Anti-B7-H3 antibodies or their fragments, and antibody-drug conjugates that target B7-H3.

Abstract

A series of novel anti-B7-H3 antibodies are provided. The antibodies have high affinity for B7-H3 and possess a stable structure. Furthermore, antibody-drug conjugates are provided that can be produced using these antibodies. These antibody-drug conjugates use a camptothecin compound with a specific structure as a toxic molecule, exhibiting significant tumor suppressor activity and bystander killing effects.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This patent application claims priority to Chinese Patent Application No. 202310655433.X filed on June 5, 2023, the contents of which the entirety of that patent application is incorporated herein by reference.

[0002] This invention belongs to the field of biotechnology and, more specifically, relates to antibody-drug conjugates that target B7-H3 and their use in tumor therapy. [Background technology]

[0003] B7-H3, also known as CD276, is a type I transmembrane protein belonging to the B7 protein family, which includes co-stimulatory and co-inhibitory members. Encoded by chromosome 15q24, B7-H3 consists of an extracellular domain, a transmembrane domain, and a short intracellular domain. Extensive immunohistochemical (IHC) analysis of normal tissues has revealed that B7-H3 is expressed at low levels or even undetectable in only a few tissues. However, B7-H3 has been found to be overexpressed in various solid tumors, including bladder cancer, prostate cancer, and melanoma. Furthermore, as research progresses, there is increasing evidence that B7-H3 exerts a co-inhibitory function primarily in immune cells, which facilitates evasion of immune surveillance by tumor cells. Therefore, B7-H3 overexpression is associated with poor prognosis in patients with tumors and correlates with tumor invasion and metastatic potential in in vitro models.

[0004] Currently, more than 20 clinical trials are underway or in progress to evaluate the safety and efficacy of B7-H3 targeted monoclonal antibody immunotherapy strategies. In addition, numerous B7-H3 targeted antibody-drug conjugates (ADCs) are in clinical trials worldwide.

[0005] DS-7300, a B7-H3 targeted ADC developed by Daiichi Sankyo Co. Ltd., is produced by conjugating the linker-payload MC-GGFG-Dxd to the anti-B7-H3 antibody hM30 via stochastic conjugation, resulting in a B7-H3 targeted ADC with a drug-to-antibody ratio (DAR) of 4. Its structure is as follows: [ka]

[0006] MGC018, a B7-H3 targeted ADC developed by MacroGenics, Inc., consists of an anti-B7-H3 humanized IgG1 monoclonal antibody conjugated to duocalmycin (DUBA) via a cleavable linker, with an average DAR of 2.7. Its structure is as follows: [ka]

[0007] BAT8009, a B7-H3 targeted ADC developed by Bio-Thera Solutions Ltd., consists of a recombinant anti-B7-H3 humanized antibody conjugated to a DNA topoisomerase inhibitor via a cleavable linker.

[0008] ABBV-155, a B7-H3 targeted ADC developed by AbbVie Inc., consists of an anti-B7-H3 antibody conjugated via a linker to a pro-apoptotic BCL-XL inhibitor, and is intended for the treatment of advanced solid tumors. Its structure is as follows: [ka]

[0009] While limited, all of the aforementioned ADC agents have demonstrated certain therapeutic efficacy. For example, in 2021, MacroGenics reported the results of a Phase I clinical trial of MGC018 for the treatment of advanced solid tumors (cohort expansion, NCT03729596). Regarding safety, 43 patients (50%) experienced treatment-related adverse events (TRAEs) of grade 3 or higher, with common serious TRAEs including neutropenia (22.1%) and thrombocytopenia (7%). In contrast, DS-7300, developed by Daiichi Sankyo Co., Ltd., employs a stochastic conjugation method, resulting in significant heterogeneity among ADC products.

[0010] Currently, the selection of B7-H3 targeted ADCs containing camptothecin remains limited. Furthermore, given the aforementioned limitations associated with existing ADCs in this class, there is still a need in the art to provide a camptothecin-containing B7-H3 targeted ADC that demonstrates improved targeting, reduced product heterogeneity, minimized side effects, and consequently, improved therapeutic efficacy.

[0011] Patent Document 1, filed on December 2, 2021, and titled "Anti-Human B7-H3 Antibody and Application Thereof," discloses an anti-human B7-H3 mouse monoclonal antibody and an anti-human B7-H3 humanized monoclonal antibody. In this application, an anti-human B7-H3 mouse monoclonal antibody capable of binding to the B7-H3 extracellular domain was created using the recombinant extracellular domain of human B7-H3 as an immunogen by hybridoma technology. Subsequently, a human-mouse chimeric antibody capable of specifically binding to B7-H3 on the cell surface was constructed based on the mouse antibody. Furthermore, humanized antibodies were developed by CDR transplantation and CDR mutagenesis. These humanized antibodies retained the ability to specifically bind to both the extracellular domain and cell membrane-bound B7-H3 of human B7-H3 and were able to undergo intracellular translocation mediated by cell membrane-bound B7-H3. However, while the mouse antibodies and humanized antibodies described in this application exhibit desirable functional properties, there is still room for further improvement. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] Chinese Patent Application No. 202111459992.0 (Chinese Patent Application Publication No. 114573695) [Overview of the project]

[0013] The inventors of this invention have found that the anti-B7-H3 humanized antibody disclosed in Patent Document 1 exhibits high affinity for human B7-H3, but is susceptible to fragmentation. In practical use of the antibody, such fragmentation leads to increased heterogeneity in the antibody-containing product, which in turn increases immunogenicity, reduces efficacy, and can cause toxic side effects. Therefore, this humanized antibody is subject to certain limitations in both direct use and further product development.

[0014] Therefore, based on this known anti-B7-H3 humanized antibody, the inventors screened for novel anti-B7-H3 antibodies that could overcome the aforementioned limitations by using site-directed mutagenesis in the variable region. Furthermore, the inventors aimed to identify antibody-drug conjugates (ADCs) that improve B7-H3 targeting and enhance cytotoxicity against B7-H3 expressing cells by conjugating the mutant antibody with a specific structure of camptothecin.

[0015] Therefore, an object of this disclosure is to provide an antibody or fragment thereof that specifically binds to B7-H3. Another object of this disclosure is to provide a B7-H3 targeted antibody-drug conjugate or salt thereof prepared using the antibody or fragment thereof.

[0016] In the context of this disclosure, halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0017] In the context of this disclosure, the terms “linker” and “linker compound” are used interchangeably.

[0018] In the context of this disclosure, the term “linker-payload” refers to a compound obtained by the direct or indirect covalent bonding of a drug (e.g., a small molecule drug such as a camptothecin compound) to a linker.

[0019] This disclosure provides the following technical solutions.

[0020] First aspect This disclosure relates to an anti-B7-H3 antibody or fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain and light chain have the following amino acid sequence combination: (i) The amino acid sequence shown in Sequence ID No. 1 and the amino acid sequence shown in Sequence ID No. 3, (ii) The amino acid sequence shown in Sequence ID No. 1 and the amino acid sequence shown in Sequence ID No. 4, or (iii) The amino acid sequence shown in Sequence ID No. 1 and the amino acid sequence shown in Sequence ID No. 5, The present invention provides an anti-B7-H3 antibody or fragment thereof comprising three heavy chain complementarity-determining regions, namely CDR-H1, CDR-H2, and CDR-H3, derived from and three light chain complementarity-determining regions, namely CDR-L1, CDR-L2, and CDR-L3.

[0021] In the context of this disclosure, the term “fragment” encompasses a variety of functional fragments of an anti-B7-H3 antibody that retain the antibody’s ability to bind to an antigen and the corresponding biological activity. It is generally known in the art that the antigen-binding ability and corresponding biological activity of an antibody can be achieved by fragments of a full-length antibody. Such fragments can be obtained using conventional techniques known to those skilled in the art and can be screened for functionality in the same way as full-length antibodies. For example, antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Exemplary fragments may include Fab fragments, F(ab')2 fragments, scFv fragments, and the like.

[0022] The above amino acid sequences correspond to the amino acid sequences of the heavy chain variable region and light chain variable region of the specific anti-B7-H3 antibody provided in this disclosure; please refer to the “Detailed Description of Preferred Embodiments” section of this disclosure. By applying generally known or conventional defining tools for complementarity-determining regions (CDRs) in the heavy chain or light chain variable region of an antibody (e.g., Kabat definition, AbM definition, Chothia definition, Contact definition, IMGT definition, or extended Chothia / AbM definition), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs included when these variable regions combine to form an antibody. For example, in Example 1 of this disclosure, the position of the amino acid sequence of the CDR within the full-length variable region is specified when defined according to the Kabat definition. Based on these generally known or conventional defining tools in the art, combinations of heavy chain CDRs and light chain CDRs can be derived, and antibodies or fragments thereof containing any combination of these heavy chain CDRs and light chain CDRs are within the scope of the present invention.

[0023] Furthermore, the anti-B7-H3 antibody or fragment thereof provided in this disclosure is 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 group consisting of: (i) CDR-H1, CDR-H2, and CDR-H3 each contain an amino acid sequence that is 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 CDR-L1, CDR-L2, and CDR-L3 each contain an amino acid sequence that is at least 75% identical to the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, (ii) CDR-H1, CDR-H2, and CDR-H3 each contain an amino acid sequence that is 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 CDR-L1, CDR-L2, and CDR-L3 each contain an amino acid sequence that is at least 75% identical to the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15, and, (iii) CDR-H1, CDR-H2, and CDR-H3 each contain an amino acid sequence that is 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 CDR-L1, CDR-L2, and CDR-L3 each contain an amino acid sequence that is at least 75% identical to the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, It may include.

[0024] In the context of this disclosure, the term “at least 75% identity” encompasses any percentage of identity from at least 75% to 100% identity, such as 75%, 80%, 85%, 90%, and further 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. Depending on the context in which the term is used, “at least 75% identity” allows for up to 25% difference in the amino acid sequence, and this difference may be located within the heavy chain CDR, the light chain CDR, or any region outside the heavy chain CDR or light chain CDR. Such regions include, but are not limited to, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or fragment provided by this disclosure, or the heavy chain constant region and the light chain constant region of the antibody or fragment provided by this disclosure. The differences may result from the deletion, addition, or substitution of amino acids at any position, and the substitutions may be conservative or non-conservative.

[0025] According to specific embodiments of the present invention, the anti-B7-H3 antibody or fragment thereof provided by this disclosure is 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 group consisting of: (i) CDR-H1, CDR-H2, and CDR-H3 each contain the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 each contain the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, (ii) CDR-H1, CDR-H2, and CDR-H3 each contain the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 each contain the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15, and, (iii) CDR-H1, CDR-H2, and CDR-H3 each contain the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 each contain the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, It may include.

[0026] The antibodies or fragments thereof provided in this disclosure may be antibodies or fragments thereof that bind to B7-H3, in particular to human B7-H3. The amino acid sequence of human B7-H3 is shown exemplarily in UniProtKB-Q5ZPR3.

[0027] The anti-B7-H3 antibody or fragment thereof provided by this disclosure may include at least a heavy chain variable region (VH) and a light chain variable region (VL). Both regions include not only the CDR described above, but also framework regions (FRs) scattered between them, which are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, in the anti-B7-H3 antibody or fragment thereof provided by this disclosure, the heavy chain variable region may include 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 shown in SEQ ID NO: 1, and / or the light chain variable region may include the amino acid sequence shown in any one of SEQ ID NOs: 3 to 5, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 5.

[0028] Preferably, in the antibody or fragment thereof provided by this disclosure, the heavy chain variable region and the light chain variable region are combinations of sequences selected from the group consisting of: (i) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3, (ii) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 4, and (iii) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 5. It may include.

[0029] According to specific embodiments of the present invention, the antibody or fragment thereof provided by this disclosure is a combination of a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 3, (ii) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 4, and (iii) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5. It may include.

[0030] The anti-B7-H3 antibody or fragment thereof provided by this disclosure may be in any form containing the domains defined above, such as a monoclonal antibody, a chimeric antibody, a partially humanized antibody, or a fully humanized antibody. The antibody or fragment thereof provided by this disclosure may also be in the form of scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, Fv, etc.

[0031] In addition to the variable region, the anti-B7-H3 antibody or fragment thereof provided by this disclosure may further include 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 of 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 this disclosure may include a heavy chain and / or a light chain, where, for example, the heavy chain may include a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and the light chain may include a κ-type or λ-type light chain constant region.

[0032] Preferably, the disclosure provides an anti-B7-H3 humanized antibody or a fragment thereof. More preferably, the disclosure provides an anti-B7-H3 humanized monoclonal antibody or a fragment thereof.

[0033] This disclosure provides isolated, structurally characterized humanized monoclonal antibodies that specifically bind to B7-H3. According to specific embodiments of the present invention, the anti-B7-H3 antibody provided by this disclosure is hz10B4m1, hz10B4m2, or hz10B4m3, as detailed in the “Detailed Description of Preferred Embodiments” section of this disclosure.

[0034] Second aspect The present invention further provides a nucleic acid molecule comprising a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain, and / or a nucleotide sequence encoding the light chain, which is contained in the anti-B7-H3 antibody or a fragment thereof according to the present invention.

[0035] Third aspect The nucleic acid molecules provided by this disclosure can be cloned into a vector, which can then be used to transform or transfect host cells. Accordingly, in a third embodiment, this disclosure provides a vector comprising the nucleic acid molecules according to the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0036] Fourth aspect The nucleic acid molecules or vectors provided in this disclosure can be used to transform or transfect host cells for purposes such as antibody storage or expression. Accordingly, in a fourth aspect, this disclosure provides host cells comprising the nucleic acid molecules and / or vectors according to the present invention, or host cells transformed or transfected with the nucleic acid molecules and / or vectors according to the present invention. The host cells may be any prokaryotic or eukaryotic cells, such as bacterial cells, insect cells, fungal cells, plant cells, or animal cells.

[0037] The anti-B7-H3 antibody or fragment thereof provided by this disclosure can be obtained using any method known in the art. For example, the host cells provided by this disclosure are cultured under conditions that allow the host cells to express the heavy and light chains of the antibody. Optionally, this method may further include a step of recovering the generated antibody. As described above, for example, the antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. The anti-B7-H3 antibody or fragment thereof provided by this disclosure can be obtained using any method known in the art. For example, the host cells provided by this disclosure are cultured under conditions that allow the host cells to express the heavy and / or light chains of the antibody so that they can be assembled to form an antibody. Optionally, this method may further include a step of recovering the generated antibody.

[0038] Fifth aspect The anti-B7-H3 antibody or its fragment, nucleic acid molecule, vector, and / or host cell provided by this disclosure can be incorporated into a pharmaceutical composition, more particularly a pharmaceutical preparation, and used for a variety of purposes as required by practice. Accordingly, in a fifth aspect, this disclosure also provides a composition comprising the anti-B7-H3 antibody or its fragment, nucleic acid molecule, vector, and / or host cell according to the present invention. Preferably, the composition is a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier, adjuvant, or additive.

[0039] Sixth aspect This disclosure provides the use of anti-B7-H3 antibodies or their fragments, nucleic acid molecules, vectors, host cells, and / or compositions in the manufacture of pharmaceuticals for the treatment of diseases associated with 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic round cell tumor.

[0040] Seventh aspect This disclosure further provides the use of anti-B7-H3 antibodies or their fragments, nucleic acid molecules, vectors, host cells, and / or compositions in the manufacture of diagnostic agents for diseases associated with 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic round cell tumor.

[0041] Eighth aspect This disclosure also provides a method for treating a disease associated with B7-H3 expression (including overexpression), comprising administering an anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition according to the present invention to a subject in need of treatment, optionally together with one of the other drugs or means. The other drugs or means refer to other drugs or means that can be administered concurrently with the antibody or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition according to the present invention, such as small molecule chemicals, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene therapies and nucleic acid therapies, and radiotherapy. The two concurrent administrations can be carried out in any form, for example, simultaneously, sequentially, or at set intervals.

[0042] 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrous round cell tumor.

[0043] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0044] The methods for treating the aforementioned diseases provided in this disclosure, where applicable, depend on a number of factors, including the specific active ingredients administered, the patient's age, weight, sex, physical and medical condition, the severity of the condition being treated, and the route of administration.

[0045] Ninth aspect This disclosure also provides a method for diagnosing diseases associated with B7-H3 expression (including overexpression), comprising contacting an anti-B7-H3 antibody or fragment thereof, nucleic acid molecules, vectors, host cells, and / or compositions according to the present invention with a sample derived 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic round cell tumor.

[0046] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0047] Tenth aspect This disclosure further provides kits comprising an anti-B7-H3 antibody or fragment thereof, nucleic acid molecules, vectors, host cells, and / or compositions according to the present invention. The kits may be intended for the therapeutic or diagnostic purposes described above. Optionally, the kits may include instructions for use.

[0048] Eleventh aspect As described above, another object of this disclosure is to provide B7-H3 targeted antibody-drug conjugates or salts thereof. Preferably, the B7-H3 targeted antibody-drug conjugates or salts thereof are prepared using the anti-B7-H3 antibody or fragment thereof provided by this disclosure. Accordingly, in the eleventh aspect, this disclosure relates to structural formula Ia and / or structural formula Ib: [ka] Ia and / or [ka] Ib The present invention provides an antibody-drug conjugate or salt thereof having a structure represented by structural formula Ia and / or structural formula Ib, where Ab is an anti-B7-H3 antibody or a fragment thereof. Preferably, the antibody or fragment thereof is an anti-B7-H3 antibody or fragment thereof as defined in the first embodiment of the present disclosure described above.

[0049] In structural formulas Ia and / or Ib, unless otherwise specified in the context of this disclosure, group M is phenylene or phenylene substituted with one or more substituents, or a chemical bond, wherein in the substituted phenylene, the phenylene is substituted with substituents (which may be more than one) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, e.g., trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano, preferably group M is halogen-substituted phenylene.

[0050] In structural formula Ia and / or structural formula Ib, unless otherwise specified in the context of this disclosure, group SP1 is selected from the group consisting of C1-C8 alkylenes, C1-C8 cycloalkylenes, and C1-C21 (preferably C1-C16, more preferably C1-C11, more preferably C5-C9) linear heteroalkylenes containing 1 to 11 (preferably 1 to 6, more preferably 3 to 5) heteroatoms selected from the group consisting of N, O, and S, where the C1-C8 alkylenes, C1-C8 cycloalkylenes, and C1-C21 linear heteroalkylenes are independently substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano groups.

[0051] In structural formula Ia and / or structural formula Ib, unless otherwise specified in the context of this disclosure, the base SP2 is -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) a A is selected from the group consisting of CO- and chemical bonds, where a is an integer in the range of 1 to 20, preferably an integer in the range of 1 to 10, and more preferably an integer in the range of 1 to 6.

[0052] In structural formula Ia and / or structural formula Ib, unless otherwise specified in the context of this disclosure, group A means a peptide group of 2 to 4 amino acids. If group A means a peptide group of 2 amino acids, it may be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO, or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO, or NH-Val-Cit-CO. If group A means a peptide group of three amino acids, it may be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO, or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO. If group A means a peptide group of four amino acids, it may be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, group A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO, or NH-Ala-Ala-Ala-CO. In structural formula Ia and / or structural formula Ib, group A is bonded to group SP2 via the amino at the amino terminus of its short peptide structure.

[0053] In structural formula Ia and / or structural formula Ib, group M is preferably halogen-substituted phenylene, and more particularly fluorine-substituted phenylene.

[0054] In structural formula Ia and / or structural formula Ib, group SP1 is preferably a C1-C11, preferably C5-C9, more preferably C7 linear heteroalkylene containing 1 to 6, preferably 3 to 5, more preferably 4 heteroatoms selected from the group consisting of N, O, and S.

[0055] In structural formula Ia and / or structural formula Ib, the group SP2 is preferably a chemical bond.

[0056] In structural formula Ia and / or structural formula Ib, unless otherwise specified in the context of this disclosure, m is in the range of 1 to 10, preferably 1 to 8 (e.g., 1 to 5), and more preferably 3 to 8. m may be an integer or a non-integer.

[0057] Furthermore, the antibody-drug conjugate or salt thereof provided by this disclosure has structural formula Ic and / or structural formula Id: [ka] I C and / or [ka] ID (In structural formula Ic and / or structural formula Id, Ab, base A, and m have the same meanings as defined above for Ab, base A, and m in structural formula Ia and / or structural formula Ib.) The structure may be represented by

[0058] In any one of structural formulas Ia, Ib, Ic, and Id, unless otherwise specified in the context of this disclosure, the group CPT refers to a camptothecin compound.

[0059] Furthermore, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT is structural formula I: [ka] Structural formula I The structure may be represented by the following, where structural formula I is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond, and preferably the amino adjacent to group G in structural formula I is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond.

[0060] In structural formula I, unless otherwise specified in the context of this disclosure, groups R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C1-C6 alkoxy, amino or substituted amino, and C1-C7 alkyl or substituted C1-C7 alkyl, or any two of groups R1, R2, R3, and R4 together with the carbon atoms to which they are bonded to form a C3-C6 (preferably C3-C5) cycloalkyl. When groups R1, R2, R3, and R4 are independently C1-C6 alkoxy, the C1-C6 alkoxy may be a linear or branched C1-C6 alkoxy, preferably a linear or branched C1-C3 alkoxy, more preferably methoxy. When groups R1, R2, R3, and R4 are independently substituted amino, the substituted amino is an amino substituted with one or more substituents selected from the group consisting of methyl and ethyl. When groups R1, R2, R3, and R4 are independently C1-C7 alkyl or substituted C1-C7 alkyl groups, examples of C1-C7 alkyl or substituted C1-C7 alkyl groups include linear or branched C1-C7 (preferably C3-C5, more preferably C4) alkyl groups or substituted C1-C7 (preferably C3-C5, more preferably C4) alkyl groups, where the substituted C1-C7 alkyl group is a C1-C7 alkyl group substituted with one or more substituents selected from the group consisting of cyclopropyl and cyclobutyl, or the linear or branched C1-C7 alkyl group or substituted C1-C7 alkyl group is preferably a C1-C3 alkyl group or substituted C1-C3 alkyl group, such as methyl and halomethyl (preferably trifluoromethyl).

[0061] In structural formula I, unless otherwise specified in the context of this disclosure, group G is hydrogen, halogen, methyl, or methoxy. Preferably, group G is hydrogen, fluorine, or chlorine.

[0062] In structural formula I, unless otherwise specified in the context of this disclosure, group Y is oxygen, sulfur, sulfone, sulfoxide, methylene, or substituted methylene. In the substituted methylene, either one of the hydrogens of the methylene may be substituted with a substituent that may be benzyl or alkyl, or both hydrogens of the methylene may be substituted with the substituent. If the substituent is alkyl, the alkyl and R3 and / or R4 may together with the carbon atom to which they are bonded to form a C3-C6 fused ring structure or a spiro-ring structure. If both hydrogens of the methylene are substituted with alkyl, the two alkyls may together with group Y to form a C3-C6 spiro-ring structure. If group Y is substituted methylene, the substituted methylene is preferably alkyl-substituted methylene, more preferably a linear or branched C1-C4 alkyl.

[0063] Preferably, group Y is oxygen, sulfur, sulfone, sulfoxide, or methylene, or preferably, group Y is oxygen, sulfur, or methylene.

[0064] In structural formula I, unless otherwise specified in the context of this disclosure, group X is oxygen or sulfur.

[0065] In structural formula I, n = 0 or 1 unless otherwise specified in the context of this disclosure.

[0066] In structural formula I, when groups R1, R2, R3, and R4 are simultaneously hydrogen, group X is oxygen, and n=0, if group Y is methylene, then group G cannot be either hydrogen or fluorine, and if group Y is oxygen or sulfur, then group G cannot be hydrogen.

[0067] Preferably, groups R1, R2, R3, and R4 are independently hydrogen, halogen (e.g., fluorine), C1-C7 alkyl or substituted C1-C7 alkyl, or any two of groups R1, R2, R3, and R4 together with the carbon atoms to which they are bonded to form a C3-C6 cycloalkyl (e.g., C3-C5 cycloalkyl). Furthermore, groups R1 and R2 may be the same, and / or groups R3 and R4 may be the same.

[0068] Preferably, group Y is an alkyl-substituted methylene group, and the alkyl group and R3 and / or R4 may together with the carbon atom to which they are bonded to form a C3-C6 fused ring structure or a spiro ring structure.

[0069] Preferably, group X may be oxygen.

[0070] Preferably, group X is oxygen, group G is hydrogen, halogen (e.g., fluorine or chlorine), methyl or methoxy, and groups Y and R1, R2, R3, and R4 are defined as described above.

[0071] Preferably, group X is oxygen, group G is hydrogen, group Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide, or sulfone, and groups R1, R2, R3, and R4 are defined as above.

[0072] Preferably, group X is oxygen, group G is fluorine, group Y is methylene or substituted methylene, oxygen, or sulfur, and groups R1, R2, R3, and R4 are defined as above.

[0073] Preferably, group X is oxygen, group G is chlorine, group Y is methylene or substituted methylene, oxygen, or sulfur, and groups R1, R2, R3, and R4 are defined as above.

[0074] Preferably, group X is oxygen, group G is methyl, group Y is methylene or substituted methylene, oxygen, or sulfur, and groups R1, R2, R3, and R4 are defined as above.

[0075] Preferably, group X is oxygen, group G is methoxy, group Y is methylene or substituted methylene, oxygen, or sulfur, and groups R1, R2, R3, and R4 are defined as above.

[0076] Preferably, group X is oxygen, group G is hydrogen, group Y is methylene, sulfoxide, sulfone, oxygen, or sulfur, groups R1 and R2 are independently hydrogen, fluorine, or methyl, and groups R3 and R4 are independently hydrogen.

[0077] Preferably, group X is oxygen, group G is fluorine, group Y is methylene, sulfoxide, sulfone, oxygen, or sulfur, groups R1 and R2 are independently hydrogen, fluorine, or methyl, and groups R3 and R4 are independently hydrogen.

[0078] Preferably, n=0.

[0079] According to a specific embodiment of the present invention, in structure I: (1) Group G is hydrogen, group Y is methylene, both groups R1 and R2 are methyl, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (2) Group G is hydrogen, group Y is methylene, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (3) Group G is hydrogen, group Y is methylene, one of groups R1 and R2 and one of groups R3 and R4 form a C3 cycloalkyl group together with the carbon atom to which they are bonded, the other of groups R1 and R2 and the other of groups R3 and R4 are hydrogen, group X is oxygen and n=0, (4) Group G is hydrogen, group Y is sulfur, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (5) Group G is hydrogen, group Y is a sulfoxide, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (6) Group G is hydrogen, group Y is sulfur, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (7) Group G is hydrogen, group Y is sulfone, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (8) Group G is hydrogen, group Y is methylene, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=1. (9) Group G is fluorine, group Y is oxygen, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (10) Group G is fluorine, group Y is sulfur, both groups R1 and R2 are hydrogen, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (11) Group G is fluorine, group Y is oxygen, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (12) Group G is fluorine, group Y is methylene, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0. (13) Group G is hydrogen, group Y is oxygen, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0, or (14) Group G is fluorine, group Y is sulfur, both groups R1 and R2 are fluorine, both groups R3 and R4 are hydrogen, group X is oxygen, and n=0.

[0080] Preferably, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT is structural formula IA: [ka] Structural formula IA The structure may be represented by the following, where structural formula IA is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond, and preferably the amino on the left-hand benzene ring in structural formula IA is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond.

[0081] In structural formula IA, groups R1, R2, R3, and R4 have the same meanings as defined above for groups R1, R2, R3, and R4 in structural formula I, but groups R1, R2, R3, and R4 are not hydrogen atoms at the same time.

[0082] Alternatively, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT is structural formula II: [ka] Structural formula II The structure may be represented by the following, where structural formula II is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond, and preferably the amino adjacent to group G in structural formula II is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond.

[0083] In structural formula II, unless otherwise specified in the context of this disclosure, group R5 is a C1-C5 alkyl group or a C1-C5 alkyl group substituted with one or more substituents, a C3-C6 cycloalkyl group or a C3-C6 cycloalkyl group substituted with one or more substituents, a phenyl group, or a substituted phenyl group. When group R5 is a C1-C5 alkyl group or a substituted C1-C5 alkyl group, examples of C1-C5 alkyl groups include linear or branched C1-C5 alkyl groups. Furthermore, group R5 is a C1-C4 linear alkyl group. If group R5 is a substituted C1-C5 alkyl or a substituted C3-C6 cycloalkyl, the substituted C1-C5 alkyl or substituted C3-C6 cycloalkyl is a C1-C5 alkyl or C3-C6 cycloalkyl substituted with substituents (may be multiple) selected from the group consisting of halogens, hydroxyl, methoxy, trifluoromethyl, amino or substituted aminos, methanesulfonyl, and C3-C6 cycloalkyls, and among the substituents (may be multiple), the substituted amino is an amino substituted with one or more substituents selected from the group consisting of methyl and ethyl. If group R5 is a substituted phenyl, the substituted phenyl is a phenyl substituted with substituents (may be multiple) selected from the group consisting of alkyls (e.g., C1-C6 alkyl, preferably C1-C3 alkyl) and halogens.

[0084] In structural formula II, unless otherwise specified in the context of this disclosure, group G is hydrogen, halogen (e.g., fluorine), methyl, or methoxy. Preferably, group G is hydrogen, fluorine, or chlorine.

[0085] In structural formula II, group X is either oxygen or sulfur.

[0086] In structural formula II, n = 0 or 1.

[0087] In structural formula II, if group X is oxygen, group G is hydrogen, and n=0, then group R5 cannot be n-butyl.

[0088] Preferably, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT is structural formula IIA: [ka] Structural formula IIA The structure may be represented by the following, where structural formula IIA is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond, and preferably the amino on the left-hand benzene ring in structural formula IIA is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond.

[0089] In structural formula IIA, group R5 has the same meaning as group R5 in structural formula II as defined above, but group R5 cannot be n-butyl.

[0090] According to a specific embodiment of the present invention, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT has the structure shown below: [ka] [ka] [ka] The structure may have such a configuration, where each structure is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond, and preferably the amino on the left-hand benzene ring in each structure is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via an amide bond.

[0091] Furthermore, in any one of structural formulas Ia, Ib, Ic, and Id, the base CPT is structural formula IV: [ka] Structural formula IV It may have a structure represented by, where the hydroxyl bonded to the same carbon as group R8 in structural formula IV is bonded to the carboxyl of group A in any one of structural formulas Ia, Ib, Ic, and Id via a self-sacrificing structure. The self-sacrificing structure is, for example, [ka] or [ka] Here, the solid line "-" indicates the bond of group A to the carboxyl in any one of structural formulas Ia, Ib, Ic, and Id, and the wavy line [ka] This refers to the bond to the hydroxyl group in structural formula IV.

[0092] In structural formula IV, unless otherwise specified in the context of this disclosure, group R8 is hydrogen, trifluoromethyl, a C1-C5 alkyl group or a C1-C5 alkyl group substituted with one or more substituents, a C3-C6 cycloalkyl group or a C3-C6 cycloalkyl group substituted with one or more substituents, or a halogen.

[0093] When group R8 is a substituted C1-C5 alkyl or a substituted C3-C6 cycloalkyl, the C1-C5 alkyl or C3-C6 cycloalkyl is substituted with substituents (may be multiple) selected from the group consisting of halogens, hydroxyl, methoxy, trifluoromethyl, amino or substituted aminos, methanesulfonyl, and C3-C6 cycloalkyls, and among the substituents, the substituted amino is an amino that is substituted with one or more substituents selected from the group consisting of methyl and ethyl.

[0094] The 12th aspect This disclosure is a general formula [ka] The present invention provides an antibody-drug conjugate or salt thereof having a structure represented by (wherein mAb is an anti-B7-H3 antibody or a fragment thereof). Preferably, mAb is an anti-B7-H3 antibody or a fragment thereof as defined in the first embodiment of the present disclosure above.

[0095] In the general formula, base M, base SP1, base SP2, base A, and base CPT have the same meanings as defined for base M, base SP1, base SP2, base A, and base CPT in the 11th embodiment described above, and N is in the range of 1 to 10, preferably 1 to 8 (e.g., 1 to 5), more preferably 3 to 8.

[0096] In that general formula, E L The following is the basis: E L -1a and / or E L -1b: [ka] and / or [ka] E L -2: [ka] EL -3:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0097] The 13th aspect The present disclosure provides a linker-payload having a structure represented by the general formula "L-A-CPT" (wherein the group L means a linker used in an antibody-drug conjugate (ADC), the group A means a peptide group of one or more amino acids, and the group CPT is a compound of camptothecin).

[0098] In the 13th aspect of the present disclosure, the linker-payload having a structure represented by the general formula "L-A-CPT" is a compound represented by Structural Formula III:

Chem.

[0099] In structural formula III, unless otherwise specified in the context of this disclosure, E is the following base: E-1: [ka] E-2: [ka] E-3: [ka] E-4: [ka] E-5: [ka] and E-6: [ka] (In the formula, the dashed line) [ka] The group consisting of (where signifies binding to group M) is selected.

[0100] In structural formula III, unless otherwise specified in the context of this disclosure, group M is phenylene or phenylene substituted with one or more substituents, or a chemical bond, wherein in the substituted phenylene, the phenylene is substituted with substituents (which may be more than one) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, e.g., trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano, and preferably, group M is halogen-substituted phenylene.

[0101] In structural formula III, unless otherwise specified in the context of this disclosure, group SP1 is selected from the group consisting of C1-C8 alkylenes, C1-C8 cycloalkylenes, and C1-C21 (preferably C1-C16, more preferably C1-C11, more preferably C5-C9) linear heteroalkylenes containing 1 to 11 (preferably 1 to 6, more preferably 3 to 5) heteroatoms selected from the group consisting of N, O, and S, where the C1-C8 alkylenes, C1-C8 cycloalkylenes, and C1-C21 linear heteroalkylenes are independently substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano groups.

[0102] In structural formula III, unless otherwise specified in the context of this disclosure, the group SP2 is -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) a A is selected from the group consisting of CO- and chemical bonds, where a is an integer in the range of 1 to 20, preferably an integer in the range of 1 to 10, and more preferably an integer in the range of 1 to 6.

[0103] In structural formula III, unless otherwise specified in the context of this disclosure, group A represents a peptide group of 2 to 4 amino acids. When group A represents a peptide group of 2 amino acids, it may be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO, or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO, or NH-Val-Cit-CO. If group A means a peptide group of three amino acids, it may be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO, or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO. If group A means a peptide group of four amino acids, it may be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, group A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO, or NH-Ala-Ala-Ala-CO. In structural formula III, group A is bonded to group SP2 via the amino at the amino terminus of its short peptide structure.

[0104] In structural formula III, group M is preferably halogen-substituted phenylene, and more particularly fluorine-substituted phenylene.

[0105] In structural formula III, group SP1 is preferably a linear heteroalkylene of C1-C11, preferably C5-C9, more preferably C7, containing 1 to 6, preferably 3 to 5, more preferably 4 heteroatoms selected from the group consisting of N, O, and S.

[0106] In structural formula III, the group SP2 is preferably a chemical bond.

[0107] In structural formula III, unless otherwise specified in the context of this disclosure, the group CPT is a compound of camptothecin.

[0108] If base E is E1, structural formula III provided by this disclosure is further structural formula IIIA: [ka] Structural formula IIIA That's fine.

[0109] In structural formula IIIA, unless otherwise specified in the context of this disclosure, groups R6 and R7 are independently hydrogen, halogen, or Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents, where phenyl is substituted with substituents (may be more than one) selected from the group consisting of 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 or phenyl substituted with 4-formylmethylamine. [ka] , or phenyl substituted with 4-formylmorpholine [ka] That is the case.

[0110] For example, in structural formulas III and IIIA, the group CPT is one of the compounds represented by structural formula I or structural formula IA, or their pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs, and the corresponding specific compounds, all of which are shown in the eleventh aspect of the present disclosure described above.

[0111] The base CPT is structural formula I as shown in the eleventh aspect of the present disclosure above: [ka] Structural formula I In the case of a compound represented by structural formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, groups G, X, Y, R1, R2, R3, R4, and n in structural formula I have the same meaning as defined for groups G, X, Y, R1, R2, R3, R4, and n in structural formula I in the 11th embodiment described above. Furthermore, in structural formula III or structural formula IIIA, structural formula I is bonded to the carboxyl of group A via an amide bond, and preferably, the amino adjacent to group G in structural formula I is bonded to the carboxyl of group A via an amide bond. That is, an amide bond is formed between the amino of the compound represented by structural formula I and the carboxyl of group A in structural formula III or structural formula IIIA.

[0112] The base CPT is structural formula IA as shown in the eleventh aspect of the present disclosure above: [ka] Structural formula IA In the case of a compound represented by structural formula IA, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, groups R1, R2, R3, and R4 in structural formula IA have the same meaning as defined for groups R1, R2, R3, and R4 in structural formula IA in the 11th embodiment described above, except that R1, R2, R3, and R4 can simultaneously be hydrogen. Furthermore, in structural formula III or structural formula IIIA, structural formula IA is bonded to the carboxyl of group A via an amide bond, preferably the amino on the benzene ring on the left side of structural formula IA is bonded to the carboxyl of group A via an amide bond. That is, an amide bond is formed between the amino of the compound represented by structural formula IA and the carboxyl of group A in structural formula III or structural formula IIIA.

[0113] In particular, when the group "CPT" in the compound represented by structural formula IIIA is the compound represented by structural formula IA, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, groups R6 and R7 may or may not be hydrogen at the same time. Similarly, groups R1, R2, R3, and R4 in structural formula IA may or may not be hydrogen at the same time. According to a specific embodiment of the present invention, when groups R6 and R7 are hydrogen at the same time, groups R1, R2, R3, and R4 in structural formula IA cannot be hydrogen at the same time, and when groups R6 and R7 are not hydrogen at the same time, groups R1, R2, R3, and R4 in structural formula IA may or may not be hydrogen at the same time.

[0114] In another example, in structural formulas III and IIIA, the group CPT is one of the compounds represented by structural formula II or structural formula IIA, or their pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs, and the corresponding specific compounds, all of which are shown in the eleventh aspect of the present disclosure above.

[0115] The base CPT is structural formula II as shown in the eleventh aspect of the present disclosure above: [ka] Structural formula II In the case of a compound represented by structural formula II, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, groups G, R5, X, and n in structural formula II have the same meanings as defined for groups G, R5, X, and n in structural formula II in the 11th embodiment described above. Furthermore, in structural formula III or structural formula IIIA, structural formula II is bonded to the carboxyl of group A via an amide bond, and preferably, the amino adjacent to group G in structural formula II is bonded to the carboxyl of group A via an amide bond. That is, an amide bond is formed between the amino of the compound represented by structural formula II and the carboxyl of group A in structural formula III or structural formula IIIA.

[0116] The base CPT is structural formula IIA as shown in the eleventh aspect of the present disclosure above: [ka] Structural formula IIA In the case of a compound represented by structural formula IIA, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, the group R5 in structural formula IIA has the same meaning as defined for the group R5 in structural formula IIA in the 11th embodiment above, except that the group R5 may be n-butyl. In this case, in structural formula III or structural formula IIIA, structural formula IIA is bonded to the carboxyl of group A via an amide bond, preferably the amino on the benzene ring on the left side of structural formula IIA is bonded to the carboxyl of group A via an amide bond. That is, an amide bond is formed between the amino of the compound represented by structural formula IIA and the carboxyl of group A in structural formula III or structural formula IIIA.

[0117] Each of the 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, and 40 shown in the 11th embodiment above, or their pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs may be used as the group "CPT" in structural formula III or structural formula IIIA, which is bonded to the carboxyl group A in structural formula III or structural formula IIIA via an amino acid by an amide bond.

[0118] In another example, in structural formulas III and IIIA, the base CPT is in structural formula IV: [ka] Structural formula IV This may be an exatecan derivative represented by , or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.

[0119] In structural formula IV, unless otherwise specified in the context of this disclosure, group R8 is hydrogen, trifluoromethyl, C1-C5 alkyl or C1-C5 alkyl substituted with one or more substituents, C3-C6 cycloalkyl or C3-C6 cycloalkyl substituted with one or more substituents, or halogen. Furthermore, in structural formulas III and IIIA, the hydroxyl bonded to the same carbon as group R8 in structural formula IV is bonded to the carboxyl of group A via a self-sacrificing structure. The self-sacrificing structure is, for example, [ka] or [ka] Here, the solid line "-" indicates the bond of group A to the carboxyl in structural formula III or structural formula IIIA, and the wavy line [ka] This refers to the bond to the hydroxyl group in structural formula IV.

[0120] When group R8 is a substituted C1-C5 alkyl or a substituted C3-C6 cycloalkyl, the C1-C5 alkyl or C3-C6 cycloalkyl is substituted with substituents (may be multiple) selected from the group consisting of halogens, hydroxyl, methoxy, trifluoromethyl, amino or substituted aminos, methanesulfonyl, and C3-C6 cycloalkyls, and among the substituents, the substituted amino is an amino that is substituted with one or more substituents selected from the group consisting of methyl and ethyl.

[0121] In particular, when the group "CPT" in the compound represented by structural formula IIIA is the compound represented by structural formula IV, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, groups R6 and R7 may be hydrogen at the same time, or not at the same time. Similarly, group R8 in structural formula IV may be hydrogen at the same time, or not. According to a specific embodiment of the present invention, when groups R6 and R7 are hydrogen at the same time, group R8 may be hydrogen at the same time, or not.

[0122] In particular, with respect to the compound represented by structural formula IIIA, when groups R6 and R7 are Ar'S, group M is preferably phenylene or substituted phenylene, while group SP1 is a C1-C21 (preferably C1-C16, more preferably C1-C11, more preferably C5-C9) linear heteroalkylene containing 1 to 11 (preferably 1 to 6, more preferably 3 to 5) heteroatoms selected from the group consisting of N, O, and S.

[0123] Preferably, the compounds represented by structural formula III or structural formula IIIA provided by this disclosure may further be structural formula V: [ka] Structural formula V (In structural formula V, groups R6 and R7 are independently Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents, and in the substituted phenyl, phenyl is substituted with substituents (may be more than one) selected from the group consisting of 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) the compound is represented by. Preferably, Ar' is phenyl, or phenyl substituted with 4-formylmethylamine [ka] , or phenyl substituted with 4-formylmorpholine [ka] That is the case.

[0124] In structural formula V, unless otherwise specified in the context of this disclosure, groups Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, e.g., trifluoromethyl), or alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, e.g., methoxy).

[0125] In structural formula V, unless otherwise specified in the context of this disclosure, m is any integer in the range of 1 to 10, preferably 1 to 5, and more preferably 3 to 5.

[0126] In structural formula V, group A represents the peptide groups of 2 to 4 amino acids as defined above.

[0127] In structural formula V, the meaning of the base CPT and its bonding relationship in structural formula V are the same as the meaning of the base CPT and its bonding relationship in structural formulas III and IIIA as defined above.

[0128] Preferably, the compound represented by structural formula V provided by this disclosure is further represented by structural formula VA: [ka] Structural formula VA (In structural formula VA, groups A, G, Y, R1, R2, R3, R4, X, and n have the same meanings as defined above for groups A, G, Y, R1, R2, R3, R4, X, and n in structural formula III or structural formula IIIA.)

[0129] Preferably, in the structural formula VA, the group G is hydrogen, fluorine, or chlorine.

[0130] Preferably, in Structural Formula V-A, group Y is methylene, sulfur, or oxygen.

[0131] In Structural Formula V-A, unless otherwise specified in the context of the present disclosure, “-A-NH-” means that an amide bond is formed between the amino group and the carboxyl group of group A.

[0132] Preferably, the compound represented by Structural Formula V-A provided by the present disclosure further has the Structural Formula V-A-1:

Chemical formula

[0133] Alternatively, the compound represented by Structural Formula V provided by the present disclosure further has the Structural Formula V-B:

Chemical formula

[0134] In Structural Formula V-B, unless otherwise specified in the context of the present disclosure, “-A-NH-” means that an amide bond is formed between the amino group and the carboxyl group of group A.

[0135] Preferably, the compound represented by Structural Formula V-B provided by the present disclosure further has the Structural Formula V-B-1:

Chemical formula

[0136] Alternatively, the compound represented by structural formula V provided in this disclosure may also be represented by structural formula VC: [ka] Structural formula VC (In structural formula VC, groups A and R8 have the same meanings as defined above for groups A and R8 in structural formula III or structural formula IIIV) This is a compound represented by

[0137] In the structural formula VC, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl group A.

[0138] In the structural formula VC, unless otherwise specified in the context of this disclosure, [ka] This is the same as the self-sacrificing structure described above when the group CPT in structural formula III or structural formula IIIV is represented by structural formula IV.

[0139] According to specific embodiments of the present invention, the compound represented by structural formula V provided by this disclosure is further represented by structural formula VI: [ka] Structural formula VI It is a compound represented by [this symbol].

[0140] In structural formula VI, group A represents the peptide groups of 2 to 4 amino acids as defined above.

[0141] In structural formula VI, the group CPT is a compound of camptothecin. The meaning of the group CPT and its bonding relationship in structural formula VI are the same as the meaning and bonding relationship of the group CPT in structural formulas III and IIIA as defined above.

[0142] Preferably, the compound represented by Structural Formula VI provided by the present disclosure further has Structural Formula VI-A:

Chemical formula

[0143] Preferably, in Structural Formula VI-A, group G is hydrogen, fluorine, or chlorine.

[0144] Preferably, in Structural Formula VI-A, group Y is methylene, sulfur, or oxygen.

[0145] In Structural Formula VI-A, unless otherwise specified in the context of the present disclosure, “-A-NH-” means that an amide bond is formed between the amino and the carboxyl of group A.

[0146] Preferably, the compound represented by Structural Formula VI-A provided by the present disclosure further has Structural Formula VI-A-1:

Chemical formula

[0147] Alternatively, the compound represented by Structural Formula VI further has Structural Formula VI-B:

Chemical formula

[0148] In structural formula VI-B, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino of the indicated group CPT and the carboxyl of group A.

[0149] More preferably, the compound represented by structural formula VI-B is further represented by structural formula VI-B-1: [ka] Structural formula VI-B-1 It is a compound represented by [this symbol].

[0150] Alternatively, the compound represented by structural formula VI provided in this disclosure may also be represented by structural formula VI-C: [ka] Structural formula VI-C This is a compound represented by (in structural formula VI-C, groups A and R8 have the same meanings as defined above for groups A and R8 in structural formula III or structural formula IIIA).

[0151] In structural formula VI-C, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl group A.

[0152] The self-sacrificing structure in structural formula VC is [ka] In that case, structural formula VC may further be structural formula VI-C.

[0153] According to a specific embodiment of the present invention, in the thirteenth aspect of this disclosure, the compound has one of the following structures: [ka]

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[0154] 14th aspect This disclosure relates to a method for producing an antibody-drug conjugate or a salt thereof, comprising the following steps: a. Antibody reduction: A reducing agent is added to a phosphate buffer containing the antibody at a concentration of 5 mg / mL to 30 mg / mL in an equivalent molar ratio of 5.5 or more:1 (reducing agent:antibody), and the reducing agent and antibody are reacted for 1.5 to 2 hours (wherein the reducing agent is one or more selected from the group consisting of TCEP, DTT, 2-MEA, and DTBA). b. Antibody conjugation and hydrolysis: The reduced antibody obtained in step a is transferred to a phosphate buffer at pH 6.5-7.8, thereby diluting the antibody to a concentration of 3.5 mg / mL-15 mg / mL in the buffer to obtain a diluted antibody solution. The linker-payload dissolved in an organic cosolvent is added to the diluted antibody solution in an equivalent molar ratio of 4.5-6.5:1 (linker-payload:antibody). The reaction system is then stirred and reacted at 15°C-35°C for 0.5 hours or more (wherein the organic cosolvent is one or more selected from the group consisting of DMA, DMSO, DMF, and ACN). Subsequently, the antibody conjugation product is transferred to a phosphate buffer at pH 7.4-9.0, and the buffer is heated at 35±10°C for 2-24 hours to obtain the hydrolysis product. c. Hydrophobic chromatography: A step to purify the obtained antibody conjugation product by hydrophobic chromatography using a hydrophobic packing material. This provides a method that includes [something].

[0155] In step a, the antibodies include, for example, 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, and AG. These are antibodies against tumor-associated antigens such as S-16, guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3, or claudin 18.2.

[0156] Preferably, the antibody is an anti-B7-H3 antibody. More preferably, the antibody is an anti-B7-H3 antibody as shown in the first embodiment of the present disclosure. For example, the anti-B7-H3 antibody may be hz10B4, hz10B4m1, hz10B4m2, or hz10B4m3 of the anti-B7-H3 antibodies shown in the examples of the present disclosure.

[0157] Preferably, in step b, the linker-payload is a compound as defined in the 13th embodiment of the present disclosure.

[0158] The 15th aspect The antibody-drug conjugates or salts thereof provided by this disclosure can be incorporated into compositions, and more specifically into pharmaceutical compositions, for example, pharmaceutical preparations, and used for a variety of purposes as required by practice. Accordingly, in a fifteenth aspect, this disclosure also provides compositions comprising the antibody-drug conjugate or salts thereof according to the present invention. Preferably, the composition is a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier, adjuvant, or excipient. The pharmaceutical compositions provided by this disclosure can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered via an appropriate route of administration.

[0159] The 16th aspect This disclosure also provides the use of antibody-drug conjugates or salts thereof and / or compositions comprising them in the manufacture of pharmaceuticals for the treatment of tumors.

[0160] Preferably, the tumor contains, for example, 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, It is associated with positive or high expression of tumor-associated antigens such as guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3, or claudin 18.2.

[0161] 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic small round cell tumor.

[0162] The 17th aspect Accordingly, the Disclosure also provides a method for treating a tumor, comprising administering an antibody-drug conjugate or a salt thereof and / or a composition comprising the same to a subject in need of treatment.

[0163] Preferably, the tumor contains, for example, 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, It is associated with positive or high expression of tumor-associated antigens such as guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3, or claudin 18.2.

[0164] 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 lung cancer, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic small round cell tumor.

[0165] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0166] The methods for treating the aforementioned diseases provided in this disclosure, where applicable, depend on a number of factors, including the specific active ingredients administered, the patient's age, weight, sex, physical and medical condition, the severity of the condition being treated, and the route of administration.

[0167] The inventors of this invention screened known anti-B7-H3 humanized antibodies using site-directed mutagenesis in the variable region to obtain a series of novel anti-B7-H3 humanized antibodies. These antibodies exhibit significantly higher purity compared to the parental antibody, while retaining the high affinity of the parental antibody for B7-H3, thereby eliminating the parental antibody's susceptibility to fragmentation and reducing toxic side effects. Furthermore, the inventors of this invention conjugated this series of novel humanized antibodies with camptothecin of a specific structure to create antibody-drug conjugates. By leveraging the stable and robust structure of these antibodies, their high targeting ability against B7-H3 expressing cells, and the potent cytotoxic effect of the specific camptothecin, the resulting ADC achieves superior tumor-killing efficacy.

[0168] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0169] [Figure 1] This figure shows the binding activity of the anti-B7-H3 antibody provided in this disclosure to B7-H3 expressed on the surface of cancer cells. [Figure 2] This figure shows the efficacy results of the antibody-drug conjugate targeting B7-H3 provided in this disclosure in a mouse model of lung cancer. [Figure 3] This figure shows the efficacy results of the B7-H3 targeted antibody-drug conjugate and anti-B7-H3 antibody provided in this disclosure in a mouse model of pancreatic cancer. [Figure 4] This figure shows the results of apoptosis induction in cancer cells by B7-H3 targeted antibody-drug conjugates and anti-B7-H3 antibodies provided in this disclosure. [Figure 5] This figure shows the bystander effect detection results (mean ± SD, n=2) for the B7-H3 targeted antibody-drug conjugate provided in this disclosure. [Figure 6]This figure (mean ± SD, n=2) shows the detection results of the ability of the B7-H3 targeted antibody-drug conjugate provided in this disclosure to induce cell cycle arrest in B7H3-positive cells, with the results for the G0 / G1 phase, G2 / M phase, and S phase arranged from top to bottom. [Modes for carrying out the invention]

[0170] Detailed description of preferred embodiments The present invention will be described below with reference to specific examples. Those skilled in the art will understand that these examples are merely illustrative of the present invention and do not limit the scope of the invention in any way.

[0171] The experimental procedures in the following examples are all conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0172] Human B7-H3: UniProtKB-Q5ZPR3 Control antibody hum30: An anti-B7-H3 antibody prepared using the method described in the U.S. Patent Application Publication No. 20160368990.

[0173] Example Group 1: Screening and Characterization of Novel Anti-B7-H3 Humanized Antibodies Example 1: Preparation of anti-B7-H3 antibody The antibody hz10B4 was prepared according to the method described in Patent Document 1. The amino acid sequences of the heavy chain variable region and light chain variable region of antibody hz10B4 are shown below (i.e., SEQ ID NOs. 20 and 22 in Patent Document 1). Here, CDR is underlined (using Kabat definition).

[0174] > hz10B4-VH(VH / CDR-H1 / CDR-H2 / CDR-H3:Sequence ID 1 / Sequence ID 8 / Sequence ID 9 / Sequence ID 10) EVQLVQSGAEVKKPGASVKVSCKASGYTFT EYIMH WVRQAPGQGLEWMG GINPASGGTTYNQKFKG RVTTMTRDTTSISTAYMELSRLRSEDTAVYYCAR KGKDYFDWYFDV WGQGTTVTVSS > hz10B4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 2 / Sequence ID 11 / Sequence ID 12 / Sequence ID 13) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSSNPLT FGGGTKVEIK

[0175] Five point mutations were designed for LCDR3 of hz10B4. A series of anti-B7-H3 antibodies were obtained by combining each of the resulting VL mutants with hz10B4-VH and referring to the method described in Example 9 of Patent Document 1 (see Table 1).

[0176] [Table 1]

[0177] > hz10B4-Lm1-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 3 / Sequence ID 11 / Sequence ID 12 / Sequence ID 14) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSSAPLT FGGGTKVEIK > hz10B4-Lm2-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 4 / Sequence ID 11 / Sequence ID 12 / Sequence ID 15) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSASPLT FGGGTKVEIK > hz10B4-Lm3-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 5 / Sequence ID 11 / Sequence ID 12 / Sequence ID 16) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSSQPLT FGGGTKVEIK > hz10B4-Lm4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 6 / Sequence ID 11 / Sequence ID 12 / Sequence ID 17) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSSGPLT FGGGTKVEIK > hz10B4-Lm5-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:Sequence ID 7 / Sequence ID 11 / Sequence ID 12 / Sequence ID 18) DIQLTQSPSSLSASVGDRVTMTC SASSSVSYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWSGSPLT FGGGTKVEIK

[0178] Example 2 Functional evaluation of anti-B7-H3 antibody 2.1 Study on the binding affinity of antibodies to human B7-H3 ECD-His The binding affinity of hz10B4, hz10B4m1-hz10B4m5, and the control antibody hum30 to human B7-H3 was determined using ForteBio's Octet® QKe.

[0179] hz10B4, hz10B4m1-hz10B4m5, and the control antibody hum30 were diluted to 4 μg / mL in PBS buffer and flowed over the surface of an AHC probe (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human B7-H3 extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1-461, C-His tag) (60 nM) was used as the mobile phase. The association time was 300 seconds and the dissociation time was 300 seconds. After the experiment, the antigen-antibody binding rate constant was calculated by fitting the data using a 1:1 Langmuir binding model with analytical software.

[0180] Table 2 shows the affinity detection results for hz10B4 (SEQ ID NO: 1 + SEQ ID NO: 2), hz10B4m1 (SEQ ID NO: 1 + SEQ ID NO: 3), hz10B4m2 (SEQ ID NO: 1 + SEQ ID NO: 4), hz10B4m3 (SEQ ID NO: 1 + SEQ ID NO: 5), hz10B4m4 (SEQ ID NO: 1 + SEQ ID NO: 6), hz10B4m5 (SEQ ID NO: 1 + SEQ ID NO: 7), and hum30 for binding to recombinant human B7-H3 extracellular domain protein. These results demonstrate that the affinity of hz10B4m1, hz10B4m2, and hz10B4m3 for recombinant human B7-H3 ECD protein is similar to that of the parent hz10B4, with a slight decrease in Koff. In particular, the affinity of hz10B4m1 was slightly stronger than that of hz10B4m2 and hz10B4m3.

[0181] [Table 2]

[0182] 2.2 Study on the binding activity of antibodies against surface antigens of SKOV3 cells The binding activity of hz10B4 and hz10B4m1~hz10B4m5 to the antigen on the surface of SKOV3 cells that naturally express human B7-H3, which is used as a target cell, was detected by FACS.

[0183] Human B7-H3 naturally expressing SKOV3 cells grown to the logarithmic growth phase were collected by centrifugation. Cell density was measured using complete medium (F-12K medium containing 10% FBS and 250 μg / mL G418) at a rate of 2 × 10⁶ cells per mL. 5 Prepared into individual cells. 1 mL of cell suspension (2 × 10⁶ 5 The cells (containing 100 cells) were divided into several EP tubes, centrifuged, and the supernatant was discarded. The cells were washed twice with PBS. 200 μL of each antibody solution (hz10B4 and hz10B4m1~hz10B4m5, each at a starting concentration of 198 nM, serially diluted 3-fold over 12 different concentrations) was added to the cell pellet in each tube. The mixture was incubated at 37°C for 30 minutes. The following controls were included: (1) Positive control (PC): control antibody hum30, (2) Negative control (NC-huIgG1): isotype control antibody. After incubation, the cells were washed three times with PBS. A 1:200 dilution of goat anti-human IgG-FITC (catalog number: F9512, Sigma) was added and incubated for 30 minutes. The cells were washed three times with PBS, and the binding ability of the test antibody to B7-H3 on the surface of SKOV3 cells was assessed by detecting the mean fluorescence intensity (MFI) using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER).

[0184] The results of the binding activity of hz10B4, hz10B4m1, hz10B4m2, hz10B4m3, hz10B4m4, hz10B4m5, and hum30 to antigens on the surface of human SKOV3 cells are shown in Table 3 and Figure 1. These results demonstrate that the binding activity of hz10B4m1, hz10B4m2, and hz10B4m3 to the surface antigens of SKOV3 cells is similar to that of the parent hz10B4.

[0185] [Table 3]

[0186] Example 3: Purity analysis of anti-B7-H3 antibody The purity of the anti-B7-H3 antibody was determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under both reducing and non-reducing conditions. The purity of the antibody product was evaluated according to the method "Determination of Molecular Size Variation of Monoclonal Antibodies (CE-SDS)" described in General Rule 3127 of Part IV of the Chinese Pharmacopoeia. The specific results are shown in Table 4.

[0187] [Table 4]

[0188] Results from both NR-CE-SDS and R-CE-SDS analyses demonstrated that the purity of hz10B4m1, hz10B4m2, and hz10B4m3 all exceeded 98%, surpassing the purity of the parent antibody hz10B4.

[0189] Example Group 2: Synthesis of Camptothecin A general method for synthesizing camptothecin: The camptothecin compounds according to the present invention can be obtained by a Friedländer reaction using the compound represented by formula A and a tricyclic compound (cyclic compound CDE), and the general reaction is shown below: [ka]

[0190] In this reaction, the tricyclic compound CDE can be purchased from MCE (MedChemExpress).

[0191] [ka]

[0192] The tricyclic compound HCDE can be obtained according to the method described in Bioorganic & Medicinal Chemistry, 2010, Vol. 18, No. 9, pp. 3140-3146: [ka]

[0193] 1. Synthesis of the compound represented by formula A Example 4 Synthesis of Compound A1 [ka]

[0194] Method 1: Compound A1 was synthesized according to the method described in the patent publication International Publication No. 2020200880. The synthesis route is as follows: [ka]

[0195] Method 2: Compound A1 was synthesized by a palladium-catalyzed coupling reaction using a zinc reagent. The synthesis route is shown below: [ka]

[0196] (1) Acetylation: 3-bromo-4-nitroaniline (25.00 g, 0.12 mol) and 200 mL of acetic acid were added to a 500 mL flask, and then 50 mL of acetic anhydride was slowly added dropwise. These starting compounds were reacted at room temperature for 16 hours. After confirmation of the completion of the reaction by TLC detection, the reaction mixture was filtered to obtain a filtrate, which was then concentrated under reduced pressure to remove the acetic acid. The resulting filtrate cake was collected, slurryed with 200 mL of MTBE, and the slurry was filtered to obtain 27.6 g of a dry yellow solid (intermediate A1-a) in 92% yield. LC-MS (ESI): m / z 259 (M+H) + .

[0197] (2) Preparation of 4-ethoxy-4-oxobutylzinc bromide Activated zinc powder (19.0 g, 0.29 mol, 2.00 equivalents) was added to a 250 mL three-necked flask (equipped with a thermometer, reflux condenser, and rubber stopper). The air in the flask was then replaced with nitrogen, and anhydrous DMF (145 mL) was added, after which the air in the flask was again replaced with nitrogen. When iodine (1.86 g, 0.015 mol, 0.1 equivalents) was added at room temperature, the color of the solution was observed to change from colorless to reddish-brown, gradually to pale yellow, and finally to colorless (in 2-3 minutes). Next, ethyl 4-bromobutyrate (28.6 g, 0.15 mol, and 1.00 equivalent) was added to the flask, and the mixture was heated to 80°C (internal temperature) and reacted for 4-5 hours. After the completion of the reaction was confirmed by TLC detection, the reaction solution was allowed to cool to room temperature and used later. The obtained supernatant was pale yellow and had a concentration of 1 mol / L.

[0198] (3) Coupling with zinc reagent Anhydrous DMF (120 mL) and intermediate A1-a (25.0 g, 1.00 equivalent) were added to a 500 mL reaction flask. The air in the flask was then replaced with nitrogen, palladium acetate (433 mg, 0.02 equivalent) was added, and the air in the flask was again replaced with nitrogen. The mixture in the flask was stirred at room temperature for 10 minutes, then S-PHOS (1.6 g, 0.04 equivalent) was added, and the air in the flask was again replaced with nitrogen. Next, the mixture in the flask was stirred for 20 minutes, and 4-ethoxy-4-oxobutylzinc bromide (145 mL, 1.50 equivalent) prepared in the above step was added dropwise at room temperature (25°C-30°C), and the reaction was maintained at 25°C-30°C for 16 hours. After complete reaction of the starting compounds was confirmed by TLC detection (DCM:EA=5:1), the reaction mixture was cooled to room temperature, and the reaction was quenched by adding ammonium chloride solution (15 mL) to the reaction mixture. Next, the reaction mixture was poured into 1 L of water, and 400 mL of ethyl acetate was added to obtain the separated phase. After filtering the phase through a Buchner funnel, the resulting aqueous phase was extracted with ethyl acetate (400 mL x 2). The resulting organic phase was washed twice with water (500 mL), then once with saturated sodium chloride solution (500 mL), dried through anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 37.0 g of a reddish-brown oily liquid (intermediate A1-b) in 130% yield. The crude product was used in the next reaction without further purification. LC-MS(ESI):[M+1] + =295.

[0199] (4) Synthesis of intermediate A1-c Intermediate A1-b (37.0 g, 1.0 equivalent) and ethanol (1.5 L) were added to a 3 L three-necked reaction flask, and the starting materials were completely dissolved in the ethanol. The reaction system was cooled to 5°C, 5% Pd / C (5.7 g, 1.0 equivalent) was added, and the temperature was raised to 25°C in a hydrogen atmosphere (at atmospheric pressure) and the reaction was carried out for 16 hours. After confirmation of the completion of the reaction by TLC detection (DCM:EA=5:1), the reaction system was filtered through Celite, and the resulting organic phase was concentrated to obtain 33.0 g of crude product (intermediate A1-c) in 130% yield. The crude product was used directly in the next reaction without further purification. LC-MS(ESI):[M+1] + = 265.

[0200] (5) Synthesis of intermediate A1-d Intermediate A1-c (33.0 g, 1.00 equivalent) and 260 mL of acetic acid were added to a 1 L flask, and then 46 mL of acetic anhydride was slowly added dropwise. The starting compounds were reacted at room temperature for 2 hours. After confirming the complete reaction of the starting compounds by TLC detection (DCM:MeOH = 10:1), the reaction mixture was filtered to obtain a filtrate, which was then concentrated under reduced pressure to remove the acetic acid. After adding 250 mL of water, the resulting aqueous phase was extracted with ethyl acetate (400 mL x 2), and the resulting organic phase was washed twice with water (500 mL), then once with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain 32 g of crude product. The crude product was slurryed with 400 mL of MTBE, filtered, and 27.0 g of a dry, pale yellow solid (intermediate A1-d) was obtained in 91% yield. The total yield of the four steps was found to be 83.7%.

[0201] LC-MS(ESI):[M+1] + =307. 1 H NMR(400MHz,DMSO)δ 9.87(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,2 H), 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).

[0202] (6) Synthesis of intermediate A1-e Intermediate A1-d (27.0 g, 88 mmol, 1.0 equivalent), water (100 mL), and tetrahydrofuran (200 mL) were added to a 500 mL three-necked reaction flask, and the starting materials were completely dissolved. Lithium hydroxide monohydrate (18.5 g, 441 mmol, 5.0 equivalents) was added at room temperature, and the reaction system was maintained at room temperature for 3 hours. Once the completion of the reaction was confirmed by TLC detection, most of the tetrahydrofuran was removed under reduced pressure, and 300 mL of water was added to the resulting residue. The resulting aqueous phase was extracted with EA (2 × 100 mL), the organic phase obtained was discarded, and the resulting aqueous phase was collected, placed in an ice bath, and pH was adjusted to 4 by adding 6 N hydrochloric acid. A solid precipitated, which was filtered to obtain 19.1 g of a white solid (intermediate A1-e) in 78% yield.

[0203] 1 H NMR(400MHz,DMSO)δ 12.09(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)

[0204] (7) Synthesis of compound A1 40 mL of polyphosphate was added to a 250 mL reaction flask and heated to 90°C. Intermediate A1-e (5.0 g, 17.97 mmol) was then added in several batches. The reaction was allowed to proceed for 5 hours while maintaining an internal temperature of 95°C to 100°C. Once the completion of the reaction was confirmed by TLC detection, the heat source was removed and the reaction system was cooled to 50°C to 60°C. Next, 15 mL of 4 M HCl (aqueous solution) was added dropwise to the reaction system to quench the reaction (the temperature rose to 100°C). Subsequently, 600 mL of 4 M NaOH aqueous solution was added dropwise to adjust the pH to 10. The resulting aqueous phase was extracted with ethyl acetate (50 mL x 3), and the resulting organic phase was collected. After washing once with saturated sodium chloride solution (50 mL), it was dried through anhydrous sodium sulfate and concentrated under reduced pressure to obtain 3.75 g of a yellow solid (intermediate A1-f) in 80.5% yield. The solid was used directly in the next reaction without purification. LC-MS(ESI):[M+1]+ =261.

[0205] The starting material, intermediate A1-f (3.75 g), was suspended in 19% hydrochloric acid (30 mL) in a 250 mL three-necked reaction flask. The reaction system was heated to 90 °C (internal temperature) and reacted for 3 hours. After confirmation of the completion of the reaction by TLC detection, the flask was cooled to below 5 °C in an ice salt bath. 4 M NaOH solution (45 mL, 30 equivalents) was added dropwise to adjust the pH to 10. The resulting aqueous phase was extracted with ethyl acetate (80 mL x 5), and the resulting organic phase was collected. After washing once with saturated sodium chloride solution (50 mL), it was dried through anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2.45 g of crude product. The crude product was purified by column chromatography using DCM as the eluent to obtain 1.93 g of a yellow solid (compound A1) in 76% yield. The total yield of the two steps was found to be 61.2%.

[0206] LC-MS(ESI):[M+1] + = 177. 1 H NMR(400MHz,DMSO)δ 6.76(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)

[0207] Method 3: Compound A1 was synthesized by lactam hydrolysis. The synthetic route is shown below: [ka]

[0208] The aminolactam compound was prepared according to the method described in the patent publication No. 106349233 of the Chinese Patent Application.

[0209] [ka]

[0210] Aminolactam compound (17.6 g, 0.1 mmol), ethanol (250 mL), and 98% sulfuric acid (5 mL) were mixed in a 500 mL three-necked flask and heated under reflux for 24 hours. The reaction mixture was collected and detected to confirm whether the reaction was complete. Once the reaction was complete, the reaction mixture was concentrated and dried under reduced pressure. Dichloromethane (200 mL) and water (100 mL) were added to the resulting residue, and the resulting mixture was cooled to below 10°C in an ice bath. After adjusting the pH to 7-8 by adding 1 N sodium hydroxide aqueous solution, the mixture was stirred to obtain the separated phase. The obtained aqueous phase was extracted with dichloromethane, the resulting organic phase was collected, washed with saturated physiological saline, dried over anhydrous sodium sulfate, aspirated, and distilled under reduced pressure to remove the organic solvent, yielding 25 g of the crude diaminoethyl ester intermediate product, which was used directly in the next reaction.

[0211] The diaminoethyl ester intermediate was dissolved in dichloromethane (200 mL), and triethylamine (20.2 g, 0.2 mol, 2 equivalents) was added. The mixture was cooled to below 10°C in an ice bath, and acetic anhydride (25.5 g, 0.25 mol, 2.5 equivalents) was added dropwise. The temperature was then maintained, and the mixture was reacted for 1 hour. The reaction solution was collected and detected to confirm whether the reaction was complete. Once the reaction was complete, the reaction solution was poured into 1N ice-cold hydrochloric acid and stirred for 15 minutes. The separated phase was obtained, and the resulting aqueous phase was extracted with dichloromethane (50 mL x 3). The resulting organic phase was collected, dried over anhydrous sodium sulfate, and removed by suction and distillation under reduced pressure to obtain the crude product. The crude product was slurryed with 400 mL of MTBE, filtered, and dried to obtain 26.9 g of intermediate A1-e as a pale yellow solid. The total yield of the two steps was found to be 87.8%. LC-MS(ESI): m / z 307(M+H) + .

[0212] Next, compound A1 was prepared from intermediate A1-e according to method 2.

[0213] Example 5 Synthesis of Compound A2 [ka]

[0214] Compound A2 was synthesized according to the method described in the patent publication International Publication No. 2021148501. The synthesis route is as follows: [ka]

[0215] Example 6 Synthesis of Compound A3 [ka]

[0216] Compound A3 was synthesized according to the method described in the U.S. Patent Publication No. 2004266803.

[0217] Example 7 Synthesis of Compound A4 Compound A4 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows: [ka]

[0218] Step 1: Intermediate A4-2 was prepared from compound A4-1 according to the method described in the literature.

[0219] Step 2: Intermediate A4-2 (3.4g) was dissolved in dichloromethane at -5°C to 5°C, triethylamine (3.0g) was added, and then AllocCl (2.8g) was slowly added dropwise. After the addition was complete, the resulting mixture was stirred and reacted for 1 to 2 hours, then the reaction was quenched by adding water. The resulting reaction product was washed once with water, then once with saturated physiological saline, dried over anhydrous sodium sulfate, concentrated and dried to obtain 5.5g of crude intermediate A4-3. LC-MS(ESI):[M+1] + = 255.7.

[0220] Step 3: Intermediate A4-3 (5.5 g) was added to TBAF (55 ml) and acrylic acid (110 ml) at room temperature. The resulting mixture was heated to 50°C-55°C and stirred, and reacted for 24 hours. The reaction solution was directly concentrated and dried, and purified by column chromatography using a petroleum ether → methanol / dichloromethane (1:50) mixture as the eluent, yielding approximately 5.2 g of crude intermediate A4-4. LC-MS(ESI):[M+1] + = 327.4.

[0221] Step 4: 5.0 g of intermediate A4-4 was added to 100 ml of ethanol / water (3:1) mixed solution at room temperature. 5.5 g of ammonium chloride and 5.5 g of iron powder were added, and the resulting mixture was heated under reflux and reacted for 1 to 2 hours. The reaction solution was cooled to room temperature, filtered, and the resulting solid was washed with ethanol. The mixture was concentrated and dried to obtain approximately 3.8 g of crude intermediate A4-5. LC-MS (ESI): [M+1] + = 297.4.

[0222] Step 5: Intermediate A4-5 (3.8 g) was added to trifluoroacetic acid (38 ml) at 20°C to 30°C, and then trifluoroacetic anhydride (38 ml) was added. The resulting mixture was stirred and reacted for 18 to 24 hours, after which the reaction product was subjected to the following workup. Specifically, it was directly concentrated and dried, and purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 4:1) mixture as the eluent to obtain 1.0 g of intermediate A4-6. LC-MS (ESI): [M+1] + = 375.3.

[0223] Step 6: Intermediate A4-6 (1.0 g) was added to methanol (20 ml) at room temperature, followed by potassium carbonate (2.0 g) and water (5 ml). The resulting mixture was stirred and reacted for 1 to 2 hours. After diluting the reaction solution with water, it was extracted with ethyl acetate. The resulting organic phase was washed with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and dried to obtain the crude product. The crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 2:1) mixture as the eluent to obtain 0.65 g of intermediate A4-7. LC-MS(ESI):[M+1] + = 279.3.

[0224] Step 7: Intermediate A4-7 (500 mg) was dissolved in tetrahydrofuran (20 ml) at room temperature, and pyrrole (120 mg) and tetrakis(triphenylphosphine)palladium (160 mg) were added under nitrogen protection. After the additions were complete, the resulting mixture was stirred and reacted for 1 to 2 hours. The reaction solution was directly concentrated and dried, and purified by column chromatography using a dichloromethane / methanol (30:1) mixture as the eluent, yielding 30 mg of compound A4 as a brown solid. LC-MS(ESI):[M+1] + = 195.4.

[0225] Example 8 Synthesis of Compound A5 Compound A5 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows: [ka]

[0226] Step 1: Compound A5-1 (25g) was added to acetic acid (100ml) at 20°C-30°C, and acetic anhydride (24.9g) was slowly added. After the addition was complete, the resulting mixture was stirred for 3-4 hours. Then, the reaction mixture was slowly placed in ice water and stirred. A solid precipitated, which was collected by filtration, washed with water, and dried in vacuum to obtain 31.0g of intermediate A5-2 as a yellow solid. LC-MS(ESI):[M+1] + = 197.2.

[0227] Step 2: Intermediate A5-2 (29g), potassium carbonate (40g), potassium iodide (5g), and bromopropanol (25g) were mixed in DMF (300ml) at 20°C-30°C. The resulting mixture was heated to 100°C-110°C and stirred for 3-4 hours. The reaction mixture was then cooled to room temperature, quenched in ice water, and extracted three times with 2L of ethyl acetate. The resulting organic phase was collected, washed with saturated saline, dried via anhydrous sodium sulfate, concentrated and dried, and then slurryed with petroleum ether to obtain a solid. The solid was dried under vacuum to obtain 33.0g of intermediate A5-3 as a yellow solid. LC-MS(ESI):[M+1] + = 255.3.

[0228] Step 3: Intermediate A5-3 (23g) was dissolved in acetonitrile (1L) at 20°C-25°C. Sodium dihydrogen phosphate solution (0.67mol, pH 6.7, 900ml), TEMPO (5g), sodium chlorite solution (52.0g dissolved in 60ml of water), and sodium hypochlorite solution (38ml mixed with 38ml of water) were added sequentially. After the additions were complete, the resulting reaction mixture turned dark brown and was stirred for 30 minutes. Next, the reaction mixture was cooled to room temperature, 2N hydrochloric acid was added to adjust the pH to 2-3, and the mixture was extracted with ethyl acetate (1000ml x 3). The resulting ethyl acetate phase was collected, washed with saturated physiological saline, dried through anhydrous sodium sulfate, filtered, concentrated and dried, and then slurryed with petroleum ether to obtain 24.0g of crude intermediate A5-4. LC-MS(ESI):[M+1] + = 269.2.

[0229] Step 4: Intermediate A5-4 (25g) and 5% palladium-supported activated carbon (5g) were mixed in methanol (500ml) at room temperature, and hydrogenation under pressure was carried out for 3 to 4 hours. The palladium-supported activated carbon was filtered off, the resulting solid was washed with methanol, the filtrate was concentrated, and the resulting residue was slurryed with petroleum ether to obtain the crude product. The crude product was dried in vacuum to obtain 18.0g of intermediate A5-5 as a yellow solid. LC-MS(ESI):[M+1] + = 239.5.

[0230] Next, intermediate A5-7 was synthesized following the same procedure as described in steps 5 and 6 of the method for synthesizing compound A4, starting from intermediate A5-5. The acetyl group was then removed by deprotection with 6N hydrochloric acid to obtain compound A5 as a yellow solid. LC-MS(ESI):[M+1] + = 179.2.

[0231] Example 9 Synthesis of Compound A6 Compound A6 was synthesized according to the method described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows: [ka]

[0232] Compound A6 is a yellow solid. LC-MS(ESI):[M+1] + = 248.9. 1 H NMR(400MHz,d6-DMSO):δ 12.289(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)

[0233] Step 1: Intermediate A1-4 (1.25 g, 5 mmol, 1 equivalent) was dissolved in 150 ml of tetrahydrofuran under nitrogen protection in a 250 mL three-necked reaction flask. The reaction system was cooled to -60°C in a dry ice bath, and then LDA (2 M in THF, 7.6 ml, 15.2 mmol, 3.04 equivalents) was added. After the addition, the resulting mixture was stirred at -60°C for 30 minutes. MeI (1.45 g, 10.21 mmol, 2.04 equivalents) was added. After the addition, the dry ice bath was removed, and the reaction mixture was allowed to rise naturally to room temperature overnight. The next day, the reaction was quenched by adding 50 ml of aqueous ammonium chloride solution and extracted with ethyl acetate (150 ml x 3). The resulting organic phase was collected, washed once with water, concentrated, and dried to obtain the crude product. The crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 5:1) mixture as the eluent, yielding 350 mg of compound A6-1 as a yellow solid in 28% yield.

[0234] 1 H NMR(CDCl3):δ 12.49(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)

[0235] Step 2: Compound A6-1 (280 mg) was mixed with 20 ml of 6N hydrochloric acid in a 100 ml single-neck flask. The resulting mixture was heated under reflux and reacted for 2 hours. Heating was stopped, and the reaction mixture was allowed to cool to room temperature. Sodium bicarbonate was then added to adjust the pH of the reaction mixture to 7-8, and the mixture was extracted with dichloromethane (30 ml x 3). The resulting organic phase was collected, concentrated, and dried to obtain 275 mg of compound A6-2 as a brownish-gray solid. The crude product was used directly in the next reaction without purification.

[0236] Step 3: Compound A6-2 (220 mg) was dissolved in acetic acid (25 ml) in a 50 ml three-necked flask, and then 1.3 g of iron powder was added. After the addition was complete, the resulting mixture was stirred at 80°C to 85°C for 1 to 2 hours. Once the reaction was complete, the reaction mixture was distilled under reduced pressure to remove the acetic acid, and water (30 ml) was added to the resulting residue. The resulting mixture was adjusted to pH 7 to 8 with the addition of sodium bicarbonate and extracted with dichloromethane (30 ml x 3). The resulting organic phase was collected and concentrated to obtain the crude product. The crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 2:1) mixture as the eluent, and 122 mg of compound A6 was obtained as a brown solid in 75% yield. LC-MS (ESI): [M+1] + =205.0.

[0237] Example 10 Synthesis of Compound A7 The synthesis pathway is shown below: [ka]

[0238] Compound A1 (2.18 g, 124 mmol, 1 equivalent) was dissolved in tetrahydrofuran (100 ml) in a 250 ml three-necked flask, and then anhydrous Boc (8.2 g, 376 mmol, 3 equivalents) was added. The resulting mixture was stirred at 40°C to 45°C for 5 hours, and the reaction solution was directly concentrated and dried. The resulting residue was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 1:3) mixture as the eluent, and 3.0 g of compound A7-1 was obtained as a yellow solid in 90% yield.

[0239] 1 H NMR(400MHz,CDCl3)δ 7.25(s,1H), 6.41(t,J=10.2Hz,3H), 5.90(s,1H), 2.73(t,J=6.2Hz,2H), 2.61~2.47(m,2H), 2.02~1.86(m,3H), 1.49~1.36(m,9H)

[0240] Compound A7-1 (3.0 g, 10.8 mmol, 1 equivalent) and DIPEA (3.5 g, 27.1 mmol, 1.5 equivalents) were mixed with dichloromethane (125 ml) in a 250 ml three-necked flask. The reaction mixture was cooled to -5°C to 0°C in an ice salt bath, and acetyl chloride (1.3 g, 16.5 mmol, 2 equivalents) was added dropwise. After the addition was complete, the ice salt bath was removed, the reaction mixture was allowed to warm naturally, and then stirred at room temperature and reacted for 4 hours. The reaction mixture was then concentrated and dried, and the resulting residue was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 1:3) mixture as the eluent to obtain 3.48 g of compound A7-2 as a pale yellow solid in 100% yield.

[0241] 1 H NMR(400MHz,CDCl3)δ 12.01(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)

[0242] Compound A7-2 (2.45 g, 7.7 mmol, 1 equivalent) was dissolved in tetrahydrofuran (200 ml) in a 500 ml three-necked flask under nitrogen protection. The reaction mixture was cooled to -70°C to -60°C in a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 equivalents) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at -70°C to -60°C for 10 minutes. Next, a solution of NFSI in tetrahydrofuran (NFSI (7.35 g, 23 mmol, 3 equivalents) dissolved in tetrahydrofuran (70 ml)) was added dropwise, and after the addition was complete, the resulting mixture was stirred at -70°C to -60°C for 10 minutes. Then, the reaction mixture was allowed to rise naturally to 20°C to 30°C and stirred for 3 hours. The reaction was quenched with saturated ammonium chloride aqueous solution (80 ml), and the reaction mixture was extracted with ethyl acetate (150 ml x 3). The resulting organic phase was collected, washed with saturated physiological saline, concentrated, and dried to obtain the crude product. The crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 1:3) mixture as the eluent, and 0.85 g of compound A7-3 was obtained as a yellow solid in 31.2% yield. LC-MS(ESI):[M+1] + =355.8

[0243] 1 H NMR(400MHz,CDCl3)δ 11.49(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)

[0244] Compound A7-3 (0.85 g, 2.4 mmol) was mixed with 6N hydrochloric acid (30 ml) in a 100 ml single-neck flask, and the resulting mixture was heated under reflux for 4 hours. Heating was then stopped, and the reaction mixture was cooled to room temperature. The pH was adjusted to 7-8 with sodium bicarbonate, and the mixture was extracted with dichloromethane (30 ml x 3). The resulting organic phase was collected, washed with saturated physiological saline, concentrated, and dried to obtain the crude product. The crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0 → 1:3) mixture as the eluent, yielding 0.25 g of compound A7 as a brown solid in 49.1% yield. LC-MS(ESI):[M+1] + = 213.9.

[0245] Example 11 Synthesis of Compound A8 The synthesis pathway is shown below: [ka]

[0246] Compound A8-1 was synthesized according to the method described in the Journal of Medicinal Chemistry, 1989, Vol. 32, No. 6, pp. 1217-1230. LC-MS:(ESI):[M+1] + = 146.3.

[0247] Compound A8-1 (1.8g) and DIEA (4.8g) were added to DCM (50ml) and cooled to 0°C. Trifluoroacetic anhydride (4.8g) was added, and the resulting mixture was warmed to room temperature and reacted for 1 hour. Water (20ml) was added to separate the phases, and the organic phase was dried by centrifugation. The resulting residue was purified by column chromatography (EA:PE=0%→30%) to obtain 2.7g of intermediate A8-2 as a white solid. LC-MS:(ESI):[M+1] + = 242.2.

[0248] 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, and the resulting 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 was complete, the resulting mixture was stirred at room temperature overnight. Water (30 mL) was added to separate the phases, and the organic phase was dried by centrifugation to obtain 2.7 g of intermediate A8-3 as an off-white solid. LC-MS(ESI):[M+1] + = 256.1.

[0249] Intermediate A8-3 (1.5 g) was added to DCM (100 ml) and acetic anhydride (30 ml) and cooled to 0°C. 65% nitric acid (2.8 g) was added, and the resulting mixture was reacted overnight at room temperature. Water (50 ml) and DCM (100 ml) were added to separate the phases, and the organic phase was centrifuged and dried in an oil pump to obtain 1.6 g of crude intermediate A8-4. The crude product was used directly in the next step. LC-MS(ESI):[M+1] + = 301.2.

[0250] Intermediate A8-4 (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 resulting mixture was warmed overnight under reflux. The reaction solution was cooled, filtered, and centrifuged to dry. The resulting residue was purified by column chromatography (EA:PE = 0% → 50%) to obtain 0.25 g of intermediate A8-5 as a brown oily substance. LC-MS (ESI): [M+1] + = 271.3.

[0251] Intermediate A8-5 (250 mg) and DIEA (400 mg) were added to DCM (10 ml) and cooled to 0°C. Trifluoroacetic anhydride (320 mg) was added, and the resulting mixture was reacted at room temperature for 1 hour. The reaction solution was washed with water (5 ml) to obtain the organic phase, which was dried, filtered, and centrifuged to obtain 340 mg of intermediate A8-6 as a yellow solid. LC-MS (ESI): [M+1] + = 367.3.

[0252] Intermediate A8-6 (150 mg) and CrO3 (400 mg) were added to acetic acid (8 ml) and acetic anhydride (4 ml), and the resulting mixture was reacted at 30°C to 40°C for 3 hours. After centrifuging the reaction solution to dry the solvent, water (5 ml) was added, and the mixture was extracted with DCM (20 ml). The organic phase was obtained, dried, filtered, and centrifuged to obtain 160 mg of crude intermediate A8-7. The crude product was used directly in the next step. LC-MS (ESI): [M+1] + = 381.3.

[0253] Intermediate A8-7 was deprotected with 6N hydrochloric acid to remove two trifluoroacetyl groups, and then purified by column chromatography to obtain compound A8. LC-MS(ESI):[M+1] + = 189.3.

[0254] The following compounds can be synthesized according to the same method as described above.

[0255] [Table 5] TIFF2026522277000106.tif247170 TIFF2026522277000107.tif237170 TIFF2026522277000108.tif37170

[0256] 2. Synthesis of camptothecin (Friedländer reaction) The camptothecin control compound MWC-1 was synthesized as follows: [ka]

[0257] Compound A1 (176 mg, 1 equivalent), cyclic compound CDE (315 mg, 1.2 equivalents), and PPTS (301 mg, 1.2 equivalents) were mixed in toluene (50 ml) at room temperature. The resulting mixture was reacted under reflux for 3 to 4 hours while separating the water. The reaction solution was then directly concentrated and dried, and the resulting residue was purified by column chromatography using a petroleum ether → methanol / dichloromethane (1:20) mixture as the eluent, yielding 260 mg of compound MWC-1 as a yellow solid. LC-MS(ESI):[M+1] + = 404.

[0258] Example 12 Synthesis of Camptothecin Compound 1 The synthesis pathway is shown below: [ka]

[0259] Compound A6 (120 mg, 0.59 mmol, 1 equivalent), tricyclic compound CDE (180 mg, 0.68 mmol, 1.2 equivalents), and PPTS (35 mg, 0.14 mmol, 0.25 equivalents) were mixed in 15 ml of a 50 ml single-necked reaction flask under nitrogen protection, and then heated under reflux and reacted for 3 to 4 hours. The reaction mixture was distilled under reduced pressure to remove most of the acetic acid, and the resulting residue was purified by column chromatography using a dichloromethane / methanol (15:1) mixture as the eluent to obtain 80 mg of crude product. The crude product was further separated and purified by preparative liquid-phase chromatography to obtain 3 mg of camptothecin compound 1 as a yellow solid. LC-MS(ESI):[M+1] + = 432.4.

[0260] Example 13 Synthesis of Camptothecin Compound 2 (2A and 2B) The synthesis pathway is shown below: [ka]

[0261] Camptothecin compound 2A (with a short retention time) and camptothecin compound 2B (with a long retention time) were obtained by separation and purification using preparative liquid-phase chromatography, following the same method as described in Example 12, but using compound A9 instead of compound A6. Camptothecin compound 2A was obtained as a yellow solid. LC-MS(ESI):[M+1] + =418.3. Camptothecin compound 2B was obtained as a yellow solid. LC-MS(ESI):[M+1] + = 418.4.

[0262] Example 14 Synthesis of Camptothecin Compound 3 The synthesis pathway is shown below: [ka]

[0263] Camptothecin compound 3 was prepared using a method similar to that described in Example 12, but with compound A7 instead of compound A6 and toluene instead of acetic acid as the solvent. This compound was obtained as an ochre-colored solid. LC-MS(ESI):[M+1] + = 440.8.

[0264] 1 H NMR (400MHz, acetone)δ 7.89(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) 1H NMR(400MHz,DMSO)δ 7.87(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=46.6H) z,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)

[0265] Example 15 Synthesis of Camptothecin Compound 4 (4A and 4B) [ka]

[0266] Camptothecin compound 4A (with a short retention time) and camptothecin compound 4B (with a long retention time) were obtained by separation and purification using preparative liquid-phase chromatography, following the same method as described in Example 12, but using compound A10 instead of compound A6. Compound 4A was obtained as an ochre-colored solid. LC-MS(ESI):[M+1] + =422.3. Camptothecin compound 4B was obtained as an ochre-colored solid. LC-MS(ESI):[M+1] + = 422.3.

[0267] Example 16 Synthesis of Camptothecin Compound 5 The synthesis pathway is shown below: [ka]

[0268] Camptothecin compound 5 was prepared using a method similar to that described in Example 12, but with compound A12 instead of compound A6, and obtained as a yellow solid. LC-MS(ESI):[M+1] + = 444.3.

[0269] Example 17 Synthesis of Camptothecin Compound 6 The synthesis pathway is shown below: [ka]

[0270] Camptothecin compound 6 was prepared using a method similar to that described in Example 12, but with compound A11 used instead of compound A6, and obtained as a yellow solid. LC-MS(ESI):[M+1] + = 430.5.

[0271] Example 18 Synthesis of Camptothecin Compound 7 The synthesis pathway is shown below: [ka]

[0272] Following a method similar to that described in Example 12, but using compound A8 instead of compound A6, 0.7 mg of camptothecin compound 7 was prepared and obtained as a yellow solid. LC-MS(ESI):[M+1] + = 416.3.

[0273] Example 19 Synthesis of Camptothecin Compound 9 The synthesis pathway is shown below: [ka]

[0274] Camptothecin compound 9 was prepared using a method similar to that described in Example 12, but with compound A3 used instead of compound A6. This compound was obtained as a reddish-brown solid. LC-MS(ESI):[M+1] + = 420.3.

[0275] Example 20 Synthesis of Camptothecin Compound 12 Compound 12 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows: [ka]

[0276] Following a method similar to that described in Example 12, but using compound A4 instead of compound A6 and toluene instead of acetic acid as the solvent, 30 mg of camptothecin compound 12 was synthesized and obtained as a brown solid. LC-MS(ESI):[M+1] + = 422.5.

[0277] Example 21 Synthesis of Camptothecin Compound 13 The synthesis pathway is shown below: [ka]

[0278] Compound 13 was synthesized using a method similar to that described in Example 12, but with compound A15 instead of compound A6 and toluene as the solvent. This compound was obtained as a brown solid. LC-MS(ESI):[M+1] + = 458.4.

[0279] Example 22 Synthesis of Camptothecin Compound 14 The synthesis pathway is shown below: [ka]

[0280] Compound 14 was synthesized using a method similar to that described in Example 12, but with compound A5 instead of compound A6 and toluene as the solvent. This compound was obtained as a yellow solid. LC-MS(ESI):[M+1] + = 406.1.

[0281] Example 23 Synthesis of Camptothecin Compound 14-P [ka]

[0282] Compound 12 (200 mg) was dissolved at room temperature in a mixed solvent of acetic acid (24 ml) and water (3 ml). The resulting mixture was cooled to 5°C under nitrogen protection, and then 30% H2O2 (580 mg) was added. The reaction mixture was stirred for 2 hours, concentrated and dried, and the resulting residue was subjected to preparative HPLC to separate compounds 14-P1 and 14-P2. These were then freeze-dried to obtain 42 mg of compound 14-P1 as a brown solid and 25 mg of compound 14-P2 as a brown solid. LC-MS(ESI):[M+1] + = 438.5.

[0283] Example 24 Synthesis of Camptothecin Compound 15 [ka]

[0284] Compound 12 (100 mg) was dissolved at room temperature in a mixed solvent of acetic acid (24 ml) and water (3 ml). The resulting mixture was cooled to 5°C under nitrogen protection, and then 30% H2O2 (750 mg) was added. The reaction mixture was stirred for 2 hours, then warmed to room temperature (25°C) and stirred overnight. After confirmation of the completion of the reaction by LC-MS analysis, the reaction mixture was concentrated and dried, and the resulting residue was subjected to preparative HPLC to separate and collect compound 15. It was freeze-dried to obtain 8 mg of compound 15 as a brown solid. LC-MS(ESI):[M+1] + = 454.4.

[0285] Example 25 Synthesis of Camptothecin Compound 16 [ka]

[0286] Intermediate 16a was synthesized according to the method described in International Publication No. 2020200880. Then, intermediate 16c, having the structure of 16a-thiocamptothecin, was prepared according to the method described in the literature (J. Med. Chem. 2008, 51, 3040-3044). Finally, deprotection was performed to remove the acetyl group, yielding compound 16. LC-MS(ESI):[M+1] + = 420.3.

[0287] Example 26 Synthesis of Camptothecin Compound 17 The synthesis pathway is shown below: [ka]

[0288] Homocamptothecin compound 17 (a mixture of S and R stereochemistrys) was obtained by following a method similar to that described in Example 12, but using compound A1 instead of compound A6, HCDE instead of CDE, and toluene as the solvent. LC-MS(ESI):[M+1] + = 418.5.

[0289] Example 27 Synthesis of Camptothecin Compound 18 The synthesis pathway is shown below: [ka]

[0290] Compound 18 was synthesized as a pale yellow solid using a method similar to that described in Example 12, but with compound A16 used instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 424.2.

[0291] Example 28 Synthesis of Camptothecin Compound 19 The synthesis pathway is shown below: [ka]

[0292] Compound 19 was synthesized as a yellow solid using a method similar to that described in Example 12, but with compound A17 used instead of compound A6 and toluene used as the solvent. LC-MS(ESI):[M+1] + = 460.5.

[0293] Example 29 Synthesis of Camptothecin Compound 20 The synthesis pathway is shown below: [ka]

[0294] Compound 20 was synthesized as a yellow solid using a method similar to that described in Example 12, but with compound A13 used instead of compound A6 and toluene used as the solvent. LC-MS(ESI):[M+1] + = 458.2.

[0295] Example 30 Synthesis of Camptothecin Compound 21 The synthesis pathway is shown below: [ka]

[0296] Compound 21 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A24 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 418.4.

[0297] Example 31 Synthesis of Camptothecin Compound 22 The synthesis pathway is shown below: [ka]

[0298] Compound 22 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A25 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 434.4.

[0299] Example 32 Synthesis of Camptothecin Compound 23 The synthesis pathway is shown below: [ka]

[0300] Compound 23 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A21 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 432.4.

[0301] Example 33 Synthesis of Camptothecin Compound 24 The synthesis pathway is shown below: [ka]

[0302] Compound 24 (isomer pair) was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A20 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 418.4.

[0303] Example 34 Synthesis of Camptothecin Compound 25 The synthesis pathway is shown below: [ka]

[0304] Compound 25 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A32 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 432.4.

[0305] Example 35 Synthesis of Camptothecin Compound 26 [ka]

[0306] Compound 26 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A18 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 440.4.

[0307] Example 36 Synthesis of Camptothecin Compound 27 [ka]

[0308] Compound 27 was obtained as a reddish-brown solid by following a method similar to that described in Example 12, but using compound A19 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 476.4.

[0309] Example 37 Synthesis of Camptothecin Compound 31 [ka]

[0310] Compound 31 was obtained as a yellow solid by following a method similar to that described in Example 12, but using compound A14 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 442.4.

[0311] Example 38 Synthesis of Camptothecin Compound 32 [ka]

[0312] Compound 32 was synthesized using a method similar to that described in Example 12, but with compound A2 instead of compound A6, HCDE instead of CDE, and toluene as the solvent. This compound was obtained as a yellow solid. LC-MS(ESI):[M+1] + = 436.4.

[0313] Example 39 Synthesis of Camptothecin Compound 33 The synthesis pathway is shown below: [ka]

[0314] Compound 33 was synthesized as a yellow solid using a method similar to that described in Example 12, but with compound A23 instead of compound A6 and toluene as the solvent. LC-MS(ESI):[M+1] + = 458.5.

[0315] Example 40 Synthesis of Camptothecin Compound 34 The synthesis pathway is shown below: [ka]

[0316] Compound 34 was synthesized as a yellow solid using a method similar to that described in Example 12, but with compound A22 used instead of compound A6 and toluene used as the solvent. LC-MS(ESI):[M+1] + = 458.5.

[0317] Example 41 Synthesis of Camptothecin Compound 35 and Compound 38 The synthetic route is shown as follows:

Chemical Structure

[0318] Following a method similar to that described in Example 12, using compound A26 instead of compound A6 and using toluene as the solvent, compound 35 was synthesized as a brownish-red solid. LC-MS (ESI): [M+1] + = 438.7.

[0319] Following a method similar to that described in Example 12, using compound A27 instead of compound A6 and using toluene as the solvent, compound 38 was synthesized and obtained as a brownish-red solid. LC-MS (ESI): [M+1] + = 474.7.

[0320] Example 42 Synthesis of Camptothecin Compounds 36 and 39 The synthetic route is shown as follows:

Chemical Structure

[0321] Following a method similar to that described in Example 12, using compound A30 instead of compound A6 and using toluene as the solvent, compound 36 was synthesized as a brownish-red solid. LC-MS (ESI): [M+1] + = 456.7.

[0322] Following a method similar to that described in Example 12, using compound A31 instead of compound A6 and using toluene as the solvent, compound 39 was synthesized and obtained as a brownish-red solid. LC-MS (ESI): [M+1] + = 492.7.

[0323] Example 43 Synthesis of Camptothecin Compounds 37 and 40 The synthesis pathway is shown below: [ka]

[0324] Compound 37 was synthesized as a reddish-brown solid using a method similar to that described in Example 12, but with compound A28 used instead of compound A6 and toluene used as the solvent. LC-MS(ESI):[M+1] + = 440.8.

[0325] Compound 40 was synthesized using a method similar to that described in Example 12, but with compound A29 instead of compound A6 and toluene as the solvent. This compound was obtained as a reddish-brown solid. LC-MS(ESI):[M+1] + = 476.7.

[0326] Example 3: Linker-payload synthesis Example 44 Synthesis of Linker-Payload MWD-L1 [ka]

[0327] The synthesis pathway is shown below: [ka]

[0328] GGFG-Dxd was synthesized according to the method described in the U.S. Patent Publication No. 20190151328.

[0329] [ka]

[0330] GGFG-Dxd was obtained as a pale yellow solid. LC-MS(ESI): M+1=841.

[0331] The linker compound BL was synthesized according to the method described in the patent publication International Publication No. 2018 / 095422.

[0332] [ka]

[0333] The linker compound BL was obtained as a yellow solid. LC-MS(ESI): M+1=858.

[0334] Linker compounds BL (857 mg, 1 mmol), GGFG-Dxd (840 mg, 1 mmol, 1 equivalent), DIPEA (323 mg, 2.5 mmol, 2.5 equivalents), and HATU (570 mg, 1.5 mmol, 1.5 equivalents) were dissolved in 30 ml of DCM and stirred for 2 hours. The reaction mixture was cooled to 5°C-10°C, 1N hydrochloric acid (20 ml) was added, and the mixture was stirred for 0.5 hours. The separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml x 2). The resulting organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dry. The resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent, and 500 mg of linker-payload MWD-L1 was obtained as a yellow solid in 29.8% yield. LC-MS (ESI): M+1=1681,(M+1) / 2=840.9.

[0335] Example 45 Synthesis of Linker-Payload MWC-L2 [ka]

[0336] Step 1: Boc-Val-Ala-OH (288 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 equivalent), DIPEA (322 mg, 2.5 mmol, 2.5 equivalents), and HATU (456 mg, 1.2 mmol, 1.2 equivalents) were dissolved in 30 ml of DCM and stirred and reacted for 2 hours. The reaction mixture was centrifuged to dry, and the resulting residue was purified by column chromatography using a PE / EA (10:1 → 5:1) mixture as the eluent to quantitatively obtain 450 mg of intermediate 2-1 as a grayish-green solid.

[0337] Step 2: Intermediate 2-1 (450 mg, 1 mmol), tricyclic compound CDE (263 mg, 1 mmol, 1 equivalent), and pyridinium p-toluenesulfonate (PPTS) (251 mg, 1 mmol, 1 equivalent) were suspended in 30 ml of toluene and reacted under reflux for 2 hours. Heating was stopped, the reaction mixture was cooled, and the solid precipitate was collected to obtain 750 mg of crude intermediate 2-2 as a brown solid. LC-MS (ESI): M+1 = 574. The crude product was used directly in the next step without purification.

[0338] Step 3: Linker compound BL (857 mg, 1 mmol), HoSu (138 mg, 1.2 mmol, 1.2 equivalents), and DCC (310 mg, 1.5 mmol, 1.5 equivalents) were dissolved in 30 ml of DCM and stirred at room temperature for 3 hours. The reaction mixture was aspirated, and the resulting filtrate was a solution of A-Osu in DCM. The filtrate was added to a mixed solution of crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 equivalents), and DCM (30 ml), and the mixture was stirred and allowed to react for 3 hours. The reaction mixture was then cooled to below 10°C, 1N hydrochloric acid (20 ml) was added, and the mixture was stirred for 0.5 hours. The separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml x 2). The resulting organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dry. The resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent, yielding 325 mg of linker-payload MWC-L2 as an orange solid in 23.0% yield. LC-MS (ESI): M+1 = 1414, (M+1) / 2 = 707.

[0339] Example 46 Synthesis of Linker-Payload MWC-L3 [ka]

[0340] Step 1: Boc-Gly-OH (175 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 equivalent), DIPEA (322 mg, 2.5 mmol, 2.5 equivalents), and HATU (456 mg, 1.2 mmol, 1.2 equivalents) were dissolved in 30 ml of DCM and stirred and reacted for 2 hours. The reaction mixture was centrifuged to dry, and the resulting residue was purified by column chromatography using a PE / EA (10:1 → 5:1) mixture as the eluent to quantitatively obtain 335 mg of intermediate 3-1 as a pale green solid.

[0341] Step 2: Intermediate 3-1 (335 mg, 1 mmol), tricyclic compound CDE (263 mg, 1 mmol, 1 equivalent), and PPTS (251 mg, 1 mmol, 1 equivalent) were suspended in 30 ml of toluene and reacted under reflux for 2 hours. Heating was stopped, the reaction mixture was cooled, and the solid precipitate was collected to obtain 560 mg of crude intermediate 3-2 as a brown solid. LC-MS (ESI): M+1 = 461. The crude product was used directly in the next step without purification.

[0342] Step 3: Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 equivalents), and HATU (456 mg, 1.2 mmol, 1.2 equivalents) were dissolved in 20 ml of DCM and stirred and reacted for 3 hours. 1N hydrochloric acid (30 ml) was added, followed by stirring for 0.5 hours. The reaction mixture was extracted with DCM (30 ml x 2). The resulting organic phase was collected, washed with saturated brine, dried through anhydrous sodium sulfate, aspirated, and centrifuged to dry. The resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent, yielding 350 mg of intermediate 3-3 as a brown solid. LC-MS (ESI): M+1 = 945.

[0343] Step 4: Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), then diethylamine (2 ml) was added. The resulting mixture was stirred and reacted for 3 hours. The reaction solution was centrifuged to dry, and the crude intermediate 3-4 product was obtained as a brownish viscous substance. LC-MS (ESI): M+1 = 722. The crude product was used directly in the next step without purification.

[0344] Step 5: Linker compound BL (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 equivalents), and DCC (114 mg, 0.56 mmol, 1.5 equivalents) were dissolved in 30 ml of DCM and stirred at room temperature for 3 hours. The reaction mixture was aspirated, and the resulting filtrate was added to a mixed solution of crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 equivalents), and DCM (30 ml), and the mixture was stirred and allowed to react for 3 hours. The reaction mixture was then cooled to below 10°C, 1N hydrochloric acid (20 ml) was added, and the mixture was stirred for 0.5 hours. The separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml x 2). The resulting organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dry. The resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent, yielding 120 mg of linker-payload MWC-L3 as an orange solid in 20.8% yield. LC-MS (ESI): M+1 = 1562, (M+1) / 2 = 781.

[0345] Example 47 Synthesis of Linker-Payload MWE-L4 [ka]

[0346] MWE-L4 was synthesized using a method similar to that described in Example 45, but with compound A3 used instead of compound A1, and was obtained as an orange solid. LC-MS(ESI): M+1=1430.

[0347] Example 48 Synthesis of Linker-Payload MWG-L5 [ka]

[0348] MWG-L5 was synthesized using a method similar to that described in Example 45, but with compound A6 used instead of compound A1, and obtained as an orange solid. LC-MS(ESI): M+1=1442.

[0349] Example 49 Synthesis of Linker-Payload MWF-L6 [ka]

[0350] MWF-L6 was synthesized using a method similar to that described in Example 45, but with compound A7 used instead of compound A1, and obtained as an orange solid. LC-MS(ESI): M+1=1450.

[0351] Example 50 Synthesis of Linker-Payload MWF-L7 [ka]

[0352] MWF-L7 was synthesized using a method similar to that described in Example 45, but with compound A7 instead of compound A1 and Alloc-Val-Ala-OH instead of Alloc-Ala-OH, and obtained as an orange solid. LC-MS(ESI): M+1 = 1493.5.

[0353] Example 51 Synthesis of Linker-Payload MWF-L8 [ka]

[0354] MWF-L8 was synthesized using a method similar to that described in Example 46, but with compound A7 used instead of compound A1, and obtained as an orange solid. LC-MS(ESI): M+1 = 1598.4.

[0355] Example 52 Synthesis of Linker-Payload MWF-L9 [ka]

[0356] MWF-L9 was synthesized using a method similar to that described in Example 45, but with compound A5 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1416.4

[0357] Example 53 Synthesis of Linker-Payload MWF-L10 [ka] MWF-L10

[0358] MWF-L10 was synthesized using a method similar to that described in Example 45, but with compound A13 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1468.4.

[0359] Example 54 Synthesis of Linker-Payload MWF-L11 [ka] MWF-L11

[0360] MWF-L11 was synthesized using a method similar to that described in Example 45, but with compound A18 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1450.4.

[0361] Example 55 Synthesis of Linker-Payload MWF-L12 [ka] MWF-L12

[0362] MWF-L12 was synthesized using a method similar to that described in Example 45, but with compound A16 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1434.4.

[0363] Example 56 Synthesis of Linker-Payload MWF-L13 [ka] MWF-L13

[0364] MWF-L13 was synthesized using a method similar to that described in Example 45, but with compound A19 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1486.4.

[0365] Example 57 Synthesis of Linker-Payload MWF-L14 [ka] MWF-L14

[0366] MWF-L14 was synthesized using a method similar to that described in Example 45, but with compound A17 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1470.4.

[0367] Example 58 Synthesis of Linker-Payload MWF-L15 [ka] MWF-L15

[0368] MWF-L15 was synthesized using a method similar to that described in Example 45, but with compound A2 used instead of compound A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1432.4.

[0369] Example 59 Synthesis of Linker-Payload MWD-L7 [ka]

[0370] The synthesis route is as follows: [ka]

[0371] Step 1: Fmoc-Val-Cit-PAB-PNP (153 mg, 0.2 mmol), exatecan mesylate (106 mg, 0.2 mmol, 1 equivalent), and DIPEA (65 mg, 0.5 mmol, 2.5 equivalents) were dissolved in 30 ml of DCM and stirred and reacted for 2 hours. The reaction mixture was centrifuged to dry, and the resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent to obtain 180 mg of intermediate 5-1 as a yellow solid. LC-MS (ESI): M+1 = 1064.

[0372] Step 2: Intermediate 5-1 (180 mg, 0.17 mmol) was dissolved in methanol (20 ml), then diethylamine (2 ml) was added. The resulting mixture was stirred and reacted for 3 hours. The reaction solution was centrifuged to dry, and the crude intermediate 5-2 was obtained as a pale yellow viscous substance. LC-MS (ESI): M+1 = 842. The crude product was used directly in the next step without purification.

[0373] Step 3: Linker compound BL (146 mg, 0.17 mmol), HoSu (25 mg, 0.21 mmol, 1.2 equivalents), and DCC (54 mg, 0.26 mmol, 1.5 equivalents) were dissolved in 20 ml of DCM and stirred at room temperature for 3 hours. The reaction mixture was aspirated, and the resulting filtrate was added to a mixed solution of crude intermediate 5-1, DIPEA (56 mg, 0.43 mmol, 2.5 equivalents), and DCM (20 ml), and the mixture was stirred and allowed to react for 3 hours. The reaction mixture was then cooled to 5°C to 10°C, 1N hydrochloric acid (20 ml) was added, and the mixture was stirred for 0.5 hours. The separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml x 2). The resulting organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dry. The resulting residue was purified by column chromatography using a DCM / MeOH (50:1 → 10:1) mixture as the eluent, yielding 67 mg of linker-payload MWD-L7 as a yellow solid in 23.1% yield. LC-MS (ESI): M+1 = 1682, (M+1) / 2 = 841.

[0374] Example 60 Synthesis of Linker-Payload MWD-L8 [ka]

[0375] A method similar to that described in Example 44 is used, but instead of compound BL. [ka] Using [the specified method], MWD-L8 was synthesized and obtained as a pale yellow solid. LC-MS(ESI): M+1 = 1645.7.

[0376] Example 61 Synthesis of Linker-Payload MWD-L9 [ka]

[0377] A method similar to that described in Example 44 is used, but instead of compound BL. [ka] Using [the specified method], MWD-L9 was synthesized and obtained as a pale yellow solid. LC-MS(ESI): M+1 = 1713.7.

[0378] Example 62 Synthesis of Linker-Payload LD [ka]

[0379] LD was synthesized using a method similar to that described in Example 48, but with MC-OSU instead of BL-OSU, and obtained as a pale yellow solid. LC-MS(ESI): M+1 = 795.9.

[0380] Example 63 Synthesis of Linker-Payload LE [ka]

[0381] LE was synthesized using a method similar to that described in Example 45, but with MC-OSU instead of BL-OSU, and obtained as a pale yellow solid. LC-MS(ESI): M+1 = 803.8.

[0382] Example 64 Synthesis of Linker-Payload LF [ka]

[0383] Following a method similar to that described in Example 45, but using A7 instead of A1 and MaL-PEG8-COOH instead of compound BL, LF was prepared and obtained as a yellow solid. LC-MS(ESI): M+1=1185.

[0384] Example 65 Synthesis of Linker-Payload LG [ka]

[0385] LG was prepared using a method similar to that described in Example 46, but with compound MC-OSu instead of compound BL-OSu, and obtained as a yellow solid. LC-MS(ESI): M+1 = 965.98.

[0386] Example 66 Synthesis of Linker-Payload LH [ka]

[0387] LH was prepared using a method similar to that described in Example 47, but with MaL-PEG8-COOH instead of compound BL, and was obtained as a pale yellow solid. LC-MS(ESI): M+1 = 1165.3.

[0388] Example 67 Synthesis of Linker-Payload LI [ka] LI

[0389] LI was prepared using a method similar to that described in Example 45, but with A13 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1203.4.

[0390] Example 68 Synthesis of Linker-Payload LJ [ka] LJ

[0391] LJ was prepared using a method similar to that described in Example 45, but with A18 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1185.4.

[0392] Example 69 Synthesis of Linker-Payload LK [ka] LK

[0393] LK was prepared using a method similar to that described in Example 45, but with A16 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1169.3.

[0394] Example 70 Synthesis of Linker-Payload LL [ka] LL

[0395] LL was prepared using a method similar to that described in Example 45, but with A19 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1221.4.

[0396] Example 71 Synthesis of Linker-Payload LM [ka] LM

[0397] LM was prepared using a method similar to that described in Example 45, but with A17 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1205.4.

[0398] Example 72 Synthesis of Linker-Payload LN [ka]

[0399] LN was prepared using a method similar to that described in Example 45, but with intermediate HCDE instead of intermediate CDE and compound BL replaced by MaL-PEG8-COOH, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1163.2 (mixture of diastereoisomer pairs).

[0400] Example 73 Synthesis of Linker-Payload MWS-L1 [ka] MWS-L1

[0401] A similar method to the one described in Example 45 is used, but A13 is used instead of A1, and the linker compound BL is used instead of [ka] MWS-L1 was prepared using [the specified method] and obtained as a yellow solid. LC-MS(ESI): M+1 = 1236.3.

[0402] Example 74 Synthesis of Linker-Payload MWS-L2 [ka] MWS-L2

[0403] MWS-L2 was prepared using a method similar to that described in Example 45, but with A18 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1218.4.

[0404] Example 75 Synthesis of Linker-Payload MWS-L3 [ka] MWS-L3

[0405] MWS-L3 was prepared using a method similar to that described in Example 45, but with A16 used instead of A1, and was obtained as a yellow solid. LC-MS(ESI): M+1 = 1202.4.

[0406] Example 76 Synthesis of Linker-Payload MWS-L4 [ka] MWS-L4

[0407] MWS-L4 was prepared using a method similar to that described in Example 45, but with A19 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1254.4.

[0408] Example 77 Synthesis of Linker-Payload MWS-L5 [ka] MWS-L5

[0409] MWS-L5 was prepared using a method similar to that described in Example 45, but with A17 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1238.4.

[0410] Example 78 Synthesis of Linker-Payload MWS-L6 [ka] MWS-L6

[0411] A method similar to the one described in Example 54 is used, but instead of Mal-PEG8-COOH. [ka] MWS-L6 was prepared using [the specified method] and obtained as a yellow solid. LC-MS(ESI): M+1 = 1302.4.

[0412] Example 79 Synthesis of Linker-Payload MWS-L7 [ka]

[0413] MWS-L7 was prepared using a method similar to that described in Example 45, but with A18 used instead of A1, and was obtained as a yellow solid. LC-MS(ESI): M+1 = 1284.4.

[0414] Example 80 Synthesis of Linker-Payload MWS-L8 [ka]

[0415] MWS-L8 was prepared using a method similar to that described in Example 45, but with A16 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1268.4.

[0416] Example 81 Synthesis of Linker-Payload MWS-L9 [ka]

[0417] MWS-L9 was prepared using a method similar to that described in Example 45, but with A19 used instead of A1, and obtained as a yellow solid. LC-MS(ESI): M+1 = 1320.4.

[0418] Example 82 Synthesis of Linker-Payload MWS-L10 [ka]

[0419] MWS-L10 was prepared using a method similar to that described in Example 45, but with A17 used instead of A1, and was obtained as a yellow solid. LC-MS(ESI): M+1 = 1304.4.

[0420] Example Group 4: Synthesis of a comparative linker-payload Example 83 Synthesis of linker-payload LA, linker-payload LB, and linker-payload LC 1. Synthesizing linker-payload for stochastic conjugation The linker-payload MC-GGFG-Dxd was synthesized according to the method described in U.S. Patent Publication No. 20190151328, and MC-GGFG-Dxd was obtained as a pale yellow solid. LC-MS(ESI): M+1=10³⁴ 1 1H NMR (CDCl3).

[0421] Linker-payload LA and linker-payload LB were synthesized according to the method described in the patent publication International Publication No. 2020200880, with LA obtained as a yellow solid (LC-MS(ESI):M+1=767) and LB obtained as a brown adhesive (LC-MS(ESI):M+1=1149).

[0422] [ka]

[0423] 2. Synthesis of other linker-payload LCs for ring crosslinking [ka]

[0424] The synthesis route is as follows: [ka]

[0425] The linker LC was synthesized as a pale yellow solid following a method similar to that used for linker-payload L-1. LC-MS(ESI): M+1=1982, (M+1) / 2=991.

[0426] Example Group 5: Preparation and physicochemical characterization of antibody-drug conjugates 1. General method for producing antibody-drug conjugates a. General-purpose method for performing site-specific conjugation [ka]

[0427] The antibody is reduced to cleave the disulfide bond, and the reduced antibody is conjugated with a linker to form a crosslinked antibody-drug conjugate. The maleimide ring is then opened via hydrolysis, and the resulting product is purified to obtain an antibody-drug conjugate with 4 DARs. The detailed method is as follows:

[0428] Antibody Reduction: A sample containing 120 mg of antibody is transferred to a buffer solution (pH 7.0) containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate / disodium hydrogen phosphate using a NAP-25 chromatography column packed with Sephadex G-25. The antibody concentration is diluted to 10 mg / mL in this buffer solution. 2.1 ml of 10 mg / mL aqueous solution of TCEP (Sigma-Aldrich) is added to 10 mL of the diluted antibody sample (total 100 mg) in an equivalent molar ratio of 1:10 (antibody:TCEP). After incubation for 2 hours, the reaction solution is subjected to buffer exchange using a Sephadex G-25 chromatography column to a buffer solution (pH 6.5) containing 50 mM NaCl and 50 mM sodium dihydrogen phosphate / disodium hydrogen phosphate.

[0429] Conjugation and hydrolysis of antibody and linker-payload: The antibody reduced as described above is diluted to 5 mg / mL, and 0.38 mL of N,N-dimethylacetamide (DMA), representing 2% of the total reaction volume as a presolvent, and a solution of the linker-payload in 10 mg / mL of DMA are sequentially added as a reaction solution in an equivalent molar ratio of 1:5.5 (antibody:linker-payload). The resulting mixture is stirred at room temperature for 30 minutes, and then subjected to buffer exchange with a buffer consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate (pH 8.0) using a NAP-25 chromatography column packed with Sephadex G-25 to remove excess linker-payload. The resulting solution is heated in a 37°C water bath for 3 hours.

[0430] Purification of antibody-drug conjugate: The sample solution is concentrated to a concentration of approximately 15 mg / mL using an AMICOM ultrafiltration centrifuge. A buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate and 3 M ammonium phosphate is added to achieve a conductivity of 100 ms / cm. This solution is then applied to a hydrophobic column packed with TOYOPEAL Butyl-650M (purchased from Tosoh Corporation (TOSOH)) using Phase A: buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate and 0.6 M ammonium sulfate, and Phase B: buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate. Gradient elution of 8 column volumes from 0% to 100% in Phase B is performed, and the main peak is collected.

[0431] The resulting final sample was subjected to buffer exchange using an AMICOM ultrafiltration centrifuge tube to a 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.4), and then filtered through a 0.22 μm filter (Sartorius stedim Ministart).

[0432] b. A general method for constructing probabilistic conjugations. [ka]

[0433] After reducing the antibody to cleave the disulfide bond, it is conjugated with a linker to form a cross-linked antibody-drug conjugate.

[0434] Specifically, the antibody-drug conjugate is obtained by preparing it according to the preparation method described in the patent publication of Chinese Patent No. 104755494.

[0435] 2. General method for analyzing antibody-drug conjugates a. Ultraviolet spectrophotometry used to determine the drug-to-antibody ratio (UV-DAR method) and concentration The concentration of the antibody-drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the absorption wavelength characteristics of the small molecule and calculating as follows.

[0436] a1. Determination of the drug-to-antibody ratio (DAR) value of the antibody-drug conjugate The DAR is known from the literature [Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70] that DAR (drug-to-antibody ratio) = (ε Ab 280 -A 280 / A z ×ε Ab z ) / (A 280 / A z ×ε D z -ε D 280 ), where ε Ab 280 is the molar absorption coefficient of the antibody at 280 nm, A 280 is the UV absorbance of the antibody-drug conjugate at 280 nm, A z is the UV absorbance of the antibody-drug conjugate at Z nm, which is the absorption wavelength characteristic of the linker-payload in the antibody-drug conjugate, and ε Ab z is the molar absorption coefficient of the antibody at Z nm, which is the absorption wavelength characteristic of the linker-payload, and ε Dz ε is the molar extinction coefficient of the linker-payload in Znm, and D 280 The molar extinction coefficient of the linker-payload at 280 nm is as follows:

[0437] [Table 6]

[0438] a2. Determination of antibody-drug conjugate concentration Since the total absorbance of a system at a specific wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additive properties of absorbance), if the molar extinction coefficients of the antibody and linker-payload do not change before and after the conjugation of the antibody and linker-payload, the concentration of the antibody-drug conjugate is assumed to follow the following relationship: A 280 =ε ADC 280 ×C ADC ×L=(ε D 280 ×DAR+ε Ab 280 )C ADC ×L.

[0439] Therefore, the molar concentration (mol / L) of the antibody-drug conjugate is C ADC =A 280 / (ε Ab 280 +ε D 280 ×DAR). Therefore, the concentration of the antibody-drug conjugate (g / L) is C ADC =A 280 / (ε Ab 280 +ε D 280 ×DAR)×MW ADC =A 280 / (ε Ab 280 +ε D 280 ×DAR)×(MW Ab +MW D ×DAR) and here MW ADCMW is the molecular weight of the antibody-drug conjugate. Ab MW is the molecular weight of the antibody. D is the molecular weight of the linker-payload, and the protein concentration can be obtained by substituting the DAR value into this equation.

[0440] b. Hydrophobic chromatography b1. Hydrophobic chromatography (HIC-HPLC) used to determine the DAR value of antibody-drug conjugates. Sample preparation: Dilute the sample to 2.0 mg / mL with mobile phase B, then centrifuge at 12000 rpm for 10 minutes, and collect the supernatant for HPLC analysis.

[0441] Chromatography column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm × 35mm Mobile phase A: 1.5M (NH4)2SO4 + 25mM PB, pH 7.0 Mobile phase B: 25 mM PB + 20% IPA, pH 7.0 Flow rate: 0.6mL / min, Detection wavelength: 280nm, Column temperature: 30℃ Loading capacity: 10 μL Chromatographic gradient used for HIC analysis:

[0442] [Table 7]

[0443] Equation for DAR calculation: DAR = Σ(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.

[0444] b2. Hydrophobic chromatography (HIC-HPLC) used to determine the DAR value of antibody-drug conjugates. Sample preparation: Dilute the sample to 2.0 mg / mL with mobile phase B, then centrifuge at 12000 rpm for 10 minutes, and collect the supernatant for HPLC analysis.

[0445] Chromatography column: TSKgel Butyl-NPR, 2.5 μm, 4.6 mm × 100 mm Mobile phase A: 1.2M (NH4)2SO4 + 25mM PB, pH 7.0 Mobile phase B: 25 mM PB + 20% IPA, pH 7.0 Flow rate: 0.6mL / min, Detection wavelength: 280nm, Column temperature: 30℃ Loading capacity: 10 μL Chromatographic gradient used for HIC analysis:

[0446] [Table 8]

[0447] Equation for DAR calculation: Same as b1.

[0448] b3. Hydrophobic chromatography (HIC-HPLC) used to determine the DAR value of antibody-drug conjugates. Sample preparation: Dilute the sample to 2.0 mg / mL with mobile phase B, then centrifuge at 12000 rpm for 10 minutes, and collect the supernatant for HPLC analysis.

[0449] Chromatography column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm × 35mm Mobile phase A: 2.5M (NH4)2SO4 + 125mM PB, pH 7.0 Mobile phase B: 125 mM PB, pH 7.0 Mobile phase C: IPA, Mobile phase D: H2O, Flow rate: 0.5mL / min, Detection wavelength: 280nm, Column temperature: 30℃ Loading capacity: 10 μL Chromatographic gradient used for HIC analysis:

[0450] [Table 9]

[0451] Equation for DAR calculation: Same as b1.

[0452] c. Mass spectrometry (LC-MS) used to determine the DAR value Sample preparation: Place an appropriate amount of sample in an ultrafiltration tube and subject it to buffer exchange with a 50 mM NH4HCO3 buffer (pH 7.1). After replenishing the buffer, the obtained sample is subjected to ultrafiltration centrifugation (13000 g × 5 min). After buffer exchange, 8 μL of PNGase F enzyme is added to the sample, and this is then incubated at 37°C for 5 hours to remove sugar. After incubation, the sample is centrifuged at 12000 rpm for 5 minutes, and the resulting supernatant is added to the sample vial as a test sample for subsequent tests.

[0453] Chromatography column: PolyLC (trademark) polyhydroxyethyl A column, 300 Å, 5 μm, 2.1 mm × 200 mm. Mobile phase: 50 mM ammonium acetate, pH 7.0 Running time: 10 minutes Flow rate: 0.1mL / min, Loading volume: 2 μL, Column temperature: 25℃ Detection wavelength: 280nm, Ionization mode: ESI positive, Dry gas temperature: 325℃, Dry gas flow rate: 8 L / min Atomizer pressure: 20 psig, Sheath gas temperature: 325℃, Sheath gas flow rate: 12 L / min Scan settings: 900m / z~8000m / z.

[0454] d. Size exclusion chromatography (SEC-HPLC) used to determine molecular size heterogeneity. Sample preparation: The sample is diluted to 1.0 mg / mL in the mobile phase, then centrifuged at 12000 rpm for 10 minutes, and the resulting supernatant is collected for analysis.

[0455] Chromatography column: Tosoh Corporation, TSKgel G3000SWXL, 5μm, 7.8mm x 300mm, Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8) Flow rate: 0.6mL / min, Detection wavelength: 280nm, Column temperature: 30℃ Loading capacity: 20 μL, Dissolution time: 20 minutes Dissolution gradient: Isocratic elution.

[0456] e. Determination of purity by non-reducing capillary electrophoresis - sodium dodecyl sulfate (NR-CE-SDS) This determination will be carried out according to the method "Monoclonal Antibody-Molecular Size Variant Determination (CE-SDS)" described in General Rule 3127, Part IV, of the Chinese Pharmacopoeia.

[0457] f. Reverse-phase high-performance liquid chromatography (RP-HPLC) used to determine the drug-antibody ratio (DAR value) of antibody-drug conjugates. The analysis will be carried out in accordance with the method described in U.S. Patent Publication No. 10227417.

[0458] Equation for DAR calculation: DAR = 2 × (Σ(weighted peak area of ​​light chain) + Σ(weighted peak area of ​​heavy chain)) / 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.

[0459] Example 84: Preparation and Characterization of Antibody-Drug Conjugates Targeting B7-H3 The anti-B7-H3 antibodies hz10B4m1, hz10B4m2, and hz10B4 (100 mg each) were conjugated with the linker-payloads MWD-L1, MWC-L2, MWC-L3, MWF-L6, MWF-L8, and MWF-L11, respectively, according to the method described in this section, to obtain crosslinked ADCs.

[0460] The anti-B7-H3 antibodies hum30 and hz10B4m1 (100 mg each) were conjugated with the linker-payloads MC-GGFG-Dxd, vcMMAE, MWS-L1, MWS-L7, LJ, and LI, respectively, according to the method described in the Chinese Patent Publication No. 104755494, to obtain probabilistically conjugated ADCs.

[0461] The concentration, DAR value, and purity of the crosslinked ADC were determined using ultraviolet spectroscopy as described in section "a. Ultraviolet spectroscopy used to determine drug-to-antibody ratio (UV-DAR method) and concentration", hydrophobic chromatography as described in section "b. Hydrophobic chromatography", and size exclusion chromatography as described in section "d. Size exclusion chromatography (SEC-HPLC) used to determine molecular size heterogeneity".

[0462] The DAR values ​​of ADCs conjugated with the linker-payload MC-GGFG-Dxd were determined using reverse-phase high-performance liquid chromatography (RP-HPLC) as described in section "f. Reverse-phase high-performance liquid chromatography (RP-HPLC) used to determine the drug-antibody ratio (DAR value) of antibody-drug conjugates". The DAR values ​​of ADCs conjugated with vcMMAE were determined using hydrophobic chromatography as described in section "b. Hydrophobic chromatography". The concentration and purity of uncrosslinked ADCs were determined using ultraviolet spectroscopy as described in section "a. Ultraviolet spectroscopy used to determine the drug-antibody ratio (UV-DAR method) and concentration" and size exclusion chromatography as described in section "d. Size exclusion chromatography (SEC-HPLC) used to determine molecular size heterogeneity".

[0463] The results are shown in Table 5.

[0464] [Table 10]

[0465] These results demonstrate that, when measured by UV spectroscopy, the DAR values ​​of the prepared low-drug-load ADCs ranged from 3.6 to 4.8, while the DAR value of the high-drug-load ADC (B7H3-ADC-5) was 6.9. SEC results showed that, with the exception of the B7H3-ADC-3 sample (95.05% purity), all other ADC samples exhibited monomer content exceeding 98%, demonstrating the high purity of the prepared ADC samples.

[0466] Example Group 6: Evaluation of Activity Example 85: Comparative study on the activity of camptothecin KB oral epithelial carcinoma cells (purchased from ATCC) were cultured in DMEM medium supplemented with 10% FBS, NCI-N87 human gastric cancer cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS, HT29 human colon cancer cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS, and BxPC-3 human pancreatic adenocarcinoma cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS.

[0467] Using the corresponding culture medium described above, the density of KB cells in the cell plate was 2 × 10⁶ cells per mL. 4 The cells were individually adjusted to a density of 5 × 10⁶ NCI-N87 cells per mL. 4 The cells were individually adjusted, and the density of HT29 cells was set to 5 × 10⁶ cells per mL. 4 Prepare the cells individually, plate them in a 96-well transparent flat-bottom plate at 100 μL per well, and culture overnight to achieve a BxPC-3 cell density of 5 × 10⁶ cells per mL. 4 I adjusted it individually.

[0468] The sample to be detected was diluted to 20 μM in the corresponding culture medium, and then a 5-fold gradient dilution was performed to obtain nine different concentrations. Wells containing cells but without any added sample were designated as wells showing maximum cell growth, and wells without cells were designated as wells representing the culture medium background. The diluted sample was added to a cell plate at a rate of 100 μL per well, and then incubated in a CO2 incubator for 96 hours. After incubation, pre-prepared CCK-8 (purchased from Dojindo Laboratories) was added to the plate at a rate of 20 μL per well, and incubated for a further 3 hours in the CO2 incubator. The OD value at 450 nm was then read using a microplate reader.

[0469] The obtained data was fitted to a four-parameter curve using the software Prism7, and the formula for calculating the maximum mortality rate is as follows: Maximum mortality rate (%) = 1 - (OD450 value of the well showing maximum mortality rate - OD450 value of the well with the culture medium background) / (OD450 value of the well showing maximum cell growth - OD450 value of cells in the culture medium background) × 100%. The results are shown in Table 6.

[0470] [Table 11] TIFF2026522277000200.tif107170

[0471] As is evident from Table 6, the novel camptothecin provided in this disclosure exhibited significantly more potent cytotoxic activity than the control molecule DXD.

[0472] Example 86: Comparative study on the in vitro activity of antibody-drug conjugates targeting B7-H3 Calu-6 human non-small cell lung cancer cell lineage (Calu-6-B7H3, developed in-house) exhibiting high B7H3 expression was isolated from liquid nitrogen and recovered. Subsequently, the cell density was increased to 5 × 10⁶ per mL in complete medium. 4 The cells were individually prepared, added to a cell culture plate at a rate of 100 μL per well, and cultured overnight.

[0473] The B7-H3 targeted ADCs prepared above were diluted to 5 μg / mL using complete medium, and then subjected to 4-fold or 2-fold gradient dilutions to obtain nine different concentrations. The diluted samples were added to cell culture plates, with two double wells for each diluted sample, and a negative control well (cells + medium) was provided. Next, the plates were placed in a cell incubator and the cells were incubated for 168 hours (7 days). After incubation, 40 μL of MTS was added to the plates per well, and the plates were returned to a 37°C incubator and allowed to react for 2 to 4 hours. Then, the cell plates were removed and the OD values ​​at 490 nm were read.

[0474] The results are shown in Table 7.

[0475] [Table 12]

[0476] In vitro cell-killing results of B7H3-targeted ADCs showed that B7H3-ADC-7 exhibited comparable in vitro 7-day cell-killing activity to the control ADC (B7H3-ADC-9, i.e., DS-7300) in Calu-6-B7H3 cells. On the other hand, B7H3-ADC-6, B7H3-ADC-11, B7H3-ADC-13, and B7H3-ADC-14 all demonstrated superior in vitro 7-day cell-killing activity compared to the control ADC (DS-7300) in Calu-6-B7H3 cells. Furthermore, the high-drug-loaded ADC (B7H3-ADC-5) showed stronger in vitro cell-killing activity than the low-drug-loaded ADC (B7H3-ADC-4).

[0477] Example 87: Comparative study on the in vivo efficacy of antibody-drug conjugates targeting B7-H3 Calu-6 human lung cancer cells (purchased from ATCC) were used. Calu-6 cells were cultured in MEM medium containing 10% fetal bovine serum supplemented with penicillin and streptomycin in 10 cm culture dishes for adherent culture at 37°C in an incubator containing 5% CO2. The cells were passaged 2-3 times per week, and when they were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated.

[0478] A comparative study was conducted between groups receiving various doses of the antibody-drug conjugate B7H3-ADC-6. Nude mice subcutaneously transplanted with Calu-6 human lung cancer cells were administered ADC via intravenous injection (IV) at two doses once weekly, while mice in the vehicle group received the same volume of vehicle (physiological saline). Tumor volume was measured twice weekly, the mice were weighed, and the data were recorded.

[0479] The experiment will be terminated when the experiment is completed, when the experiment reaches its endpoint (day 21), or when the tumor volume reaches 2000 mm³. 3 When the tumor reached a certain stage, the animals were euthanized under CO2 anesthesia, and the tumors were dissected and photographed. The results are shown in Table 8 and Figure 2.

[0480] [Table 13]

[0481] The results above show that, compared to the vehicle group, B7H3-ADC-6 demonstrated clear tumor suppressor activity in the Calu-6 human lung cancer transplant tumor model. Furthermore, the tumor suppressor activity of B7H3-ADC-6 was superior to that of DS-7300 at the same dose, and the tumor suppressor activity of B7H3-ADC-6 at 3 mg / kg was equivalent to that of DS-7300 at 10 mg / kg.

[0482] Example 88: Comparative study on the in vivo efficacy of antibody-drug conjugates (containing camptothecin) targeting B7-H3 Pan 05.04 human pancreatic cancer cells (purchased from ATCC) were used. Pan 05.04 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum supplemented with penicillin and streptomycin in 10 cm culture dishes for adherent culture at 37°C in an incubator containing 5% CO2. The cells were passaged 2-3 times per week, and when they were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated.

[0483] A comparative study was conducted between groups receiving various doses of the antibody-drug conjugate B7H3-ADC-6. Nude mice subcutaneously transplanted with Pan 05.04 human pancreatic cancer cells were administered ADC via intravenous injection (IV) at two doses once weekly, while mice in the vehicle group received the same volume of vehicle (physiological saline). Tumor volume was measured twice weekly, the mice were weighed, and the data were recorded.

[0484] The experiment will be terminated when the experiment is completed, when the experiment reaches its endpoint (day 18), or when the tumor volume reaches 2000 mm³. 3 When the tumor reached a certain stage, the animals were sacrificed under CO2 anesthesia, and the tumors were dissected and photographed. The results are shown in Table 9 and Figure 3.

[0485] [Table 14]

[0486] The results above show that, compared to the vehicle group, B7H3-ADC-6 demonstrated clear tumor suppressor activity in a transplanted tumor model of Pan 05.04 human pancreatic cancer. The tumor suppressor activity of B7H3-ADC-6 was superior to that of DS-7300 at the same dose. Furthermore, at all three dose levels (1 mg / kg, 3 mg / kg, and 10 mg / kg), B7H3-ADC-6 monotherapy showed stronger tumor suppressor activity than combination therapy with the anti-B7-H3 antibody hz10B4m1 and compound 3 (hz10B4m1 at 10 mg / kg + compound 3 at 0.11 mg / kg).

[0487] Example 89: Study on DNA damage induced by B7-H3 targeted ADCs Calu-6 cells were seeded in 6-well plates and treated for 72 hours with the following antibodies: B7H3-ADC-6 (0.6572nM, 6.572nM, 65.72nM, 657.2nM, and 1971.7nM), DS-7300 (0.6649nM, 6.649nM, 66.49nM, and 664.9nM), naked antibody hz10B4m1 (675.4nM, and 2026.3nM), and compound 3 (0.1nM, and 1nM). Subsequently, cells were collected and lysed using lysis buffer (0.1M MES, pH 6.6, 1mM MgCl2, 1mM EGTA, 0.5% Triton X-100, 10% glycerol). The lysate was centrifuged at 12,000 rpm for 5 minutes, the supernatant was removed, and the cell pellet was resuspended in 1×SDS sample buffer (50 mM Tris, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). The resulting suspension was denatured in a boiling water bath for 10 minutes, and then subjected to Western blotting analysis to detect intracellular γ-H2AX levels.

[0488] These results demonstrate that B7H3-ADC-6, DS-7300, and compound 3 all induce DNA damage in Calu-6 cells, but hz10B4m1 does not show a significant DNA damage-inducing effect. The results are shown in Figure 4.

[0489] Example 90: Study on the inhibitory effect of B7-H3 targeted ADCs on the proliferation of various tumor cells. For cells growing by adhesion, the effect of antibody-drug conjugates, antibodies, or camptothecin on the proliferation of tumor cells cultured in vitro was detected using an SRB assay. A specific number of cells in the exponential growth phase were inoculated into a 96-well cell culture plate, and the cells were grown overnight by adhesion. Then, various concentrations of antibody-drug conjugates, antibodies, or camptothecin were added to the cells. After incubation for 144 hours, the cells were fixed with trichloroacetic acid and then stained with SRB (prepared to a concentration of 4 mg / mL in 1% glacial acetic acid). 10 mM Tris solution was added to each well to solubilize the bound SRB. OD values ​​at 510 nm were read using a microplate reader.

[0490] Suppression rate (%) = ((OD value of control well - OD value of medication well) / OD value of control well) × 100%.

[0491] For cells growing in suspension, the MTT assay was used to detect the effect of antibody-drug conjugates, antibodies, or camptothecin on the proliferation of tumor cells cultured in vitro. A specific number of cells in the exponential growth phase were inoculated into a 96-well cell culture plate, and the cells were grown overnight in suspension. Then, various concentrations of antibody-drug conjugates, antibodies, or camptothecin were added to the cells. After incubation for 144 hours, MTT was added to each well of the plate, and incubation was continued for 4 hours at 37°C in a 5% CO2 saturated humidity incubator. Next, 100 μL of SDS-isobutanol-HCl solution was added to each well of the plate, and OD values ​​at 570 nm and 690 nm were read using a microplate reader.

[0492] Half-maximal inhibitory concentration (IC 50 The inhibition rate (%) was calculated from the inhibition rate (%) at various concentrations using the software Graphpad Prism 8.0. The experiment was repeated once, the data was expressed as mean ± SD, and a plot of log(concentration) vs. inhibition rate was created. The results are shown in Table 10.

[0493] [Table 15]

[0494] As demonstrated by the above studies, B7H3-ADC-6 showed significant growth inhibitory effects against tumor cells of BxPC-3, ES-2, PANC05.04, Calu-6, Calu-3, HCT-116, MDA-MB-468, and PANC-1, and these were superior to the growth inhibitory effects of the anti-B7-H3 antibody hz10B4m1.

[0495] Example 91: Study on the bystander killing effect of B7-H3 targeted ADCs. B7-H3-positive Calu-6 cells (purchased from ATCC) and B7-H3-negative MV-4-11 / luc cells (luciferase-transfected MV-4-11 cells, purchased from Nanjing Cobrier Bioscience Co., Ltd.) were co-seeded in a 1:1 ratio in 96-well plates. B7-H3-negative MV-4-11 / luc cells were seeded alone in 96-well plates as a control. Subsequently, B7H3-ADC-6 (1000 ng / mL, 3000 ng / mL, 10000 ng / mL, 30000 ng / mL, and 100000 ng / mL) were added to the wells and incubated for 144 hours. After incubation, the plates were centrifuged, the supernatant was removed, and the cells were lysed with lysis buffer. After another centrifugation step, the supernatant was collected, mixed with the substrate luciferin, and the luminescence intensity was measured using a microplate reader.

[0496] Suppression rate (%) = ((OD value of control well - OD value of medication well) / OD value of control well) × 100%.

[0497] As demonstrated by the above study, B7H3-ADC-6 exhibited a potent bystander effect. Specifically, B7H3-ADC-6 killed B7-H3-negative MV-4-11 / luc cells, but simultaneously killed B7-H3-positive Calu-6 cells. In contrast, B7H3-ADC-6 did not show any significant growth inhibitory effect on MV-4-11 / luc cells cultured alone. The results are shown in Figure 5.

[0498] Example 92: Study on cell cycle arrest induced by B7-H3 targeted ADCs. Calu-6 cells (purchased from ATCC) were seeded in 6-well plates and treated for 48 hours with the following antibodies: B7H3-ADC-6 (0.6752 nM, 6.752 nM, 67.52 nM, 675.2 nM, and 1971.7 nM), naked antibody hz10B4m1 (675.4 nM, and 2026.3 nM), and compound 3 (0.01 nM, 0.1 nM, and 1 nM). Subsequently, cells were collected and fixed overnight with ethanol. The following day, RNase and propidium iodide (PI) were added to the cells, thoroughly mixed, and stained in the dark at 37°C for 30 minutes. The DNA content of the cells was then measured using a flow cytometer (BD FACS CALIBUR 4) at a rate of 1 × 10⁶ per group. 4 Cellular samples were analyzed. Untreated Calu-6 cells were cultured alone and used as a control. Experimental data were analyzed using ModFit LT (V3.0) for Mac.

[0499] As demonstrated by the above study, both B7H3-ADC-6 and compound 3 clearly induced G2 / M phase arrest in Calu-6 cells, whereas the naked anti-B7-H3 antibody hz10B4m1 showed no significant cell cycle arrest effect. Furthermore, these results indicate that cell cycle arrest induced by B7H3-ADC-6 occurs specifically in the G2 / M phase, which is consistent with the effect of compound 3. The results are shown in Figure 6.

[0500] The foregoing description relating to embodiments of the present invention is not intended to limit the invention, and those skilled in the art can make various changes and modifications to the invention without departing from the spirit of the invention, which should be included in the appended claims.

Claims

1. Structural formula Ia and / or structural formula Ib: 【Chemistry 1】 Ia and / or 【Chemistry 2】 Ib (In the formula, Ab is an anti-B7-H3 antibody or a fragment thereof. Group M is phenylene or phenylene substituted with one or more substituents, or a chemical bond, wherein in the substituted phenylene, the phenylene is substituted with substituents (may be more than one) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, e.g., trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano. base SP １ This is selected from the group consisting of C1-C21 (preferably C1-C16, more preferably C1-C11, more preferably C5-C9) linear heteroalkylenes containing 1 to 11 (preferably 1 to 6, more preferably 3 to 5) heteroatoms selected from the group consisting of C1-C8 alkylenes, C1-C8 cycloalkylenes, and N, O, and S, where the C1-C8 alkylenes, the C1-C8 cycloalkylenes, and the C １～２１ Linear heteroalkylenes are independently substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano groups. Base SP ２ is selected from the group consisting of -NH(CH ２ CH ２ O) ａ CH ２ CH ２ CO-, -NH(CH ２ CH ２ O) ａ CH ２ CO-, -S(CH ２ ) ａ CO-, and chemical bonds, where a is an integer in the range of 1 to 20 Group A refers to peptide groups of 2 to 4 amino acids. m is in the range of 1 to 10, preferably 1 to 8 (for example, 1 to 5), more preferably 3 to 8, and, An antibody-drug conjugate or salt thereof having a structure represented by the base CPT (where CPT refers to a compound of camptothecin).

2. The anti-B7-H3 antibody or its fragment is 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 group consisting of the following: (i) CDR-H1, CDR-H2, and CDR-H3 each contain 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, and CDR-L1, CDR-L2, and CDR-L3 each contain 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: 14, (ii) CDR-H1, CDR-H2, and CDR-H3 each contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 each contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15, and, (iii) CDR-H1, CDR-H2, and CDR-H3 each contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 each contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, Includes, Preferably, the anti-B7-H3 antibody or its fragment 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 are combinations of sequences selected from the group consisting of: (i) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

3. (ii) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 4, and (iii) The heavy chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 1, and the light chain variable region includes an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No.

5. The antibody-drug conjugate or salt thereof according to claim 1, characterized by containing the above.

3. The anti-B7-H3 antibody or its fragment 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 of the constant region. Alternatively, the antibody-drug conjugate or salt thereof according to claim 1 or 2, wherein the anti-B7-H3 antibody or fragment thereof comprises a heavy chain and / or a light chain, where, for example, the heavy chain comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and the light chain comprises a κ-type or λ-type light chain constant region.

4. In structural formula Ia and / or structural formula Ib, group M is halogen-substituted phenylene, preferably fluorine-substituted phenylene. Preferably, base SP １ This is a linear heteroalkylene of C1 to C11, preferably C5 to C9, more preferably C7, containing 1 to 6, preferably 3 to 5, more preferably 4 heteroatoms selected from the group consisting of N, O, and S, and Preferably, base SP ２ The antibody-drug conjugate or salt thereof according to any one of claims 1 to 3, characterized in that the bond is preferably a chemical bond.

5. Base CPT is structural formula I: 【Transformation 3】 Structural formula I (In the formula, group R １ , group R ２ , group R ３ , and base R ４ These are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C1-C6 alkoxy, amino or substituted amino, and C1-C7 alkyl or substituted C1-C7 alkyl, or the group R １ , group R ２ , group R ３ , and base R ４ Any two of these, together with the carbon atoms to which they are bonded, form a C3-C6 (preferably C3-C5) cycloalkyl group. Group G is hydrogen, halogen, methyl, or methoxy. Group Y is oxygen, sulfur, sulfone, sulfoxide, methylene, or substituted methylene. Group X is oxygen or sulfur, and The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, having a structure represented by (n = 0 or 1), wherein structural formula I is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond, and preferably, the amino adjacent to group G in structural formula I is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond.

6. Base CPT is structural formula IA: 【Chemistry 4】 Structural formula IA (In the formula, group R １ , group R ２ , group R ３ , and base R ４ This is the base R in structural formula I. １ , group R ２ , group R ３ , and base R ４ The same meaning as defined in claim 5 applies to the base R １ , group R ２ , group R ３ , and base R ４ The antibody-drug conjugate or salt thereof according to any one of claims 1 to 5, having a structure represented by (where is not hydrogen at the same time), wherein structural formula IA is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond, preferably the amino on the left-hand benzene ring in structural formula IA is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond.

7. The base CPT is structural formula II: 【Transformation 5】 Structural formula II (In the formula, group R ５ This is a C1-C5 alkyl or a C1-C5 alkyl substituted with one or more substituents, a C3-C6 cycloalkyl or a C3-C6 cycloalkyl substituted with one or more substituents, a phenyl or a substituted phenyl. Group G is hydrogen, halogen (e.g., fluorine), methyl, or methoxy. Group X is oxygen or sulfur, and The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, having a structure represented by (n = 0 or 1), wherein structural formula II is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond, and preferably, the amino adjacent to group G in structural formula II is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond.

8. Base CPT is structural formula IIA: 【Transformation 6】 Structural formula IIA (In the formula, group R ５ This is the group R in structural formula II. ５ The same meaning as defined in claim 7 applies to the base R ５ The antibody-drug conjugate or salt thereof according to claim 7, having a structure represented by (not n-butyl), wherein structural formula IIA is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond, preferably the amino on the left-hand benzene ring in structural formula IIA is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond.

9. The base CPT has the following structure: 【Transformation 7】 【Transformation 8】 【Chemistry 9】 The antibody-drug conjugate or salt thereof according to any one of claims 1 to 8, wherein each structure is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond, and preferably, the amino on the left-hand benzene ring in each structure is bonded to the carboxyl of group A in structural formula Ia and / or structural formula Ib via an amide bond.

10. The base CPT is structural formula IV: 【Chemistry 10】 Structural formula IV (In the formula, group R ８ The structure is represented by hydrogen, trifluoromethyl, C1-C5 alkyl or C1-C5 alkyl substituted with one or more substituents, C3-C6 cycloalkyl or C3-C6 cycloalkyl substituted with one or more substituents, or halogen, where the group R in structural formula IV ８ The hydroxyl group bonded to the same carbon is bonded to the carboxyl group A in structural formula Ia and / or structural formula Ib via a self-sacrificing structure, and the self-sacrificing structure is, for example, 【Chemistry 11】 or 【Chemistry 12】 Here, the solid line "-" indicates the bond of group A to the carboxyl in structural formula Ia and / or structural formula Ib, and the wavy line 【Chemistry 13】 The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, characterized in that represents a bond to hydroxyl in structural formula IV.

11. general formula 【Chemistry 14】 It has a structure represented by the above general formula, mAb is an anti-B7-H3 antibody or a fragment thereof. Group M, Group SP １ , base SP ２ Base A and base CPT are base M and base SP in any one of claims 1 to 10. １ , base SP ２ This has the same meaning as defined for Base A and Base CPT, N is in the range of 1 to 10, preferably 1 to 8 (for example, 1 to 5), more preferably 3 to 8, and E Ｌ The following is the basis: E Ｌ -1a and / or E Ｌ -1b: 【Chemistry 15】 and / or 【Chemistry 16】 E Ｌ -2: 【Chemistry 17】 E Ｌ -3: [Chemistry 18] E Ｌ -4: 【Chemistry 19】 E Ｌ -5: 【Chemistry 20】 E Ｌ -6: 【Chemistry 21】 A group consisting of the following is selected, where, 【Chemistry 22】 This indicates the binding to cysteine ​​in mAb, and the tilde symbol 【Chemistry 23】 This refers to an antibody-drug conjugate or a salt thereof, signifying binding to group M.

12. A method for producing an antibody-drug conjugate or a salt thereof according to any one of claims 1 to 11, comprising the following steps: a. Antibody reduction: A reducing agent is added to a phosphate buffer containing an antibody at a concentration of 5 mg / mL to 30 mg / mL in an equivalent molar ratio of 5.5 or more:1 (reducing agent:antibody), and the reducing agent and the antibody are reacted for 1.5 to 2 hours (wherein the reducing agent is one or more selected from the group consisting of TCEP, DTT, 2-MEA, and DTBA). b. Antibody conjugation and hydrolysis: The reduced antibody obtained in step a is transferred to a phosphate buffer at pH 6.5 to 7.8, thereby diluting the antibody to a concentration of 3.5 mg / mL to 15 mg / mL in the buffer to obtain a diluted antibody solution. The linker-payload dissolved in an organic cosolvent is added to the diluted antibody solution in an equivalent molar ratio of 4.5 to 6.5:1 (linker-payload:antibody), and the reaction system is stirred and reacted at 15°C to 35°C for 0.5 hours or more (wherein the organic cosolvent is one or more selected from the group consisting of DMA, DMSO, DMF, and ACN). The antibody conjugation product is then transferred to a phosphate buffer at pH 7.4 to 9.0, and the buffer is heated at 35±10°C for 2 to 24 hours to obtain a hydrolysis product. c. Hydrophobic chromatography: A step in which the obtained antibody conjugation product is purified by hydrophobic chromatography using a hydrophobic packing material. Methods that include...

13. In step a, the antibodies include, for example, HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP 1, GPNMB, FGF2, FOLR 1, EGFR, EGFRvIII, Tissue Factor, c-MET, FGFR, Nectin 4, AGS-16, and Guani. The method according to claim 12, characterized in that it is an antibody against a tumor-associated antigen which is lyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3, or claudin 18.

2.

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

15. Use of an antibody-drug conjugate or salt thereof according to any one of claims 1 to 11 and / or the composition according to claim 14 in the manufacture of a pharmaceutical product for the treatment of tumors.

16. The aforementioned tumors include, for example, HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, mucin 1, STEAP 1, GPNMB, FGF2, FOLR 1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, nectin 4, AGS-16, and guanylyl cyclase C. The use according to claim 15, characterized in that it is associated with positive expression or high expression of tumor-associated antigens, which are mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3, or claudin 18.

2.

17. A method for treating a tumor, comprising administering an antibody-drug conjugate or a salt thereof according to any one of claims 1 to 11 and / or a composition according to claim 14 to a subject in need of treatment.

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