Antibody-drug conjugate

Abstract

An antibody-drug conjugate, having the structure of formula 1 described below, wherein Ab is an antibody or antibody fragment for targeting TROP2, and j is 1-8, preferably 4-8. The antibody-drug conjugate has the ability of highly specifically binding to tumor cells expressing TROP2 protein, thereby achieving excellent killing effect on the cells.

Description

An antibody drug conjugate Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an antibody drug conjugate. Background Technology

[0002] Malignant tumors (cancer) have become the leading cause of death worldwide. The fundamental reasons for the high mortality rate of cancer are the spread and metastasis of cancer cells, and the high recurrence rate and drug resistance in most patients after treatment. Currently available clinical treatments, such as surgery, radiotherapy, and chemotherapy, have minimal or no effect on cancer cell metastasis, recurrence, or drug resistance, and cannot change the long-term survival of patients. Currently, surgical resection is effective for approximately 10-20% of early-stage patients, but almost ineffective for patients who have already experienced metastasis. Radiotherapy can only treat local lesions and is often used as adjuvant therapy before and after surgery, and as a radical treatment for a few types of cancer. Chemotherapy can be used for patients with metastasis, but due to its significant side effects and the tendency to develop short-term or long-term drug resistance, it only has a significant short-term effect on approximately 20-30% of patients. Even with comprehensive treatment combining surgery, radiotherapy, and chemotherapy, the 5-year survival rate has remained around 20-30% for many years, and approximately 70-80% of patients die within 5 years after treatment due to metastasis, recurrence, and drug resistance. Even some early-stage cancer patients who have no metastasis at the time of diagnosis still die from metastasis and recurrence after treatment. Summary of the Invention

[0003] Currently, identifying tumor-associated antigens and then delivering drugs to tumor cells in the form of antibody-drug conjugates is an effective method for treating tumors. This drug form combines the targeting specificity of monoclonal antibodies to tumor cells with the powerful tumor-killing advantages of small-molecule cytotoxic agents. It can significantly reduce the toxic side effects of small-molecule toxins while improving drug efficacy, thus exhibiting significant therapeutic advantages.

[0004] TROP2 is a gene closely related to tumors, primarily promoting tumor cell growth, proliferation, and metastasis by regulating calcium signaling pathways, cyclin expression, and reducing fibronectin adhesion. TROP2 can also interact with β-catenin in the Wnt signaling cascade, thus affecting the transcription of nuclear oncogenes and cell proliferation. TROP2 protein is highly expressed in various tumors, such as pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer. It can promote tumor cell proliferation, invasion, metastasis, and spread, and its high expression is closely related to shortened survival and poor prognosis in cancer patients; however, currently available antibodies that target TROP2 alone have poor inhibitory effects on tumor cell growth. Therefore, the applicant has developed an antibody-drug conjugate with high specificity binding to TROP2-expressing tumor cells, achieving excellent cell-killing effects by targeting TROP2-positive tumor cells.

[0005] The specific technical solution of this application is as follows:

[0006] 1. An antibody-drug conjugate, comprising the structure of Formula 1:

[0007] Wherein, Ab is an antibody or antibody fragment used to target TROP2;

[0008] j can be 1 to 8, preferably 4 to 8.

[0009] 2. The antibody-drug conjugate according to item 1, wherein,

[0010] The antibody or antibody fragment for targeting TROP2 comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0011] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1;

[0012] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2;

[0013] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3;

[0014] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4;

[0015] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5;

[0016] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6.

[0017] 3. The antibody-drug conjugate according to claim 1, wherein the antibody or antibody fragment for targeting TROP2 comprises a heavy chain variable region and a light chain variable region, wherein,

[0018] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 7.

[0019] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 8.

[0020] 4. The antibody-drug conjugate according to any one of items 1-3, wherein the Ab is an antibody targeting TROP2, the heavy chain of the antibody is an IgG1 subtype or a mutated IgG1 subtype, and the light chain of the antibody is a Kappa type.

[0021] 5. The antibody-drug conjugate according to claim 4, wherein the antibody for targeting TROP2 comprises a heavy chain and a light chain, wherein,

[0022] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 9.

[0023] The amino acid sequence of the light chain is as shown in SEQ ID NO: 10 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10.

[0024] 6. A method for preparing an antibody-drug conjugate, comprising the following steps:

[0025] LD-38 is available;

[0026] Construct antibodies or antibody fragments (Abs) to target TROP2;

[0027] LD-38 is conjugated with an antibody or antibody fragment Ab targeting TROP2 to obtain an antibody-drug conjugate.

[0028] The structure of LD-38 is as follows

[0029] The antibody or antibody fragment Ab used to target TROP2 is described in references 1-5.

[0030] 7. A pharmaceutical composition comprising, wherein the antibody-drug conjugate described in any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof.

[0031] 8. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in the preparation of a medicament for the treatment and / or prevention of tumors.

[0032] 9. The use according to item 8, wherein the tumor is selected from solid tumors, hematologic malignancies, and metastatic, refractory, or recurrent lesions of cancer.

[0033] 10. The use according to item 8, wherein the tumor is selected from the group consisting of: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer.

[0034] 11. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in combination with other therapeutic agents in the preparation of a medicament for the treatment and / or prevention of tumors.

[0035] 12. According to the use described in item 11, wherein the other therapeutic agent is selected from immune checkpoint drugs, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, drugs for chemotherapy, or drugs for radiotherapy.

[0036] 13. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in the treatment and / or prevention of tumors.

[0037] 14. The use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in combination with other therapeutic agents for the treatment and / or prevention of tumors.

[0038] 15. According to the use described in item 14, wherein the other therapeutic agent is selected from immune checkpoint drugs, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, drugs for chemotherapy, or drugs for radiotherapy.

[0039] 16. A method for treating and / or preventing tumors, wherein a subject is given a therapeutically effective amount of the antibody-drug conjugate described in any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof.

[0040] 17. The method according to item 16, wherein the tumor is selected from solid tumors, hematologic malignancies, and metastatic, refractory, or recurrent lesions of cancer;

[0041] Preferably, the tumor is selected from the group consisting of: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer;

[0042] Preferably, other therapeutic drugs may also be administered simultaneously.

[0043] The antibody-drug conjugate described in this application has a high specificity binding ability to tumor cells expressing TROP2 protein, thereby achieving excellent cell killing effect. Attached Figure Description

[0044] Figure 1 is a SEC analysis diagram of BY016-LD-38 prepared in Example 2 of this application.

[0045] Figure 2 is a HIC analysis chromatogram of BY016-LD-38 prepared in Example 2 of this application.

[0046] Figure 3 shows the bystander killing effect of BY016-LD-38 in Example 5, co-cultured in 293T-Trop2 and 293T.

[0047] Figure 4 shows the tumor volume change curve of the test drug in Example 6 after administration to PDTX0501008 tumor-bearing mice with gastric cancer.

[0048] Figure 5 shows the bioluminescence signal value curve of the test drug in Example 7 in a human breast cancer JIMT-1-luc brain metastasis mouse model.

[0049] Figure 6 shows the survival rate of the test drug in Example 7 in a human breast cancer JIMT-1-luc brain metastasis mouse model.

[0050] Figure 7 shows the weight changes of the test drug in a human breast cancer JIMT-1-luc brain metastasis mouse model in Example 7.

[0051] Figure 8 shows the tumor growth curve of the JIMT-1 xenograft tumor model mice in Example 8 after administration of the test substance.

[0052] Figure 9 shows the weight changes of tumor-bearing mice in the JIMT-1 xenograft tumor model in Example 8 after administration of the test substance.

[0053] Figure 10 shows the tumor growth curve (mean ± SEM) of the gastric cancer PDX (GA2421) tumor-bearing mouse model in Example 9.

[0054] Figure 11 shows the weight growth curve (mean ± SEM) of the gastric cancer PDX (GA2421) tumor-bearing mouse model in Example 9.

[0055] Figure 12 shows the tumor growth curve (mean ± SEM) of the gastric cancer PDX (GA6844) tumor-bearing mouse model in Example 9.

[0056] Figure 13 shows the weight growth curve (mean ± SEM) of the gastric cancer PDX (GA6844) tumor-bearing mouse model in Example 9.

[0057] Figure 14 shows the tumor growth curve of the human gastric cancer N87 CDX tumor-bearing mouse model after drug treatment in Example 10.

[0058] Figure 15 shows the tumor growth curve of the human gastric cancer GSU CDX tumor-bearing mouse model after drug treatment in Example 11.

[0059] Figure 16 shows the tumor growth curve of the human colon cancer COLO205 CDX tumor-bearing mouse model after drug treatment in Example 12.

[0060] Figure 17 shows the tumor growth curve of the ENHERTU-resistant NCI-N87 CDX tumor-bearing mouse model after drug treatment in Example 13.

[0061] Figure 18 shows the concentration-time curve of total anti-ADC in rat PK of BY016-LD-38 / Dato-Dxd in Example 14.

[0062] Figure 19 shows the total toxin and free toxin content in the plasma of the NCI-N87 CDX tumor-bearing mouse model in Example 15.

[0063] Figure 20 shows the total and free toxin content in the tumor tissue of the NCI-N87 CDX tumor-bearing mouse model in Example 15. Detailed Implementation

[0064] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0065] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0066] This application provides an antibody drug conjugate, comprising the structure of Formula 1 below:

[0067] Wherein, Ab is an antibody or antibody fragment or antibody fragment used to target TROP2;

[0068] j can be 1 to 8, preferably 4 to 8, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, etc.

[0069] In this application, the antibody or antibody fragment for targeting TROP2 comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0070] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1;

[0071] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2;

[0072] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3;

[0073] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4;

[0074] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5;

[0075] The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6.

[0076] In this application, the antibody or antibody fragment for targeting TROP2 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 7.

[0077] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 8.

[0078] In this application, the Ab is an antibody targeting TROP2, the heavy chain of the antibody is IgG1 subtype or mutated IgG1 subtype, and the light chain of the antibody is Kappa type.

[0079] In this application, the antibody for targeting TROP2 comprises a heavy chain and a light chain.

[0080] In some embodiments, the heavy chain of the antibody is the IgG1 subtype, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 9.

[0081] The DNA sequence of the heavy chain is shown in SEQ ID NO: 11.

[0082] In some embodiments, the heavy chain of the antibody is a mutated IgG1 subtype, specifically with an LALA mutation. The LALA mutation refers to the replacement of leucine (Leu, L) at positions 234 and 235 with alanine (Ala, A) in the lower hinge region of the Fc segment of the IgG1 antibody, i.e., the L234A / L235A mutation.

[0083] The amino acid sequence of the light chain is as shown in SEQ ID NO: 10 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10.

[0084] The DNA sequence of the light chain is shown in SEQ ID NO: 12.

[0085] SEQ ID NO: 9-10 are all humanized sequences.

[0086] In this application, the antibody targeting TROP2 is able to bind to TROP2 with sufficient affinity, such that the antibody targeting TROP2 can be used as a diagnostic and / or therapeutic agent targeting TROP2.

[0087] The antibody targeting TROP2 in this application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to proteins other than TROP2, which is the target; and "does not bind" means that, when the binding ability of the antibody targeting TROP2 in this application to TROP2 as its target is taken as 100%, the binding ability of the antibody targeting TROP2 in this application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0088] The antibody used in this application to target human TROP2 is a humanized monoclonal antibody.

[0089] The full name of the human TROP2 mentioned in this application is human trophoblast surface glycoprotein antigen.

[0090] The amino acid sequence of human TROP2 is SEQ ID NO: 13.

[0091] In this application, "antibody" refers to a whole antibody, or may also be called a complete antibody or intact antibody. An antibody is a glycoprotein containing at least two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. In this application, the term "antibody" has its broadest meaning, encompassing immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites or domains, and can be used to refer to antigen-binding structural fragments (e.g., antigen-binding fragments) or complexes of one or more antigen-binding fragments (e.g., scFv). Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or any type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass, or of any origin (e.g., human, mouse, rabbit, camel, fish, etc.).

[0092] In this application, "antigen-binding fragment" or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a given antigen and thereby exhibit the desired antigen-binding activity. The antigen-binding function of an antibody can be performed by fragments of the complete antibody. Examples of binding fragments covered by the term "antigen-binding fragment of an antibody" include, but are not limited to, examples of the following antibody fragments, including but not limited to: Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of an antibody arm; single-domain antibody (dAb) fragments consisting of either a VH domain or a VL domain; and isolated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via artificial peptide linkers using recombinant methods, enabling them to become a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). These single-chain antibodies may include one or more antigen-binding fragments of the antibody. These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the usability of the fragments is screened in the same manner as for intact antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, large antibodies, micro antibodies, intracellular antibodies, bisomal antibodies, trisomal antibodies, tetrasomal antibodies, v-NARs, and bis-scFvs. Antigen-binding fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), forming a pair of antigen-binding regions together with a complementary light chain polypeptide. "Antibodies" include polyclonal antibodies and monoclonal antibodies.

[0093] In this application, the “Fd” fragment consists of VH and CH1 domains. The “dAb” fragment (Ward et al., (1989) Nature 341:544-546) consists of a VH domain. Separate complementarity-determining regions (CDRs) and combinations of two or more separate CDRs can optionally be joined by a synthetic linker.

[0094] In this application, the "Fv" fragment consists of the VL and VH domains of the antibody single arm. The single-chain Fv (scFv) consists of a heavy chain variable region and a light chain variable region, which are covalently linked into a single-chain polypeptide chain by a flexible peptide linker.

[0095] In this application, "monoclonal antibody" refers to an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0096] In this application, "monoclonal antibody" generally refers to a human antibody, which can be prepared using techniques known to those skilled in the art. For example, human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).

[0097] The monoclonal antibodies described in this application may also be antibody variants, for example, where improved binding affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. Thus, in some embodiments, antibody variants with one or more amino acid substitutions are provided, where the sites of interest for substitution mutation include HVR and FR. For example, amino acid substitutions can be introduced into the antibody of interest and products with desired activities can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0098] In this application, the Fc region of the antibody whose heavy chain is the IgG1 subtype is different from that of the antibody whose heavy chain is the mutated IgG1 subtype, but the variable regions are the same. That is, the variable regions of the heavy chain and the variable regions of the light chain of the two antibodies are the same.

[0099] In this application, the antibody component or antibody in the antibody-drug conjugate comprises a human-derived Fc domain (Fc region) and all other portions of a preferably human constant region. As used herein, the term "human-derived Fc domain" means an Fc domain that is the Fc domain of a human antibody of the IgG1, IgG2, IgG3, or IgG4 subclass, preferably an Fc domain of the human IgG1 subclass or a mutant Fc domain of the human IgG1 subclass. The antibody has reduced or minimal effector function. Minimal effector function is caused by an effectorless Fc mutation.

[0100] In this application, the term "Fc region" is used to define a C-terminal region in an immunoglobulin heavy chain that contains at least a portion of a constant region, including both native and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index number). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In some embodiments, the heavy chain including the Fc region (subunit) as specified herein is included in the antibody according to this application. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a “subunit” of the Fc domain refers to one of two polypeptides forming a dimer Fc domain, namely a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain that is stably self-associating. For example, the subunit of the IgG Fc domain contains an IgG CH2 constant domain and an IgG CH3 constant domain.

[0101] In this application, "variable region," also known as "variable domain," refers to the domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0102] In this application, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and progeny derived from said primary transformed cells, regardless of passage number. Progeny may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce antibodies of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as, to name just a few, HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one aspect, the host cells of the present invention are eukaryotic cells, particularly mammalian cells. In one respect, the host cell is not a cell within the human body.

[0103] In this application, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured using common methods known in the art.

[0104] This application provides a method for preparing an antibody drug conjugate, comprising the following steps:

[0105] Step 1: Provide LD-38;

[0106] Step 2: Construct an antibody or antibody fragment Ab for targeting TROP2;

[0107] Step 3: Conjugate LD-38 with an antibody or antibody fragment Ab targeting TROP2 to obtain an antibody-drug conjugate;

[0108] The structure of LD-38 is as follows

[0109] The antibody or antibody fragment Ab used to target TROP2 is as described above.

[0110] This application provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts, prodrugs, or solvates.

[0111] This application also provides the use of the aforementioned antibody-drug conjugate, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate, in the preparation of a medicament for the treatment and / or prevention of tumors.

[0112] The tumor is selected from metastatic, refractory, or recurrent lesions of solid tumors, hematologic malignancies, and cancers.

[0113] The tumors are selected from the following group: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer.

[0114] This application also provides the use of the antibody-drug conjugate, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate, in combination with other therapeutic agents in the preparation of a medicament for the treatment and / or prevention of tumors.

[0115] The other therapeutic agents are selected from immune checkpoint inhibitors, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, or drugs used for chemotherapy or radiotherapy.

[0116] This application also provides the use of the antibody-drug conjugate, or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts, prodrugs, or solvates, in the treatment and / or prevention of tumors.

[0117] This application also provides the use of the antibody-drug conjugate, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate, in combination with other therapeutic agents for the treatment and / or prevention of tumors.

[0118] The other therapeutic agents are selected from immune checkpoint inhibitors, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, or drugs used for chemotherapy or radiotherapy.

[0119] This application also provides a method for treating and / or preventing tumors, wherein a therapeutically effective amount of an antibody-drug conjugate, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, is administered to a subject.

[0120] The tumor is selected from metastatic, refractory, or recurrent lesions of solid tumors, hematologic malignancies, and cancers.

[0121] Furthermore, the tumor is selected from the group consisting of: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer.

[0122] Furthermore, other therapeutic agents may be administered concurrently when administering antibody drug conjugates, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically acceptable salts, prodrugs, or solvates.

[0123] The other therapeutic agents are selected from immune checkpoint inhibitors, antitumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, antitumor T-cell adaptor antibodies, synthetic lethal target drugs, drugs for chemotherapy, or drugs for radiotherapy. In this application, "pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0124] In this application, "treatment" (and its grammatical variations) refers to an attempt to alter the natural course of a disease in the treated individual, and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the antibodies of this application are intended to delay the development of disease or slow its progression.

[0125] In this application, "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some respects, the individual or subject is a human.

[0126] Example

[0127] The materials and test methods used in the embodiments of this application are described in a general and / or specific manner. In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0128] Example 1: Preparation of LD-38

[0129] The synthetic route for LD-38 is as follows:

[0130] Synthesis of 38-1

[0131] Fmoc-L-glutamic acid-5-tert-butyl ester (144.7 mg, 1.175 mmol) and 4-aminobenzyl alcohol (500 mg, 1.175 mmol) were dissolved in 3 mL of anhydrous DMF. DIEA (303.7 mg, 2.350 mmol) and HATU (536.19 mg, 1.410 mmol) were added, and the mixture was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete. Water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification was performed using a normal-phase silica gel column (EA / PE, 0% to 45%) to obtain 530 mg. MS(+) = 475.2.

[0132] Synthesis of 38-2

[0133] Compound 38-1 (530 mg, 0.999 mmol) was dissolved in 3 mL of anhydrous DMF, and piperidine (0.2 mL) was added to the reaction solution. The mixture was stirred at room temperature for one hour. The solution was concentrated to obtain 600 mg of crude product, which was used directly in the next step of the reaction. MS(+) = 309.2.

[0134] Synthesis of 38-3

[0135] Compound 38-2 (600 mg crude) and Fmoc-L-valine (500 mg, 1.175 mmol) were dissolved in 3 mL of anhydrous DMF. DIEA (334.13 mg, 2.585 mmol) and HATU (589.82 mg, 1.551 mmol) were added, and the mixture was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete. Water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification was performed using a normal-phase silica gel column (EA / PE, 40% to 60%) to give 450 mg, 6.MS(+) = 630.4.

[0136] Synthesis of 38-4

[0137] Compound 38-3 (450 mg, 0.715 mmol) was dissolved in 1 mL of anhydrous DCM and 4 mL of anhydrous DMF. Di(p-nitrobenzene) carbonate (869.5 mg, 2.858 mmol) and 0.354 mL of DIEA were added sequentially. After reacting at room temperature for 12 hours, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification was performed using a normal-phase silica gel column (EA / PE, 30% to 70%) to obtain 200 mg. MS(+) = 543.2.

[0138] Synthesis of 38-5

[0139] Compound 38-4 (100 mg, 0.126 mmol) and eczematidine mesylate (54.78 mg, 0.126 mmol) were dissolved in 2 mL of anhydrous DMF. DMAP (0.15 mg, 0.0001 mmol) and DIEA (32.52 mg, 0.252 mmol) were added sequentially. After reacting at room temperature for 1 hour, water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification was performed using a normal-phase silica gel column (EA / PE, 30% to 70%) to obtain 100 mg. MS(+) = 1091.4.

[0140] Synthesis of 38-6

[0141] Compound 38-5 (100 mg, 0.073 mmol) was dissolved in 3 mL of anhydrous DMF, and piperidine (0.3 mL) was added to the reaction solution. The mixture was stirred at room temperature for one hour. The solution was concentrated to obtain 110 mg of crude product, which was used directly in the next step of the reaction. MS(+) = 870.8.

[0142] Synthesis of 38-7

[0143] Compound 38-6 (110 mg crude) and Mal-amido-PEG4-acid (30.19 mg, 0.073 mmol) were dissolved in 2 mL of anhydrous DMF. DIEA (18.74 mg, 0.145 mmol) and HATU (33.08 mg, 0.087 mmol) were added, and the mixture was stirred at room temperature for 2 hours. LC-MS showed the reaction was complete. Water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification was performed using a normal-phase silica gel column (MeOH / DCM, 5% to 20%) to give 90 mg. MS(+) = 1267.6.

[0144] Synthesis of 38-8

[0145] Compound 38-7 (90 mg, 0.071 mmol) was dissolved in 3 mL of anhydrous DCM, and 0.3 mL of TFA was added. The mixture was stirred at room temperature for 2 hours. The crude product was concentrated to 80 mg and used directly in the next reaction. MS(+) = 2111.4.

[0146] Synthesis of LD-38

[0147] Compound 38-8 (80 mg, crude) and sulfonic acid quaternary ammonium salt A (13.89 mg, 0.066 mmol, CAS No.: 78276-19-4, provided by Shanghai Haoyuan Pharmaceutical Co., Ltd., synthetic method according to WO2011146595A2) were dissolved in 2 mL of anhydrous DMF. DIEA (17.07 mg, 0.132 mmol) and HATU (30.14 mg, 0.079 mmol) were added, and the mixture was stirred at room temperature for 1 hour. LC-MS showed that the reaction was complete. Purification was performed by reverse-phase preparation (mobile phase containing 0.1% formic acid) to give 13.5 mg of a white solid.

[0148] LCMS: m / z(ES+)(M+H)+=1404.6, Rt=1.314min

[0149] 1H NMR (400MHz, DMSO) δ9.89(s,1H),8.18(d,J=7.2Hz,2H),8.10–7.93(m,3H),7.78(d,J=10.8Hz,1H),7.63(d,J= 8.4Hz,2H),7.37(d,J=8.4Hz,2H),7.32(s,1H),6.99(s,2H),6.53(s,1H),5.45(s,2H),5.29(d,J=4.Hz,3H),5 .08(s,2H),4.32(dd,J=13.2,7.6Hz,1H),4.20–4.14(m,1H),3.59(dd,J=14.8,7.6Hz,6H),3.40-3.51(m,18H) ,3.14-3.20(m,4H),3.03(s,7H),2.42-2.30(m,7H),2.20-2.31(m,4H),2.03-1.82(m,8H),0.87-0.91(m,9H).

[0150] Example 2 Preparation of antibody-drug conjugates ADC (BY016-LD-38, IgG1-LD-38)

[0151] The BY016 antibody is an anti-TROP2 monoclonal antibody, with the Fc region employing the IgG1 isotype. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the DNA sequence is shown in SEQ ID NO: 11. The amino acid sequence of the light chain is shown in SEQ ID NO: 10, and the DNA sequence is shown in SEQ ID NO: 12. The BY016 antibody was prepared by constructing the heavy and light chain nucleotide sequences of the antibody into the PCDNA3.4 expression plasmid, followed by transient transfection into CHO cells. The expression supernatant was then purified using Protein A affinity.

[0152] The IgG1 Isotype antibody was purchased from Shanghai Baiying Biotechnology Co., Ltd. (Catalog No. B117901).

[0153] The antibody was transferred to pH 7.4 phosphate buffer using an ultrafiltration centrifuge tube, and 2.2 times the molar amount of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added. The reaction was carried out at 37°C for 2 hours to open the interchain disulfide bonds of the antibody. After reduction, the antibody was transferred to pH 7.4 phosphate buffer again using an ultrafiltration centrifuge tube to remove excess TCEP, and 6–8 times the molar amount of LD-38 was added. The reaction was carried out at room temperature for 1 hour. After the reaction, 2 times the molar amount of LD-38 was added to quench the reaction, and the solution was transferred to pH 5.5 acetate-sodium acetate (40 mM) buffer using an ultrafiltration centrifuge tube. The ADC sample after solution transfer was filtered through a 0.22 μm sterile filter to obtain the antibody-drug conjugate BY016-LD-38 and IgG1-LD-38. The purity of BY016-LD-38 was measured by size exclusion chromatography (SEC).

[0154] Size exclusion chromatography (SEC) was performed using a Tskgel G3000SWXL (TOSOH, 0008541) column with a mobile phase of 0.1M PB pH 7.0 and 0.1M NaCl. Isocratic elution was used at a flow rate of 1 mL / min and a column temperature of 25℃. Detection wavelengths were 280 nm and 366 nm. The SEC results are shown in Figure 1. The purity of BY016-LD-38ADC reached 99.8%.

[0155] Hydrophobic interaction chromatography (HIC) was performed using a Butyl-NPR 4.6 × 100 mm column (TOSOH, 0042168). Mobile phase A consisted of 50 mM PB pH 7.0 and 1.5 M (NH4)2SO4, while mobile phase B consisted of 50 mM PB pH 7.0 and 20% acetonitrile. The separation gradient was 2–20 min from 0% B to 100% B, with a column temperature of 30 °C and a flow rate of 0.6 mL / min. Detection wavelengths were 280 nm and 366 nm. The HIC analysis results are shown in Figure 2. The average DAR value of the BY016-LD-38 ADC was 4.23.

[0156] Example 3: Endocytosis Experiment

[0157] Experimental methods: NCI-N87 and BxPC3 cells were cultured in RPMI 1640 medium with 10% FBS, and 293T-Trop2 cells were cultured in DMEM medium with 10% FBS and 100 μg / mL Hygromycin B. All cells were cultured at 37°C in a 5% CO2 incubator. Recombinant enzyme digestion buffer (TrypLE) was used. TMNCI-N87 (Institute of Cell Biology, Chinese Academy of Sciences, SCSP-534), BxPC3 (Institute of Cell Biology, Chinese Academy of Sciences, SCSP-529), and 293T-Trop2 cells (293T cells were obtained from GNHu17 at the Institute of Cell Biology, Chinese Academy of Sciences; 293T-Trop2 is an engineered cell line with enhanced Trop2 expression obtained by transfecting 293T cells with Trop2 expression plasmid and then selecting under pressure) were digested with Express Enzyme. After counting, an appropriate amount of cell suspension was centrifuged and resuspended in the corresponding complete culture medium, and the cell density was adjusted to 1×10⁶ cells / year. 6 Cells were seeded at 100 μL / well in 96-well cell culture plates and incubated at 37°C with 5% CO2. After cell attachment, the BY016-LD-38, BY016 monoclonal antibody, Isotype ADC (IgG1-LD-38), and Isotype antibody (IgG1 antibody) provided in Example 2 were diluted to 20 μg / mL with complete culture medium and added to the cells at 100 μL / well (final drug concentration: 10 μg / mL). Cells were then cultured with pHrodo. TM The iFL Red STP ester and amine reactive dye-labeled drugs were incubated in a 37°C, 5% CO2 incubator for 0.5 h, 1 h, 3 h, 5 h, and 24 h, respectively. The cells in the 96-well plates were then digested with recombinant enzyme digestion solution, washed once with PBS + 1% FBS solution, and resuspended in 100 μL PBS + 1% FBS solution. The average fluorescence intensity signal per well was detected by flow cytometry.

[0158] The results are shown in Table 1 below, pHrodo TM After incubation with iFL Red-labeled BY016-LD-38 and BY016 monoclonal antibodies in NCI-N87, BxPC3, and 293T-Trop2 cells at 37°C and 5% CO2 for 0.5 h, 1 h, 3 h, 5 h, and 24 h, respectively, both BY016-LD-38 and BY016 monoclonal antibodies exhibited endocytosis activity, and the fluorescence intensity increased significantly with increasing time. Isotype ADC (IgG1-LD-38) and Isotype antibody (IgG1) showed no significant endocytosis activity in the above three cell types.

[0159] Table 1. Endocytosis data of BY016-LD-38 and BY016 monoclonal antibodies in NCI-N87, BxPC3, and 293T-Trop2 cells.

[0160] Example 4 Cell killing experiment

[0161] Experimental methods: 293T-Trop2 and 293T cells (293T cells were obtained from the Institute of Cell Biology, Chinese Academy of Sciences, GNHu17; 293T-Trop2 is an engineered cell line with enhanced Trop2 expression obtained by pressure selection after transfection of 293T cells with Trop2 expression plasmid) were cultured in DMEM + 10% FBS + 100 μg / mL Hygromycin B and DMEM + 10% FBS, respectively. When cell confluence was greater than 80%, the cells were passaged at a ratio of 1:3 to 1:20.

[0162] Recombinant enzyme digestion solution (TrypLE) TM 293T-Trop2 and 293T cells were digested with Express Enzyme, counted, and then a suitable amount of cell suspension was centrifuged and resuspended in DMEM + 10% FBS. The cell density was adjusted to 2 × 10⁶ cells / mL. 4 Cells were seeded at 100 μL / well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. The next day, BY016-LD-38, BY016 monoclonal antibody, isotype control ADC (IgG1-LD-38), and isotype control antibody (IgG1) provided in Example 2 were serially diluted 4-fold from 120 nM (final concentration 60 nM) using DMEM + 10% FBS, resulting in a total of 8 concentration points; a negative control with a sample concentration of 0 and a blank control with only culture medium were also set up. The diluted samples were added at 100 μL / well to 96-well cell culture plates containing cells and incubated at 37°C with 5% CO2 for 72 hours.

[0163] Add 20 μL of LamarBlue assay reagent to each well and incubate at 37°C for 4 hours. Detect the fluorescence signal intensity of each well using a multi-mode microplate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Calculate the cell viability (%) of the experimental wells using the following formula:

[0164] Cell viability (%) = (Fluorescence value of experimental wells - Fluorescence value of blank wells) / (Fluorescence value of negative control wells - Fluorescence value of blank wells) × 100%

[0165] The results are shown in Table 2 below, which presents the proliferation inhibition data of different concentrations of BY016-LD-38, BY016 monoclonal antibody, isotype control ADC (IgG1-LD-38), and isotype control antibody (IgG1) on different cells.

[0166] BY016-LD-38 inhibited the proliferation of 293T-Trop2 cells in a dose-dependent manner, with an IC50 of 0.281 nM. BY016 monoclonal antibody also inhibited the proliferation of 293T-Trop2 cells at high concentrations, but the inhibitory effect was very weak, showing less than 20% of the inhibitory activity compared to the negative control wells. Neither BY016-LD-38 nor BY016 monoclonal antibody inhibited the proliferation of 293T cells. Furthermore, the isotype control ADC (IgG1-LD-38) and isotype control antibody (IgG1) showed no inhibitory effect on the proliferation of either 293T-Trop2 or 293T cells. These results indicate that BY016-LD-38 possesses Trop2-specific killing activity in 293T-Trop2 cells.

[0167] Table 2. Data on the inhibition of 293T-Trop2 and 293T cell proliferation by BY016-LD-38 (mean cell viability ± standard deviation)

[0168] Example 5: Side-by-side killing effect

[0169] Experimental methods: 293T-Trop2 and 293T cells were cultured in DMEM + 10% FBS + 100 μg / mL Hygromycin B and DMEM + 10% FBS media, respectively, at 37℃ in a 5% CO2 incubator. Both cell types were adherent cells, and when the cell confluence was greater than 80%, they were passaged at a ratio of 1:5 to 1:20.

[0170] Labeling of 293T cells: using recombinant enzyme digestion solution (TrypLE) TM 293T cells were digested with Express Enzyme, and an appropriate amount of cell suspension was centrifuged and washed twice with PBS to adjust the density to 5 × 10⁻⁶. 6 Add an appropriate volume of CFSE to a concentration of 10 μM, mix immediately, and incubate at room temperature in the dark for 10 minutes. Then add 4–5 volumes of pre-chilled DMEM + 10% FBS and incubate on ice for 5 minutes to terminate the reaction. Wash twice with pre-chilled DMEM + 10% FBS, and adjust the cell density to 1 × 10⁶ cells / mL. 5 per mL.

[0171] Co-incubation and detection: Recombinant enzyme digestion solution (TrypLE) was used. TM 293T-Trop2 cells were digested with Express Enzyme, counted, and then a suitable amount of cell suspension was centrifuged and resuspended in DMEM + 10% FBS. The cell density was adjusted to 2 × 10⁻⁶ cells / cells. 4 cells / mL and 1.4 × 10 4The density of CFSE-labeled 293T cells was adjusted to 2 × 10⁶ cells / mL. 4 cells / mL and 2.8 × 10 4 1 mL of each of the above-mentioned 293T-Trop2 cells and CFSE-labeled 293T cells were mixed at a cell density of 1:1 or 1:2, and seeded at 2000 cells / well / 100 μL in a 96-well cell culture plate. The plates were then incubated overnight at 37°C with 5% CO2. The next day, BY016-LD-38, BY016 monoclonal antibody, isotype control ADC IgG1-LD-38, and isotype control antibody IgG1 provided in Example 2 were diluted to 8 nM (final concentration 4 nM) with DMEM + 10% FBS; an Untreated group with a sample concentration of 0 was also set up. 100 μL of the diluted sample was added to each well of a 96-well cell culture plate containing cells and incubated at 37°C with 5% CO2 for 72 hours. Discard the cell culture plate supernatant and wash once with PBS. Digest cells with recombinant enzyme digestion solution, terminate digestion with DMEM + 10% FBS medium, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash once with PBS containing 1% FBS, and resuspend cells in 250 μL or 100 μL for each sample treatment group. Count the cells in the appropriate volume of cell suspension and calculate the total number of cells in each sample treatment group. Use a Beckman CytoFLEX flow cytometer to detect the ratio of CFSE-positive to CFSE-negative cells in each sample. Calculate the number of 293T-Trop2 positive cells and 293T negative cells based on the total number of cells and the ratio of CFSE-positive to CFSE-negative cells, using the following formula:

[0172] 293T-Trop2 positive cell count = total number of cells in each sample treatment group × percentage of CFSE negative cells in each sample treatment group;

[0173] 293T negative cell count = total number of cells in each sample treatment group × percentage of CFSE positive cells in each sample treatment group.

[0174] As shown in Figure 3, BY016-LD-38 can kill both 293T-Trop2 positive cells and 293T negative cells, while BY016 monoclonal antibody, isotype control ADC (IgG1-LD-38), and isotype control antibody (IgG1) have no killing effect on either 293T-Trop2 positive or 293T negative cells. BY016-LD-38 has a specific killing effect on 293T-Trop2 cells, while the killing of 293T negative cells is caused by the effective payload released by BY016-LD-38 after endocytosis into 293T-Trop2 cells, indicating that BY016-LD-38 has a bystander killing effect.

[0175] Example 6: Tumor therapeutic effect on gastric cancer PDX

[0176] Experimental method: Resuscitated and activated human-derived gastric cancer tumor tissue (PDTX0501008) (2×2×2mm) was used. 3 The drug was subcutaneously injected into the right forelimb of mice (C-NKG, 9–10 weeks old). The tumor was inoculated when the average tumor volume reached 110 mm². 3 Around 10:00 AM, animals were randomly assigned to stratified groups for drug administration based on tumor volume and body weight. They were randomly divided into three experimental groups, with six animals in each group. After grouping, the animals received three doses of the drug. Tumor volume was measured twice weekly using calipers, measuring both the major and minor diameters. The volume was calculated using the formula: Tumor volume = 0.5 × major diameter × minor diameter. 2 Tumor volume inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. Ti: mean tumor volume in the treatment group on day i of drug administration; T0: mean tumor volume in the treatment group on day 0 of drug administration; Vi: mean tumor volume in the solvent control group on day i of drug administration; V0: mean tumor volume in the solvent control group on day 0 of drug administration. Results for each group are expressed as mean ± standard error. The T-test was used for statistical analysis of tumor volume. P < 0.05 was considered statistically significant.

[0177] The positive reference drug for BY016-ELC122 is an ADC drug that conjugates BY016 monoclonal antibody with SN38, with a conjugation DAR value of 8. The specific preparation method is based on PCT / US2021 / 030198.

[0178] The tumor volume change curves of each group of tumor-bearing mice are shown in Figure 4. The average tumor volume and significance statistics at different time points are shown in Table 3. By Day 18, the test drug BY016-ELC122 showed a certain tumor-suppressing effect, and BY016-LD-38 provided in Example 2 showed a significant tumor-suppressing effect. After the last administration on Day 14, 5 mice in the BY016-LD-38 group showed a trend of tumor volume reduction from Day 18 to Day 42. By Day 39, 4 mice showed complete tumor volume regression (0 mm) on Day 39. 3 The disease persisted until Day 42. BY016-LD-38 was significantly superior to the positive control drug BY016-ELC122, which used the same monoclonal antibody conjugated with the SN38 small molecule (DAR8). At 18 days post-administration, the BY016-LD-38 group (95.5%) > the BY016-ELC122 group (61.7%).

[0179] Table 3. Effects of the test drug on tumor volume in PDTX0501008 tumor-bearing mice with gastric cancer. Note: a: Mean ± Standard Error; b: Statistical comparison of tumor volume between the treatment group and the control group on day 18 of drug administration, T-test analysis, *p<0.05, **p<0.01, ***p<0.001; na: Not applicable

[0180] Example 7: Tumor therapeutic effect in a CDX model of breast cancer brain metastases

[0181] Experimental Methods: Human breast cancer JIMT-1-luc cells were cultured in vitro in a monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 μg / mL puromycin, and 1% penicillin-streptomycin solution, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged using routine trypsin-EDTA digestion. When cell confluence reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded. Anesthetized female Balb / c nude mice (6-8 weeks old) were fixed supine in a mouse restraint device. A pillow (a cylindrical object approximately 0.5 cm in diameter) was placed behind the mouse's neck to straighten it and further expose the neck. The neck skin was cleaned and disinfected with alcohol swabs. Make a shallow midline incision in the neck using surgical scissors and forceps, from above the clavicle to below the chin (1-1.5 cm). Carefully dissect the fat and connective tissue with micro-forceps to roughly locate the trachea. Then, further dissect the muscles on the right side of the trachea, carefully separating the connective tissue on the right side of the trachea with forceps to expose the left common carotid artery (CCA). Remove the connective tissue with micro-forceps to expose the bifurcation of the common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) and their origins. Insert two suture segments (approximately 3-4 cm each) under the external carotid artery (ECA): tie a permanent knot at the uppermost exposed end of the external carotid artery (ECA); tie a loose slipknot at the lowest point of the external carotid artery (ECA), directly above the bifurcation of the common carotid artery (CCA). Clamp the exposed common carotid artery (CCA) with an arterial hemostatic clip as low as possible. Connect a capillary tube approximately 15 cm long to a syringe containing the cells to be injected, and gently push the syringe to fill the capillary tube with the injected cells (2 x 10^6 cells). 5Before each aspiration, the cell suspension should be thoroughly mixed by pipetting or shaking to prevent cell clumping. Using microscissors, make a small incision in the vessel between the two knots in the external carotid artery (ECA). Gently insert the capillary tip (cut at an angle, being careful not to make it too sharp to avoid puncturing the vessel) into the ECA incision, then slide the capillary downwards along the vessel until it reaches the bifurcation point of the upper end of the common carotid artery (CCA) and the hemostatic clip on the CCA. Tighten the knot in the lower segment of the ECA, ensuring the knot is tight enough to allow for smooth flow of fluid within the capillary and prevent leakage during injection. Gently push the syringe plunger to infuse the cells at a rate >10 μL / s. After the cells have been infused, gently remove the capillary. Then, completely tighten the knot in the lower segment of the ECA, and finally remove the hemostatic clip from the CCA. The connective tissue and fat were replaced to cover the wound, the skin wound was sutured, and postoperative care was provided.

[0182] Mice were injected intraperitoneally with fluorescein at a dose of 150 mg / kg after inoculation. Ten minutes later, the mice were pre-anesthetized with a mixture of oxygen and isoflurane. After deep anesthesia, the mice were transferred to the imaging chamber of IVIS (Lumina II) for bioluminescence detection. Bioluminescence signals and resulting images were detected and recorded. Eleven days after tumor inoculation, the average bioluminescence signal value in the mouse brain reached 1.7 × 10⁻⁶. 7 Dosing was initiated at (photons / second), with 6 animals in each group.

[0183] Tumor volume inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. Ti: mean bioluminescence signal value in the brain of the treatment group on day i of drug administration; T0: mean bioluminescence signal value in the brain of the treatment group on day 0 of drug administration; Vi: mean bioluminescence signal value in the brain of the solvent control group on day i of drug administration; V0: mean bioluminescence signal value in the brain of the solvent control group on day 0 of drug administration. Results for each group are expressed as mean ± standard error. T-test was used for statistical analysis of tumor volume. P < 0.05 was considered statistically significant.

[0184] Tumor growth data (tumor bioluminescence signal, mean ± SE) are shown in Figure 5. After drug administration, the mean bioluminescence signal of brain tumors in the solvent control group of tumor-bearing mice on D29 reached 3.9 * 10⁹ (photons / second). Compared with the solvent control group, the mean bioluminescence signal of brain tumors in the BY016-LD-38 group of tumor-bearing mice on D29 reached 1.2 * 10⁹. 6The TGI value was 100.41%. Compared with the solvent control group, the BY016-LD-38 provided in Example 2 effectively inhibited...

[0185] Tumor growth in a human breast cancer JIMT-1-luc brain metastasis model. Survival data for mice are shown in Figure 6. At day 80, all mice in the BY016-LD-38 group were alive and in normal condition, while 5 / 6 of the mice in the control group died or were near death.

[0186] Animal weight data (Figure 7) showed that mice in the solvent control group all experienced varying degrees of weight loss as the experiment progressed; while mice bearing tumors in the JIMT-1-luc brain metastasis model tolerated the test drug BY016-LD-38 well, and no significant weight loss was observed in this group of mice.

[0187] Example 8: Tumor therapeutic effect in a breast cancer CDX model

[0188] Experimental Methods: Human breast cancer cells JIMT-1 (catalog number: ACC-589) were cultured in vitro as a monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90%, cells were harvested, counted, and seeded. 0.2 mL (5 × 10⁶ cells / mL) of the medium was used for seeding. 6 10 JIMT-1 cells were subcutaneously seeded into the right posterior dorsal region of each mouse (PBS:Matrigel = 1:1). The tumor was inoculated when the average tumor volume reached 191 mm². 3 At that time, the drug efficacy experiment began with group administration of 6 animals in each group.

[0189] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. The antitumor efficacy of the compound was evaluated using TGI (%) or tumor proliferation rate T / C (%). TGI (%) reflects the tumor growth inhibition rate. The calculation of TGI (%) is: TGI (%) = [1 - (mean tumor volume at the end of treatment in a certain treatment group - mean tumor volume at the beginning of treatment in that treatment group) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the beginning of treatment in the solvent control group)] × 100%.

[0190] Tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the tumor measurement results, using the formula RTV = Vt / V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., d0), and Vt is the average tumor volume at a specific measurement. TRTV and CRTV are based on data from the same day.

[0191] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point for each group. Tumor volume and tumor weight were compared using one-way ANOVA. Statistical analysis was performed based on data from day 40 after the start of drug administration to assess differences between groups. Because F-values ​​were considered statistically significant, the Games-Howell test was used to quantify both tumor volume and tumor weight. All data were analyzed using Prism 8.0. A p-value < 0.05 was considered statistically significant.

[0192] Results and Conclusions: The therapeutic effects of the test substance on tumors in female SCID Beige mouse models of JIMT-1 cell subcutaneous xenograft tumors are shown in Table 4 and Figure 8. The effect on body weight is shown in Figure 9. On day 40 after administration (PG-D40), the mean tumor volume of tumor-bearing mice in the solvent control group reached 1,129 mmHg. 3 Compared with the solvent control group, BY016-LD-38 (5 mg / kg, QW) provided in Example 2 showed a highly significant tumor-suppressing effect, with an average tumor volume of 84 mmHg for PG-D40. 3 (T / C = 7.33%, TGI = 111.41%, p = 0.0003). The peer control ADC (Iso-LD-38) showed no significant tumor growth inhibition.

[0193] In addition, BY016-LD-38 (5 mg / kg, QW) was re-administered to animals at PG-D75, and further administration was performed at PG-D75, D82, D89, D96, D103, D128, and D135. Starting from PG-D86, the tumor volume of BY016-LD-38 gradually decreased from 531 mm. 3 (PG-D86) reduced to 114mm 3 (PG-D117).

[0194] Tumor-bearing mice showed good tolerance to both BY016-LD-38 and the peer control ADC (Iso-LD-38).

[0195] Table 4 shows the antitumor efficacy evaluation of the test substances on the JIMT-1 xenograft tumor model.

[0196] Example 9: Tumor therapeutic effect in a gastric cancer PDX model

[0197] Experimental method: From Tumor tissue was collected from GA2421 and GA6844 tumor-bearing mice (gastric cancer xenograft models), cut into 2-3 mm diameter tumor blocks, and subcutaneously inoculated into the right anterior scapula of male BALB / c nude mice. When the average tumor volume of the tumor-bearing mice reached approximately 100-200 mm³, they were randomly assigned to groups based on tumor volume to ensure similar tumor volumes between groups, with 6 mice in each group. After the start of drug administration, the body weight and tumor size of the mice were measured twice a week. On the day of grouping, the mice were given either a solvent control via tail vein injection or BY016-LD-38, 5 mg / kg, administered via tail vein injection after a QW (quick-wash) administration, for a total of 3 times. Subsequently, the dosage was adjusted to BY016-LD-38, 10 mg / kg, administered via tail vein injection after a QW (quick-wash) administration, for a total of 2 times.

[0198] After administration, mouse body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Tumor volume inhibition rate (TGI) is calculated as follows: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration.

[0199] Results and Conclusions: In the GA2421 model (Figure 10), the mean tumor volume in the solvent control group was 1911.87 mm on Day 42. 3 The mean tumor volume in the BY016-LD-38 treatment group was 202.65 mm on Day 42. 3 The absolute tumor inhibition rate (TGI) was 89.40%. Tumor observation after drug discontinuation showed no tumor rebound even at Day 84, with a mean tumor volume of 174.61 mm. 3 .

[0200] In the GA6844 model (Figure 12), the mean tumor volume in the solvent control group was 1586.24 mm on Day 35. 3 In the BY016-LD-38 treatment group, the tumor completely disappeared on Day 35, with an absolute tumor inhibition rate (TGI) of 100%. The observation was extended to Day 85, and no rebound was observed, with the tumor completely disappearing.

[0201] The weight change curves after administration to the GA2421 and GA6844 models (Figure 11 and Figure 13) show that the mice tolerated BY016-LD-38 well and did not experience significant weight loss.

[0202] In summary, single-agent treatment with BY016-LD-38 significantly inhibited tumor growth in BALB / c nude mouse models of human gastric cancer GA2421 and GA6844 subcutaneous xenografts, even completely eliminating tumors, with no rebound effect after drug withdrawal. The tumor-bearing mice generally tolerated BY016-LD-38 well at the tested dose. No mouse deaths or significant weight loss were observed during the experiment.

[0203] Pharmacodynamics of Example 10 in a human gastric cancer N87 CDX model

[0204] Experimental Methods: N87 human gastric cancer cells were cultured in an incubator at 37°C and 5% CO2 in medium containing inactivated 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and RPMI 1640. After reaching confluence every 3-4 days, the cells were passaged into individual flasks. Tumor cells in the logarithmic growth phase were used for in vivo tumor seeding. N87 tumor cells were resuspended in a PBS:Matrigel mixture (1:1 v / v) at a concentration of 1×10⁻⁶. 8 100 μl / mouse was injected subcutaneously into the right flank of BALB / c nude mice. The inoculation was performed when the tumor grew to 147 mm. 3 Dosing was administered in groups around the left and right of day (the day was recorded as PG-D0).

[0205] The tumor volume was measured twice a week using calipers, and the mice were weighed using an electronic balance. The long and short diameters of the tumor were measured, and the volume was calculated using the formula: Volume (TV) = 0.5 × long diameter × short diameter 2 The T / C value is calculated based on tumor volume, where T is the average relative tumor volume (RTV) of each treatment group, and C is the average relative tumor volume (RTV) of the control group. RTV is the ratio of tumor volume after administration to tumor volume before administration. Tumor growth inhibition rate TGITV (%) = (1 - T / C) × 100%.

[0206] SPSS Statistics 22.0 software was used to perform intergroup statistical analysis on tumor volume and tumor weight using the One-Way ANOVA test. A p < 0.05 was considered statistically significant.

[0207] Results and Conclusions: Tumor growth curves and tumor growth inhibition data are shown in Figure 14 and Table 5. At the end of the PBS group (PG-D28), the tumor growth inhibition rates of the BY016-LD-385mg / kg group, 3mg / kg group, 1.5mg / kg group, and 0.5mg / kg group were 100%, 100%, 86%, and 67%, respectively. The tumor volume in each treatment group was significantly smaller than that in the PBS group (p<0.01), and there were significant differences between the treatment groups (except for 5mg / kg vs 3mg / kg) (p<0.01 or p<0.05). At the end of the experiment (PG-D56), the tumor volume and inter-group comparison results of each treatment group were the same as those at the end of the PBS group (PG-D28).

[0208] Table 5 shows the antitumor effect of the test substances on the N87 gastric cancer model. Note: a. Mean ± standard error; b. Compared with BY016-LD-385mg / kg group; c. Compared with BY016-LD-383mg / kg group; d. Compared with BY016-LD-381.5mg / kg group.

[0209] Pharmacodynamics of Example 11 in a human gastric cancer GSU CDX model

[0210] Experimental Methods: GSU human gastric cancer cells were cultured in RPMI 1640 medium containing inactivated 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in an incubator at 37°C and 5% CO2. After approximately two days, the cells were passaged into multiple flasks once they reached confluence. Tumor cells in the logarithmic growth phase were used for in vivo tumor seeding. The GSU tumor cells were adjusted to a concentration of 5 × 10⁻⁶ cells / mL with PBS. 7 The tumor cells were injected subcutaneously into the right flank of CB-17SCID mice at a dose of 100 μL / mouse, with a seeding volume of 5 × 10⁹ / mL. 6 / mouse. When the average tumor volume reached 151 mm. 3 Animals were divided into two groups (the day of grouping was recorded as PG-D0), with 6 animals in each group. From grouping until the end of the experiment, tumor volume (TV) was measured twice a week using calipers, measuring both the long and short diameters of the tumor. The volume was calculated using the formula: Tumor volume = 0.5 × long diameter × short diameter. 2 The T / C value is calculated based on tumor volume. The tumor volume ratio of the treatment group to the control group, T / C (%), is calculated as follows: (mean RTV of the treatment group / mean RTV of the control group) × 100%. RTV (relative tumor volume) is the ratio of tumor volume after administration to tumor volume before administration. Then, the tumor growth inhibition rate, TGITV (%), is calculated using the formula: TGITV = (1 - T / C) × 100%.

[0211] SPSS Statistics 25.0 software was used to perform one-way ANOVA to conduct statistical analysis between groups on tumor volume and tumor weight. A p < 0.05 was considered statistically significant.

[0212] Results and Conclusions: The tumor growth curves after group administration are shown in Figure 15. The tumor volume growth rate of BY016-LD-38 mice was significantly slowed down and even shrank or disappeared. The TGITV of the (PG-D25) BY016-LD-38 treatment group was 87% compared with the PBS group, while the TGITV of the peer control ADC (Iso-LD-38) was only 17%. By the end of the experiment, 4 / 6 of the mice in the BY016-LD-38 treatment group still had tumors that had disappeared.

[0213] Example 12 Pharmacodynamics in a human colon cancer COLO205 CDX model

[0214] Experimental Methods: Human colorectal cancer cell line COLO 205 (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60026) was cultured in RPMI-1640 medium containing 10% FBS (Fetal Bovine Serum) at 37℃ in a 5% CO2 incubator. When the cells reached approximately 80-90% confluence, they were trypsinized and passaged at a 1:3 ratio. COLO 205 cells in the logarithmic growth phase were collected, centrifuged at 1000 rpm for 5 minutes, and resuspended in PBS. 5 × 10⁵ cells 6 One cell was mixed with matrix gel at a 1:1 ratio and subcutaneously injected into the right flank of female BALB / c nude mice at a volume of 100 μl per mouse. The tumors were allowed to grow to approximately 130 mm. 3 The tumors were randomly divided into 5 groups of 6 each, based on their size. Measurements were taken twice weekly using calipers. The tumor volume was calculated using the formula V = 0.5a × b. 2 , a and b represent the long and wide diameters of the tumor, respectively; the tumor growth inhibition rate (TGI) (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the mean tumor volume after the start of administration in the treatment group, T0 is the mean tumor volume at the first administration in the treatment group, V0 is the mean tumor volume at the first administration in the vehicle control group, and Vi is the mean tumor volume after the start of administration in the vehicle control group. All data are expressed as mean ± SEM. Tumor volume statistical analysis: one-way ANOVA was used to statistically analyze the tumor volume on the last day; Dunnett's multiple comparisons were used to analyze the comparison between each treatment group and the vehicle group. All data were analyzed using GraphPad Prism 8.0 and above. P < 0.05 was considered statistically significant.

[0215] The positive control drug was Dato-Dxd (DS1062), an antibody-drug conjugate of an anti-Trop2 monoclonal antibody prepared according to the patented method PCT / JP2014 / 006421, purchased from Huiyou Biotechnology (Chongqing) Co., Ltd. (Catalog No. CHB270).

[0216] Results and Conclusions: The tumor growth curves after group administration are shown in Figure 16. BY016-LD-38 can inhibit tumor growth in a dose-dependent manner, even reducing tumor volume or causing complete regression. On day 17 after administration, the TGIs of BY016-LD-38 after a single dose of 5 mg / kg, 2.5 mg / kg, and 1 mg / kg were 113.36%, 78.05%, and 33.19%, respectively. The peer control ADC drug (Iso-LD-38) showed no therapeutic effect. Notably, compared with the positive control DATO-Dxd, at the same dosage (5 mg / kg), the tumor-suppressive activity of BY016-LD-38 was significantly higher than that of DATO-Dxd (TGIs were 113.36% and 61.86%, respectively, p<0.01).

[0217] Example 13 Pharmacodynamics in the ENHERTU-resistant NCI-N87 CDX model

[0218] Experimental Methods: NCI-N87 / Enhertu-R cells are a drug-resistant cell line selected for Enhertu stress in vitro. This cell line significantly increases Enhertu resistance both in vivo and in vitro, but has no significant effect on HER2 expression on the cell surface. Transcriptome sequencing and bioinformatics analysis also indicate that this cell line exhibits a multi-layered and broad-based resistance mechanism. NCI-N87 / Enhertu-R cells were cultured in a monolayer in vitro using RPMI-1640 medium supplemented with 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When the desired cell number was reached, cells were harvested, counted, and seeded. Female CB17 SCID mice, 6-8 weeks old, were seeded with 0.2 mL (8 × 10⁸ mL) of the medium. 6 100 NCI-N87 / Enhertu-R cells (with matrix gel) were subcutaneously seeded into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 150 mm². 3 Dosing will begin in groups at that time.

[0219] Experimental indicators: The experimental indicators are used to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0220] Calculation of TGI (%): TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%.

[0221] Data analysis: t-tests are used for comparisons between two groups. One-way ANOVA is used for comparisons between three or more groups. If the F-values ​​show a significant difference, multiple comparisons should be performed after ANOVA analysis. All data analyses were performed using SPSS 17.0. A p-value < 0.05 was considered statistically significant.

[0222] Results and Conclusions: The tumor growth curves after group administration are shown in Figure 17. BY016-LD-38 significantly inhibited tumor growth, with the tumor volume reaching only 232.17 mm² on day 49 after administration. 3 In contrast, the tumor volume in the control group reached 1554.17 mm. 3 The tumor inhibition of BY016-LD-38 was significantly better than that of the positive control drug Dato-Dxd (Huiyou Biotechnology (Chongqing) Co., Ltd., catalog number CHB270). At day 49 after administration, the TGI of BY016-LD-38 was 94%, which was significantly better than that of Dato-Dxd (52.20%) (p<0.01).

[0223] Example 14 Pharmacokinetic Study in Rats

[0224] Experimental Methods: Six female rats (6-8 weeks old, ~200g) were randomly divided into two groups of three. Each group received a single intravenous bolus injection of BY016-LD-38 and Dato-Dxd (Huiyou Biotechnology (Chongqing) Co., Ltd., catalog number CHB270), both at a dose of 5 mg / kg. Whole blood was collected from each group before administration and at 5 min, 1 h, 5 h, 24 h (D1), 48 h (D2), 72 h (D3), 120 h (D5), 168 h (D7), 240 h (D10), 336 h (D14), and 504 h (D21) after administration. Serum was separated. The concentrations of TotalAb (total antibody) and ADC (antibody-dependent antibody) in the serum samples were analyzed using ELISA. Pharmacokinetic parameters were calculated using a non-compartmental PK Solver (NCA) model.

[0225] Methods for determining total antibodies and ADC: The blood concentrations of total antibodies and ADC were determined using ELISA, as detailed below:

[0226] a) Dilute Trop2 protein to 1 μg / mL with PBS, coat 96-well microplates with 100 μL / well, and incubate overnight at 4°C.

[0227] b) Wash the plate 3 times, then block with 3% BSA-PBST, 300 μL / well, at room temperature for 2 h.

[0228] c) Wash the plate three times. Dilute BY016-LD-38 and Dato-Dxd at a 2-fold serial dilution using rat blank serum, starting at a concentration of 1 μg / mL, for a total of 7 concentrations. Set rat blank serum as the "0" point. Simultaneously, use rat blank serum to perform appropriate serial dilutions on samples at different time points after drug administration. Dilute all the above samples 50-fold with 1% BSA-PBST, add 100 μL / well to the microplate, and incubate at room temperature for 2 hours.

[0229] d) Wash the plate 6 times.

[0230] Total antibody assay: HRP-goat anti-human IgG-FC was diluted 1:120000 with 1% BSA-PBST, and 100 μL / well was added to the ELISA plate and incubated at room temperature for 1 h.

[0231] ADC assay: Dilute anti-DXD antibody to 0.5 μg / mL with 1% BSA-PBST, add 100 μL / well to the microplate, and incubate at room temperature for 1 h. Wash the plate 6 times, dilute HRP-goat anti-mouse IgG (H+L) with 1% BSA-PBST at a ratio of 1:40000, add 100 μL / well to the microplate, and incubate at room temperature for 1 h.

[0232] e) Wash the plate 6 times, add 100 μL of TMB to each well of the microplate. Incubate at room temperature in the dark for about 10 minutes.

[0233] f) Add 50 μL of stop solution per well. Detect using a microplate reader (wavelength 450 / 630 nm).

[0234] Biological sample analysis and data processing: GraphPad Prism 7.04 software was used to input raw pharmacokinetic sample data and calculate the concentrations of total antibody and ADC in the samples. Drug-time concentration curves were plotted based on the average concentration and sampling time. PKSolver software was used to calculate pharmacokinetic parameters such as peak concentration (Cmax), terminal elimination half-life (t1 / 2), area under the curve (AUC), steady-state apparent volume of distribution (VSS), and system clearance (Cl) using a non-compartmental model.

[0235] Results and Conclusions: The mean drug concentration-time curves in animal serum are shown in Figure 18. After a single intravenous bolus injection of the test drug at a dose of 5 mg / kg into rats, the serum concentration trends of total antibodies in BY016-LD-38 and Dato-Dxd were basically consistent. No significant separation was observed between the total antibody and ADC curves of BY016-LD-38. Even at day 21 post-treatment, the serum concentrations of both total antibodies and ADC remained above 10 μg / mL (approximately 10% of Cmax), indicating that the clearance rate of BY016-LD-38 from serum was relatively slow, and small molecules were not easily detached. The serum concentrations of Dato-Dxd, especially the ADC, decreased more rapidly. At day 21 post-treatment, the serum ADC concentration was only 2.8 μg / mL, lower than 3% of Cmax. Furthermore, a significant separation between total antibodies and ADC appeared after 72 hours, and this separation became more pronounced over time, indicating that small molecules in Dato-Dxd were easily detached.

[0236] The pharmacokinetic parameters are shown in Table 6. From the perspective of drug exposure, although the total antibody and ADC Cmax of both drugs were nearly identical, the AUC of BY016-LD-38 was significantly higher than that of Dato-Dxd. The AUC 0-t of BY016-LD-38's total antibody and ADC were 11640.4 and 10686.0 h*μg / mL, respectively, while the corresponding AUC 0-t of Dato-Dxd were 9302.2 and 5933.1 h*μg / mL, respectively. BY016-LD-38 also had a longer half-life, with its total antibody and ADC half-lives being 579.2 h and 256.7 h, respectively, while the corresponding half-lives of Dato-Dxd were 314.3 h and 212.1 h. The total antibody / ADCAUC ratios (AUCADC / AUCtotal ab) of BY016-LD-38 and Dato-Dxd were 0.92 and 0.64, respectively, indicating that BY016-LD-38 is more stable and the small molecule is less likely to detach.

[0237] Table 6. PK parameters of BY016-LD-38 and Dato-Dxd in rats

[0238] Tissue distribution in the NCI-N87 CDX model, Example 15

[0239] Experimental Methods: Six-week-old female Nude mice were selected. An appropriate amount of NCI-N87 cells was subcutaneously injected into the ventral region of each mouse. When the tumor volume reached approximately 160 mm³, BY016-LD-38 was injected once via tail vein. A total of 15 mice were used, 3 mice per time point, at a dose of 3 mg / kg. Sample Collection: Blood and tumor tissue were collected at 24 h, 72 h, and 168 h post-drug administration, with 3 mice sampled at each time point. Whole blood was collected from the orbital region of tumor-bearing mice and placed in anticoagulant tubes containing EDTA-K2. Plasma was obtained by centrifugation within 30 minutes. Mice were euthanized by CO2 asphyxiation, and tumors were then collected. Plasma and tumor tissue were stored at -80°C.

[0240] Determination of free toxins in plasma: 10.0 μL of standard curve sample, quality control sample, test sample, double blank sample (10.0 μL matrix sample), and zero concentration sample (10.0 μL matrix sample) were added to a series of tubes. 25.0 μL of internal standard working solution (Exatecan-D5 concentration 3.00 ng / mL; 25.0 μL of internal standard diluent was added to the double blank sample) was added to each well. 90.0 μL of methanol:acetonitrile (1:1, v / v) was added to each well, and the mixture was shaken at 2500 rpm for 5 min. The mixture was centrifuged at 3800 g for 5 min at 4 °C. 80.0 μL of the supernatant was collected and dried under nitrogen at 40 °C. 100 μL of 0.1% FAin 15% MeOH was added to each well, mixed thoroughly, and analyzed by LC-MS.

[0241] Determination of total toxins (bound toxins + free toxins) in blood: Add 10.0 μL of the standard curve sample, quality control sample, test sample, double blank sample (10.0 μL matrix sample), and zero concentration sample (10.0 μL matrix sample) to a series of tubes. Add 10.0 μL of papain (Adamas Life, cat: 88915D, 5 mg / ml) to each well and centrifuge at 3500 rpm for 1 min. Incubate the samples in a shaking incubator at 40℃ and 550 rpm for 6–8 hours. Add 1.00 μL of 1% FAin to each well. Centrifuge at 3500 rpm for 1 min in H2O, then incubate at 40℃ and 550 rpm for another 30 min. For zero-concentration samples, standard curve samples, quality control samples, and test samples, add 10.0 μL of internal standard working solution (Exatecan-D5, 1000 ng / ml). For double blank samples, add 10.0 μL of internal standard to prepare the solvent. Centrifuge the above samples at 3800 g for 10 seconds and vortex at 1200 rpm for 2 min. Add 100 μL of pre-cooled ACN to each well, shake at 2500 rpm for 5 min, and centrifuge at 3500 g for 5 min. Take 30.0 μL of the supernatant and add 90.0 μL of 0.5% FA in H2O to each well. Vortex for 3 min and then inject for LCMS analysis.

[0242] Determination of free toxins in tumor tissue: Homogenization of tumor tissue (administered and blank PBS groups): Weigh the tumor tissue, add 10 times the volume of 50% methanol-water, and homogenize. The homogenate from the blank PBS group was used as the matrix for standard curve and quality control sample preparation. Take 10.0 μL of standard curve sample, quality control sample, test sample, double blank sample (10.0 μL matrix sample), and zero concentration sample (10.0 μL matrix sample) and add them to a series tube. Add 25.0 μL of internal standard working solution (Exatecan-D5 concentration of 3.00 ng / mL, and add 25.0 μL of internal standard diluent to the double blank sample). Add 90.0 μL of methanol:acetonitrile (1:1, v / v) to each well and shake at 2500 rpm for 5 min. Centrifuge at 3800 g for 5 min at 4 °C. Take 80.0 μL of supernatant and dry it under nitrogen at 40 °C. Add 100 μL of 0.1% FAin 15% MeOH to each well, vortex for 3 min, and inject for LCMS analysis.

[0243] Determination of total toxins (bound toxins + free toxins) in tumor tissue: Add 10.0 μL each of the standard curve sample, quality control sample, test sample, double blank sample (10.0 μL matrix sample), and zero concentration sample (10.0 μL matrix sample) to a series of tubes; add 10.0 μL of papain (Adamas life, cat: 88915D 5 mg / ml) to each well, centrifuge at 3500 rpm for 1 min; incubate in a shaking incubator at 40℃ and 550 rpm for 6–8 hours; add 1.00 μL of 1% FAin to each well. Centrifuge at 3500 rpm for 1 min in H2O, then incubate at 40℃ and 550 rpm for another 30 min. For zero-concentration samples, standard curve samples, quality control samples, and test samples, add 10.0 μL of internal standard working solution (Exatecan-D5, 1000 ng / ml). For double blank samples, add 10.0 μL of internal standard to prepare the solvent. Centrifuge the above samples at 3800 g for 10 seconds and vortex at 1200 rpm for 2 min. Add 100 μL of pre-cooled ACN to each well and shake at 2500 rpm for 5 min. Centrifuge at 3500 g for 5 min. Take 30.0 μL of the supernatant and add 90.0 μL of 0.5% FA in H2O to each well. Vortex for 3 min and then inject for LCMS analysis.

[0244] Sample analysis: High-performance liquid chromatography (Nexera, Shimadzu) conditions: Mobile phase A: 0.1% aqueous formic acid; Mobile phase B: 0.1% acetonitrile solution; Column: ACE Excel 3μm 50 x 2.1 mm; flow rate: 0.6 mL / min; column temperature: 40 °C; mass spectrometry analysis (Triple Quad 7500, AB / Sciex); data acquisition (Analyst, Version 1.6.2, Applied Biosystems / Sciex).

[0245] Results and Conclusions: A single intravenous administration of BY016-LD-38 at a dose of 3 mg / kg was administered to the NCI-N87 CDX tumor-bearing mouse model. Figure 19 shows the total and free exatecan levels in the plasma of tumor-bearing mice. The total exatecan level in the plasma of tumor-bearing mice peaked at approximately 700 ng / mL 5 minutes after administration, and remained at 55% of the peak level at 24 hours. At 7 days post-administration, the total exatecan concentration reached 200 ng / mL, accounting for 28% of the peak value. By day 11, the total exatecan level remained at approximately 100 ng / mL (approximately 14% of the peak value). Free exatecan (LLOQ = 0.05 ng / mL) was not detected in the plasma of any animal in the administered group at any time point after administration. This indicates that BY016-LD-38 maintains stable sustainability in plasma, with minimal toxin shedding, and is relatively safe.

[0246] As shown in Figure 20, the free and total exatecan levels in tumor tissues of tumor-bearing mice both peaked at 24 hours and then gradually decreased. 24 hours after administration of BY016-LD-38, the total exatecan content in tumor tissue was approximately 42 ng / g; at 7 days post-administration, the total exatecan content decreased to approximately 45% of the peak value; and at day 11, the content decreased to 6.8 ng / g, approximately 16% of the peak value. The free exatecan content in BY016-LD-38 tumors reached 5.4 ng / g at 24 hours, approximately 12% of the total exatecan content at that time point; at 7 days post-administration, the free exatecan content reached approximately 2.9 ng / g, approximately 53% of the peak free exatecan content; even at day 11, the free exatecan content in the tumor still reached approximately 1.5 ng / g, approximately 27% of the peak free exatecan content at that time point, and approximately 22% of the total exatecan content at that time point.

[0247] In summary, after intravenous administration of BY016-LD-38 to NCI-N87 tumor-bearing mice at a dose of 3 mg / kg, the drug was mainly distributed in the plasma in the form of bound toxin (ADC), and accumulated in the tumor tissue in a continuous and stable free toxin form to fully exert the drug's tumor-killing effect.

[0248] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

[0249] sequence list

Claims

1. An antibody-drug conjugate, wherein, Includes the structure of Equation 1 as follows: Wherein, Ab is an antibody or antibody fragment used to target TROP2; j can be 1 to 8, preferably 4 to 8.

2. The antibody-drug conjugate according to claim 1, wherein, The antibody or antibody fragment for targeting TROP2 comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

6.

3. The antibody-drug conjugate according to claim 1, wherein, The antibody or antibody fragment used to target TROP2 comprises a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

7. The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

8.

4. The antibody-drug conjugate according to any one of claims 1-3, wherein, The Ab is an antibody targeting TROP2, wherein the heavy chain of the antibody is IgG1 subtype or mutated IgG1 subtype, and the light chain of the antibody is Kappa type.

5. The antibody-drug conjugate according to claim 4, wherein, The antibody targeting TROP2 comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 9 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

9. The amino acid sequence of the light chain is as shown in SEQ ID NO: 10 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

10.

6. A method for preparing an antibody-drug conjugate, wherein, Includes the following steps: LD-38 is available; Construct antibodies or antibody fragments (Abs) to target TROP2; LD-38 is conjugated with an antibody or antibody fragment Ab targeting TROP2 to obtain an antibody-drug conjugate. The structure of LD-38 is as follows The antibody or antibody fragment Ab used to target TROP2 is as described in claims 1-5.

7. A pharmaceutical composition, wherein, It includes the antibody drug conjugate as described in any one of claims 1-5, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate.

8. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in the preparation of a medicament for the treatment and / or prevention of tumors.

9. The use according to claim 8, wherein, The tumor is selected from metastatic, refractory, or recurrent lesions of solid tumors, hematologic malignancies, and cancers.

10. The use according to claim 8, wherein, The tumors are selected from the following group: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer.

11. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in combination with other therapeutic agents in the preparation of a medicament for the treatment and / or prevention of tumors.

12. The use according to claim 11, wherein, The other therapeutic agents are selected from immune checkpoint inhibitors, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, drugs used for chemotherapy, or drugs used for radiotherapy.

13. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, for the treatment and / or prevention of tumors.

14. Use of an antibody-drug conjugate comprising any one of claims 1-5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in combination with other therapeutic agents for the treatment and / or prevention of tumors.

15. The use according to claim 14, wherein, The other therapeutic agents are selected from immune checkpoint inhibitors, anti-tumor monoclonal antibody drugs, anti-angiogenic drugs, kinase inhibitors, anti-tumor T-cell binding antibodies, synthetic lethal target drugs, drugs used for chemotherapy, or drugs used for radiotherapy.

16. A method for treating and / or preventing tumors, wherein, The subject is given a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-5, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof.

17. The method according to claim 16, wherein, The tumor is selected from metastatic, refractory, or recurrent lesions of solid tumors, hematologic malignancies, and cancers. Preferably, the tumor is selected from the group consisting of: esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, oral squamous cell carcinoma, sarcoma, glioblastoma, and thyroid cancer; Preferably, other therapeutic drugs may also be administered simultaneously.

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