Anti-b7h3 and pd-l1 bispecific antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610300202.0 (22) Application Date 2023.12.27 (66) Domestic Priority Data 202211691970.1 2022.12.27 CN 202311696497.0 2023.12.11 CN (62) Divisional Application Data 202380069557.8 2023.12.27 (71) Applicant Yingen Biotechnology (Shanghai) Co., Ltd. Address Room 1106, No. 868, Yinghua Road, Pudong New Area, Shanghai 201204 (72) Inventors Li Chenggang, Yang Junjie, Zhang Yuhua, Haiqing, Zhu Zhongyuan (74) Patent Agency Shanghai Bixing Law Firm 31283 Patent Attorney Xu Jiechao (51) Int.Cl. A61K 47 / 68 (2017.01) C07K 16 / 46 (2006.01) A61K 47 / 65 (2017.01) A61K 31 / 4745 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title Bispecific Antibody-Drug Conjugate Against B7H3 and PD-L1, Preparation Method and Use Thereof (57) Abstract This invention discloses a bispecific antibody-drug conjugate, its preparation method and use, wherein the structure of the bispecific antibody-drug conjugate includes the following fragments: a bispecific antibody against B7H3 and PD-L1 or its antigen-binding fragment, a linker unit L and a cytotoxic drug. The bispecific antibody-drug conjugate of this invention has good endocytosis effect, proliferation inhibition activity and tumor growth inhibition activity. Claims (4 pages), Description (55 pages), Sequence Listing (electronic publication), Drawings (11 pages) CN 122124275 A 2026.06.02 CN 1 22 12 42 75 A 1. A bispecific antibody-drug conjugate, comprising the following fragments: a bispecific antibody against B7H3 and PD-L1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug, wherein the bispecific antibody or the antigen-binding fragment thereof comprises: 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.: 5.7. CDR3; 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; wherein the N-terminus of the two nanobodies is respectively linked to the C-terminus of the Fc fragment of the two heavy chains of the monoclonal antibody unit via a linker peptide; the linker unit L is -La-Lb-Lc-, and the Lc is linked to the cytotoxic drug, wherein -La- is, -Lb- is, and -Lc- is; the cytotoxic drug is or. 2. The bispecific antibody-drug conjugate of claim 1, wherein the light chain variable region of the monoclonal antibody unit comprises the amino acid sequence as shown in SEQ ID NO.: 4, the heavy chain variable region of the monoclonal antibody unit comprises the amino acid sequence as shown in SEQ ID NO.: 8; and the nanobody comprises the amino acid sequence as shown in SEQ ID NO.: 15. 3. The bispecific antibody-drug conjugate of claim 1, wherein the monoclonal antibody comprises an immunoglobulin constant region, which is a human IgG constant region, such as the human IgG1 constant region. 4. The bispecific antibody-drug conjugate of claim 1, wherein the light chain of the monoclonal antibody unit comprises the amino acid sequence as shown in SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises the amino acid sequence as shown in SEQ ID NO.: 10, and the nanobody comprises the 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 as shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is as shown in SEQ ID NO.: 15. 5. The bispecific antibody-drug conjugate of claim 1, wherein the amino acid sequence of the linker peptide is SEQ ID No.: 11. 6. The bispecific antibody-drug conjugate of claim 1, wherein the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 9. 7. A bispecific antibody-drug conjugate, wherein the bispecific antibody-drug conjugate is selected from any of the following structures: and; wherein p represents the average number of links, 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 which is shown in SEQ ID NO.: 16, and the light chain amino acid sequence of which 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: (Claims 2 / 4, page 3, CN 122124275 A, ..., and Claims 3 / 4, page 4, CN 122124275 A); where p represents the average number of links; DSYE001 is a bispecific antibody against B7H3 and PD-L1, the heavy chain amino acid sequence of which is shown in SEQ ID NO.: 16, and the light chain amino acid sequence of which is shown in SEQ ID NO.: 9. 9. A pharmaceutical composition comprising the 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. Claims 4 / 4 Page 5 CN 122124275 A Bispecific Antibody-Drug Conjugate Against B7H3 and PD-L1, Preparation Method Thereof, and Use Thereof

[0001] This application is a divisional application of patent application filed on December 27, 2023, with application number 202380069557.8 and invention title "Bispecific Antibody-Drug Conjugate Against B7H3 and PD-L1, Preparation Method Thereof, and Use Thereof". Technical Field

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

[0003] The B7-CD28 family, as a co-stimulatory signal for T lymphocyte activation, plays a crucial role in the immune response involving T lymphocytes. Studies have shown that different B7 molecule types have positive or negative regulatory effects on immune cell responses. B7H3 (also known as CD276) is a member of the B7 family, primarily expressed on the surface of tumor cells. It was first discovered by Chapoval AI et al.It currently exhibits co-stimulatory effects 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 the deepening research on B7H3, its inhibitory function has gradually been 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 there is substantial evidence that its high expression is associated with poor prognosis in various malignant tumors.

[0004] B7H3 is highly expressed in all tested cancer types with limited heterogeneity and is rarely expressed in normal tissues. This suggests that B7H3 can be considered a tumor antigen (TA), providing a possibility for targeted therapy against tumor cells with high B7H3 expression. 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 checkpoint PD1 / PD-L1 are undoubtedly the focus of tumor immunotherapy. PD-L1 is expressed on the surface of tumor cells, and cytotoxic PD-L1 inhibitory antibodies theoretically have better anti-tumor effects. However, among the marketed PD-L1 monoclonal antibodies, only Avelumab has been reported to mediate ADCC effects against tumors and has shown comparable safety to other PD-L1 antibodies. One important reason is that the effect of ADCC mainly depends on the abundance of antigen expression, and PD-L1 cannot be considered a typical tumor antigen, and it has great heterogeneity in tumor cells. PD1 / PD-L1 pathway blocking antibodies are often used in combination with cytotoxic antibodies to enhance the effect 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. Antibodies or their antigen-binding fragments are conjugated to small molecule drugs with biological activity, such as cytotoxicity, via cleavable or non-cleavable linkers. This fully utilizes the specificity of antibodies or their antigen-binding fragments in targeting cells of interest (target cells) or binding to highly expressed antigens, as well as the high efficiency of small molecule drugs, reducing or avoiding toxic side effects on non-target cells. This means that, compared with traditional tumor chemotherapy drugs, antibody-drug conjugates for tumors can 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 of which are currently in Phase II clinical trials. MGC018 uses a cleavable linker to attach the DNA alkylating agent...Duocarmycin is conjugated with a humanized B7H3 antibody. Early clinical trials showed that MGC018 demonstrated 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 showed antitumor activity in various tumors, including mCRPC, small cell lung cancer, squamous cell lung cancer, esophageal squamous cell carcinoma, and endometrial cancer, and demonstrated good safety.

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

[0009] The technical problem to be solved by the present invention is to overcome the deficiency of the limited number of bispecific antibody-drug conjugates in the prior art, and to provide a bispecific antibody-drug conjugate, its preparation method and application. The bispecific antibody-drug conjugate of the present invention has good endocytosis effect, proliferation inhibition activity and tumor growth inhibition activity.

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

[0011] In one aspect, the present application provides a bispecific antibody-drug conjugate, the structure of which includes 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 including 2 heavy chains and 2 light chains, a nanobody unit targeting B7H3 and including 2 identical nanobodies, wherein the N-terminus of the 2 nanobodies is respectively linked to the C-terminus of the Fc fragment of the 2 heavy chains of the monoclonal antibody unit through 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.: 7.CDR3 of 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 comprises an immunoglobulin constant region, the immunoglobulin constant region being a human IgG constant region, such as a 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 the amino acid sequence as shown in SEQ ID NO.: 9, the heavy chain of the monoclonal antibody unit comprises the amino acid sequence as shown in SEQ ID NO.: 10, and the nanobody comprises the 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 as shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as shown in SEQ ID NO.: 10; and the amino acid sequence of the nanobody is as 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, wherein the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 9.

[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 above sequences. 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 above sequences.

[0019] The Fc region of the bispecific antibody of the present invention may be a human Fc region. The Fc region of the bispecific antibody of the present invention may be any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is...IgG1 isotype. 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 Fc region variants. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0021] In some embodiments, the antibody provided in this invention may be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable 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 conjugates of the present invention are wherein the bispecific antibody is DSYE001.

[0023] In some embodiments, in the bispecific drug conjugates, compositions, uses, or methods of the present invention, wherein 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, wherein 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 as shown in SEQ ID NO.: 9, the full-length amino acid sequence of the heavy chain of the monoclonal antibody unit is as shown in SEQ ID NO.: 10, and the amino acid sequence of the nanobody is as shown in SEQ ID NO.: 15.

[0026] In some embodiments, in the bispecific drug conjugates, compositions, or uses of the present invention, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO.: 16, and the light chain amino acid sequence is as shown in SEQ ID NO.: 9. (Page 3 / 55 of the specification, CN 122124275 A)

[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 or solvate thereof; wherein, M is -L2-L1-C(O)-; L2 is -O- or -S-, and L2 is connected to the linker unit L; L1 is -(C(R1a)(R1b))m-CH2-, a C3-C6 saturated cycloalkyl group or a 3-6 member saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl group and the 3-6 member saturated heterocyclic group are each independently optionally substituted by one or more R2a; m is 1, 2, 3 or 4; the heteroatoms in the 3-6 member saturated heterocyclic group are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3; R1a and R1b are each independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R2a is halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0028] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L2 is -O-.

[0029] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is -(C(R1a)(R1b))m-CH2-; R1a is hydrogen, halogen, or C1-C6 alkyl; R1b is hydrogen, halogen, or C1-C6 alkyl.

[0030] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is -(C(R1a)(R1b))m-CH2-; R1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R1b is hydrogen or a C1-C6 alkyl group, preferably hydrogen or a C1-C3 alkyl group.

[0031] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is -(C(R1a))m-CH2-; R1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group.(R1b))m-CH2-; R1a is -CH3; R1b is hydrogen or -CH3.

[0032] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein m is 1 or 2. Specification 4 / 55 pages 9 CN 122124275 A

[0033] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is, , , or.

[0034] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is a C3-C6 saturated cycloalkyl group or a 3-6 saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl group and the 3-6 saturated heterocyclic group are each independently optionally substituted by one or more R2a, wherein each R2a is independently a halogen or a C1-C6 alkyl group.

[0035] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group is optionally substituted with one or more R2a, each of which is independently a halogen or a C1-C6 alkyl group.

[0036] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl group optionally substituted with one or more R2a, each of which is independently a halogen or a C1-C6 alkyl group.

[0037] In some embodiments, the bispecific antibody-drug conjugate of the present invention, wherein L1 is , , , , , 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 in the structure shown in formula (A-1), M is -L2-L1-C(O)-; L2 is -O-; L1 is -(C(R1a)(R1b))m-CH2- or a C3-C6 saturated cycloalkylene group, wherein the C3-C6 saturated cycloalkylene group is optionally substituted with one or more R2a; m is selected from 1 or 2; R1a and R1b are each independently selected from hydrogen, halogen and C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally substituted with one or more halogens; R2a is selected from halogen and C1-C6 alkyl, wherein the C1-C6 alkyl group is 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-La-Lb-Lc-; and the Lc is connected to the cytotoxic drug; -La- is or; connected to the Lb; -Lb- is any of the following structures: , , , , , , and; preferably or; and the right end of the above structure is preferably connected to the Lc; Specification 6 / 55 page 11 CN 122124275 A -Lc- is.

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

[0043] In some embodiments, the bispecific antibody drug conjugate of the present invention has the structure shown in formula (A-2): ; where 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 linker unit L 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): ; where, 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; for example, 4.02 or 3.95; M is -L2-L1-C(O)-; L2 is -O- or -S-, and L2 is linked to L; L1 is -(C(R1a)(R1b))m-CH2-, a C3-C6 saturated cycloalkyl group or a 3-6 member saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl group and the 3-6 member saturated heterocyclic group are each optionally substituted by one or more R2a independently; 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; R1a, R1b and R2a are each independently hydrogen, halogen, hydroxyl, amino or C1-C6 alkyl, and the C1-C6 alkyl group is optionally substituted by 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; 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; for example, 4.02 or 3.95; Ab is the bispecific antibody or its antigen-binding fragment as described in any embodiment of the present invention; L2 is -O- or -S-; preferably -O-; Specification 8 / 55 page 13 CN 122124275 A X1 is a C3-C6 saturated cycloalkyl group optionally substituted with 1, 2 or 3 R2a; X2 is -(C(R1a)(R1b))m-CH2-;m is 1 or 2; R1a, R1b and R2a are each independently hydrogen, halogen or C1-C6 alkyl; the C1-C6 alkyl is optionally 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: , , , Specification 9 / 55 page 14 CN 122124275 A, , , Specification 10 / 55 page 15 CN 122124275 A, , , Specification 11 / 55 page 16 CN 122124275 A, and; 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; 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: (See specification page 12 / 55, CN 122124275 A, and ); 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 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 being shown as SEQ ID NO.: 16, and the light chain amino acid sequence being shown as SEQ ID NO.: 9.

[0048] In some embodiments, the bispecific antibody-drug conjugate of the present invention (e.g., the bispecific antibody-drug conjugate shown in formula (A-2), formula (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 links p of the present invention can be any integer or decimal from 2 to 8. For example, the average number of links p can be any integer or decimal from 3 to 8. For example, the average number of links 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 yet another aspect, the present invention provides a bispecific antibody-drug conjugate, wherein the bispecific antibody...The drug conjugate is selected from any of the following structures (p represents the average number of links): , , and CN 122124275 A, page 14 / 55 of the specification.

[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 numbering method for the antibody CDR of the present invention is: Kabat numbering.

[0052] In another aspect, the present invention provides a bispecific antibody drug conjugate, said bispecific antibody drug conjugate being selected from any of the following structures: and; wherein, t represents the number of links, 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 numbering method for antibody CDRs of the present invention is: Kabat numbering.

[0054] In some embodiments, the number of links t in the present invention is any integer from 1 to 10, preferably any integer from 2 to 8. For example, the number of links t can be any integer from 3 to 8. For example, the number of links 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 reducing agent conventional 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 as described in 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 the present invention, the pharmaceutically acceptable carrier used in the pharmaceutical composition, examples of suitable pharmaceutically acceptable carriers are 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, various suitable dosage forms can be formulated according to the route of administration.

[0063] In other embodiments, the administration of any of the bispecific antibody-drug conjugates or pharmaceutical compositions of this invention can 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 yet another aspect, this invention provides a pharmaceutical formulation comprising any of the bispecific antibody-drug conjugates of this invention, or a pharmaceutically acceptable form thereof, or a mixture thereof, as an active ingredient, or a pharmaceutical composition of this 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, wherein the cancer is 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, wherein the cancer is 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 treating and / or preventing cancer, preferably, wherein the cancer is 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, gastric 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 this 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, this invention provides the combined administration of therapeutically effective amounts of one or more therapies (e.g., treatment methods and / or other therapeutic agents) to a subject. In some embodiments, the therapies include surgical treatment and / or radiotherapy.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 may be used alone or in combination with other therapeutic agents in a therapy. For example, they may 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, or 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 yet 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 delivery device.

[0074] Terminology Definitions

[0075] Unless otherwise stated, the present invention will be practiced 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 the present invention, certain technical terms are specifically defined as follows. 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 terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of 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 the present invention, the term “B7H3”, also known as CD276 antigen, refers to a type 1 transmembrane protein belonging to the B7 family, having an outer domain consisting 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 or human 2IgB7H3), or the extracellular domain of B7H3 (B7H3 ECD) or a fragment containing the B7H3 ECD; B7H3-ECD fusion proteins. 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 and mediating 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 method.

[0080] In this invention, the term “and / or” should be understood to mean any one of the options or a combination of any 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 the opposite terms are explicitly stated, such as "only one" or "exactly one" or "composed of..." in the claims, will refer to only one number or one element of the list.

[0082] Unless the context explicitly indicates the opposite, 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 can refer 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 has a strong ability to disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations.Tumor cells. The “cytotoxic drug” may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 or Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or derivatives thereof. For example, it may be a toxic drug, including but not limited to camptothecin derivatives, such as camptothecin derivative ethanotecan (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 is reactive to a specified protein or peptide or fragment thereof. Antibodies can be antibodies 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 may be derived from any species. The term "antibody" may 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 an antigen that is specifically recognized and bound by an antibody is called an "epitope," as described above. As mentioned above, an antigen-binding domain typically contains both a variable region (VL) and a variable region (VH) of the antibody light chain; however, it is not necessary to contain both. Fd fragments, for example, have two VH regions and typically retain some antigen-binding functionality of the complete antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments with VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab′)2 fragments, bivalent fragments with two Fab fragments connected by disulfide bridges in the hinge region; (3) Fd fragments with two VH and CH1 domains; (4) Fv fragments with VL and VH domains in the antibody single arm; and (5) dAb fragments (Ward et al., "Binding Activities of a Repertoire of Single").Immunoglobulin Variable Domains Secreted From Escherichia coli, Nature 341: 544-546 (1989), which is incorporated herein by reference in its entirety, has the following domains: (6) a separated complementarity-determining region (CDR); and (7) a single-chain Fv (scFv), for example derived from a scFV library. 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. Academia). .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 domain of the antibody heavy or light chain involved in the binding of the antibody to the antigen. In this application, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domain of the antibody vary strongly, 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 variable regions of the light and heavy chains, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. 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), most of them adopt a β-sheet configuration and are connected by three CDR structural loop regions. The CDRs in each chain are closely close 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, in the art, the variable region of an antibody or the CDR of an antibody can be encoded by a variety of methods, 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 the structural loop region (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 the germline V gene by efranc et al., as well as Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand 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 and target 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 an antibody derived from a basic homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

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

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

[0092] In this invention, the term "domain antibody" is 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 with peptide linkers 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 contain at least one, typically two, variable domains, wherein all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the human immunoglobulin constant region (Fc).

[0094] In this invention, "isotype" antibody refers to an antibody class (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4) provided by the heavy chain constant region gene. Isotypes also include modified forms of one of these classes, wherein modifications have been generated to alter Fc function, for example, to enhance or weaken effector function or binding to Fc receptors.

[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 residue (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) origin. Therefore, "cross-reactivity" should be understood as an interspecies reaction between an antigen-binding molecule (e.g., 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 represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. In some embodiments of this invention, affinity, such as the affinity between the antibody and antigen of this invention, is measured using surface plasmon resonance (SPR) technology. 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 means not binding to a protein or cell, or not binding to it with high affinity, i.e., the KD of the bound protein or cell is 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 means a KD of 1.0 × 10⁻⁶ M or lower for the antigen, 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 of the IgM isotype refers to a KD of 10⁻⁶ M or lower, preferably 10⁻⁷ M or lower, more 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 amino acid residues in a candidate amino acid sequence that are identical to those in a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) to obtain the maximum percentage sequence identity, without replacing any portion considered as sequence identity. Various methods in the art can be used for sequence alignment 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 alignment, including any algorithm required to obtain the maximum alignment of the full length of the compared sequences.

[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, which can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, for example, 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, substituent or non-substituent; for example, when substituted, the substituent can be substituted at any usable link.

[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 the 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. Methylene groups can be substituted or unsubstituted, substituent or non-substituent; for example, containing 1 to 12 carbon atoms, such as alkylene groups 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, alternative or non-substituted; for example, when substituted, the substituent can be substituted 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, cycloalkyl...The base ring contains 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, cyclohepttrienyl, cyclooctane, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. The cycloalkyl group 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 means that the ring molecules in the cyclic structure contain at least one double or triple bond. The term "partially unsaturated" covers cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined in this application. The term "unsaturated" means 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 (see page 21 / 55 of specification, CN 122124275 A), and homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. The heterocyclic ring can be fused to an aryl, heteroaryl, or cycloalkyl ring, and the ring connected to the parent structure is a heterocyclic group. The heterocyclic group can be substituted or unsubstituted; for example, when substituted, the substituent can be substituted at any usable connection point.

[0108] In this invention, the term "cyclic atom" generally refers to an atom contained in the cyclic 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 "each independently" generally means that the variable applies to any situation, regardless of whether variables with the same or different definitions exist in the same compound. For example, the variable can refer to the type or number of substituents in the compound, or the type of atoms in the compound, etc. 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 events or circumstances described below may but do not necessarily occur, and the description includes situations in which the events or circumstances occur or do not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may but does not necessarily exist, 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, for example, one to three hydrogen atoms, which are substituted independently of each other by the corresponding number of substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (such as an alkene) bond.

[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,” “cycloalkyl,” etc., can be preceded by an identifier indicating the number of atoms present in the group under a specific condition, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., and the subscript number after “C” indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); in C1-10, 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 of this invention may 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 may 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 of 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...tautomer) includes interconversion through the recombination of some bonding electrons. In this application, the term "meta-form" generally refers to a molecule containing asymmetric atoms but having symmetry factors that make the total optical rotation of the molecule zero. The term "racemic form" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0115] In this invention, the term "connector unit" or "connector structure" generally refers 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, or it may be connected to other connectors before being connected to a cytotoxic drug. The direct or indirect connection to the ligand may refer to the group being directly connected to the ligand through a covalent bond, or it may be connected to the ligand through a connector structure. For example, chemical structural fragments or bonds containing acid-instable connector structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connector structures, light-instable connector structures, dimethyl connector structures, or disulfide-containing connector structures may be used as connector structures.

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

[0117] In this invention, the term "drug loading" generally refers to the average number of cytotoxic drugs loaded on 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, for example 1-10 cytotoxic drugs linked to each ligand (Ab). 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 occur in more than one isotopic form. For example, hydrogen may exist in the form of protium (¹H), deuterium (²H), and tritium (³H), and carbon may occur naturally in three different isotopes (¹²C, ¹³C, and ¹⁴C). Examples of isotopes that may be incorporated into the compounds of this application also include, but are not limited to, ¹⁵N, ¹⁸O, ¹⁷O, ¹⁸F, ³²P, ³³P, ¹²⁹I, ¹³¹I, ¹²³I, ¹²⁴I, ¹²⁵I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of this invention may be enriched in one or more of these isotopes relative to their natural abundance. As described in this article...As is known to those skilled in the art, such isotope-enriched compounds can be used for a variety of purposes. For example, substitution with a heavy isotope such as deuterium (2H) may provide certain therapeutic advantages, which could be due to greater metabolic stability. For example, the natural abundance of deuterium (2H) is about 0.015%. Thus, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as 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 isotope-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 / pharmaceutical salts or prodrugs, along with other chemical components, such as physiologically / pharmaceutical carriers and excipients. The pharmaceutical composition may facilitate administration to an organism, promote the absorption of the active ingredient, and thereby 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 may also be used to prepare injectable formulations.

[0120] In this invention, the term "pharmaceutically acceptable salt" or "medicinal salt" generally refers to a salt of the compound or antibody-drug conjugate of this invention, or a salt of the compound or antibody-drug conjugate described in this invention. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compound or antibody-drug conjugate of this invention may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromic acid salt, 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 metabolizing 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, inhibiting, reducing, or improving the underlying barrier being treated. Furthermore, the therapeutic benefit is achieved by eradicating, 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 of obtaining a beneficial or desired outcome, 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, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults) 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 treating, curing, preventing, or improving a related medical condition or enhancing the treatment of such conditions. The amount at which a cure, prevention, or improvement is achieved. When an individual is given a single-administered active ingredient, the therapeutically effective dose refers only to...The ingredient. When administered in combination, the therapeutically effective dose refers to the combined amount of the active ingredients that cause the therapeutic effect, whether in combination, sequentially or simultaneously. The 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 benign tumor), pre-malignant, 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, which results in a corresponding increase in the size or degree of cancer.

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

[0128] The reagents and raw materials used in the present invention are all commercially available. Specification 24 / 55 pages 29 CN 122124275 A

[0129] The positive and progressive effects of the present 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 B7H3 and PD-L1 simultaneously, and can relieve the inhibition of T cells by PD-L1 while targeting tumor cells, and shows better anti-tumor activity than monoclonal antibody combination therapy.

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

[0132] The bispecific antibody-drug conjugate of this invention has one or more of the following advantages: 1. Compared with related monoclonal antibody-drug conjugates, the bispecific antibody-drug conjugate of this invention has better in vivo stability.The bispecific antibody-drug conjugate of the present invention exhibits significantly enhanced proliferative inhibitory activity against tumor cells, particularly 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 effect by reducing the expression level of the target.

[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 inhibitory activity, for example, against CT26 cells, especially against CT26 cells used to construct a homologous transplantation mouse model.

[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 against 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 against 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 good 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 a stronger tumor suppression effect in individuals with a healthy immune system; and (9) having good in vivo safety. Specification 25 / 55 pages 30 CN 122124275 A Brief Description of the Drawings

[0140] Figure 1 is an affinity screening diagram of the anti-B7H3 VHH humanized antibody prepared in the present invention with B7H3.

[0141] Figure 2 shows the bispecific antibody-drug conjugates DSYE001-X1 or DSYE001-X2 of the present invention.Figure 3 is a schematic diagram of the structure of DSYE001.

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

[0143] Figure 4 is a SEC-HPLC purity determination of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0144] Figure 5 is a Tm value determination (DSF) of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention.

[0145] Figure 6 is a binding ELISA of the anti-B7H3 and PD-L1 bispecific antibody DSYE001 of the present invention before and after heat treatment at 60℃, where a represents the binding ELISA with PD-L1 and b represents the binding ELISA with B7H3.

[0146] Figure 7 shows the binding ELISA of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention, where a represents the binding ELISA with PD-L1 and b represents the binding ELISA with B7H3.

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

[0148] Figure 9 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 shows the IFN-γ secretion induced by the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention on T cells.

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

[0151] Figure 12 shows the ADCC effect of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention on cancer cells. 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 shows the in vivo tumor-suppressing effect of the bispecific antibody DSYE001 against B7H3 and PD-L1 of the present invention.

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

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

[0155] Figure 16 shows the in vitro proliferation inhibition test of the bispecific antibody-drug conjugate DSYE001-X1 (DAR8) of the present invention on tumor cells.

[0156] Figure 17 shows 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 shows the efficacy evaluation of the bispecific antibody-drug conjugate DSYE001-X2 of the present invention in a homologous transplantation mouse model.

[0158] Figure 19 shows the efficacy evaluation 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 shows the efficacy evaluation of the bispecific antibody-drug conjugate DSYE001-X1 (DAR6) of the present invention in human breast cancer cells MDA-MB-231 tumor-bearing mice.

[0160] Figure 21 shows the efficacy evaluation 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. Instruction Manual 26 / 55 Page 31 CN 122124275 A Detailed Description of Embodiments

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

[0162] Experimental methods in the following examples that do not specify specific conditions are performed according to conventional methods and conditions or according to the product instructions.

[0163] Sample Detection

[0164] 1. ADC DAR Value Analysis Method - HIC-HPLC (Hydrophobic Chromatography)

[0165] High Performance Liquid Chromatograph: Waters e2965 High Performance Liquid Chromatography System.

[0166] Chromatographic 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); Elution was performed according to the following elution program,

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

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

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

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

[0171] Mobile phase: 50 mM NaH2PO4 + 200 mM 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 °C.

[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 the detailed description herein. For all purposes, all disclosures, patents and patent applications cited herein, including citations, are incorporated herein by reference in their entirety.

[0174] Description of Embodiments 27 / 55 pages 32 CN 122124275 A

[0175] The following embodiments 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 bispecific antibody-drug conjugate against B7H3 and PD-L1 of the present invention, the bispecific antibody against B7H3 and PD-L1 or its antigen-binding fragment is prepared according to the method of PCT / CN2022 / 125089, as follows (the CDR region of the bispecific antibody is determined according to the Kabat numbering rule): 1. Humanization of camel-derived anti-B7H3 The Germline gene of the human antibody gene is used as a template to humanize the camel-derived anti-B7H3 nanobody using the Framework shuffling method. The corresponding VHH framework shuffling library is generated by in vitro synthesis through overlapping PCR. Then, the clone of the VHH phage library is constructed, screened and identified.

[0178] More precisely, the camel-derived anti-B7H3 nanobody is humanized using a one-step strategy. Approximately 1000 clones were screened from this sub-library. Positive clones were screened for phage-level thermostability using ELISA. 5 μg / mL huB7H3 antigen was coated onto a highly absorbent 96-well ELISA plate. The supernatant from overnight amplification of the screened phages was reacted, and clones showing higher OD450 reads were selected.

[0179] The above phage clones were sequenced to obtain the B7H3 VHH gene sequence. Its C-terminus 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 2-fold. The KD value of the antibody-bound antigen was calculated. The results showed that 75-16 (its amino acid sequence is SEQ ID NO.:15, CDR1 sequence is SEQ ID NO.: 12, CDR2 sequence is SEQ ID NO.: 13, CDR3 sequence is SEQ ID NO.: 14) has the highest affinity for B7H3, and the KD reaches 3.24×10-9M (see Figure 1), so it was selected 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 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) of the PD-L1 monoclonal antibody were derived from existing humanized PD-L1 hIgG1 monoclonal antibodies, and the N-terminus of the anti-B7H3 VHH (75-16 above) was linked to the C-terminus of the Fc fragment via a linker peptide (SEQ ID NO.: 11) (structure shown in Figure 2).

[0182] The DNA sequence was synthesized and 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 by protein A column. The purified IgG1 was dialyzed with 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] Testing Section

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

[0186] Testing Method: (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Mix 5 μg of bispecific antibody with protein reduction and non-reduction buffer, and add PBS to the system to 10 μL. Heat at 100℃ for 10 minutes to fully denature the protein, and take 9 μg of the protein.μL was added to the pre-made polyacrylamide gel wells (Bio-Rad). After separation, the voltage was set to 80V for 30 minutes and then 120V for 60 minutes. The gel was stained with Coomassie brilliant blue staining solution for 30 minutes. It was then destained with decolorizing solution (acetic acid: ethanol: water = 1:3:6) for 15 minutes. The decolorization was repeated three times to remove the background. Then the image was acquired using a gel imaging system. The results showed that the B7H3 / PD-L1 bispecific antibody had good monomer purity under non-reducing conditions. Under reducing conditions, two bands appeared, one for the heavy chain and one for the light chain, due to the opening of the disulfide bonds between the light and heavy chains, and there were no impurity bands (Figure 3).

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

[0188] SEC-HPLC analysis was used to evaluate the monomer 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. The SEC chromatogram was recorded by monitoring the absorbance at 280 nm using a UV detector at a constant temperature of 25 °C, as shown in Figure 4. All detected antibodies exhibited a very high proportion of monomer peaks, with peak area ratios exceeding 95%, indicating that the bispecific antibody had good monomer purity and a low number of aggregates under PBS buffer conditions.

[0189] (3) Differential scanning fluorescence (DSF) detection of antibody Tm value

[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 times from 5000 times concentrated stock solution with ddH2O. 20 μL of sample was taken into PCR tube. SYPRO Orange working solution was added to the reaction to prevent bleaching. The liquid was collected at the bottom of the PCR tube by instant centrifugation. The qPCR instrument was turned on and the program was set to 25℃-95℃ with a temperature increase of 0.3℃ per second. Data was collected, and the temperature and signal values ​​were plotted. The melting temperature (Tm) was calculated. The data showed that the Tm of Fc and Fab of B7H3 / PD-L1 bispecific antibody was 69℃ and 90℃, respectively, which achieved good high temperature resistance and was similar to the Tm of B7H3 VHH-Fc and PD-L1 monoclonal antibody (Figure 5).

[0191] (4) Verification of antibody thermostability and binding activity by ELISA

[0192] 96-well microplate was heated at 4℃ with 2 μg / mlHistagged PD-L1 or B7H3 antigen proteins were coated overnight. 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). The antibody was then developed using 50 μL of 3,3',5,5'-tetramethylbenzidine substrate (TMB). After standing for 5 minutes, 50 μL of 2M sulfuric acid was added to stop the development. The absorbance was then measured at OD450 nm using a SpectraMax M5e (Molecular Devices) microplate reader. The antibody concentration was plotted against the OD450 reading using a 4-parameter fitting method, and the EC50 was calculated. Figures 6a and 6b show the binding curves of the bispecific antibody with PD-L1 and B7H3 before and after treatment at 60℃ for 1 hour. As can be seen from the figure, the binding curves of the bispecific antibody before and after heating are basically overlapping for both PD-L1 and B7H3. This indicates that the bispecific antibody can withstand the high temperature of 60℃ and maintain strong binding activity to 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 EC50 values ​​of the bispecific antibody, PD-L1 monoclonal antibody, and control Avelumab for binding to human PD-L1 were 0.3056 nM, 0.4407 nM, and 0.1563 nM, respectively, which are in the same order of magnitude (Figure 7a). The bispecific antibody, B7H3 VHH-Fc, and MGA271 also showed similar binding activity to human B7H3, with EC50 values ​​of 0.04105 nM, 0.02515 nM, and 0.05476 nM, respectively (Figure 7b). Instructions for Use, Pages 29 / 55, 34, CN 122124275 A

[0193] (5) Determination of the affinity between bispecific antibody and antigen by BLI method

[0194] B7H3 / PD-L1 bispecific antibody and PD-L1 monoclonal antibody, B7H3 VHH-Fc in sample buffer (0.02% tween 20 and 0.1 ...BSA was diluted to 100 nM 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 with sample buffer, and multiple antigen gradients were set up at 2-fold dilutions to bind with the antibodies to obtain 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 × 10⁻¹⁰ M and 8.11 × 10⁻⁹ M, respectively (Figure 8).

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

[0196] The ability of 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. Two × 10⁵ human PD1-CHO cells were evenly seeded in a 96-well culture plate 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 the cells at 4°C for 45 minutes. Unbound antigen was washed away with PBS. The cells were then stained with PE-streptavidin. Finally, the mean fluorescence intensity (MFI) of the PE channel in the flow cytometer was read, and the antibody concentration and MFI were plotted. The IC50 of the antibody was calculated using four-parameter fitting. The flow cytometry analysis results showed that the bispecific antibody could block the binding of PD-L1 to CHO-PD1 cells, with an IC50 of 106.4 nM and the blocking activity was similar to that of PD-L1 monoclonal antibody (IC50 of 94.20 nM) and Avelumab (IC50 of 115.0 nM) (Figure 9).

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

[0198] Mononuclear cells 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 the generation of dendritic cells (DCs). CD4+ T cells (1×10⁵) and allogeneic DCs (1.25×10⁴) were administered with RPMI containing 10% FBS.Co-culture was performed using 1640 complete medium 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 it was not as significant as the PD-L1 signal blocking antibody, it still partially activated T cells because the bispecific antibody blocked the inhibitory signal transduction of B7H3 (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 on 96-well cell plates (Corning, USA) at 4°C for 1 hour. The 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 RPMI1640 medium containing 10% FBS (Gibco) for 4 days. Four days later, the changes in T cell count were detected using the CCK8 kit. The data showed that freshly isolated human CD4+ T cells cultured in wells coated with anti-CD3 and anti-CD28 antibodies exhibited increased proliferation. When PD-L1 was added to the wells, the proliferation capacity decreased significantly, which confirmed that PD-L1 provided an inhibitory signal to T cells. Avelumab, PD-L1 monoclonal antibody and bispecific antibody could significantly promote T cell proliferation at concentrations of 100 nM and 500 nM (Figure 11). Instructions for use 30 / 55 pages 35 CN 122124275 A

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

[0202] The main antitumor effects of MGA271 and Avelumab are derived from the ADCC function of the antibodies, which is related to their IgG1 subtype. The bispecific antibody also has an IgG1 functional region. The ADCC of the antibodies was measured using the 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 × 10⁶) and MDA-MB-231 and ES-2 cells (3 × 10⁵) were co-cultured with different concentrations of bispecific antibody and Avelumab at the start of the assay. After 18 hours, lactate dehydrogenase (LDH) secretion in the culture supernatant was analyzed by ELISA. When B7H3+ PD-L1+ MDA-MB-231 cells were used as target cells, both the bispecific antibody and Avelumab showed ADCC activity, but the bispecific antibody showed stronger activity at lower concentrations. When PD-L1+ ES-2 cells were used as target cells, the bispecific antibody showed 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 (6.67×10⁶) were injected into 41 NPSG mice via the tail vein one day before inoculation with A375 tumor cells, and 5×10⁶ A375 tumor cells were injected subcutaneously the next day. Subcutaneous tumors were observed five days after tumor inoculation. Ten animals were in each group, and the groups were set as isotype control, monoclonal antibody group combined with drug group (Pembrolizumab+MGA271 and Avelumab+MGA271), monoclonal antibody group and bispecific antibody group. Drugs were administered on the 5th day after successful modeling, and twice a week until the end of the experiment. Tumor volume and animal weight were measured and recorded on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39 and 42 of the experiment. The efficacy and safety were evaluated based on the tumor growth inhibition value based on the relative tumor volume (TGIRTV) and changes in animal weight. The bispecific antibody maintained significantly stronger activity than the PD1 monoclonal antibody + MGA271 combination group throughout the experiment; as the experimental time increased, the bispecific antibody also gradually showed better effect than the Avelumab + MGA271 group, with TGI of 39.29% and 26.45% respectively at the end of the experiment (Figure 13); in addition, there was no serious weight loss in mice during the experiment, proving the safety of this bispecific antibody treatment (Figure 14).

[0205] From the test results of (1) to (10) above, it can be seen that 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 PD-L1 antibody from bispecific antibody DSYE001; anti-B7H3Antibody DSYE003 is the reference antibody in bispecific antibody DSYE001 where the PD-L1 antibody has been replaced (the variable region of the PD-L1 antibody in the bispecific antibody is replaced by 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 bispecific antibody DSYE001.

[0207] Antibodies DSYE002, DSYE003 and DSYE004 are prepared according to conventional methods, for example, after vector construction, they can be transfected into eukaryotic cells such as HEK293 cells or CHO cells for purification and expression.

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

[0209] Example 2: Preparation of Bispecific Antibody Drug Conjugate (ADC)

[0210] 2.1 Preparation of Linker-Cytoxin

[0211] Linker-Cytoxin X1: Specification 31 / 55 pages 36 CN 122124275 A

[0212] Synthetic route:

[0213] Step 1: Under nitrogen protection, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a DMF (50 mL) solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol), and reacted 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, it was washed with saturated sodium chloride aqueous solution (500 mL), dried with anhydrous Na2SO4, concentrated and passed through column chromatography (PE:EA=3:2) to obtain 5.1 g of colorless liquid, yield: 57.1%.

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

[0215] Step 3: Under hydrogen atmosphere, at 0 °C, a mixed solution of 27°C (800 mg, 1.55 mmol) of EtOH (8 mL) and EA (8 mL) was added.Add Pd / C (80 mg) to the solution and stir at 0°C for 2.5 hours. LCMS showed the reaction was complete. Filter the reaction solution through diatomaceous earth, wash the filter cake with EA (200 mL), concentrate, dissolve in THF (20 mL), and evaporate to dryness to obtain 600 mg of white solid, yield: 91%. Step 4: Under nitrogen protection, at 0°C, add DIEA (152 mg, 1.18 mmol) to a DMF (6 mL) solution of 27d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol), and react at 0°C for 2 hours. LCMS showed the reaction was complete. Add the reaction solution to a citric acid aqueous solution (pH=4) (150 mL), filter, wash the filter cake with 175 mL of water, filter dry, and pull dry with an oil pump to obtain 260 mg of brown solid, yield: 66%.

[0216] Step 5: Under nitrogen protection, at 0°C, diethylamine (8 mL) was added dropwise to a DCM (30 mL) solution containing 27e (260 mg, 0.309 mmol), and the reaction was carried out at 0°C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was added to a petroleum ether solution (600 mL) at 0°C, and a solid precipitated out. After standing for the solid to be adsorbed to the bottom of the bottle, the solution was poured out and dried with 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 a citric acid aqueous solution (30 mL) with pH=4, and flocculent solid precipitated out. After filtration, 9.2 mg of pale yellow solid X1 was obtained by preparative plate (DCM / MecOH=10 / 1), 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, 1H), 5.57 (d, 1H) , 5.43‑5.10 (m, 3H) , 4.70 (d , 2H) , 4.48‑4.39 (m , 2H) , 4.10‑4.05 (m , 1H) , 4.01‑3.75 (m , 5H) ,3.46 (t, 2H) , 3.22‑3.15 (m , 2H),3.07‑3.00 (m , 1H) , 2.75 (m , 1H) , 2.62 (m , 1H) , 2.45 (s , 3H) , 2.37‑2.20 (m , 6H) , 2.10‑2.02 (m , 2H) , 2.00‑1 .92 (m, 2H) 1.68-1.57 (m, 6H), 1.01 (t, 3H).

[0220] Connector-Cytoxin X2: Instructions for Use, Pages 33 / 55, CN 122124275 A

[0221] Synthetic Route:

[0222] Step 1

[0223] 34a (5g, 48.0mmol) and K2CO3 (19.9g, 144.0mmol) were dissolved in DMF (20mL), and benzyl bromide (12.3g, 72.0mmol) was added dropwise. The reaction was carried out at 25°C for 17 hours. The reaction mixture was detected by TLC (PE / EA=3 / 1) to indicate that the reaction was complete. The reaction mixture was added to water (200mL), extracted with EA (250mL), washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated by column chromatography (PE:EA = 2:1) to obtain 8.7g of colorless liquid 34b, with a yield of 93%. MS-ESI: m / z 195.1 [M+H]+.

[0224] Step 2

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

[0226] Step 3

[0227] Under hydrogen atmosphere, at 0°C, Pd / C (1g, 10wt.%) was added to a mixed solution of EtOH (100mL) and EA (100mL) of 34d (1.9g, 3.78mmol), and the reaction was carried out 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, 100mL×3). The filtrate was concentrated and dissolved with THF (50mL×3), then evaporated to dryness. The reaction was repeated three times to obtain 1g 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 carried out at 0 °C for 2 hours. LCMS showed that 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 out. After standing for the solid to settle, the solution was poured off and then dried with an oil pump to obtain 34 g of 120 mg white powder. LCMS showed that the product content was 70%, and the yield was 76%. MS-ESI: m / z 608.3 [M+H]+.

[0232] Step 6

[0233] Under nitrogen protection, HATU (45 mg, 0.118 mmol) of DMF (1 mL) solution was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) of DMF (1 mL) solution at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LCMS showed that the starting material reacted completely. The reaction solution was directly passed through a reverse-phase column with eluent (MeCN / MeOH = 1 / 1):H2O = 60%:40%) to purify 14.8 mg of yellow solid X2, yield 14%.

[0234] MS-ESI: m / z 1062.4 [M+H]+. H 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] Synthetic route: Specification 35 / 55 pages 40 CN 122124275 A

[0238] Step 1: Add bromopropene (960 mg, 7.92 mmol) to 32a (2.00 g, 6.6 mmol), K2CO3 (1.82 g, 13.2 mmol) in MeCN (20 mL) and stir at 20 °C for 5 hours. TLC (PE / EA = 1 / 2) showed that the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL). After drying with anhydrous sodium sulfate, the solution was purified by rotary evaporation and column chromatography (PE / EA = 2 / 1) to obtain 1.83 g of white solid 32b, yield: 81%.

[0239] Step 2: TFA (10 mL) was added to DCM (1.38 g, 4.02 mmol) of 32b, 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 obtain 0.91 g of yellow viscous substance 32c, yield negligible.

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

[0241] Step 4: Add Pd / C (600 mg) to 32 g (3 g, 5.83 mmol) of MeOH (50 mL) and stir at 25 °C under a hydrogen balloon for 5 hours. TLC (EA) showed that the reaction was complete. Filter the reaction solution and evaporate to dryness to obtain 1.9 g of white solid (32 g), yield: 77%.

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

[0243] Step 6: 32 h (1.186 g, 1.41 mmol) of DCM / diethylamine (20 mL, 20 / 1) was stirred at 25 °C for 17 h. TLC (DCM / MeOH=10 / 1) indicated the reaction was complete. The reaction solution was poured into petroleum ether (200 mL) and filtered to obtain 768 mg of white solid for 32 h, yield: 88%. MS-ESI: m / z 620.3 [M+H]+.

[0244] Step 7: Add HATU (414 mg, 1.09 mmol) 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 stir at 20 °C for 17 hours. TLC (PE / EA = 1 / 5) showed the reaction was complete. Pour the reaction solution into water (30 mL), filter, and purify the filter cake 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: Stir the solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) at 10 °C for 17 hours. TLC (EA) showed the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to obtain 301 mg of white solid 32k, yield negligible.

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

[0247] Step 10: Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32 μL (108 mg, 0.134 mmol), triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL), and stirred at 0 °C for 1 hour. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. 3 x 15 mg white solid was directly prepared from the reaction solution, yield: 12%.

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

[0249] H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 – 8.42 (m, 3H), 8.27 – 8.16 (m, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H) , 6.53 (s, 1H) , 5.61 – 5.51 (m, 1H) , 5.42 (s, 2H) , 5.20 – 5.05 (m, 2H) , 4.56 – 4.42 (m, 2H) , 4.32 – 4.22 (m, 1H) , 3.96 – 3.87 (m, 3H) , 3.79 (d, J = 5.6 Hz, 2H) , 3.70 (d, J = 5.9 Hz, 2H) , 3.25 – 3.08 (m, 2H) , 2.61 – 2.53 (m, 2H) , 2.45 – 2.36 (m, 4H) , 2.36 – 2.22 (m, 3H), 2.20 – 2.03 (m, 4H), 1.99 – 1.68 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0250] Connector-Cytoxin X4:

[0251] Synthetic route: Specification 37 / 55 pages 42 CN 122124275 A

[0252] Step 1: Pd / C (400 mg, 10 wt.%) was added to 20 mL of MeOH containing 33a (2.00 g, 2.58 mmol), 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 obtain 1.3 g of white solid 33b, yield: 74%.

[0253] Step 2: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 stirred at 0 °C for 2 hours. TLC (DCM / MeOH = 1 / 10) showed that the reaction was complete. The reaction solution was poured into water (40 mL), filtered to obtain crude product, and purified by column chromatography (DCM / MeOH = 20 / 1) to obtain 360 mg of yellow solid 33c, with a yield of 41%.

[0254] Step 3: Diethylamine (2 mL) was added to 10 mL of DCM containing 33c (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 solution was poured into PE (100 mL) and filtered to obtain 205 mg of white solid 33d, yield: 71%. MS-ESI: m / z 881.3 [M+H]+.

[0255] Step 4: Add bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) to DMF (1 mL) and water (1 mL) containing 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol), and stir at 0 degrees for 1 hour. The reaction solution directly prepared 15 mg of white solid X4, yield: 6%.

[0256] MS-ESI: m / z 1001.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.57 – 8.50 (m, 1H), 8.50 – 8.43 (m, 2H), 8.35 – 8.29 (m, 1H), 8.19 – 8.12 (m, 2H), 7.80 (d, J = 10.8 Hz, 1H), 7.27 – 7.14 (m, 7H), 6.53 (s, 1H), 5.59 – 5.51 (m, 1H), 5.44 – 5.39 (m, 2H), 5.20 – 5.07 (m, 2H), 4.56 – 4.44 (m , 3H) , 3.92 (s , 3H) , 3.80 – 3.68 (m , 5H) , 3.41 (s, 1H) , 3.21 – 3.12 (m, 2H) , 2.83 – 2.74 (m, 1H) , 2.58 – 2.55 (m, 3H) , 2.39 (s, 4H) , 2.18 – 2.03 (m, 4H) , 1.93 – 1.78 (m, 2H) , 0.87 (t, J= 7.3 Hz, 3H).

[0258] 2.2 Preparation of bispecific antibody drug conjugate

[0259] Preparation of bispecific antibody drug conjugate DSYE001-X1 (DAR4): Specification 38 / 55 pages 43 CN 122124275 A

[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 bispecific antibody DSYE001 (30 mM histidine-acetic acid + 20 mM EDTA pH5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), and placed in a constant temperature water bath shaker. The mixture was shaken at 37°C for 2 hours and then 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), added to the above antibody solution, and placed in a water bath shaker. The mixture was shaken at 22°C for 2 hours, and then 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] The DAR value was calculated by HIC DAR analysis to be p = 4.02.

[0263] Preparation of bispecific antibody-drug conjugate DSYE001-X1 (DAR6): A solution of antibody DSYE001 (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10000 mg, 20.52 mg / mL, 57.80 μmol) was added to a reaction vessel. A reducing buffer (20 mM PB, pH 6.7, 93.59 mL) was added, followed by 10 mM DTPA solution (66.67 mL). The pH of the reaction solution was adjusted to 6.72 using 0.4 M Na2HPO4 solution (7.0 mL). Then, a prepared tris(2-carbonylethyl)phosphohydrochloride solution (10 mM, 19.08 mL, 190.74 μmol) was added to the reaction vessel, and the stirring speed was 50-100 rpm. The reaction was carried out at 25°C with stirring for 3.5 h. The pH of the reaction solution was adjusted to 5.5 using 200 mL of 0.5 M NaH₂PO₄ solution. Then the reaction mixture was...The solution temperature was lowered to 20°C. After cooling, 32.06 mL of DMSO was added to the reaction system. Then, the 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), and then ultrafiltration was performed using an ultrafiltration membrane pack (30 KD, 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 was performed to detect and calculate the DAR value n = 6.1.

[0265] Preparation of bispecific antibody-drug conjugate DSYE001-X1 (DAR8): A solution of antibody DSYE001 (20 mM His / His-HCl, 90 mg / ml sucrose, 0.02% polysorbate 80, pH 6.3, 10000 mg, 20.52 mg / mL, 57.80 μmol) was added to a reaction vessel. Reduction buffer (45 mM PB, pH 7.35, 72.2 mL) was added, followed by 10 mM DTPA solution (66.67 mL). Then, the prepared tri(2-carbonylethyl) phosphate hydrochloride solution (10 mM, 40.46 mL, 404.6 μmol) was added to the reaction vessel. The stirring speed was 50-100 rpm. The reaction was carried out at 25°C with stirring for 4.5 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, and 63.40 mL of DMSO was added to the reaction system. Then, the adapter-cytotoxin X1 (651.9 mg, 606.91 μmol) was dissolved in 60.69 mL of DMSO and added to the above solution. The reaction was carried out at 22°C with stirring 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 reaction was carried out at 25°C with stirring for 4.5 h.The reaction was carried out at 22°C with stirring for 0.5 h. The solution was filtered using an activated carbon filter (Cobetter, CDFCSCSDAC01PCP, 23 cm2), and then ultrafiltration was performed 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] The DAR value n = 7.9 was obtained by HIC DAR analysis and calculation.

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

[0268] 30 mM His-HAc pH 5.5 buffer (1.845 mL) was added to the buffer of antibody DSYE003 (PBS pH 7.4; 40 mg, 5.36 mg / mL, 0.23 μmol), 100 mM EDTA solution (0.5 mL) and prepared tris(2-carbonylethyl) phosphate hydrochloride solution (6.977 mM, 0.208 mL, 1.45 μmol). The mixture was placed in a constant temperature stirrer at 500 rpm and reacted at 37°C for 2 hours, and then the reaction was stopped. 0.750 mL of DMA was added to the above solution, and then the adapter-cytotoxin X1 (2.5 mg, 2.32 μmol) was dissolved in 0.25 mL of DMA. Add the above solution to the mixture, place it in a constant temperature stirrer at 500 rpm, and react at 4°C for 1 hour. Then stop the reaction. Add 300 mg / mL activated carbon (Charcoal, Dextran Coated, manufacturer: Sigma Aldrich) suspension at 10% of the total volume of the above reaction solution, shake to mix, and then place it in a rotary mixer at 4°C for 1 hour. After 1 hour, centrifuge and collect the supernatant. Add 10% of the total volume of the supernatant to the activated carbon suspension, shake to mix, and then place it in a rotary mixer at 4°C for 1 hour. 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 four 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 detected and calculated the DAR value n = 6.32.

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

[0271] Using the adapter-cytotoxin X1 and anti-B7H3 antibody DSYE002, the preparation method is the same as that for bispecific antibody drug conjugate ADC DSYE001-X1, to prepare DSYE002-X1 (DAR6).

[0272] Preparation of anti-PD-L1 antibody drug conjugate DSYE004-X1 (DAR6) (refer to ADC-3) Specification 40 / 55 pages 45 CN 122124275 A

[0273] Using the adapter-cytotoxin X1 and anti-PD-L1 antibody DSYE004, the preparation method is the same as that for bispecific antibody drug conjugate ADC DSYE001-X1, to prepare DSYE004-X1 (DAR6).

[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 bispecific antibody DSYE001 (30 mM histidine-acetic acid + 20 mM EDTA pH 5.5; 100 mg, 10.0 mg / mL, 0.578 μmol), and placed in a constant temperature water bath shaker. The mixture was shaken at 37°C for 2 hours, and then the reaction was stopped.

[0276] The adapter-cytotoxin X2 (3.07 mg, 2.89 μmol) was dissolved in 1.0 mL LDMA (N,N-Dimethylacetamide), added to the above antibody solution, and placed in a water bath shaker. The mixture was shaken at 22°C for 2 hours, and then 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 was performed and the DAR value was calculated to be p=3.95.

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

[0279] Antibody DSYE002 is the reference antibody for removing 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 anti-B7H3 antibody DSYE002 was added to a buffer solution (30 mM histidine-acetic acid + 20 mM EDTA, pH 5.5; 60 mg, 5.5 mg / mL, 0.755) to achieve a specific concentration.Add 1.08 mL of prepared tris(2-carbonylethyl)phosphohydrochloride (7.0 mM, CN 122124275 A, page 41 / 55, CN 122124275 A, 7.55 μmol) to a water bath shaker and react at 37°C for 2 hours, then stop the reaction. Dissolve adapter-cytotoxin X2 (10.8 mg, 9.82 μmol) in 1.2 mL of DMA and add it to the above solution. Place the solution in a water bath shaker and react at 4°C for 1 hour, then stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: 30 mM histidine-hydrochloric acid, pH 5.5), and concentrate 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%), and store at 4°C protected from light.

[0281] HIC DAR analysis detected and calculated the DAR value p=3.98.

[0282] Preparation of isotype control ADC

[0283] Using isotype control antibody and adapter-cytotoxin X1, and referring to the preparation method of bispecific antibody-drug conjugate ADC DSYE001-X1, isotype control ADC (DAR4), isotype control ADC (DAR6) and isotype control ADC (DAR8) were prepared respectively.

[0284] Example 3: Cellular internalization activity of antibody-drug conjugate

[0285] Test objective

[0286] To detect the internalization effect of the bispecific antibody-drug conjugate against B7H3 and PD-L1 of the present invention on A375 and NCI-H1975 cells that simultaneously express B7H3 and PD-L1. 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 into the cells at different time points.

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

[0288] 2. The test drug and Fab-pHrodo were mixed at a ratio of 1:1.2 and incubated at 37°C and 5% CO2 for 0.5 hours. The mixture was serially diluted 4-fold from 100 nM to 0.0061 nM.

[0289] 3. The diluted sample was added to the culture plate at 50 μL / well and incubated 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 1 μg / mL Hoechst to the culture plate at a ratio of 100 μL / well.33342 and 0.5 μg / mL Calcein AM were stained with DPBS at room temperature for 15 minutes.

[0292] 6. After incubation, the supernatant was removed, and the cell culture plate was rinsed once with 1% BSA.

[0293] 7. The fluorescent spots formed by the internalized antibody in each cell were counted using the Perkin Elmer Operetta CLS High-Content Analysis System.

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

[0295] Table 1. Cell endocytosis activity of DSYE001-X2

[0296] The 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 endocytosis effect in the detected cells. For example, a higher maximum average fluorescent spot count or a lower EC50 value.

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

[0298] 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 was evaluated by incubating A375 and NCI-H1975 cells positive for B7H3 and PD-L1 for 6 days using the CellTiter-Glo® chemiluminescence immunoassay (CTG method).

[0299] Logarithmic growth phase cells were collected, and A375 and NCI-H1975 cells were plated at densities of 400 cells / well and 1000 cells / well, respectively. Each well contained 50 μL of cell plate, which was incubated overnight at 37°C in a 5% CO2 incubator. On the second day of the experiment, DSYE001-X2 and DSYE002-X2 were diluted 3-fold with complete culture medium to obtain 8 concentration gradients (starting from the highest concentration of 1000 nM). 50 μL / well of each drug was added to a cell culture plate, with the complete culture medium used as a blank control. Two replicates were set up. The plates were incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 50 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, and the plates were shaken to mix. After standing in the dark for 10 minutes, 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 to calculate the IC50 value. Cell viability was calculated using the formula: (Lum test drug - Lum blank control) / (Lum solvent blank control - Lum blank control) × 100%.

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

[0301] Example 5: In vitro proliferation inhibition test of antibody-drug conjugate on tumor cells

[0302] The inhibitory effect of anti-DSYE001-X1 (DAR8) and isotype control ADC (DAR8) on cell proliferation after incubation with human tumor cells that are positive for B7H3 and PD-L1 for 7 days was evaluated using the CellTiter-Glo® chemiluminescent cell viability assay (i.e., CTG method).

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

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

[0305] Table 2. Inhibitory effect of DSYE001-X1 (DAR8) on tumor cell proliferation in vitro 43 / 55 pages 48 CN 122124275 A

[0306] Experimental results show that, in various tumor cells, the bispecific antibody-drug conjugate DSYE001-X1 of this application has significant inhibitory activity on the proliferation of B7H3 and PD-L1 positive A375, NCI-H1975, NCI-H441, and NCI-H358 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 study the difference in the effect of bispecific antibody-drug conjugate on PD-L1 expression in tumor cells compared with its parent monoclonal antibody conjugate.

[0309] Experimental Methods

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

[0311] 2)Cells were treated with trypsin and counted to ensure cell viability was above 90.0%. 2 × 10⁶ cells / 2 mL were added to each well of a 6-well plate.

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

[0313] 4) After incubation, cells were washed twice with PBS. 500 μL of pre-chilled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphorylase inhibitor) was added to each culture plate (6-well plate), and incubated on ice for 30 min, mixing several times during this period.

[0314] 5) Cells were centrifuged at approximately 14000 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 a BCA quantitative kit. Based on the quantification results, prepare the protein sample for loading, unify the sample protein concentration to 1-2 μg / μL, and add LDS loading buffer (4X) and sample reducing agent (10X). Heat the sample at 100°C for 10 minutes. Instructions 44 / 55 pages 49 CN 122124275 A

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

[0317] 8) Thaw the loaded 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 using an iBlot2 transfer kit and transfer apparatus, run the P3 program for 7 minutes.

[0319] 10) After transfer, 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, with shaking.

[0320] 11) Blocking: Place the membrane in blocking buffer (5% skim milk prepared with 1xTBST) for 1 hour at room temperature with shaking.

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

[0322] 13) Incubation with 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 with gentle shaking.

[0324] 15) Incubation with secondary antibody: Add secondary antibody of appropriate dilution, at room temperature with gentle shaking for 1 hour.

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

[0326] 17) Chemiluminescence: Add West to the membrane.HRP substrate in the Femto ultrasensitive chemiluminescence kit.

[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. Effect 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 with 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 an immunomodulatory effect by inhibiting the binding of the corresponding receptors, but also exerts a stronger immunosuppressive relief effect by reducing the expression level of the targets.

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

[0333] In order to study 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 anti-tumor effect of the bispecific antibody-drug conjugate DSYE001-X2 was evaluated.

[0334] 1. Test Drugs 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): 12 mg / kg

[0338] G3: DSYE001-X2 (Treatment Group): 5.5 mg / kg

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

[0340] 2. Preparation Method: All samples were diluted with physiological saline.

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

[0342] 4. Experimental Methods: CT26-hPD-L1 / hB7H3 cells were resuscitated and cultured, and cell cryopreservation batches were recorded. Cells in the logarithmic growth phase were collected.CT26-hPD-L1 / hB7H3 cells (3rd-4th generation after resuscitation) were inoculated subcutaneously on the right side of mice after removing the culture medium and washing twice with DPBS (for pre- and post-tumor cell viability testing). The inoculation volume was 1×10⁶ cells / 100 μL / mouse (without matrix gel). Tumors reaching 80-120 mm³ were grouped, with the grouping day defined as D0, and drug administration began on D0. The coefficient of variation (CV) for tumor volume was not to 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 19 days after drug 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 - (Ti - T0) / (Vi - V0)] × 100). Ti: mean tumor volume of the treatment group and the positive control group on day i of administration; T0: mean tumor volume of the treatment group and the positive control group on day 0 of administration; Vi: mean tumor volume of the negative control group on day i of administration; V0: mean tumor volume of the negative control group on day 0 of administration.

[0344] The experimental results are shown in Figure 18 and Tables 4-6.

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

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

[0347] Table 5. Tumor volume inhibition rate (TGITV) of the test substance in the homologous transplantation mouse model constructed with CT26 (CT26-hPD-L1 / hB7H3)

[0348] Note: TGITV calculations are compared with G1.

[0349] Table 6. Statistical analysis of tumor volume in different groups of the homologous transplantation mouse model constructed with CT26 (CT26-hPD-L1 / hB7H3)

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

[0351] The experimental results show that, compared with DSYE002-X2, the bispecific antibody-drug conjugate DSYE001-X2 of the present invention showed significantly enhanced tumor growth inhibition 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 study the inhibitory effect of DSYE001-X1 (DAR4) on tumor formation in vivo, human lung cancer NCI-A subcutaneous xenograft model was constructed by mixing H1975 cells with human PBMCs to evaluate the in vivo antitumor effect of DSYE001-X1 (DAR4).

[0354] NCG mice, female, 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. The PBMCs were added to NCI-H1975 cells, and the PBMCs and NCI-H1975 cells were 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 collected. 3 × 10⁵ PBMCs and 2 × 10⁶ NCI-H1975 cells were injected subcutaneously into the right side of NCG mice at a dose of 0.2 mL / mouse (containing 50% Matrigel). 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 (i.v.) 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 a week for two weeks, for a total of four times. 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 a week, and the 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 (referencing ADC-2, DAR4) + anti-PD-L1 monoclonal antibody DSYE004 (treatment group): 8 mg / kg + 15 mg / kg

[0360] All samples were diluted 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 was calculated based on the tumor measurement results.Tumor volume (RTV), the calculation formula is RTV = Vt - V0, where V0 is the average tumor volume measured when the drugs are administered in groups (i.e., D0), and Vt is the average tumor volume at a certain measurement. TRTV and CRTV are taken from the same day.

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

[0363] At the end of the experiment, the mice were euthanized and the tumor weight was weighed. Instructions 47 / 55 pages 52 CN 122124275 A

[0364] The experimental results are shown in Figure 19 and Tables 7-8.

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

[0366] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.01, p < 0.05 is considered to be significant difference

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

[0368] Note: Compared with the isotype control ADC, 15 mg / kg control group, p < 0.01, p < 0.05 is considered to be significant difference

[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) on pages 48 / 55 of the instruction manual, and had a better tumor inhibition effect than the combination of B7H3 monoclonal antibody ADC and PDL1 monoclonal antibody.

[0370] Example 9: Evaluation of the efficacy of antibody-drug conjugate in human breast cancer cell MDA-MB-231 tumor-bearing mice

[0371] To study 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 anti-tumor effect of DSYE001-X1 (DAR6).

[0372] 6-8 week old female NCG mice (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 N+13 progeny after resuscitation. MDA-MB-231 cells in logarithmic growth phase were collected (seeded at passage N+17, culture medium removed, and washed twice with DPBS before seeding; cell viability before and after tumor bearing was 99.27% ​​and 97.00%, respectively), seeding density: 5 × 10⁶ / 100 cells / year.μL / mouse (1:1 PTFE). One week after tumor cell inoculation, human PBMCs were injected via tail vein at a dose of 7.5 × 10⁶ / 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, and drug administration began on D0 and continued on 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 TGITV (relative tumor inhibition rate) was calculated. The formula for calculating TGITV (relative tumor inhibition rate) is:

[0374] Where, : average RTV of the drug administration group; : average RTV of the Vehicle group (solvent group, which is the group that only administers physiological saline in this embodiment); The formula for calculating RTV is: = : tumor volume of mouse numbered n on day t; : tumor volume of mouse numbered n on day 0; : relative tumor volume of mouse numbered n on day t.

[0375] The experimental results are shown in Figure 20, Table 9 and Table 10.

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

[0377] Table 10. Relative tumor inhibition rate (TGITV) of different groups Instruction manual 49 / 55 pages 54 CN 122124275 A

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

[0379] Example 10: Evaluation of the efficacy of antibody-drug conjugate in human non-small cell lung cancer cell NCI-H1975 tumor-bearing mice

[0380] To study 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 anti-tumor effect of DSYE001-X1 (DAR6).

[0381] NCG mice, female, 6-8 weeks old, weighing approximately 18-22g. 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. Purchase frozen PBMCs, thaw and count them, and add the resuspended PBMCs to NCI-H1975 cells. PBMCs and NCI-H1975 cells are 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 are harvested.Three × 10⁵ NCI-H1975 cells and two × 10⁶ NCI-H1975 cells (containing 50% matrix gel) were subcutaneously injected into 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 three groups of five mice each. On the day of grouping (day 0), DSYE001-X1 (DAR6) and DSYE003-X1 (DAR6) in combination with pembrolizumab were administered intravenously (i.v.) twice a week for two weeks, for a total of four 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 (reference ADC-1, DAR6) + pembrolizumab (treatment group): 18 mg / kg + 15 mg / kg

[0386] All samples were prepared by dilution with PBS. Pembrolizumab was prepared using conventional 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 treatment group; CRTV: RTV of negative control group). The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = Vt - V0, where V0 is the average tumor volume measured when the drugs were administered in groups (i.e., d0), and Vt is the average tumor volume at a certain 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 value of the tumor volume of the drug group relative to the control group at a certain time point. T and C are the tumor volumes (TV) of the drug group and the control group at a certain time point, respectively.

[0389] At the end of the experiment, the mice were euthanized and the tumor weight was weighed.

[0390] The experimental results are shown in Figure 21 and Tables 11-12.

[0391] Table 11. Mean tumor volume (Mean±SEM) of each group of mice. Instructions for use, pages 50 / 55, CN 122124275 A

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

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

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

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

[0396] Example 11: Pharmacokinetics and toxicity study of antibody-drug conjugate after multiple administrations

[0397] Test objective

[0398] DSYE001-X1 (DAR6) was administered intravenously to cynomolgus monkeys once every 3 weeks for a total of 2 administrations. The nature, degree, dose-effect and time-effect relationship of the possible toxic reactions caused by the antibody-drug conjugate were observed, and the target organs or tissues of toxicity were determined to provide a reference for subsequent studies.

[0399] Experimental Methods

[0400] A total of 4 cynomolgus monkeys (2 per sex) were used in the experiment and randomly divided into 3 groups according to their body weight, with 1 monkey per sex per group. Group 1 and Group 2 animals were given 30 and 120 mg / kg of the test product (DSYE001-X1 (DAR6)), respectively, which were the low-dose and high-dose groups of the test product. The animals were administered the drug via intravenous infusion at a volume of 10 mL / kg over a period of approximately 30 min, once every 3 weeks for a total of 3 weeks (2 administrations in total). Instructions for use, pages 51 / 55, CN 122124275 A

[0401] During the experiment, animals were clinically observed, and their weight, food intake, body temperature, electrocardiogram, and clinical pathology (blood cell count, coagulation function, blood biochemistry) were recorded. One week after the last dose (D29), the animals were euthanized, and gross anatomical and histopathological (gross abnormalities, bone and bone marrow) examinations 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 analysis was performed.

[0402] Table 13. Toxicokinetic Parameters

[0403] Experimental Conclusions

[0404] During the experiment, no animals in each dose group died or were near death. No abnormal changes 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 anatomy and histopathological examination (bone and bone marrow). This indicates that the antibody-drug conjugate has good safety.

[0405] Example 12: Pharmacodynamic Evaluation of Antibody-Drug Conjugate DSYE001-X1 (DAR8) in Homologous Transplantation Mouse Model

[0406] To study the inhibitory effect of DSYE001-X1 (DAR8) on tumor growth, pharmacodynamic evaluation was carried out in BALB / c-hPD-L1 / hB7H3 mice. The specific method is described in Example 7.

[0407] The antibody-drug conjugate DSYE001-X1 of this application exhibits superior antitumor efficacy compared to 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. Although 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. In fact, various modifications to the modes of carrying out 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 Listing

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

[0411] Light chain variable region of monoclonal antibody: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSG TDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIK (SEQ ID NO:4) LCDR1: RASQSINTYLH (SEQ ID NO:1) LCDR2: YASQSIS (SEQ ID NO:2) LCDR3: QNGHSFPLT (SEQ ID NO:3) Specification 52 / 55 pages 57 CN 122124275 A Heavy chain variable region of monoclonal antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVT ITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSS (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 Nanobody: DVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIYIGSGATYYVDSVKGRF TISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSS (SEQ ID NO.: 15) HCDR1: TIWIG (SEQ ID NO.: 12) HCDR2: AIYIGSGATYYVDSVKG (SEQ ID NO.: 13)HCDR3: TGGTVGSMVRFSPVGDFGY (SEQ ID NO.: 14) Linker peptide between monoclonal antibody and nanobody: GGGGSGGGGTGGGGS (SEQ ID NO.: 11) Heavy chain of monoclonal antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVT ITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 10) Light chain of monoclonal antibody: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSG TDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 9) Heavy chain of bispecific antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVT ITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSGGGGTGGGGSDVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIYIGSGATYYVD SVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSS (SEQ ID NO.: 16) Light chain of the bispecific antibody: DIQMTQSPSSLSASVGDRVTISCRASQSINTYLHWYQQKLGQAPRLLIYYASQSISGVPSRFSGSGSG DESCRIPTION Page 53 / 55 58 CN 122124275 A TDFTLTINSLEAEDAATYYCQNGHSFPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 9) Amino acid sequence of DSYE002 (reference antibody) DVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIYIGSGATYYVDSVKGRF TISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSSGSEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 17) DSYE003 Heavy Chain QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRV TFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGTGGGGSDVQLQESGGGLVQAGGSLRLSCTASGYTLSTIWIGWFRQAPGKGLEGVAAIY IGSGATYYVDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAATGGTVGSMVRFSPVGDFGYWGQGTQVTVSS (SEQ ID NO.: 18) DSYE003 Light Chain (Human Anti‑HIV‑1 gp120 clone b12 light chain) EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGS GTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 19) PD‑L1 monoclonal antibody amino acid sequence (DSYE004) PD‑L1 mAb heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTTFGVHWVRQAPGKGLEWVGIIWPGGNTNYNSALMSRVT ITADESTSTAYMELSSLRSEDTAVYYCARENYGRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 20) PD-L1 monoclonal antibody light chain: Same as the light chain of the bispecific antibody. Pembrolizumab amino acid sequence heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAA Instruction Manual 54 / 55 Page 59 CN 122124275 A LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO.: 21) Light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSG SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 22). Instruction manual page 55 / 55, page 60, CN 122124275 A, Figure 1, Figure 2; Instruction manual figure 1 / 11, page 61, CN 122124275 A, Figure 3; Instruction manual figure 2 / 11, page 62, CN 122124275 A, Figure 4; Instruction manual figure 3 / 11, page 63, CN 122124275 A, Figure 5; Instruction manual figure 4 / 11, page 64, CN 122124275 A, Figure 6, Figure 7; Instruction manual figure 5 / 11, page 65, CN 122124275 A, Figure 8, Figure 9; Instruction manual figure 6 / 11, page 66, CN 122124275 A, Figure 10, Figure 11; Instruction manual figure 7 / 11, page 67, CN 122124275 A, Figure 12, Figure 13; Instruction manual figure 8 / 11, page 68, CN 122124275 A, Figure 14, Figure 15; Instruction manual figure 9 / 11, page 69, CN 122124275 A Figure 16 Figure 17 Appendix to the Specification 10 / 11 Page 70 CN 122124275 A Figure 18 Figure 19 Figure 20 Figure 21 Appendix to the Specification 11 / 11 Page 71 CN 122124275 A Abstract The present invention discloses a bispecific antibody-drugconjugate and a preparation method therefor and a use thereof, wherein the structure of the bispecific antibody-drug conjugate comprises the following fragments: a bispecific antibody against B7H3 and PD-L1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug. The bispecific antibody- drug conjugate of the present invention has a good internalization effect, proliferation inhibitory activity, and tumor growth inhibitory activity.

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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