of any one of claims 1 to 3, wherein the monoclonal antibody comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, for example, a human IgG1 constant region.

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

15.

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

11.

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

9.

8. The bispecific antibody-drug conjugate of 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. 【Chemistry 1】 (however, M is -L ２ -L １ -C(O)-, L ２ is —O— or —S—, preferably —O—, and L ２ is linked to said linker unit L, L １ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ -, C ３ ~C ６ saturated cycloalkyl or 3- to 6-membered saturated heterocyclyl, ３ ~C ６ The saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl each independently have one or more R ２ａ is optionally replaced by M is 1, 2, 3 or 4, and the heteroatoms in the 3- to 6-membered saturated heterocyclyl are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3; R １ａ and R １ｂ are each independently hydrogen, halogen, hydroxy, amino or C １ ~C ６ alkyl, １ ~C ６ the alkyl is optionally substituted with one or more halogens; R ２ａ is halogen, hydroxy, amino or C １ ~C ６ alkyl, １ ~C ６ The alkyl is optionally substituted with one or more halogens.

9. L １ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ - and each R １ａ are each independently hydrogen, halogen or C １ ~C ６ alkyl, and each R １ｂ are each independently hydrogen, halogen or C １ ~C ６ is alkyl, Preferably, L １ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ - and each R １ａ are each independently C １ ~C ６ alkyl, preferably C １ ~C ３ alkyl, and each R １ｂ are each independently hydrogen or C １ ~C ６ alkyl, preferably hydrogen or C １ ~C ３ is alkyl, Preferably, L １ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ - and R １ａ is -CH ３ and each R １ｂ are each independently hydrogen or —CH ３ and Preferably, L １ teeth, 【Chemistry 2】 and and / or said cycloalkyl, heterocyclyl and alkyl are each independently unsubstituted; And / or the bispecific antibody-drug conjugate according to claim 8, wherein the atoms in the bispecific antibody-drug conjugate are atoms in natural abundance.

10. L １ is C ３ ~C ６ saturated cycloalkyl or 3- to 6-membered saturated heterocyclyl, preferably L １ is C ３ ~C ６ saturated cycloalkyl, ３ ~C ６ The saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl each independently have one or more R ２ａ and R ２ａ are each independently a halogen or C １ ~C ６ is alkyl, Preferably, L １ is one or more R ２ａ cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted by R ２ａ are each independently a halogen or C １ ~C ６ is alkyl, Preferably, L １ teeth, 【Transformation 3】 The bispecific antibody-drug conjugate of claim 8, wherein

11. In the structure represented by formula (A-1), M is -L ２ -L １ -C(O)-, L ２ is —O—, L １ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ - or C ３ ~C ６ saturated cycloalkyl, ３ ~C ６ A saturated cycloalkyl is one or more R ２ａ is optionally replaced by m is selected from 1 or 2; R １ａ and R １ｂ are each independently hydrogen, halogen, and C １ ~C ６ alkyl, wherein C １ ~C ６ the alkyl is optionally substituted with one or more halogens; R ２ａ are each independently a halogen and C １ ~C ６ alkyl, wherein C １ ~C ６ 9. The bispecific antibody-drug conjugate of claim 8, wherein the alkyl is optionally substituted with one or more halogens.

12. The bispecific antibody-drug conjugate of claim 8 , wherein the cytotoxic drug is selected from any one of the following structures: 【Chemistry 4】

13. The linker unit L is -L ａ -L ｂ -L ｃ - and said L ｃ is linked to the cytotoxic drug, -L ａ -teeth, 【Transformation 5】 and -L ｂ - is one of the following structures: 【Transformation 6】 【Transformation 7】 and 【Transformation 8】 is selected from, preferably 【Chemistry 9】 and -L ｃ -teeth, 【Chemistry 10】 The bispecific antibody-drug conjugate according to any one of claims 1 to 12, wherein

14. The linker unit L is 【Chemistry 11】 and preferably 【Chemistry 12】 14. The bispecific antibody-drug conjugate of claim 13, wherein

15. The structure of the bispecific antibody-drug conjugate is as shown in formula (A-2): 【Chemistry 13】 where p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, Ab is a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, The bispecific antibody-drug conjugate of any one of claims 1 to 14, wherein L is a linker unit L according to claim 13 or 14.

16. The structure of the bispecific antibody-drug conjugate is as represented by formula (A-2a) or (A-2b): 【Chemistry 14】 however, p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number 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—, X １ is one, two or three R ２ａ C optionally substituted by ３ ~C ６ is a saturated cycloalkyl; X ２ is -(C(R １ａ ) (R １ｂ )) ｍ -CH ２ - and m is 1 or 2; R １ａ , R １ｂ and R ２ａ are each independently hydrogen, halogen or C １ ~C ６ alkyl, １ ~C ６ 16. The bispecific antibody drug conjugate of claim 15, wherein the alkyl is optionally substituted with one or more halogens.

17. The bispecific antibody-drug conjugate of any one of claims 1 to 16, wherein the bispecific antibody-drug conjugate is selected from any one of the following structures: 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 (wherein p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 2 to 8, preferably any one integer or decimal number from 4 to 8, preferably any one integer or decimal number from 6 to 8, The Ab is a bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

18. A bispecific antibody-drug conjugate selected from any one of the following structures: 【Chemistry 21】 【Chemistry 22】 (wherein p represents the average number of connections, and p is any one integer or decimal number from 1 to 10, preferably any one integer or decimal number from 3 to 8, preferably any one integer or decimal number from 4 to 8, preferably any one integer or decimal number from 6 to 8, DSYE001 is an anti-B7H3 and PD-L1 bispecific antibody, the heavy chain amino acid sequence of the bispecific antibody is as set forth in SEQ ID NO: 16, and the light chain amino acid sequence of the bispecific antibody is as set forth in SEQ ID NO:

9.

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

20. 20. Use of the bispecific antibody-drug conjugate of any one of claims 1 to 19, or the pharmaceutical composition of claim 19, in the manufacture of a medicament for treating and / or preventing cancer, wherein preferably the cancer is a B7H3 and / or PD-L1 positive-expressing cancer.

21. 21. The use of claim 20, wherein the cancer is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney cancer, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain cancer, 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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