Anti-B7-H3 antibody or fragment thereof and antibody drug conjugate targeting B7-H3

Abstract

The invention provides a series of novel anti-B7-H3 antibodies. The antibody has high affinity to B7-H3 and is stable in structure. The invention further provides an antibody drug conjugate prepared from the antibody, and the antibody drug conjugate takes the camptothecin compound with a specific structure as a toxic molecule, has remarkable tumor inhibition activity and has a bystander killing effect.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the priority benefit of the Chinese invention patent application with application number CN202310655433.X filed on June 5, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology, and in particular, the present invention relates to an antibody-drug conjugate targeting B7-H3 and application thereof in tumor treatment. Background Art

[0004] B7-H3, also known as B7H3 or CD276, is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. B7-H3 is encoded by chromosome 15q24 and consists of an extracellular region, a transmembrane region, and a short intracellular region. Immunohistochemistry (IHC) analysis of a large number of normal tissues showed that B7-H3 was only lowly expressed in a few tissues, or even undetectable. However, B7-H3 has been found to be overexpressed in a variety of solid tumors such as bladder cancer, prostate cancer, and melanoma; and as research deepens, more evidence shows that B7-H3 mainly plays a co-inhibitory role in immune cells, which helps tumor cells escape immune surveillance. Therefore, overexpression of B7-H3 is associated with poor prognosis in tumor patients and the invasion and metastasis potential of tumors in in vitro models.

[0005] Currently, more than 20 clinical trials have been or are testing the safety and effectiveness of monoclonal antibody immunotherapy strategies targeting B7-H3. In addition, several antibody-drug conjugates (ADCs) targeting B7-H3 have entered the clinical trial stage at home and abroad.

[0006] The ADC drug DS-7300 targeting B7-H3 developed by Daiichi Sankyo is prepared by randomly coupling the MC-GGFG-Dxd drug-containing linker with the hM30 antibody targeting B7H3 to obtain a B7-H3 targeting ADC drug with DAR=4. The structure is shown below:

[0007]

[0008] MGC018, an ADC drug targeting B7-H3 developed by MacroGenics, is formed by coupling anti-B7-H3 humanized IgG1 monoclonal antibody to Duocarmycin (DUBA) via a cleavable Linker, with an average DAR of 2.7. The structure is shown below:

[0009]

[0010] BAT8009, an ADC drug targeting B7-H3 developed by BioAtla, is composed of a recombinant humanized anti-B7-H3 antibody and a DNA topoisomerase inhibitor coupled via a cleavable linker.

[0011] ABBV-155, an ADC drug targeting B7-H3 developed by AbbVie, is a conjugate of an anti-B7-H3 antibody and a BCL-XL inhibitor that promotes apoptosis through a linker. It is used to treat advanced solid tumors. The structure is shown below:

[0012]

[0013] The above ADC drugs have shown certain therapeutic effects, but there are also certain problems. For example, MacroGenics announced a Phase I clinical trial (cohort expansion, NCT03729596) of MGC018 for the treatment of advanced solid tumors in 2021. In terms of safety, 43 patients experienced grade 3 or above treatment-related adverse events (TRAEs) (50%), and common serious TRAEs included neutropenia (22.1%) and thrombocytopenia (7%). The DS-7300 developed by Daiichi Sankyo uses a random coupling method, which will cause the ADC product to have greater heterogeneity.

[0014] At present, there are still relatively few available types of ADCs containing camptothecin compounds targeting B7-H3, and in view of the above-mentioned deficiencies of such existing ADC drugs, there is still a need in the art to provide an ADC drug containing camptothecin compounds that has better targeting to B7-H3 and has lower product heterogeneity, less side effects, and thus better therapeutic effects.

[0015] Chinese patent application CN202111459992.0 (publication number CN114573695A), filed on December 2, 2021 and entitled "An anti-human B7-H3 monoclonal antibody and its application", discloses mouse and humanized anti-human B7-H3 monoclonal antibodies. In this application, the recombinant human B7-H3 extracellular region was used as an immunogen, and a mouse anti-human B7-H3 monoclonal antibody capable of binding to the B7-H3 extracellular region was prepared by hybridoma technology; a human-mouse chimeric antibody capable of specifically binding to B7-H3 on the cell surface was constructed on the basis of the mouse antibody; further, a humanized antibody was constructed by CDRs transplantation and CDRs region mutation, and the humanized antibody retained the ability to specifically bind to the human B7-H3 extracellular region and B7-H3 on the cell membrane surface, and could be internalized by B7-H3 on the cell membrane surface. However, although the mouse-derived and humanized antibodies in this application have excellent effects, there is still room for further improvement. Summary of the invention

[0016] The inventors of the present invention have found that although a strain of anti-B7-H3 humanized antibody disclosed in CN114573695A has a high affinity for human B7-H3, it is easy to break. In the actual application of antibodies, the easy breakage and even fragmentation of antibodies will lead to increased heterogeneity of products containing the antibodies, thereby increasing the immunogenicity of the products, reducing the efficacy, and even causing toxic side effects. Therefore, whether it is used alone or further developed into other products, the humanized antibody has certain defects.

[0017] Therefore, based on the known anti-B7-H3 humanized antibody, the inventors of the present invention screened for a novel anti-B7-H3 antibody that can overcome the above-mentioned defects by using variable region site-directed mutagenesis; and further, attempted to couple the mutated antibody with a camptothecin compound of a specific structure to obtain an antibody-drug conjugate, in order to select an antibody-drug conjugate with better B7-H3 targeting and B7-H3-expressing cell killing effect.

[0018] Therefore, one object of the present invention is to provide an antibody or a fragment thereof that specifically binds to B7-H3. Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof that targets B7-H3 and is prepared using the antibody or a fragment thereof.

[0019] In the context of the present invention, halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0020] In the context of the present invention, "linker" is used interchangeably with "connector" or "linker compound".

[0021] In the context of the present invention, the term "drug-containing linker" refers to a compound in which a drug (eg, a small molecule drug such as a camptothecin compound) is directly or indirectly covalently bonded to a linker.

[0022] The technical solution of the present invention is as follows.

[0023] First aspect

[0024] The present invention provides an anti-B7-H3 antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise heavy chain complementary determining regions CDR-H1, CDR-H2 and CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2 and CDR-L3 from the following amino acid sequence combinations:

[0025] (i) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 3;

[0026] (ii) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 4; or

[0027] (iii) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 5.

[0028] In the context of the present invention, the term "fragment" encompasses various functional fragments of the anti-B7-H3 antibody, which retain the antigen binding ability and corresponding biological activity of the antibody. It is well known in the art that the antigen binding ability and corresponding biological activity of the antibody can be achieved by fragments of intact antibodies, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for intact antibodies. For example, the antigen-binding fragment of the antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. For example, the fragment can be a Fab, F(ab')2 or scFv fragment, etc.

[0029] The amino acid sequences of the above items are respectively the amino acid sequences of the heavy chain variable region and the light chain variable region of the specific anti-B7-H3 antibody provided by the present invention; see the "Specific Embodiments" section of the present invention. Using the definition tools of the complementary determining regions in the heavy chain or light chain variable region of antibodies known or conventional in the art (such as the Kabat scheme, the AbM scheme, the Chothia scheme, the Contact scheme, the IMGT scheme, the enhanced Chothia / Abm scheme), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained when the two are combined to form an antibody. For example, in Example 1 of the present invention, when defining CDRs according to the Kabat scheme, the position of the amino acid sequence of each CDR in the full length of the variable region is provided. Heavy chain CDRs and light chain CDRs combinations can be obtained according to the definition tools known or conventional in the art, and antibodies or fragments thereof containing each CDR combination in these heavy chain CDRs and light chain CDRs combinations are within the scope of protection of the present invention.

[0030] Further, the anti-B7-H3 antibody or fragment thereof provided by the present invention comprises a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3; and, CDR-L1, CDR-L2 and CDR-L3) selected from the following:

[0031] (i) the CDR-H1, CDR-H2 and CDR-H3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:14, respectively;

[0032] (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:15, respectively; and

[0033] (iii) the CDR-H1, CDR-H2 and CDR-H3 respectively comprise amino acid sequences that are at least 75% identical to the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and the CDR-L1, CDR-L2 and CDR-L3 respectively comprise amino acid sequences that are at least 75% identical to the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:16.

[0034] In the context of the present invention, the term "at least 75% identity" encompasses any percentage of identity between at least 75% identity and 100% identity, such as 75%, 80%, 85%, 90%, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, even 100% identity. Depending on the use of the term, the up to 25% difference in amino acid sequence caused by the "at least 75% identity" may exist in the heavy chain or light chain CDRs, or in any region outside the heavy chain or light chain CDRs, such as the framework region of the heavy chain variable region and the light chain variable region of the antibody of the present invention or its fragment, or the heavy chain constant region and the light chain constant region of the antibody of the present invention or its fragment. The difference may be caused by deletion, addition or substitution of amino acids at any position, wherein the substitution may be a conservative substitution or a non-conservative substitution.

[0035] According to a specific embodiment of the present invention, the anti-B7-H3 antibody or fragment thereof provided by the present invention comprises a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3; and, CDR-L1, CDR-L2 and CDR-L3) selected from the following:

[0036] (i) the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:14, respectively;

[0037] (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:15, respectively; and

[0038] (iii) the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:16, respectively.

[0039] The antibody or fragment thereof provided by the present invention is an antibody or fragment thereof against B7-H3, particularly human B7-H3. The amino acid sequence of human B7-H3 is exemplarily provided in UniProtKB-Q5ZPR3.

[0040] The anti-B7-H3 antibody or fragment thereof provided by the present invention comprises at least a heavy chain variable region (VH) and a light chain variable region (VL), both of which include the above-mentioned CDRs and the framework region (FR) therebetween, and the arrangement of each region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further, in the anti-B7-H3 antibody or fragment thereof provided by the present invention, the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3-5 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0041] Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following:

[0042] (i) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 3;

[0043] (ii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:4; and

[0044] (iii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:5.

[0045] According to a specific embodiment of the present invention, the antibody or fragment thereof provided by the present invention comprises a combination of a heavy chain variable region and a light chain variable region selected from the following:

[0046] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3;

[0047] (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; and

[0048] (iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5.

[0049] The anti-B7-H3 antibody or fragment thereof provided by the present invention may be any form of antibody or fragment thereof comprising the domains defined above, such as monoclonal antibodies, chimeric antibodies, partially or fully humanized antibodies, etc. The antibody or fragment thereof provided by the present invention may also be in the form of scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0050] In addition to the variable region, the anti-B7-H3 antibody or fragment thereof provided by the present invention may further comprise a constant region, such as a heavy chain constant region (CH) and / or a light chain constant region (CL), or any one or more domains in the constant region, such as any one or more of CH1, CH2, CH3, CH4, etc. Alternatively, the anti-B7-H3 antibody or fragment thereof provided by the present invention may comprise a heavy chain and / or a light chain, wherein the heavy chain may comprise, for example, a heavy chain constant region of IgG, IgA, IgM, IgD or IgE, and the light chain may comprise a light chain constant region of κ or λ type.

[0051] Preferably, the present invention provides an anti-B7-H3 humanized antibody or a fragment thereof. More preferably, the present invention provides an anti-B7-H3 humanized monoclonal antibody or a fragment thereof.

[0052] The present invention provides a humanized monoclonal antibody that specifically binds to B7-H3 and has been isolated and structurally characterized. According to a specific embodiment of the present invention, the anti-B7-H3 antibody provided by the present invention is hz10B4m1, hz10B4m2 or hz10B4m3, see the "Specific Embodiment" section of the present invention for details.

[0053] Second aspect

[0054] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable region, heavy chain variable region, heavy chain and / or light chain contained in the anti-B7-H3 antibody or fragment thereof described in the present invention.

[0055] The third aspect

[0056] The nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in a third aspect, the present invention also provides a vector, wherein the vector comprises the nucleic acid molecule of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0057] The fourth aspect

[0058] The vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, in a fourth aspect, the present invention provides a host cell, the host cell comprising the nucleic acid molecule and / or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and / or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

[0059] The anti-B7-H3 antibodies or fragments thereof provided by the present invention can be obtained by any method known in the art. For example, the host cells provided by the present invention are cultured while allowing the host cells to express the heavy and light chains of the antibodies. Optionally, the method further comprises the step of recovering the produced antibodies. As described above, for example, antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. The anti-B7-H3 antibodies or fragments thereof provided by the present invention can be obtained by any method known in the art. For example, the host cells provided by the present invention are cultured while allowing the host cells to express the heavy and / or light chains of the antibodies to assemble into the antibodies. Optionally, the method further comprises the step of recovering the produced antibodies.

[0060] Fifth Aspect

[0061] The anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector and / or host cell provided by the present invention can be included in a pharmaceutical composition, more particularly in a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Therefore, in a fifth aspect, the present invention also provides a composition, comprising the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector and / or host cell described in the present invention; preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0062] The sixth aspect

[0063] The present invention provides the use of the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating a disease associated with B7-H3 expression (including overexpression). Preferably, the disease is a tumor. More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0064] Seventh Aspect

[0065] The present invention also provides the use of the anti-B7-H3 antibody or its fragment, nucleic acid molecule, vector, host cell and / or composition in the preparation of a reagent for diagnosing a disease related to B7-H3 expression (including overexpression). Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0066] Eighth Aspect

[0067] The present invention also provides a method for treating a disease related to B7-H3 expression (including overexpression), the method comprising administering an anti-B7-H3 antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention to a subject in need thereof, and optional other drugs or means. The optional other drugs or means refer to other drugs or means that can be administered in combination with the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present invention, such as small molecule drugs, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADC, oncolytic viruses, gene and nucleic acid therapeutic drugs and radiotherapy. The combined administration of the two can be carried out in any form, such as simultaneously, continuously or at intervals of a certain time.

[0068] Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0069] Preferably, the subject is a mammal, preferably a primate, more preferably a human.

[0070] The above-mentioned disease treatment methods provided by the present invention depend on many factors when applied, including the specific active ingredient administered, the patient's age, weight, gender or physical and medical condition, the severity of the condition to be treated, the route of administration, etc.

[0071] Ninth Aspect

[0072] The present invention also provides a method for diagnosing a disease related to B7-H3 expression (including overexpression), the method comprising contacting an anti-B7-H3 antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention with a sample from a subject. Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0073] Preferably, the subject is a mammal; more preferably, the subject is a human.

[0074] Tenth Aspect

[0075] The present invention provides a kit comprising the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition. The kit is used for the treatment or diagnosis as described above. Optionally, the kit may also include instructions for use.

[0076] Aspect 11

[0077] As described above, another object of the present invention is to provide an antibody-drug conjugate or a salt thereof targeting B7-H3, wherein preferably, the antibody-drug conjugate or a salt thereof is prepared using the anti-B7-H3 antibody or a fragment thereof provided by the present invention. Therefore, in the eleventh aspect, the present invention provides an antibody-drug conjugate or a salt thereof, wherein the antibody-drug conjugate or a salt thereof has a structure as shown in structural formula Ia and / or Ib:

[0078]

[0079] and / or

[0080]

[0081] In formula Ia and / or Ib, Ab represents an anti-B7-H3 antibody or a fragment thereof. Preferably, the antibody or a fragment thereof is an anti-B7-H3 antibody or a fragment thereof as defined above in the first aspect of the present invention.

[0082] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in a substituted phenylene group, the substituents are selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., haloC1-6 alkyl, preferably haloC1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester, amide and cyano; preferably, M is a halogen-substituted phenylene group.

[0083] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight chain heteroalkylene, wherein the C1-21 straight chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.

[0084] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-、-NH(CH2CH2O) a CH2CO-, -S(CH2) a CO— or chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6.

[0085] In formula Ia and / or Ib, and in the context of the present invention unless otherwise indicated, A represents a short peptide structure formed by 2 to 4 amino acids. Among them, when A represents a short peptide structure formed by two amino acids, it can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg- or -Gly-Val-, preferably -Phe-Lys-, -Val-Ala- or -Val-Cit-; when A represents a short peptide structure formed by three amino acids, it can be -Glu-Val-Ala-, -Glu-Val-Cit- or -Ala-Ala-Ala-, preferably -Glu-Val-Ala- or -Ala-Ala-Ala-; when A represents a short peptide structure formed by four amino acids, it can be -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-, preferably -Gly-Gly-Phe-Gly-. Preferably, A is -Val-Ala-, -Gly-Gly-Phe-Gly- or -Ala-Ala-Ala-. In formula Ia and / or Ib, group A is connected to SP2 via the amino group at the amino terminal of its short peptide structure.

[0086] In the formula Ia and / or Ib, M is preferably a halogen-substituted phenylene group, in particular a fluorine-substituted phenylene group.

[0087] In formula Ia and / or Ib, SP1 is preferably a C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene group containing 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S.

[0088] In formula Ia and / or Ib, SP2 is preferably a chemical bond.

[0089] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, m is 1 to 10, preferably 1 to 8 (eg 1 to 5), more preferably 3 to 8. m may be an integer or a non-integer.

[0090] Furthermore, the antibody-drug conjugate or its salt provided by the present invention has a structure as shown in structural formula Ic and / or Id:

[0091]

[0092] and / or

[0093]

[0094]

[0095] In formula Ic and / or Id, Ab, group A and m are as defined in formula Ia and / or Ib.

[0096] In any of formulae Ia, Ib, Ic and Id, and unless otherwise indicated in the context of the present invention, CPT is a camptothecin compound.

[0097] Further, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in structural formula I, wherein structural formula I is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id through an amide bond, preferably the amino group adjacent to the group G in structural formula I is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id through an amide bond:

[0098]

[0099] In formula I, and in the context of the present invention, unless otherwise specified, R1, R2, R3, R4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, R4 together with the carbon atoms to which they are attached constitute a C3-6 (preferably C3-5) cyclic alkyl. When R1, R2, R3, R4 are independently C1-6 alkoxy, the C1-6 alkoxy includes a straight or branched C1-6 alkoxy, preferably a straight or branched C1-3 alkoxy, more preferably a methoxy. When R1, R2, R3, R4 are independently substituted amino, the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R1, R2, R3, and R4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 (preferably C3-5, more preferably C4) alkyl or substituted C1-7 (preferably C3-5, more preferably C4) alkyl, and the substituted C1-7 alkyl is a C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably a C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl).

[0100] In formula I, and in the context of the present invention unless otherwise stated, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0101] In formula I, and in the context of the present invention, unless otherwise specified, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene may be a methylene group in which one hydrogen is substituted or two hydrogens are substituted simultaneously, and the substituent may be a benzyl or an alkyl group; when the substituent is an alkyl group, the alkyl group and R3 and / or R4 and the carbon atoms to which they are attached may form a C3-6-membered cyclic or spirocyclic structure; or, when the substituent is two alkyl groups, the two alkyl groups may form a C3-6-membered spirocyclic structure together with the group Y. When Y is a substituted methylene group, the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group.

[0102] Preferably, Y is oxygen, sulfur, sulfone, sulfoxide or methylene; or, preferably, Y is oxygen, sulfur or methylene.

[0103] In formula I, and unless otherwise stated in the context of the present invention, X is oxygen or sulfur.

[0104] In formula I, and in the context of the present invention unless otherwise stated, n=0 or 1.

[0105] In structural formula I, when R1, R2, R3, and R4 are all hydrogen, X is oxygen, and n=0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is oxygen or sulfur, G cannot be hydrogen.

[0106] Preferably, R1, R2, R3, and R4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl). Further, R1 and R2 may be the same; and / or, R3 and R4 may be the same.

[0107] Preferably, Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and / or R4, and the carbon atoms to which they are connected can form a C3-6-membered cyclic or spirocyclic structure.

[0108] Preferably, X may be oxygen.

[0109] Preferably, X is oxygen, G is hydrogen, halogen (eg fluorine or chlorine), methyl or methoxy, and Y and R1, R2, R3, R4 are as defined above.

[0110] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, and R1, R2, R3, R4 are as defined above.

[0111] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0112] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0113] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0114] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0115] Preferably, X is oxygen, G is hydrogen, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0116] Preferably, X is oxygen, G is fluorine, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0117] Preferably, n=0.

[0118] According to a specific embodiment of the present invention, in structural formula I:

[0119] G is hydrogen, Y is methylene, R1 and R2 are methyl, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0120] G is hydrogen, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0121] G is hydrogen, Y is methylene, one of R1 and R2 and one of R3 and R4 together with the carbon atom to which they are attached form a C3 cyclic alkyl group, the other of R1 and R2 and the other of R3 and R4 are hydrogen, X is oxygen, and n=0;

[0122] G is hydrogen, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0123] G is hydrogen, Y is sulfoxide, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0124] G is hydrogen, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0125] G is hydrogen, Y is sulfone, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0126] G is hydrogen, Y is methylene, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=1;

[0127] G is fluorine, Y is oxygen, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0128] G is fluorine, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0129] G is fluorine, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0130] G is fluorine, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0131] G is hydrogen, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0; or

[0132] G is fluorine, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0.

[0133] Preferably, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in the structural formula IA, wherein the structural formula IA is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond, preferably the amino group on the left benzene ring in the structural formula IA is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond:

[0134]

[0135] In formula IA, the groups R1, R2, R3, and R4 are the same as those defined in formula I above, but R1, R2, R3, and R4 are not hydrogen at the same time.

[0136] Alternatively, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in structural formula II, wherein structural formula II is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond, preferably the amino group adjacent to the group G in structural formula II is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond:

[0137]

[0138] In formula II, and in the context of the present invention, unless otherwise specified, R5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl. When R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes a straight chain or branched C1-5 alkyl. Further, R5 is a C1-4 straight chain alkyl. When R5 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, mesyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R5 is a substituted phenyl, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-3) or halogen.

[0139] In formula II, and unless otherwise specified in the context of the present invention, G is hydrogen, halogen (eg fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0140] In formula II, X is oxygen or sulfur.

[0141] In structural formula II, n=0 or 1.

[0142] In structural formula II, when X is oxygen, G is hydrogen, and n=0, R5 cannot be n-butyl.

[0143] Preferably, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in the structural formula IIA, wherein the structural formula IIA is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond, preferably the amino group on the left benzene ring in the structural formula IIA is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id via an amide bond:

[0144]

[0145] In formula IIA, the group R5 is the same as the definition of the group R5 in formula II above, but R5 cannot be n-butyl.

[0146] According to a specific embodiment of the present invention, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of the CPT is as follows, wherein each structural formula is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id through an amide bond, preferably the amino group on the left benzene ring in each structural formula is connected to the carboxyl group in the group A in any one of the structural formulas Ia, Ib, Ic and Id through an amide bond:

[0147]

[0148]

[0149]

[0150] Further, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in structural formula IV, wherein the hydroxyl group connected to the same carbon as R8 in structural formula IV is connected to the carboxyl group of group A in any one of the structural formulas Ia, Ib, Ic and Id via a self-releasing structure, and the self-releasing structure is, for example The solid line represents the site of attachment to the carboxyl group of group A in any of formulas Ia, Ib, Ic and Id, and the wavy line represents the site of attachment to the hydroxyl group in formula IV:

[0151]

[0152] In formula IV, and unless otherwise specified in the context of the present invention, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.

[0153] When R8 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.

[0154] Aspect 12

[0155] The present invention provides an antibody-drug conjugate or a salt thereof, wherein the antibody-drug conjugate or a salt thereof has the general formula The structure shown, wherein mAb represents an anti-B7-H3 antibody or a fragment thereof. Preferably, mAb is an anti-B7-H3 antibody or a fragment thereof as defined above in the first aspect of the present invention.

[0156] In this general formula, the groups M, SP1, SP2, A and CPT are the same as those defined in the eleventh aspect above; N is 1-10, preferably 1-8 (eg 1-5), more preferably 3-8.

[0157] In this general formula, E L is selected from the following groups, wherein, Indicates that it is linked to the cysteine ​​in mAb. Indicates connection with M:

[0158] E L -1a and / or EL -1b: and / or E L -2: E L -3: E L -4: E L -5: E L -6:

[0159] Aspect 13

[0160] The present invention provides a drug-containing linker (linker-payload) having a structure shown in the general formula "LA-CPT", wherein L represents a linker for an antibody drug conjugate (ADC), A represents one or more amino acids, and CPT is a camptothecin compound.

[0161] In the thirteenth aspect of the present invention, the drug-containing linker having the structure shown in the general formula LA-CPT is a compound having the structure shown in structural formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0162]

[0163] In formula III, and unless otherwise specified in the context of the present invention, E is selected from the following groups, wherein, Indicates the attachment site to M:

[0164] E-1: E-2: E-3: E-4: E-5: and E-6:

[0165] In formula III, and in the context of the present invention, unless otherwise specified, M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in a substituted phenylene group, the substituent is selected from an alkyl group (e.g., a C1-6 alkyl group, preferably a C1-4 alkyl group), a haloalkyl group (e.g., a haloC1-6 alkyl group, preferably a haloC1-4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-6 alkoxy group, preferably a C1-4 alkoxy group, preferably a methoxy group), a halogen, an ester group, an amide group, and a cyano group; preferably, M is a halogen-substituted phenylene group.

[0166] In formula III, and unless otherwise specified in the context of the present invention, SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight chain heteroalkylene, wherein the C1-21 straight chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.

[0167] In structural formula III, and unless otherwise specified in the context of the present invention, SP2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, and more preferably an integer of 1-6.

[0168] In formula III, and unless otherwise specified in the context of the present invention, A represents a short peptide structure formed by 2 to 4 amino acids. Among them, when A represents a short peptide structure formed by two amino acids, it can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg- or -Gly-Val-, preferably -Phe-Lys-, -Val-Ala- or -Val-Cit-; when A represents a short peptide structure formed by three amino acids, it can be -Glu-Val-Ala-, -Glu-Val-Cit- or -Ala-Ala-Ala-, preferably -Glu-Val-Ala- or -Ala-Ala-Ala-; when A represents a short peptide structure formed by four amino acids, it can be -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-, preferably -Gly-Gly-Phe-Gly-. Preferably, A is -Val-Ala-, -Gly-Gly-Phe-Gly- or -Ala-Ala-Ala-. In formula III, group A is connected to SP2 via the amino group at the amino terminal of its short peptide structure.

[0169] In formula III, M is preferably a halogen-substituted phenylene group, particularly a fluorine-substituted phenylene group.

[0170] In formula III, SP1 is preferably a C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene group containing 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S.

[0171] In formula III, SP2 is preferably a chemical bond.

[0172] In formula III, and unless otherwise indicated in the context of the present invention, CPT is a camptothecin compound.

[0173] Wherein, when the group E is E1, the structural formula III provided by the present invention is further structural formula IIIA:

[0174]

[0175] In formula IIIA, and in the context of the present invention, unless otherwise specified, R6, R7 are independently hydrogen, halogen or Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl

[0176] For example, in structural formula III and structural formula IIIA, CPT is the compound shown in structural formula I or structural formula IA provided in the eleventh aspect of the present invention above, or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, and the corresponding specific compound.

[0177] When CPT is a compound of formula I provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0178]

[0179] In the structural formula I, the groups G, X, Y, R1, R2, R3, R4, and n are the same as the definitions of the groups G, X, Y, R1, R2, R3, R4, and n in the structural formula I in the eleventh aspect above. And in the structural formula III or the structural formula IIIA, the carboxyl group in the structural formula I and the group A are connected through an amide bond, preferably the amino group adjacent to the group G in the structural formula I is connected to the carboxyl group of A through an amide bond, that is, the amino group of the compound represented by the structural formula I forms an amide bond with the carboxyl group of A in the structural formula III or IIIA.

[0180] When CPT is a compound represented by structural formula IA provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0181]

[0182]

[0183] In the structural formula IA, the groups R1, R2, R3, and R4 are the same as the definitions of the groups R1, R2, R3, and R4 in the structural formula IA in the eleventh aspect above, but R1, R2, R3, and R4 can be hydrogen at the same time. And in the structural formula III or the structural formula IIIA, the structural formula IA is connected to the carboxyl group in the group A through an amide bond, preferably the amino group on the left benzene ring in the structural formula IA is connected to the carboxyl group of the group A through an amide bond, that is, the amino group of the compound represented by the structural formula IA forms an amide bond with the carboxyl group of A in the structural formula III or IIIA.

[0184] In particular, when "CPT" in the compound of formula IIIA is a compound of formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 may be hydrogen at the same time or may not be hydrogen at the same time. Similarly, R1, R2, R3, and R4 in formula IA may be hydrogen at the same time or may not be hydrogen at the same time. According to a specific embodiment of the present invention, when R6 and R7 are hydrogen at the same time, R1, R2, R3, and R4 in formula IA may not be hydrogen at the same time; when R6 and R7 are not hydrogen at the same time, R1, R2, R3, and R4 in formula IA may be hydrogen at the same time or may not be hydrogen at the same time.

[0185] For example, in structural formula III and structural formula IIIA, CPT is the compound shown in structural formula II or structural formula IIA provided in the eleventh aspect of the present invention above, or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, and the corresponding specific compound.

[0186] When CPT is a compound of formula II provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0187]

[0188] In the structural formula II, the groups G, R5, X, and n are the same as the definitions of the groups G, R5, X, and n in the structural formula II in the eleventh aspect above. And in the structural formula III or the structural formula IIIA, the carboxyl group in the structural formula II and the group A are connected through an amide bond, preferably the amino group adjacent to the group G in the structural formula II is connected to the carboxyl group of the group A through an amide bond, that is, the amino group of the compound represented by the structural formula II forms an amide bond with the carboxyl group of A in the structural formula III or IIIA.

[0189] When CPT is a compound represented by structural formula IIA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof provided in the eleventh aspect of the present invention above:

[0190]

[0191] In the structural formula IIA, the group R5 is the same as the definition of the group R5 in the structural formula IIA in the eleventh aspect above, but can be an n-butyl group. And in the structural formula III or the structural formula IIIA, the structural formula IIA is connected to the carboxyl group in the group A through an amide bond, preferably the amino group on the left benzene ring in the structural formula IIA is connected to the carboxyl group of the group A through an amide bond, that is, the amino group of the compound represented by the structural formula IIA forms an amide bond with the carboxyl group of A in the structural formula III or IIIA.

[0192] Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14-P, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs thereof set forth in the eleventh aspect above can be used as "CPT" in formula III or IIIA, and are linked to the carboxyl group of group A in formula III or IIIA through an amide bond via its amino group.

[0193] For example, in structural formula III and structural formula IIIA, CPT can be an exteacan derivative shown in structural formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0194]

[0195] In formula IV, and in the context of the present invention, unless otherwise specified, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen. And in formula III and formula IIIA, formula IV is connected to the carboxyl group in group A via a self-releasing structure via the hydroxyl group connected to the same carbon as R8, and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in formula III or formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in formula IV.

[0196] When R8 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.

[0197] In particular, when "CPT" in the compound of formula IIIA is a compound of formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 may be hydrogen at the same time or may not be hydrogen at the same time. Similarly, R8 in formula IV may be hydrogen or may not be hydrogen. According to a specific embodiment of the present invention, when R6 and R7 are hydrogen at the same time, R8 may be hydrogen or may not be hydrogen.

[0198] In particular, for the compound shown in structural formula IIIA, when R6 and R7 are Ar'S, M is preferably a phenylene or a substituted phenylene, and SP1 is a C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight chain heteroalkylene, wherein the straight chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S.

[0199] Preferably, the compounds represented by structural formula III and structural formula IIIA provided by the present invention are further represented by structural formula V:

[0200]

[0201] In formula V, R6 and R7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide and cyano. Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl

[0202] In formula V, and in the context of the present invention unless otherwise specified, Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

[0203] In formula V, and in the context of the present invention, unless otherwise specified, m is any integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.

[0204] In structural formula V, A represents a short peptide structure formed by 2-4 amino acids, as defined above.

[0205] In Formula V, the definition of CPT and its linkage in Formula V are the same as CPT in Formula III and Formula IIIA, as defined above.

[0206] Preferably, the compound represented by structural formula V provided by the present invention is further represented by structural formula VA:

[0207]

[0208] In structural formula VA, the groups A, G, Y, R1, R2, R3, R4, X, and n are the same as the definitions of the groups A, G, Y, R1, R2, R3, R4, X, and n in structural formula III or structural formula IIIA above.

[0209] Preferably, in formula VA, G is hydrogen, fluorine or chlorine.

[0210] Preferably, in formula VA, Y is methylene, sulfur or oxygen.

[0211] In formula VA, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0212] Preferably, the compound represented by structural formula VA provided by the present invention is further represented by structural formula VA-1:

[0213]

[0214] Alternatively, the compound represented by structural formula V provided by the present invention is further represented by structural formula VB:

[0215]

[0216] In the structural formula VB, the groups A, G, R5, X, and n are the same as the definitions of the groups A, G, R5, X, and n in the structural formula III or IIIA above.

[0217] In formula VB, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0218] Preferably, the compound represented by structural formula VB provided by the present invention is further represented by structural formula VB-1:

[0219]

[0220] Alternatively, the compound represented by structural formula V provided by the present invention is further represented by structural formula VC:

[0221]

[0222] In the structural formula VC, the groups A and R8 are the same as the definitions of the groups A and R8 in the structural formula III or IIIV above.

[0223] In the structural formula VC, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0224] In structural formula VC, and unless otherwise specified in the context of the present invention, '-' is the same as the self-releasing structure when the CPT in structural formula III or structural formula IIIV above is structural formula IV.

[0225] According to a specific embodiment of the present invention, the compound represented by structural formula V provided by the present invention is further represented by structural formula VI:

[0226]

[0227] In formula VI, A represents a short peptide structure formed by 2-4 amino acids, as defined above.

[0228] In formula VI, CPT is a camptothecin compound. The definition of CPT and its connection relationship in formula VI are the same as those of CPT in formula III and formula IIIA, as defined above.

[0229] Preferably, the compound represented by structural formula VI provided by the present invention is further represented by structural formula VI-A:

[0230]

[0231] In structural formula VI-A, the groups A, G, Y, R1, R2, R3, R4, X, and n have the same definitions as the groups A, G, Y, R1, R2, R3, R4, X, and n in the above structural formula III or structural formula IIIA.

[0232] Preferably, in formula VI-A, G is hydrogen, fluorine or chlorine.

[0233] Preferably, in formula VI-A, Y is methylene, sulfur or oxygen.

[0234] In formula VI-A, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0235] Preferably, the compound represented by structural formula VI-A provided by the present invention is further represented by structural formula VI-A-1:

[0236]

[0237] Alternatively, the compound represented by structural formula VI is further represented by structural formula VI-B:

[0238]

[0239] In structural formula VI-B, the groups A, G, R5, X, and n are the same as the definitions of the groups A, G, R5, X, and n in the above structural formula III or structural formula IIIA.

[0240] In formula VI-B, and unless otherwise specified in the context of the present invention, "-A-NH-" indicates that the amino group in the CPT shown forms an amide bond with the carboxyl group in A.

[0241] More preferably, the compound represented by structural formula VI-B is further represented by structural formula VI-B-1:

[0242]

[0243] Alternatively, the compound represented by structural formula VI provided by the present invention is further represented by structural formula VI-C:

[0244]

[0245] In the structural formula VI-C, the definitions of the groups A and R8 are the same as those of the groups A and R8 in the structural formula III or IIIA above.

[0246] In formula VI-C, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group and the carboxyl group in A form an amide bond.

[0247] When the self-releasing structure in the structural formula VC is When , structural formula VC can be further structured as shown in structural formula VI-C.

[0248] According to a specific embodiment of the present invention, in the thirteenth aspect of the present invention, the compound has the following structure:

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] Aspect 14

[0260] The present invention provides a method for preparing an antibody drug conjugate or a salt thereof, the method comprising the following steps:

[0261] a. Antibody reduction: adding ≥5.5 times the molar equivalent of the antibody to a phosphate buffer containing 5-30 mg / mL antibody, and reacting for 1.5-2 h, wherein the reducing agent is selected from TCEP, DTT, 2-MEA and DTBA;

[0262] b. Antibody coupling and hydrolysis: replace the reduced antibody in step a into a phosphate buffer at pH 6.5-7.8, and dilute the antibody to a concentration of 3.5-15 mg / mL, add 4.5-6.5 times the molar equivalent of the antibody-containing linker dissolved in an organic co-solvent to the antibody dilution, and stir the reaction at 15-35° C. for ≥0.5 h, wherein the organic co-solvent is one or more selected from DMA, DMSO, DMF and ACN; replace the antibody coupling product into a phosphate buffer at pH 7.4-9.0, and heat at 35±10° C. for 2-24 h to obtain a hydrolyzate;

[0263] c. Hydrophobic chromatography: Use hydrophobic filler to purify the antibody-coupled product by hydrophobic chromatography.

[0264] Wherein, in step a, the antibody is an antibody against tumor-associated antigens, such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, guanylate cyclase C, cyclaseC), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, etc.

[0265] Preferably, the antibody is an anti-B7-H3 antibody. More preferably, the antibody is the anti-B7-H3 antibody provided in the first aspect of the present invention. For example, the anti-B7-H3 antibody may be the anti-B7-H3 antibody hz10B4, hz10B4m1, hz10B4m2 or hz10B4m3 provided in the embodiments of the present invention.

[0266] Preferably, in step b, the drug-containing linker is a compound defined in the thirteenth aspect of the present invention.

[0267] Aspect 15

[0268] The antibody drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Therefore, in the fifteenth aspect, the present invention also provides a composition, which comprises the antibody drug conjugate or its salt described in the present invention; preferably, the composition is a pharmaceutical composition, which optionally also comprises a pharmaceutically acceptable carrier, adjuvant or excipient. The pharmaceutical composition provided by the present invention can be prepared into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0269] Aspect 16

[0270] The present invention also provides use of the antibody-drug conjugate or its salt and / or a composition comprising the same in preparing a drug for treating tumors.

[0271] Preferably, the tumor is associated with a tumor-associated antigen such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

[0272] Preferably, the tumor is associated with B7-H3 expression. More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0273] Aspect 17

[0274] Accordingly, the present invention also provides a method for treating tumors, comprising administering the antibody-drug conjugate or its salt and / or a composition comprising the same provided by the present invention to a subject in need thereof.

[0275] Preferably, the tumor is associated with a tumor-associated antigen such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

[0276] Preferably, the tumor is associated with B7-H3 expression (including overexpression). More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0277] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0278] The above-mentioned disease treatment methods provided by the present invention depend on many factors when applied, including the specific active ingredient administered, the patient's age, weight, gender or physical and medical condition, the severity of the condition to be treated, the route of administration, etc.

[0279] Based on a known anti-B7-H3 humanized antibody, the inventors of the present invention screened and obtained a series of novel anti-B7-H3 humanized antibodies by site-directed mutagenesis of the variable region. Compared with the parent antibody, these antibodies retain the high affinity of the parent antibody for B7-H3 while also having significantly higher purity, thereby overcoming the defect of the parent antibody being easily broken and reducing toxic side effects. Furthermore, the inventors of the present invention coupled this series of novel humanized antibodies with camptothecin compounds of specific structures to obtain antibody-drug conjugates, and utilized the stable and robust structure of these antibodies and the high targeting to B7-H3 expressing cells and the strong cytotoxic effect of the camptothecin compounds of specific structures to achieve excellent tumor killing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0280] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0281] Figure 1 The binding activity of the anti-B7H3 antibody of the present invention to B7H3 expressed on the surface of cancer cells is shown.

[0282] Figure 2 The results show the efficacy of the B7H3-targeting antibody-drug conjugate of the present invention in a lung cancer mouse model.

[0283] Figure 3 The results show the efficacy of the B7H3-targeting antibody-drug conjugate and anti-B7H3 antibody of the present invention in a pancreatic cancer mouse model.

[0284] Figure 4 The results of inducing cancer cell apoptosis by the antibody-drug conjugate targeting B7H3 and the anti-B7H3 antibody of the present invention are shown.

[0285] Figure 5 The bystander effect detection results of the antibody drug conjugate targeting B7H3 of the present invention are shown (mean±SD, n=2).

[0286] Figure 6 The results of the test of the ability of the B7H3-targeting antibody-drug conjugate of the present invention to induce B7H3-positive cell cycle arrest are shown (mean±SD, n=2); the results of G0 / G1, G2 / M, and S phases are arranged from top to bottom.

[0287] Best Mode for Carrying Out the Invention

[0288] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0289] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0290] Human B7H3: UniProtKB-Q5ZPR3;

[0291] Control antibody hum30: an antibody targeting B7H3, prepared according to the patent method of US20160368990A1.

[0292] (I) Screening and characterization of novel anti-B7-H3 humanized antibodies

[0293] Example 1 Preparation of anti-B7H3 antibody

[0294] The antibody hz10B4 was prepared according to the method provided in patent application CN202111459992.0 (CN114573695A). The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody hz10B4 are shown below (i.e., SEQ ID NO: 20 and SEQ ID NO: 22 in CN114573695A), and the CDRs are underlined (the division scheme is Kabat).

[0295] >hz10B4-VH(VH / CDR-H1 / CDR-H2 / CDR-H3:SEQ ID NOs:1 / 8 / 9 / 10)

[0296] EVQLVQSGAEVKKPGASVKVSCKASGYTFT EYIMH WVRQAPGQGLEWMG GINPASGGTTYNQKFKG RVTMTRDTSISTAYMELSRLRSEDTAVYYCAR KGKDYFDWYFDV WGQGTTVTVSS

[0297] >hz10B4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:2 / 11 / 12 / 13)

[0298] DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSNPLT FGGGTKVEIK

[0299] Five point mutations were designed for hz10B4 LCDR3, and the VLs obtained after the point mutations were combined with hz10B4-VH, respectively, and a series of anti-B7-H3 antibodies were obtained by referring to the method described in Example 9 of CN114573695A (see Table 1).

[0300] Table 1. Point mutations in hz10B4 LCDR3

[0301] Anti-B7-H3 Antibody VH×VL Point mutations in LCDR3 hz10B4 hz10B4-VH×hz10B4-VL(SEQ ID NOs:1×2) / hz10B4m1 hz10B4-VH×hz10B4-Lm1-VL(SEQ ID NOs:1×3) SSN → SSA hz10B4m2 hz10B4-VH×hz10B4-Lm2-VL(SEQ ID NOs:1×4) SSN → SAS hz10B4m3 hz10B4-VH×hz10B4-Lm3-VL(SEQ ID NOs:1×5) SSN → SSQ hz10B4m4 hz10B4-VH×hz10B4-Lm4-VL(SEQ ID NOs:1×6) SSN → SSG hz10B4m5 hz10B4-VH×hz10B4-Lm5-VL(SEQ ID NOs:1×7) SSN → SGS

[0302] >hz10B4-Lm1-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:3 / 11 / 12 / 14)

[0303] DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSAPLT FGGGTKVEIK

[0304] >hz10B4-Lm2-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:4 / 11 / 12 / 15)

[0305] DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSASPLT FGGGTKVEIK

[0306] >hz10B4-Lm3-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:5 / 11 / 12 / 16)

[0307] DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSQPLT FGGGTKVEIK

[0308] >hz10B4-Lm4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:6 / 11 / 12 / 17)

[0309] DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSGPLT FGGGTKVEIK

[0310] >hz10B4-Lm5-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:7 / 11 / 12 / 18)DIQLTQSPSSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPS

[0311] RFSGSGSGTDFTLTISSLQPEDFATYYC QQWSGSPLT FGGGTKVEIK

[0312] Example 2 Functional evaluation of anti-B7H3 antibodies

[0313] 2.1 Study on the binding affinity of antibodies to human B7H3 ECD-His

[0314] The binding affinity of hz10B4, hz10B4m1-m5 to human B7H3 was determined using Fortebio Octet QKe.

[0315] hz10B4, hz10B4m1-m5 and control antibody hum30 were diluted to 4 μg / ml with PBS buffer and flowed over the surface of AHC probe (Cat: 18-0015, PALL) for 120 s. Human B7-H3 extracellular domain recombinant protein (sequence number: UniProtKB-Q5ZPR3, 1aa-461aa, C-His tag) (60 nM) was used as mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the software was used to fit the 1:1 Langmuir binding mode and calculate the kinetic constants of antigen-antibody binding.

[0316] The results of affinity test of hz10B4 (SEQ ID NOs: 1+2), hz10B4m1 (SEQ ID NOs: 1+3), hz10B4m2 (SEQ ID NOs: 1+4), hz10B4m3 (SEQ ID NOs: 1+5), hz10B4m4 (SEQ ID NOs: 1+6), hz10B4m5 (SEQ ID NOs: 1+7) and hum30 to human B7-H3 extracellular region recombinant protein are shown in Table 2. The results showed that the affinity of hz10B4m1, hz10B4m2 and hz10B4m3 to human B7-H3 extracellular region recombinant protein was similar to that of parent hz10B4, with a slightly decreased Koff, among which the affinity of hz10B4m1 was slightly better than that of hz10B4m2 and hz10B4m3.

[0317] Table 2. Affinity test results of antibodies and human B7-H3 extracellular domain recombinant protein

[0318] Anti-B7H3 Antibody ka(1 / Ms) kd(1 / s) KD(nM) hz10B4 6.65E+05 1.43E-04 2.16E-10 hz10B4m1 6.39E+05 2.25E-04 3.52E-10 hz10B4m2 7.55E+05 2.87E-04 3.81E-10 hz10B4m3 6.66E+05 2.81E-04 4.22E-10 hz10B4m4 5.05E+05 4.20E-04 8.31E-10 hz10B4m5 5.92E+05 3.36E-04 5.68E-10 hum30 9.41E+05 3.24E-04 3.44E-10 NC NA NA NA

[0319] 2.2 Study on the binding activity of antibodies to SKOV3 cell surface antigens

[0320] Using SKOV3 cells naturally expressing human B7H3 as target cells, the binding activity of hz10B4, hz10B4m1-m5 to SKOV3 cell surface antigens was detected by FACS.

[0321] SKOV3 cells naturally expressing human B7H3 grown to the logarithmic phase were collected by centrifugation and the cell density was adjusted to 2×10 5 Pipette 1 ml of cell suspension into EP tubes, i.e. 2×10 5 The cells were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. 200 μL of antibodies hz10B4 and hz10B4m1-m5 (concentration of 198 nM, 3-fold gradient dilution, 12 concentrations) were added to the cells and incubated at 37°C for 30 min. In addition, the following controls were set: (1) positive control (PC): control antibody hum30; (2) negative control (NC-huIgG1): isotype control antibody. After incubation, the cells were washed 3 times with PBS, and 1:200 diluted goat anti-human IgG-FITC (Cat: F9512, Sigma) was added and incubated for 30 min. After washing the cells 3 times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the ability of the test antibodies to bind to B7-H3 on the surface of SKOV3 cells.

[0322] The results of the binding activity test of hz10B4, hz10B4m1, hz10B4m2, hz10B4m3, hz10B4m4, hz10B4m5 and hum30 with human SKOV3 cell surface antigen are shown in Tables 3 and Figure 1 The results showed that the binding activities of hz10B4m1, hz10B4m2 and hz10B4m3 to SKOV3 cell surface antigens were similar to those of the parent hz10B4.

[0323] Table 3. Binding activity of antibodies to SKOV3 cell surface antigens

[0324] Anti-B7H3 Antibody EC50(nM) hz10B4m1 1.893 hz10B4m2 1.449 hz10B4m3 1.589 hz10B4m4 16.9 hz10B4m5 2.247 hum30 1.218 hz10B4 1.585

[0325] Example 3 Purity analysis of anti-B7H3 antibodies

[0326] The purity of anti-B7H3 antibody was determined under reducing and non-reducing conditions using capillary electrophoresis of sodium dodecyl sulfate (CE-SDS). The purity of the antibody product was determined according to General Chapter 3127 of the Chinese Pharmacopoeia, "Determination of Monoclonal Antibody Size Variants (CE-SDS Method)". The specific results are shown in Table 4.

[0327] Table 4. Anti-B7H3 antibody purity analysis results

[0328]

[0329] The results of NR-CE-SDS and R-CE-SDS analysis showed that the purity of hz10B4m1, hz10B4m2, and hz10B4m3 were all above 98%, and were all superior to the parent antibody hz10B4.

[0330] (II) Synthesis of Camptothecin Compounds

[0331] General method for the synthesis of camptothecin compounds:

[0332] The camptothecin compounds of the present invention can be obtained by reacting the compound represented by formula A with a tricyclic compound via Friedlander reaction. The general reaction formula is as follows:

[0333]

[0334] Among them, CDE tricyclic compound was purchased from MCE:

[0335]

[0336] The HCDE tricyclic compound was prepared according to the method of Bioorganic and Medicinal Chemistry, 2010, vol. 18, #9, p. 3140-3146:

[0337]

[0338] 1. Synthesis of the compound shown in formula A

[0339] Example 4 Synthesis of Compound A1

[0340]

[0341] Method 1: Compound A1 was synthesized according to the method of patent publication document WO2020200880A1. The synthesis route is as follows:

[0342]

[0343] Method 2: Compound A1 was synthesized by zinc reagent palladium catalyzed coupling method. The synthetic route is as follows:

[0344]

[0345] (1) Acetylation

[0346] 3-Bromo-4-nitroaniline (25.00 g, 0.12 mol) and 200 mL of acetic acid were added to a 500 mL flask, and 50 mL of acetic anhydride was slowly added dropwise, and the reaction was carried out at room temperature for 16 hours. After TLC detected that the raw material reaction was complete, the reaction solution was filtered, the reaction solution was concentrated under reduced pressure to remove acetic acid, the combined filter cake was pulped with 200 mL of MTBE, and 27.6 g of the dried yellow solid (A1-a) was filtered. The yield was 92%. LC-MS (ESI): m / z 259 (M+H) +.

[0347] (2) Preparation of 4-ethoxy-4-oxobutylzinc bromide

[0348] In a 250mL three-necked flask (thermometer, reflux condenser, rubber stopper), activated zinc powder (19.0g, 0.29mol, 2.00eq) was added, and anhydrous DMF (145mL) was added after replacing nitrogen. The nitrogen was replaced again, and iodine (1.86g, 0.015mol, 0.1eq) was added at room temperature. It can be observed that the solution changes from colorless to brown-red, then gradually changes to light yellow and finally returns to colorless (2-3min), and then ethyl 4-bromobutyrate (28.6g, 0.15mol, 1.00eq) is added, and the temperature is raised to 80℃ (internal temperature) for 4-5 hours. After the reaction is complete by TLC detection, the reaction solution is allowed to stand and cooled to room temperature for use. The supernatant is light yellow with a concentration of 1mol / L.

[0349] (3) Zinc reagent coupling

[0350] Anhydrous DMF (120 mL) and intermediate A1-a (25.0 g, 1.00 eq) were added to a 500 mL reaction bottle, and palladium acetate (433 mg, 0.02 eq) was added after replacing nitrogen. After stirring at room temperature for 10 min, S-PHOS (1.6 g, 0.04 eq) was added, and nitrogen was replaced again. After stirring for 20 min, 4-ethoxy-4-oxobutyl zinc bromide (145 mL, 1.50 eq) prepared in the above step was added dropwise at room temperature (25-30 ° C). After the addition, the temperature was kept at 25-30 ° C for 16 hours. After the reaction of the raw materials was complete by TLC detection (DCM: EA = 5: 1), the reaction solution was cooled to room temperature, and ammonium chloride solution (15 mL) was added to the reaction solution to quench the reaction. The reaction solution was poured into 1L of water, 400mL of ethyl acetate was added, the solution was separated, and the solution was filtered through a Buchner funnel. The aqueous phase was extracted with ethyl acetate (400mL*2), the organic phase was washed twice with water (500mL), washed once with a saturated sodium chloride solution (500mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 37.0g of a reddish brown oily liquid (A1-b), with a yield of 130%. The crude product was used directly in the next step without further purification. LC-MS (ESI): [M+1]+=295.

[0351] (4) Synthesis of Intermediate A1-c

[0352] Add intermediate A1-b (37.0 g, 1.0 eq) and ethanol (1.5 L) to a 3L three-necked reaction flask, and the raw materials are completely dissolved. The reaction system is cooled to 5°C, 5% Pd / C (5.7 g, 1.0 eq) is added, and the temperature is raised to 25°C under a hydrogen atmosphere (normal pressure) for 16 hours. After TLC detection (DCM:EA=5:1), the reaction is complete, the reaction system is filtered under diatomaceous earth, and the organic phase is concentrated to obtain 33.0 g of the crude product (A1-c), with a yield of 130%. The crude product is used directly in the next step without further purification. LC-MS (ESI): [M+1]+=265.

[0353] (5) Synthesis of Intermediate A1-d

[0354] Add intermediate A1-c (33.0g, 1.00eq) and 260mL acetic acid to a 1L flask, slowly add 46mL acetic anhydride, and react at room temperature for 2 hours. After TLC detection (DCM: MeOH = 10: 1) the raw material reaction is complete, the reaction solution is filtered, the reaction solution is concentrated under reduced pressure to remove acetic acid, 250mL water is added, the aqueous phase is extracted with ethyl acetate (400mL*2), the organic phase is washed twice with water (500mL), washed once with saturated sodium chloride solution (500mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 32g of crude product. The crude product is slurried with 400mL MTBE, and the dried light yellow solid (A1-d) 27.0g is filtered, with a yield of 91%. The total yield of the four-step reaction is 83.7%.

[0355] LC-MS (ESI): [M+1]+=307.

[0356] 1H NMR (400MHz, DMSO) δ9.87(s,1H),9.19(s,1H),7.41(s,1H),7.37(d,J=8.7Hz,1H),7.20(d,J=8.5Hz,1H),4.06(q, J=7.0Hz,2H),2.52(d,J=8.5Hz,2H),2.29(t,J=7.3Hz,2H),2.02(s,6H),1.79–1.64(m,2H),1.18(t,J=7.1Hz,3H).

[0357] (6) Synthesis of Intermediate A1-e

[0358] Add intermediate A1-d (27.0 g, 88 mmol, 1.0 eq), water (100 mL) and tetrahydrofuran (200 mL) to a 500 mL three-necked reaction flask, and the raw materials are completely dissolved. Add lithium hydroxide monohydrate (18.5 g, 441 mmol, 5.0 eq) at room temperature, and maintain the reaction system at room temperature for 3 hours. TLC detection shows that the reaction is complete, and most of the tetrahydrofuran is evaporated under reduced pressure. Add 300 mL of water to the residue, extract the aqueous phase with EA (2×100 mL), and discard the organic phase. Adjust the pH of the aqueous phase to 4 with 6N hydrochloric acid under ice bath, and solid precipitates. Filter to obtain a white solid (intermediate A1-e), 19.1 g, with a yield of 78%.

[0359] 1H NMR (400MHz, DMSO) δ12.09(s,1H),9.86(s,1H),9.18(s,1H),7.38(d,J=15.5Hz,2H),7.23(d ,J=8.5Hz,1H),2.51(d,J=8.1Hz,2H),2.23(t,J=7.1Hz,2H),2.02(s,6H),1.76–1.61(m,2H)

[0360] (7) Synthesis of Compound A1

[0361] Add 40ml of polyphosphoric acid to a 250mL reaction bottle, heat to 90°C, add intermediate A1-5 (5.0g, 17.97mmol) in batches, and keep the internal temperature at 95-100°C for 5 hours. After the reaction is complete by TLC detection, remove the heating, cool the system to 50-60°C, and add 15mL of 4M HCl (aq) (the temperature will rise to 100°C) to the reaction system to quench the reaction. After the dropwise addition, add 600mL of 4M NaOH aqueous solvent under ice bath to adjust the pH to 10. The aqueous phase is extracted with ethyl acetate (50mL*3), the organic phases are combined, washed once with saturated sodium chloride solution (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 3.75g of a yellow solid (intermediate A1-f), with a yield of 80.5%. Directly react in the next step without purification. LC-MS (ESI): [M+1]+=261.

[0362] The raw material intermediate A1-f (3.75 g) was suspended in 19% hydrochloric acid (30 mL) in a 250 mL three-necked reaction bottle. The reaction system was heated to 90 ° C (internal temperature) for 3 hours. TLC detected that the raw material reaction was complete, and the temperature was cooled to below 5 ° C in an ice-salt bath. 4M NaOH solution (45 mL, 30 eq) was added dropwise to adjust the pH to 10. The aqueous phase was extracted with ethyl acetate (80 mL * 5), and the organic phases were combined and washed once with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.45 g of crude product. The crude product was purified by column chromatography, and the eluent was DCM to obtain a yellow solid (Compound A1), 1.93 g, with a yield of 76%. The total yield of the two-step reaction was 61.2%.

[0363] LC-MS (ESI): [M+1]+=177.

[0364] 1H NMR(400MHz,DMSO)δ6.76(d,J=8.7Hz,1H),6.68(s,2H),6.42(d,J=8.7Hz,1H) ,4.17(s,2H),2.55(t,J=5.9Hz,2H),2.46(t,J=6.2Hz,2H),2.00–1.80(m,2H)

[0365] Method 3: Compound A1 was prepared by lactam hydrolysis. The synthetic route is as follows:

[0366]

[0367] The aminolactam compound is prepared according to the method of patent publication document CN106349233A.

[0368]

[0369] In a 500ml three-necked flask, aminolactam compound (17.6g, 0.1moml), ethanol (250ml), 98% sulfuric acid (5ml) were mixed and heated under reflux for 24 hours. Samples were taken for testing. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. The residue was added with dichloromethane (200ml) and water (100ml), cooled to below 10°C in an ice bath, adjusted to pH 7-8 with 1N sodium hydroxide aqueous solution, stirred and separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to remove the organic solvent to obtain a crude diaminoethyl ester intermediate, 25g, which was directly reacted in the next step.

[0370] The diaminoethyl ester intermediate was dissolved in dichloromethane (200ml), triethylamine (20.2g, 0.2mol, 2eq) was added, the mixture was cooled to below 10°C in an ice bath, and acetic anhydride (25.5g, 0.25mol, 2.5eq) was added dropwise. After the addition, the mixture was kept warm for 1 hour, and samples were taken for testing. After the reaction was complete, the reaction solution was poured into 1N hydrochloric acid on ice, stirred for 15 minutes, separated, and the aqueous phase was extracted with dichloromethane (50ml*3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to remove the organic solvent. The crude product was slurried with 400mL MTBE, filtered and dried to obtain the intermediate A1-e as a light yellow solid, 26.9g. The total yield of the two-step reaction was 87.8%. LC-MS (ESI): m / z 307 (M+H) +.

[0371] According to method 2, compound A1 is prepared from intermediate A1-e.

[0372] Example 5 Synthesis of Compound A2

[0373]

[0374] According to the method of patent publication WO2021148501, compound A2 was synthesized, and the synthesis route is as follows:

[0375]

[0376] Example 6 Synthesis of Compound A3

[0377]

[0378] According to the method of patent publication document US2004266803A, compound A3 was synthesized.

[0379] Example 7 Synthesis of Compound A4

[0380] Compound A4 was synthesized according to a method similar to that of Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0381]

[0382] Step 1:

[0383] Intermediate A4-2 was prepared from compound A4-1 according to the literature method.

[0384] Step 2:

[0385] At -5 to 5°C, the intermediate A4-2 (3.4 g) was dissolved in dichloromethane, triethylamine (3.0 g) was added, and AllocCl (2.8 g) was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted for 1 to 2 hours. Water was added to quench the mixture, and the mixture was washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 5.5 g of crude intermediate A4-3. LC-MS (ESI): [M+1]+=255.7.

[0386] Step 3:

[0387] At room temperature, the intermediate A4-3 (5.5 g) was mixed with TBAF (55 ml) and acrylic acid (110 ml), heated to 50-55°C and stirred for 24 hours; the reaction mixture was directly concentrated to dryness, column chromatography, and petroleum ether was eluted to methanol: dichloromethane = 1:50 to obtain about 5.2 g of the crude intermediate A4-4. LC-MS (ESI): [M+1]+ = 327.4.

[0388] Step 4:

[0389] At room temperature, the intermediate A4-4 (5.0 g) was mixed in 100 ml of a mixed solution of ethanol: water = 3:1, and ammonium chloride (5.5 g) and iron powder (5.5 g) were added, and heated to reflux for 1 to 2 hours; the reaction was also cooled to room temperature, filtered, and the solid was washed with ethanol and concentrated to dryness to obtain about 3.8 g of the crude intermediate A4-5, LC-MS (ESI): [M+1]+ = 297.4.

[0390] Step 5:

[0391] At 20-30°C, mix intermediate A4-5 (3.8 g) in trifluoroacetic acid (38 ml), add trifluoroacetic anhydride (38 ml), and stir to react for 18-24 hours; post-treatment: directly concentrate to dryness, column chromatography, elute with petroleum ether to ethyl acetate: petroleum ether = 1:4 to obtain intermediate A4-6: 1.0 g; LC-MS (ESI): [M+1]+ = 375.3.

[0392] Step 6:

[0393] At room temperature, the A4-6 intermediate (1.0 g) was mixed with methanol (20 ml), potassium carbonate (2.0 g) and water (5 ml) were added, and the mixture was stirred for 1 to 2 hours; water was added to dilute the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a crude product. The crude product was eluted with petroleum ether by column chromatography until ethyl acetate: petroleum ether = 1:2 to obtain the A4-7 intermediate: 0.65 g, LC-MS (ESI): [M+1]+ = 279.3.

[0394] Step 7:

[0395] At room temperature, the intermediate A4-7 (500 mg) was dissolved in tetrahydrofuran (20 ml). Under nitrogen protection, pyrrole (120 mg) and tetrakistriphenylphosphine palladium (160 mg) were added, and the mixture was stirred for 1 to 2 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with dichloromethane: methanol = 30:1 to obtain A4 as a brown solid, 30 mg; LC-MS (ESI): [M+1]+ = 195.4.

[0396] Example 8 Synthesis of Compound A5

[0397] Compound A5 was synthesized according to a method similar to that of Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0398]

[0399] Step 1:

[0400] At 20-30°C, mix compound A5-1 (25 g) in acetic acid (100 ml), slowly add acetic anhydride (24.9 g), and stir for 3-4 hours after the addition is completed; slowly transfer the reaction solution into ice water, stir to precipitate solid, filter, collect the solid, wash with water, and vacuum dry to obtain intermediate A5-2 as a yellow solid, 31.0 g, LC-MS (ESI): [M+1]+=197.2.

[0401] Step 2:

[0402] At 20-30°C, A5-2 intermediate (29 g), potassium carbonate (40 g), potassium iodide (5 g) and bromopropanol (25 g) were mixed in DMF (300 ml), heated to 100-110°C, and stirred for 3-4 hours; the reaction solution was cooled to room temperature, quenched in ice water, extracted three times with 2 L of ethyl acetate, combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to dryness, slurried with petroleum ether to obtain a solid, and dried in vacuo to obtain A5-3 intermediate as a yellow solid: 33.0 g, LC-MS (ESI): [M+1]+=255.3.

[0403] Step 3:

[0404] At 20-25°C, the A5-3 intermediate (23 g) was dissolved in acetonitrile (1 L), and sodium dihydrogen phosphate solution (0.67 mol, pH 6.7, 900 ml), TEMPO (5 g), sodium chlorite solution (52.0 g dissolved in 60 ml water) and sodium hypochlorite solution (38 ml mixed with 38 ml water) were added in sequence. After the addition was completed, the reaction solution turned dark brown and was stirred for 30 minutes. The reaction solution was cooled to room temperature, the pH was adjusted to 2-3 with 2N hydrochloric acid, and extracted with ethyl acetate (1000 ml×3). The ethyl acetate layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and slurried with petroleum ether to obtain 24.0 g of crude intermediate A5-4, LC-MS (ESI): [M+1]+=269.2.

[0405] Step 4:

[0406] At room temperature, the intermediate A5-4 (25 g) and 5% palladium on carbon (5 g) were mixed in methanol (500 ml) and subjected to pressure hydrogenation reaction for 3 to 4 hours; the palladium on carbon was filtered off, the solid was washed with methanol, the filtrate was concentrated, the residue was slurried with petroleum ether to obtain a crude product, and vacuum dried to obtain the intermediate A5-5 as a yellow solid: 18.0 g, LC-MS (ESI): [M+1]+=239.5.

[0407] According to the method similar to step 5 and step 6 in the synthesis of compound A4, intermediate A5-7 was synthesized from intermediate A5-5, and finally the acetyl protection was removed with 6N hydrochloric acid to prepare compound A5 as a yellow solid, LC-MS (ESI): [M+1]+=179.2.

[0408] Example 9 Synthesis of Compound A6

[0409] Compound A6 was synthesized according to the method of Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthesis route is as follows:

[0410]

[0411] Compound A6 is a yellow solid, LC-MS (ESI): [M+1]+=248.9.

[0412] 1 H NMR (400MHz, d6-DMSO): δ12.289 (1H, s), 8.666-8.648 (1H, d), 8.198-8.1 (1H, d ), 3.138-3.108(2H, t), 2.768-2.734(2H, t), 2.219(3H, s), 2.050-1.991(2H, m)

[0413] Step 1:

[0414] In a 250ml three-necked flask, under nitrogen protection, compound A1-4 (1.25g, 5mmol, 1eq) was dissolved in 150ml tetrahydrofuran, cooled to -60℃, and LDA (2M in THF, 7.6ml, 15.2mmol, 3.04eq) was added. After the addition, the mixture was stirred at -60℃ for 30 minutes. MeI (1.45g, 10.21mmol, 2.04eq) was added. After the addition, the dry ice bath was removed, the temperature was naturally raised, and the reaction was allowed to proceed overnight at room temperature. 50ml of aqueous ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (150ml*3). The organic phases were combined, washed once with water, and the organic phases were concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography, and the eluent was petroleum ether:ethyl acetate = 1:0-5:1 to obtain compound A6-1 as a yellow solid, 350mg, with a yield of 28%.

[0415] 1 H NMR (CDCl3): δ12.49 (1H, s), 8.77 (1H, d), 8.03 (1H, d), 3.21 (2H, m), 2.22 (3H, s), 1.90 (2H, t), 1.20 (6H, s)

[0416] Step 2:

[0417] In a 100ml single-necked flask, compound A6-1 (280mg) was mixed with 20ml 6 N hydrochloric acid, heated under reflux and stirred for 2 hours; heating was stopped, and after the reaction solution was cooled to room temperature, the pH of the reaction solution was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined and concentrated to dryness to obtain compound A6-2, a brown-grey solid, 275mg. The crude product was directly used for the next step without purification.

[0418] Step 3:

[0419] In a 50ml three-necked flask, dissolve the A6-2 compound (220mg) in acetic acid (25ml), add 1.3g iron powder, stir at 80-85℃ for 1-2 hours after the reaction is complete, remove the acetic acid by vacuum distillation, add water (30ml) to the residue, adjust the pH to 7-8 with sodium bicarbonate, extract with dichloromethane (30ml*3), concentrate to dryness to obtain a crude product, purify the crude product by column chromatography, eluent is petroleum ether: ethyl acetate = 1:0-2:1, obtain A6 compound, brown solid, 122mg, two-step reaction yield 75%. LC-MS (ESI): [M+1]+ = 205.0

[0420] Example 10 Synthesis of Compound A7

[0421] The synthetic route is as follows:

[0422]

[0423] In a 250 ml three-necked flask, compound A1 (2.18 g, 124 mmol, 1 eq) was dissolved in tetrahydrofuran (100 ml), and Boc anhydride (8.2 g, 376 mmol, 3 eq) was added. The mixture was stirred at 40-45 °C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether: ethyl acetate = 1:0-1:3 as the eluent to obtain compound A7-1 as a yellow solid (3.0 g). The yield was 90%.

[0424] 1H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J=6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

[0425] In a 250ml three-necked flask, A7-1 compound (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in dichloromethane (125ml), and the reaction solution was cooled to -5~0°C with an ice-salt bath, and acetyl chloride (1.3g, 16.5mmol, 2eq) was added dropwise. After the addition was completed, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours; the reaction solution was concentrated to dryness, and the residue was purified by column chromatography with petroleum ether: ethyl acetate = 1:0~1:3 as the eluent to obtain A7-2 compound as a light yellow solid, 3.48g, with a yield of 100%.

[0426] 1H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J=9.1Hz,1H),7.57(t,J=36.2Hz,1H),6.09(s, 1H),2.80(t,J=6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

[0427] In a 500 ml three-necked flask, under nitrogen protection, compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C with a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. q) was dissolved in tetrahydrofuran (70 ml), and after the addition, the mixture was stirred at -70 to -60 °C for 10 minutes, and the temperature was naturally raised to 20 to 30 °C and stirred for 3 hours; a saturated aqueous solution of ammonium chloride (80 ml) was added to quench the reaction, and the reaction solution was extracted with ethyl acetate (150 ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether:ethyl acetate = 1:0-1:3 to obtain compound A7-3 as a yellow solid, 0.85 g, with a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0428] 1H NMR (400MHz, CDCl3) δ11.49(s,1H),8.64(d,J=9.2Hz,1H),7.74(d,J=8.5Hz,1H) ,6.06(s,1H),3.01(t,J=6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

[0429] In a 100ml single-necked flask, compound A7-3 (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. The heating was stopped, and after the reaction solution was cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound A7, a brown solid of 0.25g, with a yield of 49.1%. LC-MS (ESI): [M+1]+ = 213.9

[0430] Embodiment 11 Synthesis of Compound A8

[0431] The synthetic route is as follows:

[0432]

[0433] Compound A8-1 was synthesized according to the method of Journal of Medicinal Chemistry, 1989, vol. 32, #6, p. 1217-1230. LC-MS: (ESI): [M+1]+=146.3.

[0434] Compound A8-1 (1.8 g) and DIEA (4.8 g) were added to DCM (50 ml). The mixture was cooled to 0°C. Trifluoroacetic anhydride (4.8 g) was added, the mixture was warmed to room temperature, and the reaction was continued for 1 hour. Water (20 ml) was added, the organic layer was separated, and the mixture was dried by spin drying. The intermediate A8-2 was purified by column chromatography (EA:PE = 0-30%) to obtain a white solid (2.7 g). LC-MS: (ESI): [M+1]+ = 242.2.

[0435] Zn(Et)2 (33.6 ml) was added to DCM (70 ml) and cooled to 0°C. TFA (3.8 g) and CH2I2 (9.0 g) were added. The mixture was reacted for 15 minutes. A solution of compound A8-2 (2.7 g) in DCM (30 ml) was added dropwise. After the addition, the mixture was stirred at room temperature overnight. Water (30 ml) was added, the organic layer was separated, dried, and spin-dried to obtain intermediate A8-3 as an off-white solid, 2.7 g. LC-MS (ESI): [M+1]+=256.1.

[0436] A8-3 intermediate (1.5 g) was added to DCM (100 ml) and acetic anhydride (30 ml), cooled to 0°C, 65% nitric acid (2.8 g) was added, and the mixture was reacted at room temperature overnight. Water (50 ml) and DCM (100 ml) were added. The organic layer was separated and spin-dried. The crude intermediate A8-4 (1.6 g) was obtained by oil pump drying and used directly in the next step. LC-MS (ESI): [M+1]+=301.2.

[0437] A8-4 intermediate (1.6 g crude product), iron powder (3.2 g), and ammonium chloride (6.4 g) were added to anhydrous ethanol (100 ml), and the mixture was heated to reflux overnight. The mixture was cooled, filtered, dried, and purified by column chromatography (EA:PE = 0-50%) to obtain A8-5 intermediate as a brown oil, 0.25 g. LC-MS (ESI): [M+1]+ = 271.3.

[0438] A8-5 intermediate (250 mg) and DIEA (400 mg) were added to DCM (10 ml), cooled to 0°C, trifluoroacetic anhydride (320 mg) was added, and the mixture was reacted at room temperature for 1 hour. Water (5 ml) was added for washing. The organic layer was separated, dried, filtered, and dried to obtain A8-6 intermediate as a yellow solid, 340 mg. LC-MS (ESI): [M+1]+=367.3.

[0439] A8-6 intermediate (150 mg) and CrO3 (400 mg) were added to acetic acid (8 ml) and acetic anhydride (4 ml), and reacted at 30-40°C for 3 hours. The solvent was dried by spin drying. Water (5 ml) was added and extracted with DCM (20 ml). The organic layer was separated, dried, filtered, and spin dried to obtain 160 mg of crude intermediate A8-7, which was used directly in the next step. LC-MS (ESI): [M+1]+=381.3.

[0440] The intermediate A8-7 was deprotected from the bis-trifluoroacetyl group by 6N hydrochloric acid and purified by column chromatography to obtain compound A8. LC-MS (ESI): [M+1]+=189.3.

[0441] The following compounds can be synthesized according to the above similar method

[0442]

[0443]

[0444]

[0445]

[0446] 2. Synthesis of Camptothecin Compounds (Friedlander Reaction) Synthesis of Control Camptothecin Compound MWC-1:

[0447]

[0448] At room temperature, compound A1 (176 mg, 1 eq) and CDE ring compound (315 mg, 1.2 eq) and PPTS (301 mg, 1.2 eq) were mixed in toluene (50 ml), and refluxed to separate water for 3 to 4 hours; the reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether as the eluent and eluted to methanol / dichloromethane = 1:20 to obtain MWC-1 as a yellow solid, 260 mg, LC-MS (ESI): [M+1]+ = 404.

[0449] Example 12 Synthesis of Camptothecin Compound 1

[0450] The synthetic route is as follows:

[0451]

[0452] In a 50 ml single-mouth reaction bottle, under nitrogen protection, compound A6 (120 mg, 0.59 mmol, 1 eq), CDE tricyclic compound (180 mg, 0.68 mmol, 1.2 eq) and PPTS (35 mg, 0.14 mmol, 0.25 eq) were mixed in 15 ml and heated under reflux for 3 to 4 hours; most of the acetic acid was distilled off under reduced pressure, and the residue was crudely separated by column chromatography with an eluent of dichloromethane: methanol = 15:1 to obtain a crude product of 80 mg, which was then separated and purified by preparative liquid phase separation to obtain camptothecin compound 1 as a yellow solid, 3 mg.

[0453] LC-MS (ESI): [M+1]+=432.4.

[0454] Example 13 Synthesis of Camptothecin Compounds 2 (2A and 2B)

[0455] The synthetic route is as follows:

[0456]

[0457] The product was synthesized by a method similar to Example 12, using A9 instead of A6. The product was separated and purified by preparative liquid phase to obtain compound 2A (early retention time), a yellow solid, LC-MS (ESI): [M+1]+=418.3; compound 2B (late retention time), a yellow solid, LC-MS (ESI): [M+1]+=418.4.

[0458] Embodiment 14 Synthesis of Camptothecin Compound 3

[0459] The synthetic route is as follows:

[0460]

[0461] The camptothecin compound 3 was prepared in a similar manner to Example 12, using A7 instead of A6 and toluene instead of acetic acid as solvent, as a yellowish brown solid. LC-MS (ESI): [M+1]+=440.8

[0462] 1H NMR (400MHz, Acetone) δ7.89(d,J=9.1Hz,1H),7.48(d,J=9.1Hz,1H),7.37(s,1H),7.31(d,J=8.9Hz,0H), 5.59–5.26(m,5H),3.11–3.05(m,4H),2.73–2.59(m,3H),1.98(dt,J=14.0,7.0Hz,1H),1.05–0.98(m,3H)

[0463] 1H NMR (400MHz, DMSO) δ7.87(d,J=9.1Hz,0H),7.41(d,J=9.1Hz,0H),7.21(s,0H),6.50(s,0H),6.15(s,1H),5.36(d ,J=46.6Hz,1H),2.93(t,J=6.5Hz,1H),2.66–2.54(m,1H),1.97–1.68(m,3H),1.24(s,1H),0.88(t,J=7.3Hz,3H)

[0464] Embodiment 15 Synthesis of Camptothecin Compounds 4 (4A and 4B)

[0465]

[0466] According to the similar synthesis method of Example 12, A10 was used instead of A6 to prepare the product, and the product was separated and purified by preparative liquid phase to obtain compound 4A (early retention time), which was a khaki solid, LC-MS (ESI): [M+1]+=422.3; compound 4B (late retention time), which was a khaki solid, LC-MS (ESI): [M+1]+=422.3.

[0467] Example 16 Synthesis of Camptothecin Compound 5

[0468] The synthetic route is as follows:

[0469]

[0470] According to the similar method of Example 12, camptothecin compound 5 was prepared as a yellow solid by using A12 instead of A6. LC-MS (ESI): [M+1]+=444.3.

[0471] Embodiment 17 Synthesis of Camptothecin Compound 6

[0472] The synthetic route is as follows:

[0473]

[0474] The camptothecin compound 6 was synthesized by a similar method to Example 12, using A11 instead of A6, as a yellow solid. LC-MS (ESI): [M+1]+=430.5.

[0475] Embodiment 18 Synthesis of Camptothecin Compound 7

[0476] The synthetic route is as follows:

[0477]

[0478] According to the similar synthesis method of Example 12, A8 was used instead of A6 to obtain compound 7 as a yellow solid, 0.7 mg. LC-MS (ESI): [M+1]+=416.3.

[0479] Embodiment 19 Synthesis of Camptothecin Compound 9

[0480] The synthetic route is as follows:

[0481]

[0482] According to the similar synthesis method of Example 12, A3 was used instead of A6 to obtain camptothecin compound 9 as a brown-red solid. LC-MS (ESI): [M+1]+=420.3.

[0483] Embodiment 20 Synthesis of Camptothecin Compound 12

[0484] Compound 12 was synthesized according to a method similar to that of Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0485]

[0486] According to the similar synthesis method of Example 12, using A4 instead of A6 and using toluene instead of acetic acid as solvent, compound 12 was synthesized as a brown solid, 30 mg; LC-MS (ESI): [M+1]+=422.5.

[0487] Embodiment 21 Synthesis of Camptothecin Compound 13

[0488] The synthetic route is as follows:

[0489]

[0490] According to the synthetic method similar to Example 12, A15 was used instead of A6, and toluene was used as solvent to synthesize compound 13, which was a brown solid. LC-MS (ESI): [M+1]+=458.4.

[0491] Embodiment 22 Synthesis of Camptothecin Compound 14

[0492] The synthetic route is as follows:

[0493]

[0494] According to the synthetic method similar to Example 12, using A5 instead of A6 and using toluene as solvent, compound 14 was prepared as a yellow solid. LC-MS (ESI): [M+1]+=406.1.

[0495] Embodiment 23 Synthesis of Camptothecin Compound 14-P

[0496]

[0497] At room temperature, compound 12 (200 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml), cooled to 5°C under nitrogen protection, 30% H2O2 (580 mg) was added, stirred and reacted for 2 hours, the reaction solution was concentrated to dryness, and HPLC was used to prepare liquid phase separation to obtain compounds 14-P1 and 14-P2. Freeze-dried to obtain 42 mg of brown solid compound 14-P1 and 25 mg of brown solid compound 14-P2; LC-MS (ESI): [M+1]+=438.5.

[0498] Embodiment 24 Synthesis of Camptothecin Compound 15

[0499]

[0500] At room temperature, compound 12 (100 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml), cooled to 5°C under nitrogen protection, 30% H2O2 (750 mg) was added, stirred and reacted for 2 hours, returned to room temperature 25°C and stirred and reacted overnight, and LCMS analysis showed that the reaction was complete, the reaction solution was concentrated to dryness, separated by HPLC preparative liquid phase, collected, and freeze-dried to obtain compound 15 as a brown solid 8 mg. LC-MS (ESI): [M+1]+=454.4.

[0501] Embodiment 25 Synthesis of Camptothecin Compound 16

[0502]

[0503] Intermediate 16a was synthesized according to the patent method of WO2020200880A1, and then 16a-thiocamptothecin structure intermediate 16c was prepared according to the method of the literature (J.Med.Chem.2008,51,3040-3044). Finally, the acetyl protection was removed to obtain compound 16, LC-MS (ESI): [M+1]+=420.3.

[0504] Embodiment 26 Synthesis of Camptothecin Compound 17

[0505] The synthetic route is as follows:

[0506]

[0507] According to the synthesis method similar to Example 12, A1 was used instead of A6, HCDE was used instead of CDE, and toluene was used as solvent to obtain homocamptothecin compound 17 (S, R mixture), LC-MS (ESI): [M+1]+=418.5.

[0508] Embodiment 27 Synthesis of Camptothecin Compound 18

[0509] The synthetic route is as follows:

[0510]

[0511] According to the synthetic method similar to Example 12, A16 was used instead of A6, and toluene was used as solvent to synthesize compound 18, which was a light yellow solid. LC-MS (ESI): [M+1]+=424.2.

[0512] Embodiment 28 Synthesis of Camptothecin Compound 19

[0513] The synthetic route is as follows:

[0514]

[0515] According to the synthetic method similar to Example 12, A17 was used instead of A6, and toluene was used as solvent to synthesize compound 19 as a yellow solid. LC-MS (ESI): [M+1]+=460.5.

[0516] Embodiment 29 Synthesis of Camptothecin Compound 20

[0517] The synthetic route is as follows:

[0518]

[0519] According to the synthetic method similar to Example 12, A13 was used instead of A6, and toluene was used as solvent to synthesize compound 20 as a yellow solid. LC-MS (ESI): [M+1]+=458.2.

[0520] Embodiment 30 Synthesis of Camptothecin Compound 21

[0521] The synthetic route is as follows:

[0522]

[0523] According to the synthetic method similar to Example 12, using A24 instead of A6 and using toluene as solvent, compound 21 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=418.4.

[0524] Embodiment 31 Synthesis of Camptothecin Compound 22

[0525] The synthetic route is as follows:

[0526]

[0527] According to the synthetic method similar to Example 12, using A25 instead of A6 and using toluene as solvent, compound 22 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=434.4.

[0528] Embodiment 32 Synthesis of Camptothecin Compound 23

[0529] The synthetic route is as follows:

[0530]

[0531] According to the synthetic method similar to Example 12, using A21 instead of A6 and using toluene as solvent, compound 23 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=432.4.

[0532] Embodiment 33 Synthesis of Camptothecin Compound 24

[0533] The synthetic route is as follows:

[0534]

[0535] According to the synthetic method similar to Example 12, using A20 instead of A6 and using toluene as solvent, compound 24 (a pair of isomers) was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=418.4.

[0536] Embodiment 34Synthesis of Camptothecin Compound 25

[0537] The synthetic route is as follows:

[0538]

[0539] According to the synthetic method similar to Example 12, using A32 instead of A6 and using toluene as solvent, compound 25 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=432.4.

[0540] Embodiment 35 Synthesis of Camptothecin Compound 26

[0541]

[0542] According to the synthetic method similar to Example 12, A18 was used instead of A6, and toluene was used as solvent to obtain compound 26 as a brown-red solid. LC-MS (ESI): [M+1]+=440.4.

[0543] Embodiment 36 Synthesis of Camptothecin Compound 27

[0544]

[0545] According to the synthetic method similar to Example 12, using A19 instead of A6 and using toluene as solvent, compound 27 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=476.4.

[0546] Embodiment 37 Synthesis of Camptothecin Compound 31

[0547]

[0548] According to the synthetic method similar to Example 12, using A14 instead of A6 and using toluene as solvent, compound 31 was obtained as a yellow solid. LC-MS (ESI): [M+1]+=442.4.

[0549] Embodiment 38 Synthesis of Camptothecin Compound 32

[0550]

[0551] According to the synthetic method similar to Example 12, A2 was used instead of A6, HCDE was used instead of CDE, and toluene was used as solvent to synthesize compound 32 as a yellow solid. LC-MS (ESI): [M+1]+=436.4.

[0552] Embodiment 39 Synthesis of Camptothecin Compound 33

[0553] The synthetic route is as follows:

[0554]

[0555] According to the synthesis method similar to Example 12, A23 was used instead of A6, and toluene was used as solvent. Compound 33 was a yellow solid. LC-MS (ESI): [M+1]+=458.5.

[0556] Embodiment 40 Synthesis of Camptothecin Compound 34

[0557] The synthetic route is as follows:

[0558]

[0559] According to the synthetic method similar to Example 12, using A22 instead of A6 and using toluene as solvent, compound 34 was synthesized as a yellow solid, LC-MS (ESI): [M+1]+=458.5.

[0560] Embodiment 41 Synthesis of Camptothecin Compounds 35 and 38

[0561] The synthetic route is as follows:

[0562]

[0563] According to the synthetic method similar to Example 12, using A26 instead of A6 and toluene as solvent, compound 35 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=438.7.

[0564] According to the synthetic method similar to Example 12, A27 was used instead of A6, and toluene was used as solvent to synthesize compound 38 as a brown-red solid. LC-MS (ESI): [M+1]+=474.7.

[0565] Embodiment 42 Synthesis of Camptothecin Compounds 36 and 39

[0566] The synthetic route is as follows:

[0567]

[0568] According to the synthetic method similar to Example 12, using A30 instead of A6 and toluene as solvent, compound 36 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=456.7.

[0569] According to the synthetic method similar to that of Example 12, A31 was used instead of A6, and toluene was used as solvent to obtain compound 39 as a brown-red solid. LC-MS (ESI): [M+1]+=492.7

[0570] Embodiment 43 Synthesis of Camptothecin Compounds 37 and 40

[0571] The synthetic route is as follows:

[0572]

[0573] According to the synthetic method similar to Example 12, using A28 instead of A6 and toluene as solvent, compound 37 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=440.8.

[0574] According to the synthetic method similar to Example 12, A29 was used instead of A6, and toluene was used as solvent to synthesize compound 40 as a brown-red solid. LC-MS (ESI): [M+1]+=476.7.

[0575] (III) Synthesis of drug-containing linkers

[0576] Embodiment 44 Synthesis of drug-containing linker MWD-L1

[0577]

[0578] The synthetic route is as follows:

[0579]

[0580] GGFG-Dxd was synthesized according to the method described in the patent publication (US20190151328A1):

[0581]

[0582] GGFG-Dxd is a light yellow solid, LC-MS (ESI): M+1=841;

[0583] The BL linker compound was synthesized according to the method described in patent publication WO2018 / 095422A1:

[0584]

[0585] BL linker compound is a yellow solid, LC-MS (ESI): M+1=858

[0586] BL linker compound (857 mg, 1 mmol), GGFG-Dxd (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), HATU (570 mg, 1.5 mmol, 1.5 eq), dissolved in 30 ml DCM, stirred for 2 h. The reaction solution was cooled to 5-10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1-10: 1, the drug-containing linker MWD-L1 was obtained as a yellow solid, 500 mg, yield 29.8%, LC-MS (ESI): M+1 = 1681, (M+1) / 2 = 840.9.

[0587] Embodiment 45 Synthesis of drug-containing linker MWC-L2

[0588]

[0589] Step 1

[0590] Boc-Val-Ala-OH (288 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was spin-dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and 450 mg of intermediate 2-1 was quantitatively obtained as a gray-green solid.

[0591] Step 2

[0592] Intermediate 2-1 (450 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), p-toluenesulfonic acid pyridinium salt (PPTS) (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 2-2, brown solid 750 mg. LC-MS (ESI): M+1=574. The product was directly used for the next step without purification.

[0593] Step 3

[0594] BL linker compound (857 mg, 1 mmol), HoSu (138 mg, 1.2 mmol, 1.2 eq), DCC (310 mg, 1.5 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was the DCM solution of A-Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq) and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phase was combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, the linker MWC-L2 was obtained as an orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1 = 1414, (M+1) / 2 = 707 Embodiment 46 Synthesis of drug-containing linker MWC-L3

[0595]

[0596] Step 1

[0597] Boc-Gly-OH (175 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and intermediate 3-1 was quantitatively obtained as a light green solid 335 mg.

[0598] Step 2

[0599] Intermediate 3-1 (335 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), PPTS (251 mg, 1 mmol, 1 eq) were suspended in 30 ml toluene and heated under reflux for 2 hours. Heating was stopped, and after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 3-2, 560 mg of brown solid. LC-MS (ESI): M+1=461. The product was directly used for the next step without purification.

[0600] Step 3

[0601] Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq), dissolved in 20 ml DMF, stirred for 3 h. Add 1N hydrochloric acid (30 ml) and stir for 0.5 h. Extract with DCM (30 ml*2) and combine the organic phases. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried. Purified by column chromatography, eluent: DCM: MeOH = 50: 1 ~ 10: 1, intermediate 3-3 was obtained as a brown solid, 350 mg, LC-MS (ESI): M+1 = 945

[0602] Step 4

[0603] Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), and diethylamine (2 ml) was added, and the mixture was stirred for 3 h. The reaction solution was spin-dried to obtain a crude intermediate 3-4 as a brown viscous substance, LC-M (ESI): M+1=722, and the crude product was directly used for the next step without purification.

[0604] Step 5

[0605] BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, to obtain linker MWC-L3 as an orange-red solid 120 mg, yield 20.8%, LC-MS (ESI): M+1 = 1562, (M+1) / 2 = 781

[0606] Embodiment 47 Synthesis of drug-linked MWE-L4

[0607]

[0608] MWE-L4 was synthesized by a similar method to Example 45 using A3 instead of A1 as an orange-red solid. LC-MS (ESI): M+1=1430

[0609] Embodiment 48 Synthesis of drug-containing linker MWG-L5

[0610]

[0611] MWG-L5 was synthesized by a similar method to Example 45 using A6 instead of A1 as an orange-red solid. LC-MS (ESI): M+1=1442

[0612] Embodiment 49 Synthesis of drug-containing linker MWF-L6

[0613]

[0614] MWF-L6 was synthesized by a similar method to Example 45 using A7 instead of A1 as an orange-red solid. LC-MS (ESI): M+1=1450

[0615] Embodiment 50 Synthesis of drug-containing linker MWF-L7

[0616]

[0617] MWF-L7 was synthesized by a similar method to Example 45, using A7 instead of A1 and Alloc-Ala-Ala-Ala-OH instead of Alloc-Val-Ala-OH. The product was an orange-red solid. LC-MS (ESI): M+1=1493.5

[0618] Embodiment 51 Synthesis of drug-containing linker MWF-L8

[0619]

[0620] According to the method of Example 46, MWF-L8 was synthesized by replacing A1 with A7 as an orange-red solid. LC-MS (ESI): M+1=1598.4.

[0621] Embodiment 52 Synthesis of drug-containing linker MWF-L9

[0622]

[0623] According to the method of Example 45, MWF-L9 was synthesized by substituting A5 for A1 as a yellow solid. LC-MS (ESI): M+1=1416.4.

[0624] Embodiment 53 Synthesis of drug-containing linker MWF-L10

[0625]

[0626] According to the method of Example 45, MWF-L10 was synthesized by replacing A1 with A13 as a yellow solid. LC-MS (ESI): M+1=1468.4.

[0627] Embodiment 54 Synthesis of drug-containing linker MWF-L11

[0628]

[0629] According to the method of Example 45, MWF-L11 was synthesized by replacing A1 with A18 as a yellow solid. LC-MS (ESI): M+1=1450.4.

[0630] Embodiment 55 Synthesis of drug-containing linker MWF-L12

[0631]

[0632] According to the method of Example 45, MWF-L12 was synthesized by replacing A1 with A16 as a yellow solid. LC-MS (ESI): M+1=1434.4.

[0633] Embodiment 56 Synthesis of drug-containing linker MWF-L13

[0634]

[0635] According to the method of Example 45, MWF-L13 was synthesized by replacing A1 with A19 as a yellow solid. LC-MS (ESI): M+1=1486.4.

[0636] Embodiment 57 Synthesis of drug-containing linker MWF-L14

[0637]

[0638] According to the method of Example 45, MWF-L14 was synthesized by using A17 instead of A1 as a yellow solid. LC-MS (ESI): M+1=1470.4.

[0639] Embodiment 58 Synthesis of drug-containing linker MWF-L15

[0640]

[0641] According to the method of Example 45, MWF-L15 was synthesized by using A2 instead of A1 as a yellow solid. LC-MS (ESI): M+1=1432.4.

[0642] Embodiment 59 Synthesis of drug-containing linker MWD-L7

[0643]

[0644] The synthetic route is as follows:

[0645]

[0646] Step 1

[0647] Fmoc-Val-Cit-PAB-PNP (153 mg, 0.2 mmol), exatecan mesylate (106 mg, 0.2 mmol, 1 eq), DIPEA (65 mg, 0.5 mmol, 2.5 eq), dissolved in 30 ml DCM, stirred for 2 h. The reaction solution was spin-dried and purified by column chromatography, eluent: DCM: MeOH = 50: 1-10: 1, to obtain intermediate 5-1 as a light yellow solid 180 mg. LC-MS (ESI): M+1 = 1064.

[0648] Step 2

[0649] Intermediate 5-1 (180 mg, 0.17 mmol) was dissolved in methanol (20 ml), diethylamine (2 ml) was added, and the mixture was stirred for 3 h. The reaction solution was spin-dried to obtain a crude intermediate 5-2 as a light yellow viscous substance, LC-M (ESI): M+1=842, and the crude product was directly used for the next step without purification.

[0650] Step 3

[0651] BL linker compound (146 mg, 0.17 mmol), HoSu (25 mg, 0.21 mmol, 1.2 eq), DCC (54 mg, 0.26 mmol, 1.5 eq) were dissolved in 20 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was added to a mixed solution of the crude intermediate 5-1, DIPEA (56 mg, 0.43 mmol, 2.5 eq), and DCM (20 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 5-10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH=50:1-10:1, to obtain 67 mg of drug-containing linker MWD-L7 as a yellow solid, with a yield of 23.1%, LC-MS (ESI): M+1=1682, (M+1) / 2=841.

[0652] Embodiment 60 Synthesis of drug-containing linker MWD-L8

[0653]

[0654] According to the similar synthesis method of Example 44, MWD-L8 was synthesized by replacing BL compound as a light yellow solid, LC-MS (ESI): M+1=1645.7.

[0655] Embodiment 61 Synthesis of drug-containing linker MWD-L9

[0656]

[0657] According to the similar synthesis method of Example 44, MWD-L9 was synthesized by replacing BL compound as a light yellow solid, LC-MS (ESI): M+1=1713.7.

[0658] Embodiment 62 Synthesis of drug-containing linker LD

[0659]

[0660] According to the similar synthesis method of Example 48, MC-OSU was used instead of BL-OSU compound to synthesize LD as a light yellow solid, LC-MS (ESI): M+1=795.9.

[0661] Embodiment 63 Synthesis of drug-containing linker LE

[0662]

[0663] According to the similar synthesis method of Example 45, MC-OSU was used instead of BL-OSU compound to synthesize LE as a light yellow solid, LC-MS (ESI): M+1=803.8.

[0664] Embodiment 64 Synthesis of drug-containing linker LF

[0665]

[0666] According to the similar method of Example 45, LF was prepared by replacing A1 with A7 and replacing BL with MaL-PEG8-COOH as a yellow solid. LC-MS (ESI): M+1=1185.

[0667] Embodiment 65 Synthesis of drug-containing linker LG

[0668]

[0669] According to Example 46, MC-OSu was used instead of BL-OSu to prepare LG as a yellow solid. MS (ESI): M+1=965.98.

[0670] Embodiment 66 Synthesis of drug-containing linker LH

[0671]

[0672] According to the similar synthesis method of Example 47, MaL-PEG8-COOH was used instead of BL compound to synthesize LH as a light yellow solid. LC-MS (ESI): M+1=1165.3.

[0673] Embodiment 67 Synthesis of drug-containing linker LI

[0674]

[0675] Using a similar method to Example 45, substituting A13 for A1, LI was prepared as a yellow solid. LC-MS (ESI): M+1=1203.4.

[0676] Embodiment 68 Synthesis of drug-containing linker LJ

[0677]

[0678] Using a similar method to Example 45, using A18 instead of A1, LJ was prepared as a yellow solid. LC-MS (ESI): M+1=1185.4.

[0679] Embodiment 69 Synthesis of drug-containing linker LK

[0680]

[0681] Using a similar method to Example 45, using A16 instead of A1, LK was prepared as a yellow solid. LC-MS (ESI): M+1=1169.3.

[0682] Embodiment 70 Synthesis of drug-containing linker LL

[0683]

[0684] Using a similar method to Example 45, using A19 instead of A1, LL was prepared as a yellow solid. LC-MS (ESI): M+1=1221.4.

[0685] Embodiment 71 Synthesis of drug-containing linker LM

[0686]

[0687] Using the method of Example 45, substituting A17 for A1, LM was prepared as a yellow solid. LC-MS (ESI): M+1=1205.4.

[0688] Embodiment 72 Synthesis of drug-containing linker LN

[0689]

[0690] Synthesized according to a method similar to Example 45, using HCDE intermediate instead of CDE intermediate; using MaL-PEG8-COOH instead of BL compound to prepare LN, a yellow solid, LC-MS (ESI): M+1=1163.2 (a mixture of a pair of diastereomers).

[0691] Embodiment 73 Synthesis of drug-containing linker MWS-L1

[0692]

[0693]

[0694] According to the similar method of Example 45, A13 is used to replace A1.

[0695] MWS-L1 was prepared by replacing the BL linker compound as a yellow solid, LC-MS (ESI): M+1=1236.3.

[0696] Embodiment 74 Synthesis of drug-containing linker MWS-L2

[0697]

[0698] Using a similar method to Example 45, substituting A18 for A1, MWS-L2 was prepared as a yellow solid. LC-MS (ESI): M+1=1218.4.

[0699] Embodiment 75 Synthesis of drug-containing linker MWS-L3

[0700]

[0701] Using a similar method to Example 45, substituting A16 for A1, MWS-L3 was prepared as a yellow solid. LC-MS (ESI): M+1=1202.4.

[0702] Embodiment 76 Synthesis of drug-containing linker MWS-L4

[0703]

[0704] Using a similar method to Example 45, substituting A19 for A1, MWS-L4 was prepared as a yellow solid. LC-MS (ESI): M+1=1254.4.

[0705] Embodiment 77 Synthesis of drug-containing linker MWS-L5

[0706]

[0707] According to the similar method of Example 45, using A17 instead of A1, MWS-L5 was prepared as a yellow solid. LC-MS (ESI): M+1=1238.4.

[0708] Embodiment 78 Synthesis of drug-containing linker MWS-L6

[0709]

[0710] According to the method similar to Example 54

[0711] MWS-L6 was prepared by replacing Mal-PEG8-COOH as a yellow solid, LC-MS (ESI): M+1=1302.4

[0712] Embodiment 79 Synthesis of drug-containing linker MWS-L7

[0713]

[0714] Using a similar method to Example 45, substituting A18 for A1, MWS-L7 was prepared as a yellow solid. LC-MS (ESI): M+1=1284.4.

[0715] Embodiment 80 Synthesis of drug-containing linker MWS-L8

[0716]

[0717] Using a similar method to Example 45, substituting A16 for A1, MWS-L8 was prepared as a yellow solid. LC-MS (ESI): M+1=1268.4.

[0718] Embodiment 81 Synthesis of drug-containing linker MWS-L9

[0719]

[0720] Using a similar method to Example 45, substituting A19 for A1, MWS-L9 was prepared as a yellow solid. LC-MS (ESI): M+1=1320.4.

[0721] Embodiment 82 Synthesis of drug-containing linker MWS-L10

[0722]

[0723] Using a similar method to Example 45, using A17 instead of A1, MWS-L10 was prepared as a yellow solid. LC-MS (ESI): M+1=1304.4.

[0724] (IV) Comparison of the synthesis of drug-containing linkers

[0725] Embodiment 83 Synthesis of drug-containing linkers LA, LB and LC

[0726] 1. Synthesis of random conjugated drug-containing linkers

[0727] The drug-containing linker MC-GGFG-Dxd was synthesized according to the method described in the patent disclosure document (US20190151328A1), and MC-GGFG-Dxd was obtained as a light yellow solid, LC-MS (ESI): M+1=1034, 1 H NMR (CDCl3).

[0728] The drug-containing linkers LA and LB were synthesized according to the method described in the patent disclosure document (WO2020200880), and LA was obtained as a yellow solid, LC-MS (ESI): M+1=767; LB was a brown viscous substance, LC-MS (ESI): M+1=1149.

[0729]

[0730] 2. Synthesis of other ring-bridged drug-containing linkers LC

[0731]

[0732] The synthetic route is as follows:

[0733]

[0734] The linker LC was synthesized in a similar manner to the drug-containing linker L-1. LC was a light yellow solid. LC-MS (ESI): M+1=1982, (M+1) / 2=991.

[0735] (V) Preparation and physicochemical characterization of antibody-drug conjugates

[0736] 1. General preparation method of antibody drug conjugates

[0737] a. General preparation method for site-directed coupling

[0738]

[0739] The antibody is reduced to open the disulfide bond, then coupled with the linker to form a bridged antibody-drug conjugate, then hydrolyzed to open the maleimide ring, and finally purified to obtain an antibody-drug conjugate with DAR=4. The details are as follows:

[0740] Reduction of antibodies: 120 mg of antibody sample was replaced with a buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate at pH 7.0 using a NAP-25 column with a Sephadex G25 carrier, and the antibody concentration was diluted to 10 mg / ml. Take 10 ml of a total of 100 mg of antibody sample, add an aqueous solution of 10 mg / ml TCEP (Sigma-Aldrich) at an antibody-TCEP molar ratio of 1 to 10 equivalents, and the volume of the added TCEP aqueous solution is 2.1 ml. After incubation for 2 hours, the reaction solution was replaced with a Sephadex G25 column, and replaced with a buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate at pH 6.5.

[0741] Coupling and hydrolysis of antibody and drug-containing linker: dilute the above reduced antibody to 5 mg / mL, add 0.38 ml of N, N-dimethylacetamide (DMA) as a presolvent at 2% of the total reaction volume, and add 0.67 ml of DMA-drug-containing linker mixed solution containing 10 mg / mL drug-containing linker at a molar ratio of 1 to 5.5 times of antibody-small molecule drug as the reaction solution. Stir at room temperature for 30 minutes. Use the NAP-25 column with Sephadex G25 carrier to replace the reaction solution with a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 8.0, remove excess drug-containing linker, and heat in a water bath at 37°C for 3 hours.

[0742] Purification of antibody drug conjugates: The above samples were concentrated using AMICOM ultrafiltration centrifuge tubes to about 15 mg / mL. Add 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution until the conductivity is 100 ms / cm. Load the sample onto a TOYOPEAL Butyl-650M filler (purchased from TOSOH) hydrophobic column, phase A is 50 mm disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6 M ammonium sulfate, and phase B is 50 mm disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Phase B was eluted with a gradient of 0-100% for 8 times the column volume, and the main peak was collected.

[0743] The final sample was replaced with 50 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 um filter membrane (Sartorius stedim Ministart).

[0744] b. General preparation method for random coupling

[0745]

[0746] The antibody is reduced to open the disulfide bonds and then coupled with the linker to form a bridged antibody drug conjugate.

[0747] Specifically, it is prepared according to the preparation method disclosed in patent CN104755494B.

[0748] 2. General analytical methods for antibody-drug conjugates

[0749] a. Determination of drug-antibody coupling ratio (UV-DAR method) and concentration by ultraviolet spectrophotometry

[0750] The concentration of the antibody drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of the small molecule, and then performing the following calculation.

[0751] a1. Determination of Drug-Antibody Conjugate Ratio (DAR) of Antibody Drug Conjugates

[0752] According to the literature [Clin Cancer Res. 2004 Oct 15; 10(20):7063-70] in, is the molar absorption coefficient of the antibody at 280 nm, A 280 is the UV absorption value of the antibody-drug conjugate at 280 nm, A z is the UV absorption value of the antibody-drug conjugate at the characteristic absorption wavelength Znm of the drug-containing linker, is the molar absorption coefficient of the antibody at the characteristic absorption wavelength Z nm of the drug-containing linker, is the molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm, is the molar absorption coefficient of the drug-containing linker at 280 nm.

[0753] in:

[0754]

[0755] a2. Determination of drug concentration

[0756] Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additivity of absorbance), assuming that the molar absorptivity of the antibody and the drug-containing linker does not change before and after the antibody is conjugated to the drug-containing linker, the concentration of the antibody-drug conjugate has the following relationship:

[0757]

[0758] therefore, From this we get

[0759] Substituting the DAR value into the formula will give the protein concentration.

[0760] Among them, MW ADC is the molecular weight of the antibody drug conjugate, MW Ab is the molecular weight of the antibody, MW D is the molecular weight of the drug-containing linker.

[0761] b. Hydrophobic chromatography

[0762] b1. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0763] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis;

[0764] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0765] Mobile phase A: 1.5M (NH4)2SO4 + 25mM PB, pH 7.0

[0766] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0767] Flow rate: 0.6 mL / min;

[0768] Detection wavelength: 280nm;

[0769] Column temperature: 30°C;

[0770] Sample volume: 10 μL;

[0771] HIC Chromatographic Gradient

[0772] time ％A ％B 0 100 0 10 80 20 20 40 60 25 0 100 25.1 100 0 35 100 0

[0773] DAR calculation formula:

[0774] DAR = ∑(weighted peak area) / 100, that is, DAR = (D0 peak area ratio*0+D1 peak area ratio*1+D2 peak area ratio*2+D3 peak area ratio*3+D4 peak area ratio*4+D5 peak area ratio*5+D6 peak area ratio*6+D7 peak area ratio*7+D8 peak area ratio*8) / 100.

[0775] b2. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0776] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis;

[0777] Chromatographic column: TSKgel Butyl-NPR, 2.5μm, 4.6mm*100mm;

[0778] Mobile phase A: 1.2M (NH4)2SO4 + 25mM PB, pH 7.0

[0779] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0780] Flow rate: 0.6 mL / min;

[0781] Detection wavelength: 280nm;

[0782] Column temperature: 30°C;

[0783] Sample volume: 10 μL;

[0784] HIC Chromatographic Gradient

[0785] time ％A ％B 0 100 0 1 100 0 20 0 100 20.1 100 0 30 100 0

[0786] DAR calculation formula: same as b1.

[0787] b3. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0788] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis;

[0789] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0790] Mobile phase A: 2.5M (NH4)2SO4 + 125mM PB, pH 7.0

[0791] Mobile phase B: 125 mM PB, pH 7.0

[0792] Mobile phase C: IPA

[0793] Mobile phase D: H2O

[0794] Flow rate: 0.5 mL / min;

[0795] Detection wavelength: 280nm;

[0796] Column temperature: 30°C;

[0797] Sample volume: 10 μL;

[0798] HIC Chromatographic Gradient

[0799] time ％A ％B ％C ％D 0 80 0 0 20 20 5 45 3 47 22 0 50 3 47 22.1 80 0 0 20 30 80 0 0 20

[0800] DAR calculation formula: same as b1.

[0801] c. Determination of DAR value by mass spectrometry LC-MS

[0802] Sample treatment: Take an appropriate amount of sample in an ultrafiltration tube, replace it with 50mM NH4HCO3 replacement buffer (pH7.1), add buffer, and ultrafiltration centrifuge (13000g*5min). Add 8μl of PNGase F enzyme to the replaced sample, incubate at 37℃ for 5h for desugaring, and after incubation, centrifuge at 12000rpm for 5min, and take the supernatant into the injection vial as the test sample for later use.

[0803] Chromatographic column: PolyHYDROXYETHYLA Column, 5um, 2.1mm×200mm;

[0804] Mobile phase: 50 mM ammonium acetate, pH 7.0;

[0805] Running time: 10 minutes;

[0806] Flow rate: 0.1 mL / min;

[0807] Injection volume: 2 μL;

[0808] Column temperature: 25°C;

[0809] Detection wavelength: 280nm;

[0810] Ionization method: ESI positive

[0811] Drying gas temperature: 325℃

[0812] Drying gas flow rate: 8L / min

[0813] Atomizer pressure: 20psig

[0814] Sheath gas temperature: 325°C;

[0815] Sheath gas flow rate: 12L / min;

[0816] Scan settings: 900-8000 m / z.

[0817] d. Size Exclusion Chromatography SEC-HPLC to Determine Molecular Size Heterogeneity

[0818] Sample treatment: The sample was diluted to about 1.0 mg / ml with mobile phase, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for analysis.

[0819] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5μm, 7.8mm*300mm;

[0820] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0821] Flow rate: 0.6 mL / min;

[0822] Detection wavelength: 280nm;

[0823] Column temperature: 30°C;

[0824] Sample volume: 20uL;

[0825] Elution time: 20min;

[0826] Elution gradient: isocratic.

[0827] e. Purity determination by non-reducing capillary electrophoresis NR-CE-SDS

[0828] The method was determined according to the monoclonal antibody molecular size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.

[0829] f. Determination of small molecule / antibody coupling ratio (DAR value) of antibody-drug conjugates by reversed-action chromatography (RP-HPLC)

[0830] The analysis was performed according to the method described in patent publication US10227417 B2.

[0831] The DAR value was calculated according to the formula: DAR = 2 × (∑ light chain weighted peak area + ∑ heavy chain weighted peak area) / 100. That is: DAR = 2 × (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100.

[0832] Embodiment 84 Preparation and characterization of antibody-drug conjugates targeting B7H3

[0833] According to the coupling method in this section, 100 mg each of the B7H3-targeting antibodies hz10B4m1, hz10B4m2, and hz10B4 were coupled with drug-containing linkers MWD-L1, MWC-L2, MWC-L3, MWF-L6, MWF-L8, and MWF-L11 to prepare bridged ADCs.

[0834] According to the method of patent CN104755494B, 100 mg each of the antibodies hum30 and hz10B4m1 targeting B7H3 were taken and coupled with MC-GGFG-Dxd, vcMMAE, MWS-L1, MWS-L7, LJ, and LI to prepare randomly coupled ADCs.

[0835] The concentration, DAR value and purity of the bridged ADC were determined using 2.a UV spectrophotometry, 2.b hydrophobic chromatography and 2.d size exclusion chromatography in this section, respectively.

[0836] Use the reverse-action chromatography method 2.f in this section to determine the DAR value of the ADC sample coupled with the MC-GGFG-Dxd drug-containing linker; use the hydrophobic chromatography method 2.b in this section to determine the DAR value of the ADC sample coupled with the vcMMAE drug-containing linker; use the ultraviolet spectrophotometry method 2.a and the size exclusion chromatography method 2.d in this section to determine the concentration and purity of the non-bridged ADC drug, respectively.

[0837] The results are shown in Table 5.

[0838] Table 5. Characterization results of antibody drug conjugates

[0839]

[0840]

[0841] The results showed that the UV-DAR of the prepared low-drug-loaded ADCs were between 3.6 and 4.8, and the UV-DAR of the high-drug-loaded ADC (B7H3-ADC-5) was 6.9. The SEC results showed that, except for the B7H3-ADC-3 sample (purity 95.05%), the monomer content of the remaining ADC samples was above 98%, indicating that the prepared ADC samples were of high purity.

[0842] (VI) Activity Assessment

[0843] Embodiment 85 Comparative study on the activity of camptothecin compounds

[0844] Oral epithelial cancer cells KB (purchased from ATCC) were cultured in DMEM medium supplemented with 10% FBS; human gastric cancer cells NCI-N87 (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS; human colon cancer cells HT29 (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS; human pancreatic adenocarcinoma cells BxPC-3 (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS.

[0845] Use the above corresponding culture medium to adjust the cell density of KB cells to 2×10 4 / mL; NCI-N87 cells were adjusted to a cell density of 5×10 4 / mL; HT29 cells were adjusted to a cell density of 5×10 4 / mL, 100 μL / well was plated in a 96-well transparent flat-bottom plate and cultured overnight; the cell density of BxPC-3 cells was adjusted to 5×10 4 Pieces / mL.

[0846] Use culture medium to dilute the sample to be tested to 20 μM, then dilute 5 times, set 9 gradients (set the well without sample, i.e. the well with maximum cell growth, and the well without cell, i.e. the culture base). Add 100 μL / well to the cell culture plate and incubate in a carbon dioxide incubator for 96 hours. After incubation, add the prepared CCK-8 (purchased from Tongren Chemical) at 20 μL / well, incubate in a carbon dioxide incubator for 3 hours, and read the OD450 value with an enzyme plate reader.

[0847] The data were fitted with a 4-parameter curve using Prism7 software, and the maximum killing calculation formula was: 1-(OD450 value of the maximum killing well-OD450 value of the culture base) / (OD450 value of the cell maximum growth well-OD450 value of the culture base)×100%. The results are shown in Table 6.

[0848] Table 6. Comparative study results of the activity of camptothecin compounds

[0849]

[0850]

[0851]

[0852] As shown in Table 6, the cell killing activity of the novel camptothecin compound provided by the present invention is significantly better than that of the control molecule DXD.

[0853] Embodiment 86 Comparative study of in vitro activity of antibody-drug conjugates targeting B7H3

[0854] Calu-6-B7H3 (homemade), a human non-small cell lung cancer cell line that highly expresses B7H3, was taken out of liquid nitrogen and revived. The cell density was adjusted to 5×10 4 After the concentration of 100 μl / well was reached, 100 μl / well was added to the cell culture plate and cultured overnight.

[0855] The ADC targeting B7H3 prepared above was diluted to 5ug / ml using complete medium, and then diluted 4-fold or 2-fold gradients, for a total of 9 gradients, with 2 replicates for all samples; a negative control (cells + medium) was set. Then, the cells were placed in a cell culture incubator and incubated for 168h (7 days). The cell culture plate was removed, and MTS was added at 40μl / well, and the cells were reacted in a 37°C incubator for 2-4h; the cell plate was removed and the OD value was read at 490nm.

[0856] The results are shown in Table 7.

[0857] Table 7. In vitro cell killing effect of ADC targeting B7H3

[0858]

[0859]

[0860] The in vitro cell killing results of ADCs targeting B7H3 showed that the 7-day in vitro killing activity of B7H3-ADC-7 on Calu-6-B7H3 cells was equivalent to that of the control ADC (B7H3-ADC-9, i.e., DS-7300), and the 7-day in vitro killing activity of B7H3-ADC-6, B7H3-ADC-11, B7H3-ADC-13, and B7H3-ADC-14 on Calu-6-B7H3 cells was better than that of the control ADC (DS-7300). In addition, the in vitro killing activity of the high-drug-loaded ADC (B7H3-ADC-5) was better than that of the low-drug-loaded ADC (B7H3-ADC-4).

[0861] Embodiment 87 Comparative study on the in vivo efficacy of camptothecin-antibody drug conjugates targeting B7H3

[0862] Human lung cancer cells Calu-6 (purchased from ATCC) were used. Calu-6 cells were cultured in 10-cm culture dishes, MEM culture medium was supplemented with 10% fetal bovine serum and penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. The cells were passaged 2-3 times a week, and when the cells were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated.

[0863] The antibody drug conjugate B7H3-ADC-6 was compared under different dose groups. Nude mice with subcutaneous transplantation of human lung cancer Calu-6 cells were given intravenous (IV) injections once a week for two injections; the solvent group was given the same volume of solvent (normal saline). Tumor volume was measured twice a week, the mouse body weight was weighed, and the data was recorded.

[0864] The experiment was completed, the experimental endpoint (D21) was reached, or the tumor volume reached 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed and photographed. The results are shown in Table 8 and Figure 2 .

[0865] Table 8. Efficacy of B7H3-ADC-6 on human lung cancer Calu-6 subcutaneous xenograft tumors in nude mice (TGI% calculated based on tumor volume).

[0866]

[0867]

[0868] The above results show that in the Calu-6 human lung cancer transplant tumor model, B7H3-ADC-6 has significant tumor inhibitory activity compared with the solvent group; at the same dose, B7H3-ADC-6 has better tumor inhibitory activity than DS-7300, and the tumor inhibitory activity of B7H3-ADC-6 at a dose of 3 mg / kg is equivalent to that of DS-7300 at a dose of 10 mg / kg.

[0869] Embodiment 88 Comparative study on the in vivo efficacy of camptothecin-antibody drug conjugates targeting B7H3

[0870] Human pancreatic cancer cells Pan 05.04 (purchased from ATCC) were used. Pan 05.04 cells were cultured in 10-cm culture dishes, RPMI 1640 culture medium was supplemented with 10% fetal bovine serum and penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. The cells were passaged 2-3 times a week, and when the cells were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated.

[0871] The antibody drug conjugate B7H3-ADC-6 and the anti-B7-H3 antibody therein were compared under different dose groups. Nude mice transplanted with subcutaneous human pancreatic cancer Pan 05.04 cells were intravenously injected (IV) once a week for two injections; the solvent group was given the same volume of solvent (normal saline). Tumor volume was measured twice a week, the mouse body weight was weighed, and the data were recorded.

[0872] The experiment was completed, the experimental endpoint (D18) was reached, or the tumor volume reached 2000mm3 The animals were killed by CO2 anesthesia, and then the tumors were removed and photographed. The results are shown in Table 9 and Figure 3 .

[0873] Table 9. Efficacy of B7H3-ADC-6 on subcutaneous tumors in mice (TGI% calculated based on tumor volume).

[0874]

[0875] The above results show that in the Pan05.04 human pancreatic cancer transplant tumor model, B7H3-ADC-6 has significant tumor inhibitory activity compared with the solvent group; at the same dose, B7H3-ADC-6 has better tumor inhibitory activity than DS-7300; in three different dose groups (1 mg / kg, 3 mg / kg and 10 mg / kg), the tumor inhibitory activity of B7H3-ADC-6 alone is better than that of the combined administration group of anti-B7H3 antibody hz10B4m1 + compound 3 (B7H3 antibody 10 mg / kg + compound 3 0.11 mg / kg).

[0876] Embodiment 89 Study on ADC targeting B7H3 inducing cellular DNA damage

[0877] Calu-6 cells were seeded in 6-well plates and treated with drugs B7H3-ADC-6 (0.6572, 6.572, 65.72, 657.2, 1971.7 nM), DS-7300 (0.6649, 6.649, 66.49, 664.9 nM), B7H3-ADC-6 antibody hz10B4m1 (675.4, 2026.3 nM) and compound 3 (0.1, 1 nM) for 72 h. The cells were collected and lysed with lysis buffer (0.1 M MES, pH 6.6, 1 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol), centrifuged at 12000 rpm for 5 min, the supernatant was removed, and 1× SDS sample buffer (50 mM Tris, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), denatured in a boiling water bath for 10 min, and then the γ-H2AX level in the cells was detected by Western blot.

[0878] The experimental results showed that B7H3-ADC-6, DS-7300 and compound 3 could induce DNA damage in Calu-6 cells, while hz10B4m1 had no obvious DNA damage induction effect. Figure 4 .

[0879] Embodiment 90Study on the inhibition of proliferation of different tumor cells by ADC drugs targeting B7H3

[0880] The SRB method was used to detect the effect of drugs on the proliferation of tumor cells cultured in vitro. A certain number of cells in logarithmic growth phase were inoculated into a 96-well culture plate. After the cells were grown overnight, different concentrations of drugs were added. After 144 hours of drug action, the cells were fixed with trichloroacetic acid. After staining with SRB solution (prepared with 1% glacial acetic acid, concentration of 4 mg / mL), 10 mM Tris solution was added to each well to dissolve. The OD value was read at a wavelength of 510 nm using an enzyme reader.

[0881] Inhibition rate (%) = (OD value control well - OD value drug well) / OD value control well × 100%

[0882] The MTT method was used to detect the effect of drugs on the proliferation of tumor cells cultured in vitro. A certain number of cells in logarithmic growth phase were inoculated in 96-well culture plates. After overnight adherence, different concentrations of drugs were added. After 144 hours of drug action, MTT was added to each well and cultured for 4 hours in a 37°C 5% CO2 saturated humidity incubator. 100 μL of the triple solution was added to each well, and the OD value was measured at 570nm and 690nm wavelengths on the microplate reader.

[0883] According to the inhibition rate of each concentration, the half inhibitory concentration IC was calculated using Graphpad Prism 8.0 software. 50 The experiment was repeated once, and the data were expressed as mean ± SD, and a concentration (logarithm)-inhibition rate graph was generated. The results are shown in Table 10.

[0884] Table 10. Inhibition results of ADC drugs targeting B7H3 on proliferation of different tumor cells

[0885]

[0886]

[0887] The above studies show that B7H3-ADC-6 has a significant inhibitory effect on the proliferation of BxPC-3, ES-2, PANC05.04, Calu-6, Calu-3, HCT-116, MDA-MB-468, and PANC-1 tumor cells, which are superior to the B7H3 antibody hz10B4m1.

[0888] Embodiment 91 Study on the bystander killing effect of ADC targeting B7H3

[0889] B7H3-positive Calu-6 cells (purchased from ATCC) and B7H3-negative MV-4-11 / luc (MV-4-11 transferred with luciferase, purchased from Nanjing Kebai Biotechnology Co., Ltd.) cells were plated in 96-well plates at a ratio of 1:1 or B7H3-negative MV-4-11 / luc cells were inoculated in 96-well plates alone (control). After that, drug B7H3-ADC-6 (1000, 3000, 10000, 30000, 100000 ng / mL) was added to each well for treatment for 144 hours, followed by centrifugation, removal of supernatant, addition of lysis buffer to lyse cells, centrifugation, removal of supernatant, addition of substrate luciferin, and detection of fluorescence intensity by microplate reader.

[0890] Inhibition rate (%) = (OD value control well - OD value drug well) / OD value control well × 100%

[0891] The above studies show that B7H3-ADC-6 has a strong bystander effect: it kills B7H3-positive Calu-6 cells and B7H3-negative MV-4-11 / luc cells at the same time; but it has no significant effect on the proliferation of MV-4-11 / luc cells cultured alone. Figure 5 .

[0892] Embodiment 92 Study on cell cycle arrest induced by ADC targeting B7H3

[0893] Calu-6 cells (purchased from ATCC) were seeded in 6-well plates, and the drug B7H3-ADC-6 (0.6752, 6.752, 67.52, 675.2, 1971.7 nM), B7H3-ADC-6 antibody hz10B4m1 (675.4, 2026.3 nM) and compound 3 (0.01, 0.1, 1 nM) were added. After 48 hours of treatment, the cells were collected and fixed with ethanol overnight. Then RNase and PI were added, mixed, and stained at 37°C in the dark for 30 minutes. Finally, the DNA content in the cells was detected by flow cytometry (BD FACS CALIBUR 4), and 1×10 4 Calu-6 cells cultured alone were used as controls. The experimental results were analyzed using ModFit Lt Mac V3.0 software.

[0894] The above studies show that B7H3-ADC-6 and compound 3 both significantly induced G2 / M phase arrest in Calu-6 cells; the anti-B7H3 antibody hz10B4m1 had no significant cell cycle arrest effect. In addition, the results showed that B7H3-ADC-6 induced cell cycle arrest in the G2 / M phase, which is consistent with the action characteristics of compound 3. Figure 6 .

[0895] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. An antibody-drug conjugate or a salt thereof, wherein the antibody-drug conjugate or a salt thereof has a structure as shown in structural formula Ia and / or Ib: and / or in, Ab represents an anti-B7-H3 antibody or a fragment thereof; M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituents are selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., haloC1-6 alkyl, preferably haloC1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester group, amide group and cyano group; SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight chain heteroalkylene, wherein the C1-21 straight chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano; SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-、-NH(CH2CH2O) a CH2CO-, -S(CH2) a CO- or chemical bond, wherein a is an integer from 1 to 20; A represents a short peptide structure formed by 2-4 amino acids; m is 1 to 10, preferably 1 to 8 (e.g. 1 to 5), more preferably 3 to 8; CPT is a camptothecin compound.

2. The antibody-drug conjugate or a salt thereof according to claim 1, characterized in that: The anti-B7-H3 antibody or fragment thereof comprises a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3; and, CDR-L1, CDR-L2 and CDR-L3) selected from the following: (i) the CDR-H1, CDR-H2 and CDR-H3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:14, respectively; (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:15, respectively; and (iii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:16, respectively; Preferably, The anti-B7-H3 antibody or fragment thereof comprises at least a heavy chain variable region (VH) and a light chain variable region (VL); Preferably, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following: (i) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 3; (ii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:4; and (iii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:

5.

3. The antibody-drug conjugate or a salt thereof according to claim 1 or 2, characterized in that: The anti-B7-H3 antibody or fragment thereof further comprises a constant region, such as a heavy chain constant region (CH) and / or a light chain constant region (CL), or any one or more domains in the constant region; Alternatively, the anti-B7-H3 antibody or fragment thereof comprises a heavy chain and / or a light chain; for example, the heavy chain comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE, and the light chain comprises a light chain constant region of κ or λ type.

4. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 3, characterized in that: In formula Ia and / or Ib, M is a halogen-substituted phenylene group, preferably a fluorine-substituted phenylene group; Preferably, SP1 is C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene, which contains 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S; Preferably, SP2 is a chemical bond.

5. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in Structural Formula I, wherein Structural Formula I is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond, preferably the amino group adjacent to Group G in Structural Formula I is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond: in, R1, R2, R3, and R4 are independently hydrogen, halogen, hydroxy, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms to which they are attached form a C3-6 (preferably C3-5) cyclic alkyl; G is hydrogen, halogen, methyl or methoxy; Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene; X is oxygen or sulfur; n=0 or 1.

6. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 5, characterized in that: The structure of CPT is shown in Structural Formula IA, wherein Structural Formula IA is connected to the carboxyl group in Group A in Structural Formulas Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in Structural Formula IA is connected to the carboxyl group in Group A in Structural Formulas Ia and / or Ib via an amide bond: Wherein, the groups R1, R2, R3, and R4 have the same definitions as the groups R1, R2, R3, and R4 in the structural formula I in claim 5, but R1, R2, R3, and R4 are not hydrogen at the same time.

7. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in structural formula II, wherein structural formula II is connected to the carboxyl group in group A in structural formula Ia and / or Ib via an amide bond, preferably the amino group adjacent to group G in structural formula II is connected to the carboxyl group in group A in structural formula Ia and / or Ib via an amide bond: Wherein, R5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl; G is hydrogen, halogen (e.g. fluorine), methyl or methoxy; X is oxygen or sulfur; n=0 or 1.

8. The antibody-drug conjugate or a salt thereof according to claim 7, characterized in that: The structure of CPT is shown in Structural Formula IIA, wherein Structural Formula IIA is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in Structural Formula IIA is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond: The group R5 has the same definition as the group R5 in the structural formula II in claim 7, but R5 cannot be n-butyl.

9. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 8, characterized in that: The structure of the CPT is shown below, wherein each structural formula is connected to the carboxyl group in the group A in the structural formula Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in each structural formula is connected to the carboxyl group in the group A in the structural formula Ia and / or Ib via an amide bond:

10. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in formula IV, wherein the hydroxyl group connected to the same carbon as R8 in formula IV is connected to the carboxyl group of group A in formula Ia and / or Ib via a self-releasing structure, such as The solid line indicates the site of attachment to the carboxyl group of group A in formula Ia and / or Ib, and the wavy line indicates the site of attachment to the hydroxyl group in formula IV: Wherein, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.

11. An antibody drug conjugate or a salt thereof, wherein the antibody drug conjugate or the salt thereof has the general formula The structure shown, In the general formula, mAb represents an anti-B7-H3 antibody or a fragment thereof; The groups M, SP1, SP2, A and CPT are the same as those defined in any one of claims 1 to 10; N is 1 to 10, preferably 1 to 8 (e.g. 1 to 5), more preferably 3 to 8; E Lis selected from the following groups, wherein, Indicates that it is linked to the cysteine ​​in mAb. Indicates connection with M: E L -1a and / or E L -1b: and / or E L -2: E L -3: E L -4: E L -5: E L -6:

12. A method for preparing an antibody drug conjugate or a salt thereof according to any one of claims 1 to 11, comprising the following steps: a. Antibody reduction: adding ≥5.5 times the molar equivalent of the antibody to a phosphate buffer containing 5-30 mg / mL antibody, and reacting for 1.5-2 h, wherein the reducing agent is selected from TCEP, DTT, 2-MEA and DTBA; b. Antibody coupling and hydrolysis: replace the reduced antibody in step a into a phosphate buffer at pH 6.5-7.8, and dilute the antibody to a concentration of 3.5-15 mg / mL, add 4.5-6.5 times the molar equivalent of the antibody-containing linker dissolved in an organic co-solvent to the antibody dilution, and stir the reaction at 15-35° C. for ≥0.5 h, wherein the organic co-solvent is one or more selected from DMA, DMSO, DMF and ACN; replace the antibody coupling product into a phosphate buffer at pH 7.4-9.0, and heat at 35±10° C. for 2-24 h to obtain a hydrolyzate; c. Hydrophobic chromatography: Use hydrophobic filler to purify the antibody-coupled product by hydrophobic chromatography.

13. The preparation method according to claim 12, characterized in that: In step a, the antibody is an antibody against tumor-associated antigens, such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.

2. 14 . A composition comprising the antibody drug conjugate or a salt thereof according to any one of claims 1 to 11 .

15. Use of the antibody-drug conjugate or a salt thereof according to any one of claims 1 to 11 and / or the composition according to claim 14 in the preparation of a medicament for treating tumors.

16. The use according to claim 15, characterized in that The tumor and tumor-associated antigens such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

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