Bispecific antibody drug conjugates against b7h3 and pd-l1 and methods of making and uses thereof

Abstract

The application discloses a kind of bispecific antibody drug conjugates and its preparation method and purposes, and wherein, the structure of the bispecific antibody drug conjugates includes the following fragments: the bispecific antibody or its antigen binding fragment of anti-B7H3 and PD-L1, linker unit L and cytotoxic drug.The bispecific antibody drug conjugates of the application has good endocytosis effect, multiplication inhibitory activity and tumor growth inhibitory activity.

Description

[0001] This application is a divisional application of the patent application filed on December 27, 2023, with application number 202380069557.8 and invention title "Bispecific antibody-drug conjugate against B7H3 and PD-L1 and its preparation method and use". Technical Field

[0002] This invention provides a bispecific antibody-drug conjugate against B7H3 and PD-L1, its preparation method, uses, and pharmaceutical compositions comprising it. Background Technology

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

[0004] B7H3 was highly expressed in all tested cancer types with limited heterogeneity and was rarely expressed in normal tissues. This suggests that B7H3 can be considered a tumor antigen (TA), offering potential for targeted therapy against B7H3-overexpressing tumor cells. Currently, B7H3-targeted therapy strategies mainly include blocking monoclonal antibodies, radioimmunotherapy, antibody-drug conjugates (ADCs), cytotoxic monoclonal antibodies, and bispecific antibodies (BsAbs).

[0005] Inhibitors targeting the immune checkpoints PD1 / PD-L1 are undoubtedly a focal point of tumor immunotherapy. PD-L1 is expressed on the surface of tumor cells, and cytotoxic PD-L1 inhibitory antibodies theoretically possess better anti-tumor activity. However, among marketed PD-L1 monoclonal antibodies, only Avelumab has been reported to mediate ADCC effects against tumors and has shown comparable safety profiles to other PD-L1 antibodies. A key reason is that ADCC efficacy largely depends on the abundance of antigen expression, and PD-L1 cannot be considered a typical tumor antigen, and it exhibits significant heterogeneity within tumor cells. PD1 / PD-L1 pathway blocking antibodies are often used in combination with cytotoxic antibodies to enhance the efficacy of immunotherapy.

[0006] Antibody-drug conjugates (ADCs) consist of three parts: an antibody or its antigen-binding fragment (target), a linker, and a small molecule drug. The antibody or its antigen-binding fragment is conjugated to a small molecule drug with biological activity, such as cytotoxicity, via a cleavable or non-cleavable linker. This fully utilizes the specificity of the antibody or its antigen-binding fragment in targeting cells of interest (target cells) or binding to highly expressed antigens, as well as the high efficiency of the small molecule drug, reducing or avoiding toxic side effects on non-target cells. This means that, compared to traditional chemotherapy drugs for tumors, antibody-drug conjugates for tumors 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 have made the most progress, both currently in Phase II clinical trials. MGC018 conjugates the DNA alkylating agent duocarmycin to a humanized B7H3 antibody via a cleavable linker. Early clinical trials have shown that MGC018 exhibits preliminary antitumor activity and manageable toxicity in patients with advanced metastatic castration-resistant prostate cancer (mCRPC) and melanoma. DS7300 conjugates an irinotecan derivative to a B7H3 antibody via a cleavable linker. Phase I clinical trials have shown antitumor activity in various tumors, including mCRPC, small cell lung cancer, squamous cell lung cancer, esophageal squamous cell carcinoma, and endometrial cancer, with a good safety profile.

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

[0009] The technical problem to be solved by this invention is to overcome the deficiency of limited bispecific antibody-drug conjugates in existing technologies, and to provide a bispecific antibody-drug conjugate, its preparation method, and its applications. The bispecific antibody-drug conjugate of this invention exhibits excellent endocytosis effect, proliferation inhibition activity, and tumor growth inhibition activity.

[0010] The present invention mainly solves the above-mentioned technical problems through the following technical means.

[0011] On one hand, this application provides a bispecific antibody-drug conjugate, the structure of which comprises the following fragments: a bispecific antibody against B7H3 and PD-L1 or its antigen-binding fragment, a linker unit L, and a cytotoxic drug, wherein the bispecific antibody or its antigen-binding fragment comprises: The monoclonal antibody unit targets PD-L1 and comprises two heavy chains and two light chains; the nanobody unit targets B7H3 and comprises two identical nanobodies. The N-terminus of each of the two nanobodies is connected to the C-terminus of the Fc fragment of each of the two heavy chains of the monoclonal antibody unit via a linker peptide.

[0012] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein, The light chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO.: 1, CDR2 with the amino acid sequence SEQ ID NO.: 2, and CDR3 with the amino acid sequence SEQ ID NO.: 3; the heavy chain variable region of the monoclonal antibody unit includes CDR1 with the amino acid sequence SEQ ID NO.: 5, CDR2 with the amino acid sequence SEQ ID NO.: 6, and CDR3 with the amino acid sequence SEQ ID NO.: 7; and the nanobody includes CDR1 with the amino acid sequence SEQ ID NO.: 12, CDR2 with the amino acid sequence SEQ ID NO.: 13, and CDR3 with the amino acid sequence SEQ ID NO.: 14.

[0013] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein, The light chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO.: 4; the heavy chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO.: 8; and the nanobody includes an amino acid sequence as shown in SEQ ID NO.: 15.

[0014] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein, The monoclonal antibody contains an immunoglobulin constant region, which is a human IgG constant region, such as the human IgG1 constant region.

[0015] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein, The light chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 10, and the nanobody comprises an amino acid sequence as shown in SEQ ID NO.: 15; or The full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9; the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO.: 15.

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

[0017] In some embodiments, the bispecific antibody-drug conjugate of the present invention comprises, wherein the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.: 9.

[0018] In some embodiments, the CDR amino acid sequence may have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences described above. In some embodiments, the amino acid sequence of the variable region may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences described above.

[0019] The Fc region of the bispecific antibody of the present invention can be a human Fc region. The Fc region of the bispecific antibody of the present invention can be any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc regions are all IgG1 isotypes. In some embodiments, the Fc regions are all IgG4 isotypes.

[0020] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided in this invention to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0021] In some embodiments, the antibodies provided in this invention may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0022] In some embodiments, the bispecific antibody-drug conjugate of the present invention is wherein the bispecific antibody is DSYE001.

[0023] In some embodiments, in the bispecific drug conjugates, compositions, uses, or methods of the present invention, the bispecific antibody comprises a monoclonal antibody unit and a nanobody unit, wherein the CDR sequence of the monoclonal antibody unit comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and the CDR sequence of the nanobody unit comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0024] In some embodiments, in the bispecific drug conjugates, compositions, uses, or methods of the present invention, the light chain variable region of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 4, the heavy chain variable region of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 8, and the nanobody comprises an amino acid sequence as shown in SEQ ID NO.: 15.

[0025] In some embodiments, in the bispecific drug conjugates, compositions, uses, or methods of the present invention, the full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10, and the amino acid sequence of the nanobody is shown in SEQ ID NO.: 15.

[0026] In some embodiments, in the bispecific drug conjugates, compositions, or uses described in this invention, the heavy chain amino acid sequence of said bispecific antibody is shown as SEQ ID NO.: 16, and the light chain amino acid sequence is shown as SEQ ID NO.: 9.

[0027] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the cytotoxic drug is a structure of formula (A-1), its tautomers, enantiomers, diastereomers, or mixtures of isomers, or a pharmaceutically usable salt thereof or a solvate thereof. ; in, M is -L 2 -L 1 -C(O)-; L 2 It is -O- or -S-, and L 2 Connect to the aforementioned connector unit L; L 1 -(C(R) 1a (R) 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 saturated heterocyclic group are each independently and optionally converted by one or more R 2a replace; m is 1, 2, 3 or 4; the heteroatoms in the 3-6 saturated heterocyclic groups are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3; R 1a and R 1b Each is independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a It is a halogen, hydroxyl, amino, or C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally substituted with one or more halogens.

[0028] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 2 It is -O-.

[0029] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 -(C(R) 1a (R) 1b )) m -CH2-;R 1a It is hydrogen, halogen, or C1-C6 alkyl; R 1b It is hydrogen, halogen, or C1-C6 alkyl.

[0030] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 -(C(R) 1a (R) 1b )) m -CH2-;R 1a It is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R 1b It is hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C3 alkyl.

[0031] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 -(C(R) 1a (R) 1b )) m -CH2-;R 1a -CH3; R 1b It is hydrogen or -CH3.

[0032] In some embodiments, the bispecific antibody-drug conjugate of the present invention is wherein m is 1 or 2.

[0033] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 for , , , or .

[0034] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 It is a C3-C6 saturated cycloalkyl group or a 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl group and the 3-6 membered saturated heterocyclic group are each independently and optionally converted by one or more R 2a Replace, R 2a Each is independently a halogen or a C1-C6 alkyl group.

[0035] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1It is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group is optionally surrounded by one or more R 2a Replace, R 2a Each is independently a halogen or a C1-C6 alkyl group.

[0036] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 For optional use by one or more R 2a Substituted cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, R 2a Each is independently a halogen or a C1-C6 alkyl group.

[0037] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L 1 for , , , , , or .

[0038] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein M is... or .

[0039] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the structure shown in formula (A-1), M is -L 2 -L 1 -C(O)-; L 2 -O-; L 1 -(C(R) 1a (R) 1b )) m -CH2- or C3-C6 saturated cycloalkylene groups, wherein the C3-C6 saturated cycloalkylene group is optionally surrounded by one or more R 2a replace; m is selected from 1 or 2; R 1a and R 1b Each is independently selected from hydrogen, halogens, and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with one or more halogens; R 2a Selected from halogens and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with one or more halogens.

[0040] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the cytotoxic drug is selected from any of the following structures: , , , , , , , , , , and .

[0041] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the linker unit L is -L a -L b -L c -; and the L c Connected to the cytotoxic drug described above; -L a -for or ; and the L b connect; -L b -Any of the following structures: , , , , , and Preferred or Furthermore, the right end of the above structure is preferably connected to the L. c connect; -L c -for .

[0042] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein the linker unit L is , or Preferred .

[0043] In some embodiments, the bispecific antibody-drug conjugate of the present invention has the structure shown in formula (A-2): ; in, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95; Ab and M are defined as in any embodiment of the present invention; L is the connector unit L described in any embodiment of the present invention.

[0044] In some embodiments, the bispecific antibody-drug conjugate of the present invention has the structure shown in formula (A-2): ; in, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95; M is -L 2 -L 1 -C(O)-; L 2 It is -O- or -S-, and L 2 Connect to L; L 1 -(C(R) 1a (R) 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 saturated heterocyclic group are each independently and optionally converted by one or more R 2a replace; m is 1, 2, 3 or 4; the heteroatoms in the 3-6 saturated heterocyclic groups are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3; R 1a R 1b and R 2a Each of the elements is independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl may optionally be substituted with one or more halogens.

[0045] In some embodiments, the bispecific antibody-drug conjugate of the present invention has the structure shown in formula (A-2a) or (A-2b): or ; in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95; Ab is a bispecific antibody or its antigen-binding fragment as described in any embodiment of the present invention; L 2 It can be -O- or -S-; preferably -O-; X1 is any one of 1, 2, or 3 R's. 2aSubstituted C3-C6 saturated cycloalkyl groups; X2 is -(C(R) 1a (R) 1b )) m -CH2-; m is 1 or 2; R 1a R 1b and R 2a Each is independently hydrogen, halogen, or C1-C6 alkyl; the C1-C6 alkyl group may optionally be substituted with one or more halogens.

[0046] In some embodiments, the bispecific antibody-drug conjugate of the present invention is selected from any of the following structures: , , , , , , , , , , and ; in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8; for example, 4.02 or 3.95; Ab is a bispecific antibody or its antigen-binding fragment as described in any embodiment of the present invention.

[0047] In another aspect, the present invention provides a bispecific antibody-drug conjugate selected from any of the following structures: , and ; in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; for example, 4.02, 3.95, 6.1 or 7.9; DSYE001 is a bispecific antibody against B7H3 and PD-L1. The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.: 9.

[0048] In some embodiments, the bispecific antibody-drug conjugates of the present invention (e.g., the bispecific antibody-drug conjugates shown in formula (A-2), (A-2a), or (A-2b) of the present invention) wherein p represents the average number of links, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 2 to 8, preferably any integer or decimal from 4 to 8, preferably any integer or decimal from 6 to 8; for example, 4.02, 3.95, 6.1, or 7.9.

[0049] In some embodiments, the average number of connections p of the present invention can be any integer or decimal from 2 to 8. For example, the average number of connections p can be any integer or decimal from 3 to 8. For example, the average number of connections p can be any integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10.

[0050] In another aspect, the present invention provides a bispecific antibody-drug conjugate selected from any of the following structures (p represents the average number of links): , , and .

[0051] The amino acid sequence of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention is shown in the sequence listing. The CDR numbering method for the antibodies of the present invention is: Kabat numbering.

[0052] In another aspect, the present invention provides a bispecific antibody-drug conjugate selected from any of the following structures: and ; in, t represents the number of connections, and t is any integer from 1 to 10, preferably any integer from 2 to 8, preferably any integer from 4 to 8, for example 4, 6 or 8.

[0053] The amino acid sequence of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention is shown in the sequence listing. The CDR numbering method for the antibodies of the present invention is: Kabat numbering.

[0054] In some embodiments, the number of connections t in this invention is any integer from 1 to 10, preferably any integer from 2 to 8. For example, the number of connections t can be any integer from 3 to 8. For example, the number of connections t is any integer from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0055] In another aspect, the present invention provides a method for preparing a bispecific antibody-drug conjugate, comprising the following steps: under the action of a reducing agent, the bispecific antibody dissolved in a buffer solution is mixed with the linker-cytotoxin dissolved in a solvent to obtain the bispecific antibody-drug conjugate.

[0056] In some embodiments, the reducing agent is a common reducing agent for such reactions in the art, such as tris(2-carbonylethyl)phosphohydrochloride.

[0057] In some embodiments, the buffer solution is a buffer solution conventional for such reactions in the art.

[0058] In some embodiments, the solvent is a solvent conventional for such reactions in the art, such as dimethylacetamide.

[0059] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody-drug conjugate as described in any one of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0060] A further object of the present invention is to provide a method for preparing the pharmaceutical composition of the present invention, the method comprising 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.

[0061] In this invention, the pharmaceutically acceptable carrier used in the pharmaceutical composition is, for example, described in Remington's Pharmaceutical Sciences (2005).

[0062] In this invention, the pharmaceutical composition can be administered in any form, as long as it achieves the prevention, relief, prevention, or cure of symptoms in human or animal patients. For example, it can be formulated into various suitable dosage forms depending on the route of administration.

[0063] In other embodiments, the administration of any of the bispecific antibody-drug conjugates or pharmaceutical compositions described in this invention may be combined with other treatment methods. These other treatment methods may be selected from, but are not limited to, radiotherapy, chemotherapy, immunotherapy, or combinations thereof.

[0064] In another aspect, the present invention provides a pharmaceutical formulation comprising, as an active ingredient, any of the bispecific antibody-drug conjugates described in any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof, or a pharmaceutical composition described in any one of the present invention. In some embodiments, the formulation is in the form of a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form.

[0065] In another aspect, the present invention provides the use of a bispecific antibody-drug conjugate as described in any one of the present invention, or a pharmaceutical composition as described in any one of the present invention, in the preparation of a medicament for treating and / or preventing cancer, preferably, said cancer being a cancer expressing B7H3 and / or PD-L1 positively.

[0066] In another aspect, the present invention provides a method for treating and / or preventing cancer, comprising administering to a subject in need a bispecific antibody-drug conjugate as described in any one of the present invention, or a pharmaceutical composition as described in any one of the present invention, preferably, the cancer being a cancer expressing B7H3 and / or PD-L1 positively.

[0067] In another aspect, the present invention provides a bispecific antibody-drug conjugate as described in any one of the present invention, or a pharmaceutical composition as described in any one of the present invention, for the treatment and / or prevention of cancer, preferably, said cancer being a cancer expressing B7H3 and / or PD-L1 positively.

[0068] In some embodiments, the cancer described in this invention is selected from one or more of lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma. Preferably, the cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0069] In some embodiments, the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-articular, intra-articular (e.g., in arthritic joints), inhalation, aerosol delivery, or intratumoral administration.

[0070] In some embodiments, the present invention provides the combined administration of therapeutically effective amounts of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. In some embodiments, the therapies include surgical treatment and / or radiation therapy.

[0071] In some embodiments, the methods or uses provided by the present invention further include administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. The antibody-drug conjugates of the present invention, or pharmaceutically acceptable salts thereof, can be used alone or in combination with other therapeutic agents in a therapy. For example, they can be co-administered with at least one additional therapeutic agent.

[0072] In another aspect, the present invention provides a pharmaceutical combination comprising a bispecific antibody-drug conjugate against B7H3 and PD-L1 as described herein, a pharmaceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0073] In another aspect, the present invention provides a kit comprising a bispecific antibody-drug conjugate against B7H3 and PD-L1 as described in any one of the present invention, or a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described in any one of the present invention, preferably further comprising a drug delivery device.

[0074] Terminology Definition

[0075] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0076] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise explicitly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0077] In this invention, the term "B7H3," also known as the CD276 antigen, refers to a type 1 transmembrane protein belonging to the B7 family, possessing an extracellular domain composed of a single IgV-IgC domain. B7 family proteins contain extracellular IgV-like and IgC-like domains and have short cytoplasmic tails. B7H3 is an immune checkpoint molecule that is aberrantly overexpressed in various cancers. The amino acid sequence of the B7H3 protein includes the full-length B7H3 protein (such as human 4IgB7H3 protein or human 2IgB7H3 protein), or the extracellular domain of B7H3 (B7H3ECD), or a fragment containing the B7H3 ECD; or a B7H3-ECD fusion protein. Exemplary sequences of the B7H3 protein can be found 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.

[0078] In this invention, the term "PD-L1," Programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type I transmembrane protein. PD-L1 is the surface glycoprotein ligand of PD-1, a key immune checkpoint receptor expressed by activated T cells and B cells that mediates immunosuppression.

[0079] In this invention, the term “about” when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0080] In this invention, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0081] In this 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 inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as "only one" or "exactly one" or when used in the claims as "consisting of...", will it refer to only one number or one element of the list.

[0082] Unless the context clearly indicates otherwise, the words “a” and “an” in this invention shall be understood as “at least one”.

[0083] In this invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this application, "antibody-drug conjugate" can be a bispecific antibody-drug conjugate, which refers to a bispecific antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment via a stable linker unit.

[0084] In this invention, the term "cytotoxic drug" generally refers to a toxic drug that possesses chemical molecules within tumor cells that strongly disrupt their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 (or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or their derivatives, for example, can be toxic drugs, including but not limited to camptothecin derivatives, for example, can be camptothecin derivative essanotecan (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).

[0085] In this invention, the term "antibody" generally refers to an immunoglobulin that reacts to a specified protein or peptide or fragment thereof. Antibodies can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of this application can be derived from any species. The term "antibody" can include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, provided that these antibodies exhibit the desired biological activity.

[0086] In this invention, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope," as described above. As mentioned above, the antigen-binding domain typically includes a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain; however, it is not necessary to include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding functionality of the complete antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab′)2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges of hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of antibody single arms; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichiacoli”, Nature 341: 544-546 (1989), which are incorporated herein by reference in their entirety), having a VH domain; (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), for example derived from scFV libraries.Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be conjugated using a recombination method via a synthetic linker, which allows it to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)) (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); (8) “VHH” refers to the variable antigen-binding domain of heavy chain antibodies from camelids (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be called nanobody (Nb).

[0087] In this invention, the term "variable region" or "variable domain" generally refers to the structural domain of the antibody heavy or light chain involved in antibody-antigen binding. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of the antibody vary significantly, resulting in various specific antibody binding and specificity to their specific antigens. This variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the light chain and heavy chain variable regions, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, connected by three CDR structural loop regions. The CDRs in each chain are closely packed together through the FR region and together with the CDRs from the other chain, they form the antigen-binding site of the antibody.

[0088] In this invention, and in the art, various methods can be used to encode the variable region of an antibody or to delineate the antibody's CDR, such as the Kabat numbering scheme and definition rules based on sequence variability (see Kabat et al., Immunological Protein Sequence, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia numbering scheme and definition rules based on the location of structural loop regions (see Al-Lazikani et al., JMol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V gene by franc et al., as well as Honneger's numbering scheme (AHo's), Martin's numbering scheme, Gelfand's numbering scheme, etc., see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018.

[0089] In this invention, the term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies by any particular method.

[0090] In this invention, the term "multispecific antibody" refers to an antibody containing two or more antigen-binding domains, capable of binding to two or more different epitopes (e.g., two, three, four or more different epitopes), the epitopes being on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind to two different antigens or two different epitopes. Bispecific antibodies targeting B7H3 and PD-L1 may be referred to, for example, as "anti-B7H3 / PD-L1," "anti-B7H3x PD-L1," or "B7H3x PD-L1" bispecific molecules, or other similar terms.

[0091] In this invention, the term "nanobody" refers to a variable domain of heavy chain antibody (VHH), which contains only one heavy chain variable region (VHH) and CH2 and CH3 regions, and naturally lacks the light chain compared to other antibodies. It is composed of the variable domain of the heavy chain from camels (camels, llamas, alpacas, and their close relatives). Nanobody crystals are 2.5 nm in diameter and 4 nm in length, representing the smallest naturally occurring fragment capable of binding to antigens.

[0092] In this invention, the term "domain antibody" refers to an immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0093] In this invention, the term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally include at least a portion of the constant region (Fc) of human immunoglobulins.

[0094] In this invention, "isotype" antibody refers to a type of antibody (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4) provided by the heavy chain constant region gene. Isotype also includes modified forms of one of these types, wherein the modification has been generated to alter Fc function, for example, to enhance or weaken effector function or binding to the Fc receptor.

[0095] In this 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. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is based on the EU numbering system, also known as the EU index, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0096] In this invention, the term "cross-reactivity" refers to the binding of antigen fragments to the same target molecule in human, monkey, and / or mouse (mouse or rat) sources. Therefore, "cross-reactivity" should be understood as the interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a similar molecule (e.g., BDCA2) expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human BDCA2, monkey, and / or mouse BDCA2 (mouse or rat) can be determined by FACS analysis.

[0097] In this invention, "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K). D The equilibrium dissociation constant is represented by the dissociation rate constant and the binding rate constant (k, k ... dis and k on The ratio of affinity to antigen. Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity is measured using surface plasmon resonance (SPR) technology, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0098] In this invention, the term "non-binding" protein or cell refers to a protein or cell that does not bind to itself, or does not bind to it with high affinity, i.e., K-type proteins or cells that bind to it. D 1.0×10 -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0099] In this invention, the term "high affinity" for IgG antibodies refers to the affinity for the K+ of the antigen. D 1.0×10 -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding in the IgM subtype refers to K... D 10 -6 M or lower, preferably 10 -7 M or lower, preferably 10-8 M or lower.

[0100] In this invention, the term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to those in a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0101] In this invention, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine; for example, it can be fluorine or chlorine.

[0102] In this invention, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. It can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted; for example, when substituted, the substituent can be substituted at any usable connection point.

[0103] In this invention, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from the removal of two hydrogen atoms from a group containing one carbon atom. The methylene group can be substituted or unsubstituted, substituted or non-substituted; for example, containing 1 to 12 carbon atoms, such as an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). Alkylene groups can be substituted or unsubstituted, and when substituted, the substituent can be replaced at any usable connection point.

[0104] In this invention, the term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described herein. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy.

[0105] In this invention, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring comprises 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptanetrienyl, cyclooctane, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. Cycloalkyl groups can be substituted or unsubstituted; for example, when substituted, the substituent can be substituted at any usable connection point.

[0106] In this invention, the term "partially unsaturated" generally refers to a cyclic structure in which the cyclic molecules contain at least one double or triple bond. The term "partially unsaturated" encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined in this application. The term "unsaturated" indicates that a portion has one or more degrees of unsaturation.

[0107] In this invention, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic light substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms, wherein 1 to 3 are heteroatoms; even more preferably, it comprises 3 to 6 ring atoms, wherein 1 to 3 are heteroatoms; most preferably, it comprises 5 or 6 ring atoms, wherein 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, and the ring connected to the parent structure is the heterocyclic group. Heterocyclic groups can be substituted or unsubstituted; for example, when substituted, the substituent can be replaced at any usable connection point.

[0108] In this invention, the term "cyclic atom" generally refers to an atom contained in a ring structure. For example, a cyclic atom can be a carbon atom on a benzene ring or a nitrogen atom on a pyridine ring. When a hydrogen atom is attached to a cyclic atom, the cyclic atom can be substituted or unsubstituted; for example, when substituted, the substituent can be substituted at any usable connection point.

[0109] In this invention, the term "independently" generally means that the variable applies to any situation, regardless of whether the variable has the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, when R appears twice in a compound and R is defined as "independently carbon or nitrogen", both Rs can be carbon, both Rs can be nitrogen, or one R can be carbon and the other R can be nitrogen.

[0110] In this invention, the terms "optional" or "optionally" generally mean that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0111] In this invention, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, independently substituted by the corresponding number of substituents. Substituents are only considered in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0112] In this invention, unless otherwise specified, the “connection” between groups can generally be in any orientation; the “connection” between group X and group Y can generally be in any orientation, and any orientation generally means that when group X is used to connect group Y and group Z, two or more connection sites of group X can be arbitrarily connected to group Y or group Z.

[0113] In this invention, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" can be preceded by an identifier indicating the number of atoms present in the group under specific conditions, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., where the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In this context, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0114] In this invention, the cytotoxic drug can be its tautomer, meso compound, racemic compound, enantiomer, and / or diastereomer. 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 can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this application, the terms "tautomer" or "tautomer form" are used interchangeably and generally refer to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. In this application, the term "meta-polymorph" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0115] In this invention, the terms "connector unit" or "connector structure" generally refer to a chemical structural fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. Alternatively, it may be connected to other connectors before being linked to a cytotoxic drug. The direct or indirect connection to the ligand can refer to the group directly connecting to the ligand via a covalent bond, or it can refer to the connection of the ligand via a connector structure. For example, chemical structural fragments or bonds containing acid-labile connector structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connector structures, light-labile connector structures, dimethyl connector structures, or disulfide-containing connector structures can be used as connector structures.

[0116] In this invention, the term "optionally connected to other molecular parts" generally means that the structure is not connected to any other chemical structure, or that the structure is connected to one or more other chemical structures different from the structure (e.g., ligands described in this application) (e.g., connected by chemical bonds or by a linker structure).

[0117] In this invention, the term "drug loading" typically refers to the average number of cytotoxic drugs loaded onto each ligand, or it can be expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 cytotoxic drugs linked to each ligand (Ab), for example, 1-10 cytotoxic drugs. In embodiments of this application, drug loading is expressed as p, t, or n, and can be, for example, an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0118] In this invention, certain atoms of the compounds or antibody-drug conjugates of this invention may appear in more than one isotopic form. For example, hydrogen may appear as protium (… 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that may be incorporated into the compounds of this application include, but are not limited to, those that exist naturally. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of the present invention can be enriched in one or more of these isotopes relative to their natural abundance. As those skilled in the art will know, such isotope-enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium (I, or similar isotopes). 2 H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. For example, deuterium (H) 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Thus, the deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as the case may be). Unless otherwise specified, the structures described in the present invention may also include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody-drug conjugates that are otherwise identical to the structure of the present invention, except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of the present invention.

[0119] In this invention, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described in this application or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The pharmaceutical composition may facilitate administration to the organism, promote the absorption of the active ingredient, and thus exert its biological activity. Conventional preparation methods for pharmaceutical compositions can be found in the Chinese Pharmacopoeia. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For example, any blended fixative oil, including synthetic mono- or diglycerides, may be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0120] In this invention, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to salts of compounds or antibody-drug conjugates of this invention, or salts of compounds or antibody-drug conjugates described in this invention, which are safe and / or effective when used in mammals and have the desired biological activity. The compounds or antibody-drug conjugates of this invention can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, 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.

[0121] In this invention, the term "pharmaceutically acceptable carrier" generally refers to a carrier or delivery system for administering therapeutic agents, such as antibodies or peptides, genes, and other therapeutic agents. This 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 causing excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol, and ethanol. These carriers may also contain excipients, such as wetting agents or emulsifiers, pH buffers, etc.

[0122] In this invention, the terms "treatment" and "treating" generally refer to a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefits. Therapeutic benefits include, but are not limited to, eradicating, suppressing, reducing, or improving the underlying disorder being treated. Furthermore, therapeutic benefits are achieved by eradicating, suppressing, reducing, or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the patient, although the patient may still have the underlying disorder.

[0123] In this invention, the terms "prevention" and "preventing" generally refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. For the purpose of preventing benefits, a pharmaceutical composition may be administered to a patient at risk of developing a specific disease or to a patient who reports having one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0124] In this invention, the terms “subject” or “patient” generally refer to humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged or elderly people) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quails and / or turkeys.

[0125] In this invention, the terms "therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount by which the compound or antibody-drug conjugate of this invention, alone or in combination with other therapeutic agents, effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions, when administered to cells, tissues, or subjects. A therapeutic effective dose also refers to a dose sufficient to result in symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or accelerates the treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of the therapeutic agent will result in an increase of at least 10% in diagnostic criteria or parameters, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0126] In this invention, the term "cancer" refers to a group of cells exhibiting an abnormally high level of proliferation and growth. Cancer may be benign (also called a benign tumor), premalignant, or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. In this invention, the term "tumor" refers to one or more cells containing cancer. In this invention, the term "tumor growth" refers to the proliferation or growth of one or more cells containing cancer, resulting in a corresponding increase in the size or extent of the cancer.

[0127] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0128] The reagents and raw materials used in this invention are all commercially available.

[0129] The positive and progressive effects of this invention are as follows: The bispecific antibody-drug conjugate of the present invention, wherein the bispecific antibody has the following advantages: 1. It can bind to both B7H3 and PD-L1 simultaneously, and can relieve the inhibition of T cells by PD-L1 while targeting tumor cells, thus showing superior anti-tumor activity compared to combination therapy with monoclonal antibodies.

[0130] 2. Compared with related monoclonal antibody combination therapy, the bispecific antibody of this application has the advantages of good compliance and controllable quality.

[0131] 3. The stability characterization of the bispecific antibody in this invention is mainly reflected in the study of monomer purity and thermal stability. After a single affinity purification, the monomer content of the bispecific antibody can reach 95%, even better than the purity achieved after multiple secondary purifications in the industry. The structural and activity analysis results of the antibody after heat treatment demonstrate that the antibody can maintain a good molecular conformation and complete biological activity under harsh environments, which is beneficial for the industrial production and packaging storage of the antibody. Overall, this invention constructs a B7H3 / PD-L1 bispecific antibody in the form of IgG-VHH2; it exhibits good molecular stability and has significantly better in vitro activity (binding molecular level and cellular level) than Avelumab and MGA271. In vivo data demonstrate that, in B7H3 + In A375 tumor cells, the bispecific antibody exhibited superior antitumor activity compared to the combination therapy with the B7H3 monoclonal antibody. Therefore, the bispecific antibody of this invention, due to its excellent developability and activity, has broad application prospects.

[0132] The bispecific antibody-drug conjugates of the present invention have one or more of the following advantages: 1. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugate of the present invention has significantly enhanced proliferative inhibitory activity against tumor cells in vitro, especially against A375, NCI-H1975, NCI-H441 and NCI-H358 cells that are positive for B7H3 and PD-L1.

[0133] 2. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugate of the present invention can more significantly induce the downregulation of PD-L1 expression, indicating that the bispecific antibody drug conjugate of the present invention can exert a stronger immunosuppressive relief effect by reducing the target expression level.

[0134] 3. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugate of the present invention has significantly enhanced in vivo tumor growth inhibition activity, such as in CT26 cells, and especially in the construction of homologous transplantation mouse models using CT26 cells.

[0135] 4. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugate of the present invention can significantly inhibit the growth of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, especially melanoma A375 cells, esophageal squamous cell carcinoma KYSE-150 cells, small cell lung cancer NCI-H292 cells, liver cancer Huh7 cells, MDA-MB-231 breast cancer cells, or NCI-H1975 non-small cell lung cancer.

[0136] 5. Compared with related monoclonal antibody-drug conjugates and monoclonal antibody combination therapy, the bispecific antibody-drug conjugate of the present invention can significantly inhibit the growth of melanoma, esophageal squamous cell carcinoma, small cell lung cancer, liver cancer, breast cancer cells, and non-small cell lung cancer, especially MDA-MB-231 breast cancer cells and NCI-H1975 non-small cell lung cancer.

[0137] 6. Compared with related monoclonal antibody drug conjugates, the bispecific antibody drug conjugate of the present invention has a significantly enhanced endocytosis effect.

[0138] 7. The bispecific antibody-drug conjugate of the present invention has excellent safety.

[0139] The bispecific antibody-drug conjugate of any one of the present invention may have one or more effects selected from the group consisting of: (1) having inhibitory activity against the in vitro proliferation of tumor cells; (2) having targeted inhibition; (3) having plasma stability; (4) having in vivo tumor suppression effect; (5) having bystander effect; (6) having antitransporter transport capability; (7) having in vivo tumor targeting capability; (8) having stronger tumor suppression effect in individuals with a healthy immune system; and (9) having good in vivo safety. Attached Figure Description

[0140] Figure 1 This is a screening diagram showing the affinity between the anti-B7H3 VHH humanized antibody prepared in this invention and B7H3.

[0141] Figure 2 This is a schematic diagram of the structure of the bispecific antibody DSYE001 in the bispecific antibody-drug conjugates DSYE001-X1 or DSYE001-X2 of the present invention.

[0142] Figure 3 This is an SDS-polyacrylamide gel electrophoresis image of DSYE001, the bispecific antibody against B7H3 and PD-L1 of the present invention.

[0143] Figure 4 The purity of DSYE001, the bispecific antibody against B7H3 and PD-L1 of the present invention, was determined by SEC-HPLC.

[0144] Figure 5 The Tm value (DSF) of DSYE001, the bispecific antibody against B7H3 and PD-L1 of the present invention, was determined.

[0145] Figure 6The present invention provides a bispecific antibody DSYE001 against B7H3 and PD-L1, which is bound to an ELISA before and after heat treatment at 60°C. Here, a represents the ELISA binding to PD-L1, and b represents the ELISA binding to B7H3.

[0146] Figure 7 The present invention relates to a bispecific antibody DSYE001 binding ELISA against B7H3 and PD-L1, wherein a represents the binding ELISA against PD-L1 and b represents the binding ELISA against B7H3.

[0147] Figure 8 The graphs show the affinity of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention at five different concentrations of BLI at either B7H3-his or PD-L1-his. a represents the affinity curve with PD-L1, and b represents the affinity curve with B7H3.

[0148] Figure 9 The image shows the PD-L1 / CHO-PD1 blocking curve of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention.

[0149] Figure 10 The present invention provides a bispecific antibody against B7H3 and PD-L1, DSYE001, which induces T cells to secrete IFN-γ.

[0150] Figure 11 The bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention promotes T cell proliferation.

[0151] Figure 12 The ADCC effect of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention on cancer cells is shown in Figure a, 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.

[0152] Figure 13 This invention demonstrates the in vivo tumor-suppressing effect of DSYE001, a bispecific antibody against B7H3 and PD-L1.

[0153] Figure 14 The effect of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention on mouse body weight.

[0154] Figure 15 The results of the endocytosis activity test of the bispecific antibody-drug conjugate DSYE001-X2 of the present invention are shown.

[0155] Figure 16This study presents an in vitro assay of the bispecific antibody-drug conjugate DSYE001-X1 (DAR8) of the present invention to inhibit the proliferation of tumor cells.

[0156] Figure 17 The effect of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention on PD-L1 expression in NCI-H1975 cells.

[0157] Figure 18 This study evaluates the efficacy of the bispecific antibody-drug conjugate DSYE001-X2 in a homologous transplanted mouse model.

[0158] Figure 19 This study evaluates the efficacy of the bispecific antibody-drug conjugate DSYE001-X1 (DAR4) of the present invention in human non-small cell lung cancer cells NCI-H1975 tumor-bearing mice.

[0159] Figure 20 This study evaluates the efficacy of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention in human breast cancer cell MDA-MB-231 tumor-bearing mice.

[0160] Figure 21 This study evaluates the efficacy of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention in human non-small cell lung cancer cells NCI-H1975 tumor-bearing mice. Detailed Implementation

[0161] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0162] For experimental methods in the following examples where specific conditions are not specified, follow conventional methods and conditions, or select according to the product instructions.

[0163] Sample testing

[0164] 1. ADC DAR value analysis method – HIC-HPLC (hydrophobic chromatography)

[0165] High Performance Liquid Chromatography (HPLC): Waters e2965 HPLC system.

[0166] Column: MabPac™ HIC-Butyl 5μm 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); Perform elution according to the following procedure.

[0167] Detection conditions: The mobile phase flow rate was set to 1 mL / min, the detection wavelength to 280 nm, and the column temperature to 30℃.

[0168] 2. SEC purity analysis – SEC-HPLC (size exclusion chromatography)

[0169] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0170] Column: Waters Xbridge BEH200 SEC (7.8 × 300 mm, 3.5 μm)

[0171] Mobile phase: 50mM NaH2PO4 + 200mM arginine (pH 6.80) + 10% isopropanol

[0172] Detection conditions: The mobile phase flow rate was set to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 ℃.

[0173] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as 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 in quotation marks, are incorporated herein by reference in their entirety.

[0174] Example

[0175] The following examples are provided to demonstrate and further explain some preferred embodiments and aspects of the invention, and should not be construed as limiting its scope.

[0176] Example 1: Preparation and testing of bispecific antibody DSYE001

[0177] In the anti-B7H3 and PD-L1 bispecific antibody-drug conjugate of the present invention, the anti-B7H3 and PD-L1 bispecific antibody or its antigen-binding fragment is prepared according to the method in PCT / CN2022 / 125089, as shown below (the CDR region of the bispecific antibody is determined according to the Kabat numbering rules): 1. Humanization of camel-derived anti-B7H3 Camel-derived anti-B7H3 nanobodies were humanized using the Germline gene of the human antibody gene as a template via the framework shuffling method. The corresponding VHH morphology libraries were generated through whole-body in vitro synthesis via overlap PCR. Then, clones of the VHH phage library were constructed, screened, and identified.

[0178] More precisely, a one-step strategy was used to humanize camel-derived anti-B7H3 nanobodies. Approximately 1000 clones were screened from this sub-library, and the selected positive clones were screened for phage-level thermostability using ELISA. 5 μg / mL huB7H3 antigen was coated onto highly absorbent 96-well ELISA plates, and the supernatant from overnight amplification of the screened phages was reacted. Clones exhibiting higher OD450 reads were selected.

[0179] The above-mentioned phage clones were sequenced to obtain the B7H3 VHH gene sequence. The C-terminus of this sequence was fused with the human Fc protein gene to construct and express B7H3-Fc. Using the biomembrane interference method, a Protein A probe was used to capture 100 nM of B7H3 VHH-Fc and bind it to a 200 nM initial concentration of B7H3 antigen diluted twofold. The KD value of the antibody-antigen binding was calculated. The results showed that 75-16 (amino acid sequence SEQ ID NO.: 15, CDR1 sequence SEQ ID NO.: 12, CDR2 sequence SEQ ID NO.: 13, CDR3 sequence SEQ ID NO.: 14) had the highest affinity for B7H3, with a KD reaching 3.24 × 10⁻⁶. -9 M (see) Figure 1 Therefore, it was chosen for the next step of constructing bispecific antibodies.

[0180] 2. Construction and expression of bispecific antibodies against B7H3 and PD-L1

[0181] 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) are derived from existing PD-L1hIgG1 humanized monoclonal antibodies. Furthermore, the N-terminus of the anti-B7H3 VHH (75-16 above) is linked to the C-terminus of the Fc fragment via a linker peptide (SEQ ID NO.: 11) (structure as shown). Figure 2 (As shown).

[0182] The DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in *E. coli*. The purified plasmid was transfected into HEK293 cells via PEI. The cells were then cultured in OPM-CD05 expression medium. After 6 days of culture, the cell culture supernatant was collected, and the antibody was purified using a protein A column. The purified IgG1 was dialyzed against phosphate-buffered saline (PBS), flash-frozen, and stored at -80°C.

[0183] The heavy chain amino acid sequence of the purified anti-B7H3 and PD-L1 bispecific antibody DSYE001 is SEQ ID NO.: 16, and the light chain amino acid sequence is SEQ ID NO.: 9.

[0184] Test section

[0185] Unless otherwise specified, the term "bispecific antibody" in this invention refers to the bispecific antibody against B7H3 and PD-L1, and is also referred to as "B7H3 / PD-L1 bispecific antibody", "B7H3 / PD-L1 bispecific antibody" or "bispecific antibody".

[0186] Test method: (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 5 μg of bispecific antibody was mixed with protein reduction and non-reduction buffer, and the mixture was brought to a final volume of 10 μL with PBS. The mixture was heated at 100 °C for 10 minutes to fully denature the protein. 9 μL of this mixture was added to pre-made polyacrylamide gel wells (Bio-Rad). Separation was performed by first applying 80V for 30 minutes, then 120V for 60 minutes. The gel was stained with Coomassie Brilliant Blue for 30 minutes, and then destained with a destaining solution (acetic acid:ethanol:water = 1:3:6) for 15 minutes. This destaining process was repeated three times to remove the background. Images were then acquired using a gel imaging system. The results showed that the B7H3 / PD-L1 bispecific antibody exhibited good monomer purity under non-reduction conditions. Under reduction conditions, two bands appeared (heavy and light chains) due to the opening of disulfide bonds between the light and heavy chains, with no impurities. Figure 3 ).

[0187] (2) Size exclusion chromatography (SEC-HPLC)

[0188] SEC-HPLC analysis was used to assess the monomeric purity of the bispecific antibody. The B7H3 / PD-L1 bispecific antibody was analyzed using a 1260 HPLC system (Agilent, Santa Clara, CA) on a ThermoMAbPac SEC-1, 5 μm, (7.8 × 300 mm) P / N 088460, and compared with PD-L1 monoclonal antibody and B7H3 VHH-Fc. The mobile phase used was phosphate-buffered saline (PBS). The flow rate was set to 0.7 mL / min; the injection volume was 15 μL. SEC chromatograms were recorded by monitoring the absorbance at 280 nm using a UV detector at a constant temperature of 25 °C. Figure 4 As shown, all the antibodies tested had a very high proportion of monomer peaks, with the peak area ratio of monomer peaks reaching over 95%. This indicates that the bispecific antibodies have good monomer purity and a small number of aggregates under PBS buffer conditions.

[0189] (3) Differential scanning fluorescence (DSF) method for detecting the Tm value of antibody

[0190] DSF was detected using a real-time PCR instrument (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 5000-fold concentrated stock solution with ddH2O. 20 μL of sample was placed in a PCR tube, and SYPRO Orange working solution was added to the reaction to prevent bleaching. The mixture was briefly centrifuged, and the liquid was collected at the bottom of the PCR tube. The qPCR instrument was turned on, and the program was set to 25℃-95℃ with a temperature increase of 0.3℃ per second. Data were collected, and temperature and signal values ​​were plotted. The melting temperature (Tm) was calculated. The data showed that the Tm of the Fc and Fab of the B7H3 / PD-L1 bispecific antibody were 69℃ and 90℃, respectively, demonstrating excellent heat resistance, and were similar to the Tm of the B7H3 VHH-Fc and PD-L1 monoclonal antibody. Figure 5 ).

[0191] (4) Verify the thermostability and binding activity of the antibody using ELISA.

[0192] 96-well microplates were coated overnight at 4°C with 2 μg / ml histagged PD-L1 or B7H3 antigen protein. The next day, 100 μL of Casein blocking buffer was added to each well and the plates were blocked at 37°C for 1 hour. Three-fold serial dilutions of B7H3 / PD-L1 bispecific antibody (for thermostability testing, the bispecific antibody was treated in a 60°C water bath for 1 hour) and monoclonal antibodies (for testing bispecific antibody binding to B7H3 using ELISA, the control monoclonal antibodies used were MGA271, Isotype hIgG1, and B7H3 VHH-Fc; for testing binding to PD-L1, the control monoclonal antibodies were PD-L1 monoclonal antibody, Avelumab, and Isotype hIgG1) were added to the wells. After incubation at 37°C for 1 hour, unbound antibodies were washed away with 0.1% PBST. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, USA). A 50 μL solution of 3,3',5,5'-tetramethylbenzidine substrate (TMB) was used for color development. After standing for 5 minutes, 50 μL of 2M sulfuric acid was added to stop the color development. The absorbance was then measured at OD450 nm using a SpectraMax M5e (Molecular Devices) microplate reader. A 4-parameter fitting method was used to plot the antibody concentration against the OD450 reading, and the EC50 was calculated. 50 . Figure 6Figures a and b show the binding curves of the bispecific antibody with PD-L1 and B7H3 before and after treatment at 60°C for 1 hour. As can be seen from the figures, the binding curves of the bispecific antibody before and after heating largely overlap for both PD-L1 and B7H3, indicating that the bispecific antibody can withstand the high temperature of 60°C and maintain strong binding activity against both PD-L1 and B7H3 targets. On the other hand, the bispecific antibody, positive antibody, PD-L1 monoclonal antibody, and B7H3 VHH-Fc showed similar binding abilities to both targets. The bispecific antibody, PD-L1 monoclonal antibody, and control Avelumab bound to human PD-L1 ECGs... 50 The values ​​are 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which are in the same order of magnitude. Figure 7 a) Bispecific antibodies, B7H3 VHH-Fc, and MGA271 also exhibit similar binding activity to human B7H3, EC 50 The values ​​were 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively. Figure 7 b).

[0193] (5) Determination of the affinity of bispecific antibodies for antigens using the BLI method

[0194] B7H3 / PD-L1 bispecific antibody, PD-L1 monoclonal antibody, and B7H3 VHH-Fc were diluted to 100 nM in sample buffer (0.02% Tween 20 and 0.1% BSA in PBS). The affinity of the bispecific antibody for specific human B7H3 and human PD-L1 antigens was analyzed using OCTET 96. Protein A probes were used to immobilize the antibodies. B7H3-his and PD-L1-his antigens were diluted to an initial concentration of 200 nM in sample buffer, and multiple antigen gradients were set up at 2-fold dilutions to bind with the antibodies, obtaining rate constants and affinity. Kon and Koff values ​​were calculated using software provided by the supplier, and the KD value of the antibody was obtained. As shown in the figure, the bispecific antibody has a high affinity for PD-L1 and B7H3, with KD values ​​reaching 5.85 × 10⁻⁶. -10 M and 8.11×10 -9 M ( Figure 8 ).

[0195] (6) Flow cytometry detection of the ability of bispecific antibodies to block the PD1 / PD-L1 pathway

[0196] The ability of the B7H3 / PD-L1 bispecific antibody to block the binding of human PD-L1 and human PD1-CHO cells was evaluated by flow cytometry and compared with Avelumab and PD-L1 monoclonal antibody. 2×10 5Personal PD1-CHO cells were evenly seeded in 96-well plates and incubated at room temperature for 30 minutes with a mixture of 400 nM starting, serially diluted antibodies (B7H3 / PD-L1 bispecific antibody, Avelumab, PD-L1 monoclonal antibody, Isotype hIgG1, B7H3 VHH-Fc) and biotinylated PD-L1 antigen (50 nM). The mixture was then incubated with cells at 4°C for 45 minutes. Unbound antigen was washed away with PBS. Cells were then stained with PE-streptavidin for fluorescence. The mean fluorescence intensity (MFI) of the PE channel in the flow cytometer was read. Antibody concentration and MFI were plotted, and the IC50 of the antibody was calculated using a four-parameter fitting method. 50 Flow cytometry analysis showed that the bispecific antibody could block the binding of PD-L1 to CHO-PD1 cells, with an IC50 value of [missing information]. 50 The concentration was 106.4 nM and the blocking activity was similar to that of PD-L1 monoclonal antibodies (IC50). 50 (94.20 nM), Avelumab (IC) 50 (115.0 nM) similar to ( Figure 9 ).

[0197] (7) Mixed lymphocyte reaction (MLR) to detect the ability of bispecific antibodies to activate T cells

[0198] Monocytes isolated from peripheral blood mononuclear cells (PBMCs) were cultured in vitro 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 to induce dendritic cells (DCs). CD4+ T cells (1×10⁻⁶) were also cultured. 5 ) and allogeneic DCs (1.25×10 4 Co-culture was performed using RPMI 1640 complete medium containing 10% FBS under 5% CO2 and constant temperature of 37℃. Groups were established with no antibody and with different concentrations of B7H3 / PD-L1 bispecific antibody, PD-L1 monoclonal antibody, Avelumab, B7H3 VHH-Fc, MGA271, and Isotype hIgG1. After 5 days, the IFN-γ concentration in the culture supernatant was analyzed using an IFN-γ ELISA kit. MLR results showed that the bispecific antibody could stimulate CD4+ T cells to secrete IFN-γ, and the T cell activation ability of the bispecific antibody was superior to that of PD-L1 monoclonal antibody and Avelumab at both low and high concentrations. Furthermore, when only B7H3 antibody was present, although there was no significant effect compared to PD-L1 signal blocking antibody, the bispecific antibody partially activated T cells due to the blocking of B7H3 inhibitory signal transduction. Figure 10).

[0199] (8) T cell proliferation experiment

[0200] 1 μg / mL CD3 antibody (Clone HIT3a), 1 μg / mL CD28 antibody (Clone CD28.2), and 5 μg / mL human PD-L1 were coated onto 96-well cell culture plates (Corning, USA) at 4°C for 1 hour. Control wells were coated with mouse IgG2a isotype control alone, or with CD3 and CD28 antibodies using the same method. 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 at 37°C in RPMI 1640 medium containing 10% FBS (Gibco) for 4 days. After 4 days, changes in T cell count were detected using a CCK8 assay kit. Data showed that freshly isolated human CD4+ T cells cultured in wells coated with anti-CD3 and anti-CD28 antibodies significantly improved cell count. + T cells exhibited increased proliferation, but the proliferation capacity significantly decreased when PD-L1 was added to the wells, confirming that PD-L1 provided an inhibitory signal to T cells; Avelumab, PD-L1 monoclonal antibody, and bispecific antibody significantly promoted T cell proliferation at concentrations of 100 nM and 500 nM. Figure 11 ).

[0201] (9) Antibody-dependent cytotoxicity (ADCC)

[0202] The primary antitumor activity of MGA271 and Avelumab derives from the antibody ADCC function, which is related to their IgG1 subtype; the bispecific antibody also possesses an IgG1 functional region. The antibody ADCC was measured using an LDH cytotoxicity assay kit. Human PBMCs were purified from leukocyte packages using Ficoll gradient centrifugation, and NK cells were isolated from human PBMCs using negative-selective magnetic beads (Miltenyi Biotec, Auburn, CA). NK cells (3 × 10⁻⁶) 6 ) and MDA-MB-231, ES-2 cells (3×10 5 At the start of the assay, different concentrations of bispecific antibody and Avelumab were added or not added, and the culture was co-cultured. After 18 hours, the secretion of lactate dehydrogenase (LDH) in the culture supernatant was analyzed by ELISA. When using B7H3... + PD-L1 +When MDA-MB-231 was used as the target cell, both the bispecific antibody and Avelumab exhibited ADCC activity, but the bispecific antibody showed stronger activity at low concentrations; when PD-L1 was used... + Using ES-2 cells as target cells, the bispecific antibody was observed to have activity comparable to Avelumab at a dose of 100 nM, and also exhibited strong ADCC activity at low concentrations. Figure 12 ).

[0203] (10) Study on the in vivo antitumor activity of bispecific antibodies

[0204] Human PBMCs (6.67 × 10⁻⁶) 6 One day before inoculation with A375 tumor cells, 41 NPSG mice were injected via the tail vein, and the following day, 5 × 10⁵ cells were injected subcutaneously. 6 A375 tumor cells were inoculated. Subcutaneous tumor formation was observed five days after tumor inoculation. Ten animals were used in each group, divided into isotype control, monoclonal antibody combination therapy (Pembrolizumab + MGA271 and Avelumab + MGA271), monoclonal antibody, and bispecific antibody groups. Drug administration began on day 5 after successful model establishment 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. Efficacy and safety were evaluated based on tumor growth inhibition values ​​based on relative tumor volume (TGIRTV) and changes in animal body weight. The bispecific antibody maintained significantly stronger activity than the PD1 monoclonal antibody + MGA271 combination therapy throughout the experiment; with increasing experimental time, the bispecific antibody also gradually showed superior efficacy compared to the Avelumab + MGA271 group, with TGIs of 39.29% and 26.45% at the end of the experiment, respectively. Figure 13 Furthermore, no significant weight loss was observed in mice during the experiment, demonstrating the safety of this bispecific antibody treatment. Figure 14 ).

[0205] As can be seen from the test results (1) to (10) above, the bispecific antibody constructed in this invention can bind to B7H3 and PD-L1 at the same time, and can relieve the inhibition of T cells by PD-L1 while targeting tumor cells, and at the same time show anti-tumor activity superior to monoclonal antibody combination therapy.

[0206] Anti-B7H3 antibody DSYE002 is the reference antibody for removing the PD-L1 antibody from the bispecific antibody DSYE001; anti-B7H3 antibody DSYE003 is the reference antibody for replacing the PD-L1 antibody in the bispecific antibody DSYE001 (the variable region of the PD-L1 antibody in the bispecific antibody is replaced with Human Anti-HIV-1 gp120 clone b12 VH and VL); anti-PD-L1 antibody DSYE004 is the reference antibody for the PD-L1 antibody in the bispecific antibody DSYE001.

[0207] Antibodies DSYE002, DSYE003, and DSYE004 were prepared using conventional methods, such as vector construction followed by transfection into eukaryotic cells like HEK293 and CHO cells for purification and expression.

[0208] The amino acid sequences of the bispecific antibodies against B7H3 and PD-L1 DSYE001, DSYE002, DSYE003, and DSYE004 are shown in the sequence listing section.

[0209] Example 2: Preparation of Bispecific Antibody Drug Conjugates (ADCs)

[0210] 2.1 Preparation of linker-cytotoxin

[0211] Connector - Cytotoxin X1:

[0212] Synthesis route:

[0213] first step: Under nitrogen protection, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a DMF (50 mL) solution containing 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol), and the reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). After separation, the solution was washed with saturated sodium chloride aqueous solution (500 mL), dried over anhydrous Na2SO4, concentrated, and column filtered (PE:EA = 3:2) to give 5.1 g of colorless liquid, yield: 57.1%.

[0214] Step Two: Under nitrogen protection, a solution of 27b (4.50 g, 21.8 mmol) in THF (30 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol), TsOH (800 mg, 4.65 mmol), and reacted at 0 °C for 2 hours at 25 °C. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction solution was added to 200 mL of water, extracted twice with EA (200 mL), dried over anhydrous Na2SO4, and concentrated by column chromatography (PE / EA = 3 / 2) to give 1.56 g of a white solid, yield: 26%.

[0215] Step 3: Under hydrogen atmosphere and at 0°C, Pd / C (80 mg) was added to a mixed solution of EtOH (8 mL) and EA (8 mL) at 27°C (800 mg, 1.55 mmol), and the mixture was stirred at 0°C for 2.5 hours. LC-MS showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with EA (200 mL), concentrated, dissolved in THF (20 mL), and evaporated to dryness to obtain 600 mg of white solid, yield: 91%. Step 4: Under nitrogen protection, at 0°C, DIEA (152 mg, 1.18 mmol) was added to a DMF (6 mL) solution of 27 d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol), and the reaction was carried out at 0°C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was added to an aqueous citric acid solution (pH=4) (150 mL), filtered, and the filter cake was washed with 175 mL of water. After drying, the solution was pulled dry with an oil pump to obtain 260 mg of brown solid, yield: 66%.

[0216] Step 5: Under nitrogen protection, diethylamine (8 mL) was added dropwise to a 30 mL solution of DCM (27°C, 260 mg, 0.309 mmol) at 0 °C, and the reaction was carried out at 0 °C for 3 hours. LC-MS showed that the reaction was complete. The reaction solution was added to a 600 mL solution of petroleum ether at 0 °C, and a solid precipitated out. After standing for the solid to be adsorbed to the bottom of the flask, the solution was poured out and dried using an oil pump to obtain 90 mg of brown solid, yield: 47.1%.

[0217] Step 6: Under nitrogen protection, at 0°C, HATU (74 mg, 0.19 mmol) was added to a DMF (2.5 mL) solution containing 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol), and the reaction was carried out at 0°C for 2 hours. LCMS showed that the basic reaction was complete. At 0°C, the reaction solution was added to an aqueous solution of citric acid (pH=4) (30 mL), and flocculent solid precipitated. After filtration, the solid was analyzed by preparative agar (DCM / MecOH=10 / 1) to give 9.2 mg of a pale yellow solid (X1), yield: 6%.

[0218] MS m / z (ESI): 1074 [M+1].

[0219] 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, 1 H), 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).

[0220] Connector - Cytotoxin X2:

[0221] Synthesis route:

[0222] first step

[0223] 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 reaction was carried out at 25 °C for 17 hours. The reaction mixture was analyzed by TLC (PE / EA = 3 / 1) to confirm the completeness 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 by column chromatography (PE:EA = 2:1) to give 8.7 g of colorless liquid 34b, with a yield of 93%. MS-ESI: m / z 195.1 [M+H]+.

[0224] Step 2

[0225] Dissolve 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) in THF (20 mL), protect under nitrogen atmosphere and cool to 0 °C. Add 43b (7.7 g, 39.6 mmol) in THF (10 mL) dropwise. After addition, react at 0 °C for 2 hours. TLC (PE / EA = 2 / 1) shows that most of the starting material has reacted. Pour the reaction solution into 100 mL of water, extract with DCM (100 mL), separate the layers, wash with saturated NaCl, dry with anhydrous Na2SO4, and pass through a column (PE / EA = 1 / 1) to give 3.9 g of colorless viscous 34d, yield: 39%. MS-ESI: m / z 503.3 [M+H]+.

[0226] Step 3

[0227] Under hydrogen atmosphere and at 0°C, Pd / C (1 g, 10 wt.%) was added to a mixed solution of EtOH (100 mL) and EA (100 mL) containing 34d (1.9 g, 3.78 mmol) and reacted at 0°C for 3 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated, dissolved in THF (50 mL × 3), and evaporated to dryness. This process was repeated three times to obtain 1 g of gray solid 34e, yield: 64%. MS-ESI: m / z 435.2 [M+Na]+.

[0228] Step 4

[0229] Under nitrogen protection, DIEA (303 mg, 2.35 mmol) was added dropwise to a DMF (20 mL) solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) at 0 °C. After the addition was complete, the reaction was allowed to proceed at 0 °C for 2 hours. LC-MS showed the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, and allowed to stand for 5 minutes. After filtration, the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution, dried, and stirred. Column chromatography (EA:MeOH = 30:1) yielded 600 mg of yellow solid 34f, yield: 77%. MS-ESI: m / z 830.3 [M+H]+.

[0230] Step 5

[0231] Under nitrogen protection, diethylamine (5 mL) was added dropwise to a 34f (150 mg, 0.18 mmol) DCM (5 mL) solution at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LCMS showed that the reaction was complete. Petroleum ether solution (100 mL × 6) was added to the reaction solution, and a solid precipitated. After standing to allow the solid to settle, the solution was poured off and then dried using an oil pump, yielding 34 g of 120 mg white powder. LCMS showed that the product content was 70%, yield: 76%. MS-ESI: m / z 608.3 [M+H]+.

[0232] Step 6

[0233] Under nitrogen protection, HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) solutions at 0 °C, and the reaction was carried out at 0 °C for 2 h. LC-MS showed that the starting material reacted completely. The reaction solution was directly passed through a reversed-phase column with eluent (MeCN / MeOH = 1 / 1):H2O = 60%:40%) to purify 14.8 mg of yellow solid x 2, yield 14%.

[0234] MS-ESI: m / z 1062.4 [M+H]+.

[0235] 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).

[0236] Connector - Cytotoxin X3:

[0237] Synthesis route:

[0238] first step: Allyl bromide (960 mg, 7.92 mmol) was added to 20 mL of MeCN containing 32a (2.00 g, 6.6 mmol), K2CO3 (1.82 g, 13.2 mmol), and stirred at 20 °C for 5 hours. 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 extract was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography (PE / EA = 2 / 1) to give 1.83 g of white solid 32b, yield: 81%.

[0239] Step Two: TFA (10 mL) was added to 10 mL of DCM containing 32b (1.38 g, 4.02 mmol), and the mixture was stirred at 25 °C for 17 hours. TLC (PE / EA = 1 / 3) showed that the reaction was complete. The reaction solution was evaporated to dryness to give 0.91 g of yellow viscous substance 32c, yield negligible.

[0240] Step 3: 41d (1.92 g, 4.87 mmol) was added to a mixture of 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol) in DME / H2O (20 mL / 10 mL), and stirred at 25 °C for 3 hours. TLC (DCM / MeOH = 1 / 1) showed the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5 with aq.HCl (1 N), extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (DCM / MeOH = 20 / 1) to give 1.53 g of white solid 32e, yield: 67%. MS-ESI: m / z 467.4 [M+H]+.

[0241] Step 4: Pd / C (600 mg) was added to 50 mL of MeOH at 32°C (3 g, 5.83 mmol), and the mixture was stirred for 5 hours under a hydrogen balloon at 25°C. TLC (EA) showed the reaction was complete. The reaction solution was filtered and evaporated to dryness to give 32 g of a white solid (1.9 g), yield: 77%.

[0242] Step 5: HATU (707 mg, 1.86 mmol) was added to 10 mL of DMF containing 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol). The mixture was stirred at 0 °C for 3.5 h. TLC (EA) showed that the reaction was complete. The reaction mixture was poured into 80 mL of H2O, extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (EA) to give 1.186 g of white solid after 32 h (yield: 83%). MS-ESI: m / z 842.3 [M+H]+.

[0243] Step 6: The DCM / diethylamine (20 mL, 20 / 1) solution was stirred at 25 °C for 17 hours (32 h, 1.186 g, 1.41 mmol). 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 white solid 32i, yield: 88%. MS-ESI: m / z 620.3 [M+H]+.

[0244] Step 7: HATU (414 mg, 1.09 mmol) was added to 10 mL of DMF containing 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol), and the mixture was stirred at 20 °C for 17 hours. TLC (PE / EA = 1 / 5) showed the reaction was complete. The reaction mixture was poured into water (30 mL), filtered, and the filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to obtain 511 mg of white solid 32j, yield: 44%. MS-ESI: m / z 1068.3 [M+H]+.

[0245] Step 8: A solution of 32 J (482 mg, 0.451 mmol) of diethylamine / DCM (10 mL, 1 / 5) was stirred at 10 °C for 17 hours. TLC (EA) showed that the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to give 301 mg of white solid 32 K, yield negligible.

[0246] Step 9: Morpholine (93 mg, 1.07 mmol) was added to 5 mL of THF containing 32 kJ (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol), and the mixture was stirred at 25 °C for 5 hours. LC-MS showed that the reaction was complete. 108 mg of a white solid (32 mL) was obtained from the reaction mixture, yield: 38%. MS-ESI: m / z 806.3 [M+H]+.

[0247] Step 10: Add 27 mg of acetyl bromide (0.134 mmol) to 32 mL of THF (2 mL) and DMF (2 mL) containing 108 mg (0.134 mmol) of triethylamine (41 mg (0.402 mmol) and stir at 0 °C for 1 hour. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. The reaction solution was directly used to prepare 15 mg of white solid x 3, yield: 12%.

[0248] MS-ESI: m / z 926.3 [M+H]+.

[0249] 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 = 5.9Hz, 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 = 7.3Hz, 3H).

[0250] Connector - Cytotoxin X4:

[0251] Synthesis route:

[0252] first step: Pd / C (400 mg, 10 wt.%) was added to 20 mL of MeOH containing 2.00 g (2.58 mmol) of 33a, and the mixture was stirred at 20 °C for 5 hours. TLC (EA) showed that the reaction was complete. The reaction solution was filtered and evaporated to dryness to give 1.3 g of white solid 33b, yield: 74%.

[0253] Step Two: HATU (305 mg, 0.802 mmol) was added to 5 mL of DMF containing 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol), and the mixture was stirred at 0 °C for 2 hours. TLC (DCM / MeOH = 1 / 10) showed the reaction was complete. The reaction mixture was poured into 40 mL of water, filtered to obtain a crude product, and purified by column chromatography (DCM / MeOH = 20 / 1) to give 360 ​​mg of a yellow solid 33c, with a yield of 41%.

[0254] Step 3: Diethylamine (2 mL) was added to 10 mL of DCM at 33°C (360 mg, 0.326 mmol). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) showed that the reaction was complete. The reaction mixture was poured into PE (100 mL) and filtered to give 205 mg of white solid at 33°C, yield: 71%. MS-ESI: m / z 881.3 [M+H]+.

[0255] Step 4: A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to a mixture of 33 d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL). The mixture was stirred at 0°C for 1 hour. The reaction solution directly yielded 15 mg of white solid X4, yield: 6%.

[0256] MS-ESI: m / z 1001.2 [M+H]+.

[0257] 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).

[0258] 2.2 Preparation of Bispecific Antibody-Drug Conjugates

[0259] Preparation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR4):

[0260] At 37°C, the prepared tris(2-carbonylethyl) phosphate hydrochloride (7.0 mM, 0.215 mL, 1.503 μmol) was added to the buffer solution of the 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 constant temperature water bath shaker and shaken at 37°C for 2 hours, after which the reaction was stopped.

[0261] The adapter-cytotoxin X1 (3.10 mg, 2.89 μmol) was dissolved in 1.0 mL of DMA (N,N-Dimethylacetamide) and added to the above antibody solution. The mixture was placed in a water bath and shaken at 22 °C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid, pH 5.5), and then concentrated by ultrafiltration to obtain a solution of the bispecific antibody-drug conjugate ADC DSYE001-X1 (DAR4) (30 mM histidine-hydrochloric acid, pH 5.5; 93.8 mg, 12.7 mg / mL, yield: 93.8%), which was stored at 4 °C protected from light.

[0262] HIC DAR analysis and calculation yielded a DAR value of p=4.02.

[0263] Preparation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6): The antibody DSYE001 solution (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10000 mg, 20.52 mg / mL, 57.80 μmol) was added to the reaction vessel. Reduction buffer (20 mM PB, pH 6.7, 93.59 mL) was then added, followed by 10 mM DTPA solution (66.67 mL). The pH of the reaction solution was adjusted to 6.72 using 0.4 M Na2HPO4 solution (7.0 mL). Then, the prepared tris(2-carbonylethyl) phosphate hydrochloride solution (10 mM, 19.08 mL, 190.74 μmol) was added to the reaction vessel. The mixture was stirred at 50-100 rpm and reacted at 25°C for 3.5 h. The pH of the reaction solution was adjusted to 5.5 using 200 mL of 0.5 M NaH₂PO₄ solution. The reaction solution was then cooled to 20°C. After cooling, 32.06 mL of DMSO was added to the reaction system. Then, adapter-cytotoxin X1 (465.7 mg, 433.56 μmol) was dissolved in 43.35 mL of DMSO and added to the above solution. The mixture was stirred at 50-100 rpm and reacted at 22°C for 1.25 h. Then, the prepared NAC solution (50 mM, 43.35 mL, 2.17 mM) was added to the reaction solution, and the mixture was stirred at 50-100 rpm and reacted at 22°C for 0.5 h. The feed solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm2), followed by ultrafiltration using an ultrafiltration membrane pack (30KD, 0.11 m2) 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.

[0264] HIC DAR analysis and calculation yielded a DAR value of n = 6.1.

[0265] Preparation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR8): The antibody DSYE001 solution (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10000 mg, 20.52 mg / mL, 57.80 μmol) was added to the reaction vessel. Reduction buffer (45 mM PB, pH 7.35, 72.2 mL) was then added, followed by 10 mM DTPA solution (66.67 mL). Next, the prepared tris(2-carbonylethyl) phosphate hydrochloride solution (10 mM, 40.46 mL, 404.6 μmol) was added to the reaction vessel. The mixture was stirred at 50-100 rpm and reacted at 25°C for 4.5 h. The pH of the reaction solution was adjusted to 5.5 using 0.5 M NaH₂PO₄ solution (200 mL). The reaction solution was then cooled to 20°C. After cooling, 63.40 mL of DMSO was added to the reaction system. Then, adapter-cytotoxin X1 (651.9 mg, 606.91 μmol) was dissolved in 60.69 mL of DMSO and added to the above solution. The mixture was stirred at 50-100 rpm and reacted at 22°C for 1.25 h. Then, the prepared NAC solution (50 mM, 60.69 mL, 3.03 mM) was added to the reaction solution, and the mixture was stirred at 50-100 rpm and reacted at 22°C for 0.5 h. The feed solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm2), followed by ultrafiltration using an ultrafiltration membrane pack (30 KD, 0.11 m2) 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.

[0266] HIC DAR analysis and calculation yielded a DAR value of n = 7.9.

[0267] Preparation of anti-B7H3 antibody-drug conjugate DSYE003-X1 (DAR6) (refer to ADC-1)

[0268] Add 1.845 mL of 30 mM His-HAc pH 5.5 buffer to the buffer solution of antibody DSYE003 (PBS pH 7.4; 40 mg, 5.36 mg / mL, 0.23 μmol), then add 0.5 mL of 100 mM EDTA solution and the prepared tris(2-carbonylethyl)phosphohydrochloride solution (6.977 mM, 0.208 mL, 1.45 μmol). Place the solution in a constant temperature stirrer at 500 rpm and react at 37°C for 2 hours, then stop the reaction. Add 0.750 mL of DMA to the above solution, then dissolve the adapter-cytotoxin X1 (2.5 mg, 2.32 μmol) in 0.25 mL of DMA and add it to the above solution. Place the solution in a constant temperature stirrer at 500 rpm and react at 4°C for 1 hour with shaking, then stop the reaction. Add 10% of the total volume of the above reaction solution to a suspension of 300 mg / mL activated carbon (Charcoal, Dextran Coated, manufacturer: Sigma Aldrich), shake to mix, and then place in a rotary mixer at 4 ℃ for 1 h. After 1 h, centrifuge and collect the supernatant. Add another 10% of the total volume of the supernatant to the activated carbon suspension, shake to mix, and then place in a rotary mixer at 4 ℃ for 1 h. After centrifugation at 4300 rcf for 10 min, the supernatant was collected and filtered through a 0.22 μm syringe filter (Merck Millipore). Then, it was concentrated by ultrafiltration for 4 DV cycles in a 30 KD ultrafiltration tube using a 30 mM His-HAc pH 5.5 buffer to obtain a solution of the 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.

[0269] HIC DAR analysis and calculation yielded a DAR value of n = 6.32.

[0270] Preparation of anti-B7H3 antibody-drug conjugate DSYE002-X1 (DAR6) (refer to ADC-2)

[0271] DSYE002-X1 (DAR6) was prepared using the linker-cytotoxin X1 and the anti-B7H3 antibody DSYE002, following the preparation method of the bispecific antibody-drug conjugate ADC DSYE001-X1.

[0272] Preparation of anti-PD-L1 antibody-drug conjugate DSYE004-X1 (DAR6) (refer to ADC-3)

[0273] DSYE004-X1 (DAR6) was prepared using the linker-cytotoxin X1 and the anti-PD-L1 antibody DSYE004, following the preparation method of the bispecific antibody-drug conjugate ADC DSYE001-X1.

[0274] Preparation of bispecific antibody-drug conjugate DSYE001-X2:

[0275] At 37°C, the prepared tris(2-carbonylethyl) phosphate hydrochloride (7.0 mM, 0.207 mL, 1.449 μmol) was added to the buffer solution of the 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 constant temperature water bath shaker and shaken at 37°C for 2 hours, after which the reaction was stopped.

[0276] The adapter-cytotoxin X2 (3.07 mg, 2.89 μmol) was dissolved in 1.0 mL of DMA (N,N-Dimethylacetamide) and added to the above antibody solution. The mixture was placed in a water bath and shaken at 22 °C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid, pH 5.5), and then concentrated by ultrafiltration to obtain a solution of the bispecific antibody-drug conjugate ADCDSYE001-X2 (30 mM histidine-hydrochloric acid, pH 5.5; 96.5 mg, 15.8 mg / mL, yield: 96.5%), which was stored at 4 °C protected from light.

[0277] HIC DAR analysis and calculation yielded a DAR value of p=3.95.

[0278] Preparation of anti-B7H3 antibody-drug conjugate DSYE002-X2 (refer to ADC-4)

[0279] Antibody DSYE002 is a reference antibody for the removal of PD-L1 antibody from the bispecific antibody DSYE001. The amino acid sequence of DSYE002 is shown in the sequence listing.

[0280] At 37°C, the prepared tris(2-carbonylethyl) phosphate hydrochloride (7.0 mM, 1.08 mL, 7.55 μmol) was added to the buffer solution of anti-B7H3 antibody DSYE002 (30 mM histidine-acetic acid + 20 mM EDTA pH 5.5; 60 mg, 5.5 mg / mL, 0.755 μmol), and the solution was placed in a constant temperature water bath shaker and shaken at 37°C for 2 hours, after which the reaction was stopped. The adapter-cytotoxin X2 (10.8 mg, 9.82 μmol) was dissolved in 1.2 mL of DMA and added to the above solution. The mixture was placed in a water bath and shaken at 4 °C for 1 hour, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid, pH 5.5), and then concentrated by ultrafiltration to obtain a solution of the exemplary product DSYE002-X2 (30 mM histidine-hydrochloric acid, pH 5.5; 30.5 mg, 7.5 mg / mL, yield: 50.8%), which was stored at 4 °C protected from light.

[0281] HIC DAR analysis and calculation yielded a DAR value of p=3.98.

[0282] Preparation of isotype control ADC

[0283] Using isotype control antibody and adaptor-cytotoxin X1, and referring to the preparation method of bispecific antibody-drug conjugate ADCDSYE001-X1, isotype control ADC (DAR4), isotype control ADC (DAR6), and isotype control ADC (DAR8) were prepared respectively.

[0284] Example 3: Intracellular activity of antibody-drug conjugates

[0285] Test objective

[0286] The endocytosis effect of the bispecific antibody-drug conjugate against B7H3 and PD-L1 of this invention in A375 and NCI-H1975 cells simultaneously expressing B7H3 and PD-L1 was detected. Cells were co-incubated with a fixed concentration of the test drug and the endocytosis indicator pHrodo. The endocytosis capacity of the test drug was evaluated by observing the fluorescence signal generated by pHrodo accompanying the antibody-drug entry into the cells at different time points.

[0287] Experimental methods: 1. Add A375 and NCI-H1975 cells to 95-well black culture plates pretreated with 8 μg / ml at a density of 1.2 E4 / well and incubate overnight at 37°C with 5% CO2.

[0288] 2. Mix the test drug with Fab-pHrodo at a ratio of 1:1.2 and incubate at 37°C and 5% CO2 for 0.5 hours; start the mixture at 100 nM and serially dilute it 4-fold to 0.0061 nM.

[0289] 3. Add the diluted sample to the culture plate at a rate of 50 μL / well and incubate at 37°C and 5% CO2 for 24 hours.

[0290] 4. After incubation, remove the supernatant and rinse the cell culture plate once with 1% BSA.

[0291] 5. Add 100 μL of DPBS containing 1 μg / mL Hoechst 33342 and 0.5 μg / mL Calcein AM to the culture plate and stain at room temperature for 15 minutes.

[0292] 6. After incubation, remove the supernatant and rinse the cell culture plate once with 1% BSA.

[0293] 7. Fluorescent spots formed by internalized antibodies in each cell were statistically analyzed using the Perkin Elmer Operetta CLS High-Content Analysis System.

[0294] The results are shown in Table 1 and... Figure 15 .

[0295] Table 1. DSYE001-X2 cellular endocytosis activity

[0296] Experimental results show that, compared with the anti-B7H3 antibody-drug conjugate DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of this application has a better endocytic effect in the detection cells. For example, a higher maximum average number of fluorescent spots or a lower EC50 value.

[0297] Example 4: In vitro assay of antibody-drug conjugates inhibiting tumor cell proliferation

[0298] The CellTiter-Glo® chemiluminescence immunoassay (CTG method) was used to evaluate the inhibitory effect of anti-B7H3 / PD-L1 bispecific antibody-drug conjugates DSYE001-X2 and anti-B7H3 antibody-drug conjugates DSYE002-X2 on cell proliferation after incubation for 6 days in B7H3 and PD-L1 positive A375 and NCI-H1975 cells.

[0299] Logarithmic growth phase cells were collected and seeded at densities of 400 cells / well for A375 and 1000 cells / well for NCI-H1975, with 50 μL per well. The cells were incubated overnight at 37°C with 5% CO2. On the second day, DSYE001-X2 and DSYE002-X2 were diluted 3-fold with complete culture medium to obtain eight concentration gradients (starting with the highest concentration of 1000 nM). 50 μL of each drug was added to the cell culture plate, with the complete culture medium serving as a blank control. Two replicates were set up. The cells were incubated at 37°C with 5% CO2 for 6 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 50 μL of CTG assay reagent (Promega, Cat#: G7573) was added to each well, vortexed, and incubated in the dark for 10 minutes before the signal value was read using a microplate reader. Using GraphPad Prism software, a nonlinear regression model was used to plot an S-shaped dose-response curve and calculate the IC50. 50 Value. Cell viability calculation formula = (Lum) 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 ) × 100%.

[0300] Experimental results show that, compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of this application has comparable inhibitory activity on the proliferation of A375 and NCI-H1975 cells that are positive for B7H3 and PD-L1.

[0301] Example 5: In vitro assay of antibody-drug conjugates inhibiting tumor cell proliferation

[0302] The CellTiter-Glo® chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of incubating human tumor cells positive for B7H3 and PD-L1 for 7 days on cell proliferation.

[0303] Logarithmic growth phase cells were collected and seeded at a density of 1000-3000 cells / well in cell culture plates. The plates were incubated overnight at 37°C with 5% CO2. On the second day of the experiment, DSYE001-X1 (DAR8) was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting from the highest concentration of 1000 nM). 50 μL / well of each drug was added to the cell culture plate, with complete culture medium used as a blank control. Three replicates were set up. The plates were incubated at 37°C with 5% CO2 for 6 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 50 μL of CTG assay reagent was added to each well, vortexed, and incubated in the dark for 10 minutes before the signal value was read using a microplate reader. GraphPad Prism software was used to plot an S-shaped dose-response curve using a nonlinear regression model and the IC50 was calculated. 50 Value. Cell viability calculation formula = (Lum 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 )×100%.

[0304] The results are shown in Table 2. Figure 16 .

[0305] Table 2. Inhibitory effect of DSYE001-X1 (DAR8) on tumor cell proliferation in vitro

[0306] Experimental results show that the bispecific antibody-drug conjugate DSYE001-X1 of this application has significant inhibitory activity on the proliferation of A375, NCI-H1975, NCI-H441 and NCI-H358 cells that are positive for B7H3 and PD-L1 in various tumor cells.

[0307] Example 6: Effect of antibody-drug conjugate on PD-L1 expression in NCI-H1975 cells

[0308] The purpose of this experiment is to investigate the difference in the effect of bispecific antibody-drug conjugates on PD-L1 expression in tumor cells compared to their parent monoclonal antibody conjugates.

[0309] Experimental methods

[0310] 1) NCI-H1975 tumor cells were cultured in RPMI 1640 medium with 10% FBS in a 37°C 5% CO2 incubator.

[0311] 2) Treat the cells with trypsin and count them to ensure that the cell viability is above 90.0%. Add 2×106 cells / 2mL to each well of a 6-well plate.

[0312] 3) On the second day, add culture medium (blank control), isotype control antibody, isotype control antibody ADC, DSYE001-X1 (DAR6), DSYE003-X1 (DAR6) and Anti-PDL1-X1 (final concentration of 100 nM) and incubate at 37°C for 72 hours.

[0313] 4) After incubation, wash the cells twice with PBS, add 500 μL of RIPA lysis buffer (pre-chilled on ice with 1% protease inhibitor and 1% phosphorylase inhibitor) to each culture plate (six-well plate), and incubate on ice for 30 min, mixing several times during the incubation period.

[0314] 5) Centrifuge at approximately 14,000 rpm for 10 minutes at 4°C to remove cell debris. Transfer the supernatant to a new tube for protein concentration determination and subsequent experiments.

[0315] 6) Perform protein quantification using the BCA quantitative kit. Based on the quantification results, prepare the protein sample for loading, standardize the protein concentration to 1-2 μg / μL, and add LDS loading buffer (4X) and sample reducing agent (10X). Heat the sample at 100℃ for 10 minutes.

[0316] 7) Alternatively, store the denatured sample in a -80°C freezer.

[0317] 8) Thaw the sample.

[0318] 9) Western blot: Load 10 μL of sample into each well of an SDS-PAGE gel (the amount depends on the antibody titer); Electrophoresis: 80 V for 30 minutes, then 120 V for 90 minutes; Transfer membrane: Use the iBlot2 transfer kit and transfer instrument to transfer membranes, run the P3 program for 7 minutes.

[0319] 10) After the transfer is complete, cut the membrane to the molecular weight of the protein to be detected, wash the membrane with 1xTBST 3 times, 5 minutes each time, at room temperature, and shake.

[0320] 11) Blocking: The membrane was placed in blocking solution (5% skim milk prepared with 1xTBST) at room temperature, shaken, for 1 hour.

[0321] 12) Wash the membrane with 1xTBST 3 times, 5 minutes each time, at room temperature, shaking.

[0322] 13) Incubate primary antibody: Add primary antibody of appropriate dilution (diluted with 5% bovine serum albumin prepared with 1xTBST), incubate overnight at 4°C with gentle shaking.

[0323] 14) Wash the membrane with 1xTBST 3 times, 10 minutes each time, at room temperature, shaking.

[0324] 15) Incubate the secondary antibody: Add the appropriate dilution of the secondary antibody, at room temperature, shake slowly, for 1 hour.

[0325] 16) Wash the membrane with 1xTBST 3 times, 10 minutes each time, at room temperature, shaking.

[0326] 17) Chemiluminescence: Add HRP substrate from the West Femto Ultrasensitive Chemiluminescence Kit to the membrane.

[0327] 18) Detect chemiluminescence and take pictures on a Bio-Rad ChemiDoc™ xrs+ or Tanon 5200 Multi instrument.

[0328] 29) Detection indicators: PD-L1, β-actin.

[0329] The results are shown in Table 3 and... Figure 17 .

[0330] Table 3. Effects of DSYE001-X1 (DAR6) on PD-L1 expression in NCI-H1975 cells

[0331] The results showed that the bispecific antibody-drug conjugate DSYE001-X1 induced a more significant downregulation of PD-L1 expression compared to the parental B7H3 single-arm antibody-drug conjugate DSYE003-X1 and the parental PD-L1 antibody-drug conjugate anti-PD-L1-X1 (DSYE004-X1). This suggests that the bispecific antibody-drug conjugate DSYE001-X1, by binding to two targets, not only exerts its immunomodulatory effect by inhibiting the binding of the corresponding receptors, but also exerts a stronger immunosuppressive effect by reducing the expression level of the targets.

[0332] Example 7: Evaluation of the efficacy of antibody-drug conjugates in a homologous transplantation mouse model

[0333] To investigate the inhibitory effect of the bispecific antibody-drug conjugate against B7H3 and PD-L1 on tumor growth, a homologous transplantation mouse model was constructed in humanized BALB / c mice (BALB / c-hPD-L1 / hB7H3) using CT26 (CT26-hPD-L1 / hB7H3) that stably expresses human B7H3 and PD-L1, and the antitumor effect of the bispecific antibody-drug conjugate DSYE001-X2 was evaluated.

[0334] 1. Test drug and materials

[0335] Isotype control ADC: 10 mg / kg

[0336] G1: Blank control group (control group): physiological saline

[0337] G2:DSYE002-X2 (refer to ADC-4, treatment group): 12mg / kg

[0338] G3:DSYE001-X2 (treatment group): 5.5mg / kg

[0339] Note: Each test substance was administered in equimolar amounts.

[0340] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0341] 3. Experimental animals: BALB / c-hPD-L1 / hB7-H3 mice, female, 6-8 weeks old, weighing approximately 18-22g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0342] 4. Test methods: CT26-hPD-L1 / hB7H3 cells were revived and cultured, and cell cryopreservation batches were recorded. CT26-hPD-L1 / hB7H3 cells in logarithmic growth phase (3rd-4th passages after revival) were collected, the culture medium was removed, and the cells were washed twice with DPBS before seeding (for pre- and post-tumor cell viability testing). Seeding density: 1×10⁻⁶ 6 100 μL of cells per mouse (without matrix gel) was subcutaneously injected into the right side of mice. Tumor volume reached 80-120 mm. 3 Groups were formed, with the grouping day defined as D0, and drug administration began on D0. The coefficient of variation (CV) for tumor volume did not exceed 1 / 3. Isotype controls ADC, DSYE001-X2, and DSYE002-X2 were administered intravenously (iv) twice weekly at doses of 10 mg / kg, 12 mg / kg, and 5.5 mg / kg, respectively. The experiment ended after 19 days of administration. Tumor volume and body weight were measured twice weekly, and data were recorded. The tumor inhibition rate was calculated based on tumor volume measurements.

[0343] 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 Ti: Mean tumor volume on day i of drug administration in the treatment group and the positive control group; T0: Mean tumor volume on day 0 of drug administration in the treatment group and the positive control group; V i Vi: Mean tumor volume of the negative control group on day i of drug administration; V0: Mean tumor volume of the negative control group on day 0 of drug administration.

[0344] The experimental results are shown in Figure 18 See Table 4-6.

[0345] Table 4. Tumor volume at different time points in each group 1 (mm³)

[0346] Note: 1. Tumor volume is expressed as mean ± standard error; 2. Number of days after starting medication.

[0347] Table 5. Changes in tumor volume inhibition rate (TGI) of the test substance in a mouse model constructed from CT26 (CT26-hPD-L1 / hB7H3) homologous transplantation TV )

[0348] Note: TGI TV All calculations are compared with G1.

[0349] Table 6. Statistical analysis of tumor volume in different groups of CT26 (CT26-hPDL1 / hB7H3) homologous transplantation mouse models.

[0350] Note: Independent samples t-test was used. P < 0.05; P < 0.01; P < 0.001.

[0351] Experimental results show that, compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of this invention exhibits significantly enhanced tumor growth inhibitory activity after administration.

[0352] Example 8: Efficacy evaluation of antibody-drug conjugates in human non-small cell lung cancer cells NCI-H1975 tumor-bearing mice

[0353] To investigate the inhibitory effect of DSYE001-X1 (DAR4) on tumor formation in vivo, a subcutaneous xenograft model was constructed in mice using human lung cancer NCI-H1975 cells mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1 (DAR4).

[0354] Female NCG mice, 6-8 weeks old, weighing approximately 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. NCI-H1975 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. NCI-H1975 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. Frozen PBMCs were purchased, revived, counted, and resuspended. These PBMCs were then added to the NCI-H1975 cells, and co-cultured for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0355] After co-culturing PBMCs and NCI-H1975 cells for 5 days, PBMCs and freshly digested NCI-H1975 cells were harvested, with PBMCs at a concentration of 3 × 10⁶ cells / cell. 5 2 × 10⁶ NCI-H1975 cells 6 0.2 mL / mouse (containing 50% matrix gel) was injected subcutaneously on the right side of NCG mice. When the tumor volume reached approximately 100-200 mm³, 20 mice were selected based on tumor volume and randomly divided into 4 groups of 5 mice each. On the day of grouping (day 0), mice were intravenously (iv) injected with the isotype control ADC, DSYE001-X1 (DAR4), the reference ADC DSYE002-X1 (DAR4), and a combination of DSYE002-X1 (DAR4) and PDL1 monoclonal antibody, twice weekly for 2 weeks, for a total of 4 injections. The dosage was 18 mg / kg for the bispecific ADC and 15 mg / kg for both the isotype control ADC and PDL1 monoclonal antibody (equimolar doses). The experiment ended on day 28. Experimental grouping and administration are as follows. Tumor volume and mouse body weight were measured twice weekly, and data were recorded.

[0356] Isotype control ADC: 15 mg / kg

[0357] DSYE001-X1 (DAR4) (treatment group): 18 mg / kg

[0358] DSYE002-X1 (DAR4) (treatment group): 8 mg / kg

[0359] DSYE002-X1 (refer to ADC-2, DAR4) + anti-PD-L1 monoclonal antibody DSYE004 (treatment group): 8 mg / kg + 15 mg / kg

[0360] All samples were prepared by diluting with PBS.

[0361] Relative tumor proliferation rate (T / C) (%): The calculation formula is as follows: T / C % = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the tumor measurement results. The calculation formula is RTV = Vt - V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., D0), and Vt is the average tumor volume at a specific measurement. TRTV and CRTV are based on data from the same day.

[0362] Tumor growth inhibition rate (TGI) (%) = (1 - T / C) × 100%. T / C % is the relative tumor proliferation rate, which is the percentage of tumor volume in the treatment group relative to the control group at a certain time point. T and C are the tumor volumes (TV) of the treatment group and the control group at a specific time point, respectively.

[0363] At the end of the experiment, the mice were euthanized and the tumors were weighed.

[0364] Experimental results are as follows Figure 19 As shown in Table 7-8.

[0365] Table 7. Mean tumor volume (Mean ± SEM) in each group of mice

[0366] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.01, p A value < 0.05 is considered statistically significant.

[0367] Table 8. Mean tumor weight (Mean ± SEM) in each group of mice

[0368] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.01, p A value < 0.05 is considered statistically significant.

[0369] The results showed that DSYE001-X1 could significantly inhibit tumor growth, and its anti-tumor activity was stronger than that of B7H3 monoclonal antibody ADC (DSYE002-X1). Compared with the combination of B7H3 monoclonal antibody ADC and PDL1 monoclonal antibody, it had a better tumor inhibition effect.

[0370] Example 9: Efficacy evaluation of antibody-drug conjugates in human breast cancer cell MDA-MB-231 tumor-bearing mice

[0371] To investigate the inhibitory effect of DSYE001-X1 (DAR6) on tumor formation in vivo, a subcutaneous xenograft model was constructed in mice using human breast cancer cells MDA-MB-231 mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1 (DAR6).

[0372] Female NCG mice aged 6-8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used as experimental animals. MDA-MB-231 cells (provided by Jicui Yaokang, mycoplasma test results were negative) were resuscitated and passaged, with passage number N+13. MDA-MB-231 cells in logarithmic growth phase were collected (inoculated at passage number N+17, after removing the culture medium and washing twice with DPBS before inoculation (cell viability before and after tumor bearing was 99.27% ​​and 97.00%, respectively), inoculation amount: 5 × 10⁶ cells / year. 6 / 100μL / animal (1:1 Substrate gel inoculation). One week after tumor cell inoculation, human PBMCs were injected via tail vein, dose: 7.5×10⁻⁶. 6 / 100 μL / mouse. On day 9 post-inoculation, when the average tumor volume reached 85.44 mm3, 12 mice were randomly divided into two groups of 6 mice each based on tumor volume. The day of grouping was defined as D0. Drug administration began on D0, and the administration dates were D0 and D7. The experiment ended on day 41. Tumor volume and mouse weight were measured twice a week, and the data were recorded.

[0373] At the end of the experiment, the mice were euthanized, and the TGI was calculated. TV (Relative tumor inhibition rate). TGI TV The formula for calculating the relative tumor inhibition rate is:

[0374] in, : Mean RTV of the treatment group; : Average RTV of the Vehicle group (solvent group, which in this example is the group that only received physiological saline); The formula for calculating RTV is: = : Tumor volume of mouse number n on day t; : Tumor volume of mouse number n on day 0; : The relative tumor volume of mouse numbered n on day t.

[0375] Experimental results are as follows Figure 20 As shown in Tables 9 and 10.

[0376] Table 9. Tumor volume changes in different groups

[0377] Table 10. Relative tumor inhibition rate (TGI) of different groups TV )

[0378] The results showed that DSYE001-X1 could significantly inhibit the growth of breast cancer cells.

[0379] Example 10: Efficacy evaluation of antibody-drug conjugates in human non-small cell lung cancer cells NCI-H1975 tumor-bearing mice

[0380] To investigate the inhibitory effect of DSYE001-X1 (DAR6) on tumor formation in vivo, a subcutaneous xenograft model was constructed in mice using human lung cancer NCI-H1975 cells mixed with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1 (DAR6).

[0381] Female NCG mice, 6-8 weeks old, weighing approximately 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. NCI-H1975 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. NCI-H1975 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. Frozen PBMCs were purchased, revived, counted, and resuspended. These PBMCs were then added to the NCI-H1975 cells, and co-cultured for 5 days in RPMI 1640 medium containing IL-2 and 10% FBS.

[0382] After co-culturing PBMCs and NCI-H1975 cells for 5 days, PBMCs and freshly digested NCI-H1975 cells were harvested, with PBMCs at a concentration of 3 × 10⁶ cells / cell. 5 2 × 10⁶ NCI-H1975 cells 60.2 mL / mouse (containing 50% matrix gel) was injected subcutaneously on the right side of NCG mice. When the tumor volume reached approximately 100-200 mm³, 15 mice were selected based on tumor volume and randomly divided into 3 groups of 5 mice each. On the day of grouping (day 0), DSYE001-X1 (DAR6) and DSYE003-X1 (DAR6) in combination with pembrolizumab were administered intravenously (iv) twice weekly for 2 weeks, for a total of 4 administrations. The dosage was 18 mg / kg for the bispecific antibody ADC and 15 mg / kg for the isotype control ADC and pembrolizumab (equimolar doses). The experiment ended on day 21. Experimental grouping and administration are as follows. Tumor volume and mouse body weight were measured twice weekly, and data were recorded.

[0383] Isotype control ADC: 15 mg / kg

[0384] DSYE001-X1 (DAR6) (treatment group): 18 mg / kg

[0385] DSYE003-X1 (refer to ADC-1, DAR6) + pembrolizumab (treatment group): 18 mg / kg + 15 mg / kg

[0386] All samples were diluted with PBS. Pembrolizumab was prepared using standard methods, and the sequence was obtained from Recommended INN list R72 (2014).

[0387] Relative tumor proliferation rate (T / C) (%): The calculation formula is as follows: T / C % = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the tumor measurement results. The calculation formula is RTV = Vt - V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., d0), and Vt is the average tumor volume at a specific measurement. TRTV and CRTV are based on data from the same day.

[0388] Tumor growth inhibition rate (TGI) (%) = (1 - T / C) × 100%. T / C % is the relative tumor proliferation rate, which is the percentage of tumor volume in the treatment group relative to the control group at a certain time point. T and C are the tumor volumes (TV) of the treatment group and the control group at a specific time point, respectively.

[0389] At the end of the experiment, the mice were euthanized and the tumors were weighed.

[0390] Experimental results are as follows Figure 21 As shown in Table 11-12.

[0391] Table 11. Mean tumor volume (Mean ± SEM) in each group of mice

[0392] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.0001 is considered to be a significant difference.

[0393] Table 12. Mean tumor weight (Mean ± SEM) in each group of mice

[0394] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.0001 is considered to be a significant difference.

[0395] The results showed that DSYE001-X1 could significantly inhibit tumor growth, and had a better tumor suppression effect than the combination of B7H3 monoclonal antibody ADC and pembrolizumab.

[0396] Example 11: Pharmacokinetic and toxicity studies of antibody-drug conjugates after multiple doses

[0397] Test objective

[0398] Cynomolgus monkeys were administered DSYE001-X1 (DAR6) intravenously once every 3 weeks for a total of 2 doses. The nature, extent, dose-response, and time-response relationship of potential toxic reactions caused by the antibody-drug conjugate were observed to determine the target organs or tissues for toxicity, providing a reference for subsequent studies.

[0399] Experimental methods

[0400] Four cynomolgus macaques (2 per sex) were used in the experiment and randomly divided into three groups according to body weight, with one macaque per sex per group. Groups 1 and 2 were administered the test drug (DSYE001-X1 (DAR6)) at doses of 30 and 120 mg / kg, respectively, representing the low and high dose groups. The drugs were administered intravenously at a volume of 10 mL / kg over approximately 30 minutes, once every 3 weeks for a total of 3 weeks (2 administrations in total).

[0401] During the trial, animals were clinically observed, and their weight, food intake, body temperature, electrocardiogram, and clinical pathology (blood cell count, coagulation function, blood biochemistry) were monitored. One week after the last dose (D29), the animals were euthanized, and gross anatomical and histopathological examinations (gross abnormalities, bone and bone marrow) were performed. At the same time, the serum total antibody protein and DSYE001-X1 (DAR6) concentrations and plasma small molecule concentrations of animals administered on D1 and D22 were measured, and toxicokinetics were analyzed.

[0402] Table 13. Toxicokinetic Parameters

[0403] Experimental conclusions

[0404] During the trial, no animals in any dosage group died or were near death. No abnormalities related to the test product were observed in body weight, food intake, body temperature, electrocardiogram parameters and waveforms, clinical pathology (blood cell count, coagulation function, and blood biochemistry), gross anatomical examination, or histopathological examination (bone and bone marrow). This indicates that the antibody-drug conjugate has good safety.

[0405] Example 12: Efficacy evaluation of antibody-drug conjugate DSYE001-X1 (DAR8) in a homologous transplantation mouse model

[0406] To investigate the inhibitory effect of DSYE001-X1 (DAR8) on tumor growth, pharmacodynamic evaluation was conducted in BALB / c-hPD-L1 / hB7H3 mice. The specific methods are described in Example 7.

[0407] The antibody-drug conjugate DSYE001-X1 of this application has a better anti-tumor effect than B7H3 monoclonal antibody ADC or its combination with PD-L1 antibody.

[0408] Various modifications and variations of the methods and systems described in this invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be limited to such specific embodiments. Indeed, various modifications to the modes of implementation of the invention will be apparent to those skilled in molecular biology, immunology, or related fields and are intended to fall within the scope of the appended claims.

[0409] sequence list

[0410] The amino acid sequence of DSYE001 (underlined portion is CDR)

[0411] Light chain variable region of monoclonal antibodies: DIQMTQSPSSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIK (SEQ ID NO: 4) LCDR1: RASQSINTYLH (SEQ ID NO:1) LCDR2: YASQSIS (SEQ ID NO:2) LCDR3: QNGHSFPLT (SEQ ID NO:3) Heavy chain variable region of monoclonal antibodies: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVTITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSS (SEQ ID NO.: 8) HCDR1: TFGVH (SEQ ID NO.: 5) HCDR2: IIWPGGNTNYNSALMS (SEQ ID NO.: 6) HCDR3: ENYGRAMDY (SEQ ID NO.: 7) Heavy chain variable region of nanobodies: DVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIYIGSGATYYVDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSS (SEQ ID NO.: 15) HCDR1: TIWIG (SEQ ID NO.: 12) HCDR2: AIYIGSGATYYVDSVKG (SEQ ID NO.: 13) HCDR3: TGGTVGSMVRFSPVGDFGY (SEQ ID NO.: 14) Linking peptide between monoclonal antibodies and nanobodies: GGGGSGGGGTGGGGS (SEQ ID NO.: 11) Heavy chain of monoclonal antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVTITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.:10) Light chain of monoclonal antibody: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO.: 9) Heavy chain of bispecific antibody: (SEQ ID NO.: 16) Double-specific antibody link: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.: 9) DSYE002 amino acid sequence DVQLQESGGGLVQAGGSLRLSCTASGYTLS TIWIG WFRQAPGKGLEGVA AIYIGSGATYYVDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAA TGGTVGSMVRFSPVGDFGY WGQGTQVTVSSGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 17) Heavy chain of DSYE003 QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGTGGGGSDVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIYIGSGATYYVDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSS (SEQ ID NO.: 18) DSYE003 light chain (Human Anti-HIV-1 gp120 clone b12 light chain) EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO.: 19) PD-L1 monoclonal antibody amino acid sequence (DSYE004) PD-L1 monoclonal antibody heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVTITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 20) PD-L1 monoclonal antibody light chain: identical to the light chain of bispecific antibodies. Pembrolizumab amino acid sequence Heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO.: 21) Light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 22).

Claims

1. A bispecific antibody-drug conjugate, comprising the following fragments: a bispecific antibody against B7H3 and PD-L1 or its antigen-binding fragment, a linker unit L, and a cytotoxic drug, wherein, The bispecific antibody or its antigen-binding fragment includes: A monoclonal antibody unit targeting PD-L1 and comprising two heavy chains and two light chains, wherein the variable region of the light chain of the monoclonal antibody unit comprises CDR1 with the amino acid sequence SEQ ID NO.: 1, CDR2 with the amino acid sequence SEQ ID NO.: 2, and CDR3 with the amino acid sequence SEQ ID NO.: 3, and the variable region of the heavy chain of the monoclonal antibody unit comprises CDR1 with the amino acid sequence SEQ ID NO.: 5, CDR2 with the amino acid sequence SEQ ID NO.: 6, and CDR3 with the amino acid sequence SEQ ID NO.: 7; A nanobody unit targeting B7H3 and comprising two identical nanobodies, the nanobodies comprising CDR1 with the amino acid sequence SEQ ID NO.: 12, CDR2 with the amino acid sequence SEQ ID NO.: 13, and CDR3 with the amino acid sequence SEQ ID NO.: 14; The N-terminus of each of the two nanobodies is connected to the C-terminus of the Fc fragment of each of the two heavy chains of the monoclonal antibody unit via a linker peptide. The connector unit L is -L a -L b -L c - and the L c Linked to the cytotoxic drug, wherein -L a -for -L b -for -L c -for ; The cytotoxic drug is or .

2. The bispecific antibody-drug conjugate as described in claim 1, wherein, The light chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO.: 4; the heavy chain variable region of the monoclonal antibody unit includes an amino acid sequence as shown in SEQ ID NO.: 8; and the nanobody includes an amino acid sequence as shown in SEQ ID NO.:

15.

3. The bispecific antibody-drug conjugate as described in claim 1, wherein, The monoclonal antibody contains an immunoglobulin constant region, which is a human IgG constant region, such as the human IgG1 constant region.

4. The bispecific antibody-drug conjugate as described in claim 1, wherein, The light chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises an amino acid sequence as shown in SEQ ID NO.: 10, and the nanobody comprises an amino acid sequence as shown in SEQ ID NO.: 15; or The full-length amino acid sequence of the light chain of the monoclonal antibody unit is shown in SEQ ID NO.: 9; the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is shown in SEQ ID NO.:

15.

5. The bispecific antibody-drug conjugate as described in claim 1, wherein, The amino acid sequence of the linker peptide is SEQ ID No.:

11.

6. The bispecific antibody-drug conjugate as described in claim 1, wherein, The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence is shown in SEQ ID NO.:

9.

7. A bispecific antibody-drug conjugate, wherein, The bispecific antibody-drug conjugate is selected from any of the following structures: and ； Where p represents the average number of connections, and p is any integer or decimal from 3 to 4, 4 to 5, 5 to 6, 6 to 7, or 7 to 8; DSYE001 is a bispecific antibody against B7H3 and PD-L1. The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.:

9.

8. A bispecific antibody-drug conjugate, wherein, The bispecific antibody-drug conjugate is selected from any of the following structures: 、 、 and ； Where p represents the average number of connections; DSYE001 is a bispecific antibody against B7H3 and PD-L1. The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.: 16, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.:

9.

9. A pharmaceutical composition comprising a bispecific antibody-drug conjugate as described in any one of claims 1-8, and a pharmaceutically acceptable carrier or excipient.

10. Use of the bispecific antibody-drug conjugate of any one of claims 1-8 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating and / or preventing cancer; preferably, the cancer is a cancer expressing B7H3 and / or PD-L1 positively; more preferably, the cancer is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, renal tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, and Ewing's sarcoma; even more preferably, the cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

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