Antibody-drug conjugates containing antibodies against human TROP2 and uses thereof

Abstract

The present disclosure relates to antibody-drug conjugates (ADCs) targeting human TROP2 (tumor-associated calcium signaling factor 2, TACSTD2) and uses thereof, more particularly, ADCs comprising an antibody or antigen-binding fragment thereof that binds to human TROP2, and the use of the ADCs for generating and / or treating a drug for the treatment of a disease, more particularly a hyperproliferative and / or angiogenic disease, such as a cancer disease.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to Korean Patent Application No. 10-2022-0043049, filed on April 6, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Cancer refers to diseases caused by abnormally grown masses resulting from autonomous overgrowth of body tissues, and is the result of uncontrolled cell growth in various tissues. Early stage tumors can be removed by surgical and radiotherapy techniques, while metastatic tumors are generally treated using chemotherapy.

[0003] Most chemotherapeutic agents administered parenterally can induce undesirable side effects and even severe toxicity as a result of systemic administration. Therefore, the focus of development has been on developing treatments to achieve increased efficacy and / or reduced minimal toxicity / side effects, for example through selective application of these chemotherapeutic agents in tumor cells or directly adjacent tissues. Summary of the Invention

[0004] In certain aspects, the present disclosure provides an antibody-drug conjugate comprising an anti-TROP2 antibody that binds to TROP2.In certain embodiments, the antibody disclosed herein can be used to bind to TROP expressed in tumors and deliver drugs to tumors.In certain embodiments, the antibody-drug conjugate disclosed herein has improved stability compared to antibody-drug conjugates known in the art.

[0005] In certain aspects, the present disclosure provides a conjugate having a structure represented by general formula I, or a pharma- ceutically acceptable salt thereof: [General formula I] Ab-[L-(B) l ] m During the ceremony, the Ab is an anti-TROP2 (tumor-associated calcium signal transducer 2, also known as TACSTD2) antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13; L is a linker, B is an active drug moiety; l and m are each independently an integer selected from 1 to 20. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a schematic diagram showing a method for preparing antibody-drug conjugate ADC1 according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram showing a method for preparing antibody-drug conjugate ADC2 according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 is a schematic diagram showing a method for preparing antibody-drug conjugate ADC3 according to an embodiment of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram showing a method for preparing antibody-drug conjugate ADC4 according to an embodiment of the present disclosure. [Figure 5A] Through a first in vivo experiment, the results confirm the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 5A) and a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 5B). [Figure 5B]Through a first in vivo experiment, the results confirm the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 5A) and a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 5B). [Figure 6A] Through a second in vivo experiment, results are shown confirming the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 6A), a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 6B), a mouse model implanted with gastric cancer cell line NCI-N87 (FIG. 6C), and a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 6D). [Figure 6B] Through a second in vivo experiment, results are shown confirming the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 6A), a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 6B), a mouse model implanted with gastric cancer cell line NCI-N87 (FIG. 6C), and a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 6D). [Figure 6C] Through a second in vivo experiment, results are shown confirming the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 6A), a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 6B), a mouse model implanted with gastric cancer cell line NCI-N87 (FIG. 6C), and a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 6D). [Figure 6D] Through a second in vivo experiment, results are shown confirming the tumor growth inhibitory efficacy of antibody-drug conjugates according to embodiments of the present disclosure in a mouse model implanted with breast cancer cell line MDA-MB-468 (FIG. 6A), a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 6B), a mouse model implanted with gastric cancer cell line NCI-N87 (FIG. 6C), and a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 6D). [Figure 7A] The results confirm the tumor growth inhibitory efficacy of an antibody-drug conjugate according to an embodiment of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 7A), a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 7B), a mouse model implanted with pancreatic cancer cell line Capan-1 (FIG. 7C), a mouse model implanted with non-small cell lung cancer cell line NCI-H2170 (FIG. 7D), and a mouse model implanted with colon cancer cell line HT29 (FIG. 7E). [Figure 7B] The results confirm the tumor growth inhibitory efficacy of an antibody-drug conjugate according to an embodiment of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 7A), a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 7B), a mouse model implanted with pancreatic cancer cell line Capan-1 (FIG. 7C), a mouse model implanted with non-small cell lung cancer cell line NCI-H2170 (FIG. 7D), and a mouse model implanted with colon cancer cell line HT29 (FIG. 7E). [Figure 7C] The results confirm the tumor growth inhibitory efficacy of an antibody-drug conjugate according to an embodiment of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 7A), a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 7B), a mouse model implanted with pancreatic cancer cell line Capan-1 (FIG. 7C), a mouse model implanted with non-small cell lung cancer cell line NCI-H2170 (FIG. 7D), and a mouse model implanted with colon cancer cell line HT29 (FIG. 7E). [Figure 7D] The results confirm the tumor growth inhibitory efficacy of an antibody-drug conjugate according to an embodiment of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 7A), a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 7B), a mouse model implanted with pancreatic cancer cell line Capan-1 (FIG. 7C), a mouse model implanted with non-small cell lung cancer cell line NCI-H2170 (FIG. 7D), and a mouse model implanted with colon cancer cell line HT29 (FIG. 7E). [Figure 7E]The results confirm the tumor growth inhibitory efficacy of an antibody-drug conjugate according to an embodiment of the present disclosure in a mouse model implanted with pancreatic cancer cell line BxPC-3 (FIG. 7A), a mouse model implanted with non-small cell lung cancer cell line HCC827 (FIG. 7B), a mouse model implanted with pancreatic cancer cell line Capan-1 (FIG. 7C), a mouse model implanted with non-small cell lung cancer cell line NCI-H2170 (FIG. 7D), and a mouse model implanted with colon cancer cell line HT29 (FIG. 7E). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Antibody-drug conjugates (ADCs) are a targeted technology that conjugates a toxin or drug to an antibody that binds to an antigen, releasing the toxin inside the cell to cause the death of cancer cells, etc. ADCs enable precise delivery of drugs to targeted cancer cells while minimizing the effect on healthy cells, and enable the drug to be released only under specific conditions, and therefore have superior efficacy compared to antibody therapeutic agents themselves, and can significantly reduce the risk of side effects compared to existing anticancer drugs.

[0008] The basic structure of these antibody-drug conjugates is "antibody-linker-small molecule drug (toxin)". In this structure, the linker plays a functional role in linking the antibody and the drug, but in some cases also ensures that the drug is released from the antibody at an appropriate time, for example after reaching the target cell. That is, the stability of the linker can play a very important role in the efficacy and safety, such as systemic toxicity, of the antibody-drug conjugate (Discovery Medicine 2010,10(53):329-39).

[0009] The use of monoclonal antibodies for cancer therapy has achieved considerable success. For example, monoclonal antibodies are suitable for target-directed addressing of tumor tissues and tumor cells. Antibody-drug conjugates have become a new and powerful option for the treatment of lymphomas and solid tumors, and immunomodulatory antibodies have also recently achieved considerable success in clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering and its action, mechanisms of resistance, and the interaction between the immune system and cancer cells.

[0010] Antigens expressed on the surface of human cancer cells are defined as a broad range of targets that are overexpressed, mutated, and selectively expressed compared to normal tissues. A key challenge is to identify antigens suitable for antibody-based therapy. These therapeutics mediate changes in antigen or receptor function (i.e., function as stimulators or antagonists), modulate the immune system through Fc and T cell activation, and exert efficacy through the delivery of specific drugs that bind to antibodies that target specific antigens. Molecular technologies that can alter antibody pharmacokinetics, mechanism of action, size, and immune stimulation are emerging as key factors in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of approaches for selecting optimized antibodies, including target antigen and antibody affinity and binding, choice of antibody structure, and therapeutic approach (signaling blockade or immune function).

[0011] Human TROP2 (tumor-associated calcium signaling factor 2, TACSTD2) is a 35-46 kDa transmembrane calcium signaling glycoprotein with 323 amino acids (274 extracellular domain, 23 transmembrane domain, and 26 intracellular domain), which belongs to the epithelial cell adhesion molecule (EpCAM) family and is an important component in cell signaling, proliferation, and differentiation. Although TROP2 expression has been associated with tumor induction or tumor suppression in various cancers, TROP2 is generally known to act as a tumor-inducing factor.

[0012] Cleavage of the region between R87 and T88 of the 274 extracellular domain induces rearrangement of the TROP2 structure, resulting in changes in biological activity. This truncation does not occur in normal tissues, but TROP2 truncation occurs in most cancers, including skin, ovarian, colon, and breast cancers, and therefore TROP2 acts as a tumor inducer.

[0013] In certain aspects, the present disclosure provides antibody-linker-drug (e.g., toxin) systems that are more stable in circulation (e.g., in plasma), allowing the drug to be easily released within cancer cells to maximize efficacy, and allowing the drug and / or toxin to stably reach target cells, optionally by applying a linker that includes a self-immolative group.

[0014] Thus, in certain embodiments, the present disclosure provides antibody-drug conjugates targeting human TROP2 (tumor-associated calcium signaling factor 2, TACSTD2), as well as pharma- ceutically acceptable salts thereof.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0016] In certain aspects, the present disclosure provides a conjugate represented by the following general formula I, or a pharma- ceutically acceptable salt or solvate thereof: [General formula I] Ab-[L-(B) l ] m During the ceremony, the Ab is an anti-TROP2 (tumor-associated calcium signaling 2, also known as TACSTD2) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13; L is a linker, B is an active drug moiety;

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or 20, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or 20, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15 or 20, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or 20, a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20 while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15 or 20, a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO:16, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:16.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the amino acid sequence of SEQ ID NO: 16, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain variable region consisting of a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the amino acid sequence of SEQ ID NO: 16, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and specifically binds to human TROP2.

[0028] In a particular embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and specifically binds to human TROP2.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and specifically binds to human TROP2.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and specifically binds to human TROP2.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, while maintaining at least 80% sequence identity, at least 81% sequence identity, at least 82% sequence identity, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 100% sequence identity, at least 101% sequence identity, at least 102% sequence identity, at least 103% sequence identity, at least 104% sequence identity, at least 105% sequence identity, at least 106% sequence identity, at least 107% sequence identity, at least 108% sequence identity, at least 109% sequence identity, at least 110% sequence identity, at least 1110% sequence identity, at least 112% sequence identity, at least 1130% sequence identity, at least 114% sequence identity, at least 1150 ... identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 99.5% and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, while maintaining at least 80% sequence identity, at least 81% sequence identity, at least 82% sequence identity, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, or at least 87% sequence identity to the amino acid sequence of SEQ ID NO:16. , at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 99.5% sequence identity, and specifically binds to human TROP2.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, while maintaining at least 80% sequence identity, at least 81% sequence identity, at least 82% sequence identity, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 100% sequence identity, at least 101% sequence identity, at least 102% sequence identity, at least 103% sequence identity, at least 104% sequence identity, at least 105% sequence identity, at least 106% sequence identity, at least 107% sequence identity, at least 108% sequence identity, at least 109% sequence identity, at least 110% sequence identity, at least 1110% sequence identity, at least 112% sequence identity, at least 1130% sequence identity, at least 114% sequence identity, at least 1150 ... identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 99.5% and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, while maintaining at least 80% sequence identity, at least 81% sequence identity, at least 82% sequence identity, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity to the amino acid sequence of SEQ ID NO:16. The antibody comprises a light chain variable region consisting of an amino acid sequence having at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 99.5% sequence identity, and specifically binds to human TROP2.

[0033] In certain embodiments, the antibody is a humanized antibody.

[0034] In certain embodiments, a humanized antibody comprises (a) (i) a variable heavy chain framework region from a heavy chain of a human antibody or from a human consensus framework, and (ii) a variable light chain framework region from a light chain of a human antibody or from a human consensus framework. In some embodiments, at least one amino acid in the variable domain framework region from the heavy chain is replaced with a corresponding amino acid from a heavy chain of a mouse antibody or a mouse consensus framework, or the variable domain framework region from the light chain is replaced with a corresponding amino acid from a light chain of a mouse antibody or a mouse consensus framework. In some embodiments, the amino acid substitution in the heavy chain is selected from the following amino acid substitutions: Substitution of Y (human) with F (mouse) at position 27; Substitution of T (human) with S (mouse) at position 30; Substitution of V (human) with L (mouse) at position 37; Substitution of M (human) with I (mouse) at position 48; Substitution of G (human) for A (mouse) at position 49; Substitution of I (human) with L (mouse) at position 70, and Substitution of R (human) with V (mouse) at position 72; (ii) the amino acid substitution in the light chain is an S (mouse) to Y (human) substitution at position 49;

[0035] In certain embodiments, the amino acid substitutions in the heavy chain may further include the following amino acid substitutions: Substitution of R (human) with K (mouse) at position 67, and / or Substitution of V (human) with A (mouse) at position 68.

[0036] In certain embodiments, the amino acid substitutions in the light chain may further include the following amino acid substitutions: Substitution of S (human) with Y (mouse) at position 67; substitution of I (human) for T (mouse) at position 2 and substitution of S (human) for Y (mouse) at position 67; substitution of T (human) for R (mouse) at position 22 and substitution of S (human) for Y (mouse) at position 67; Substitution of K (human) for E (mouse) at position 42 and substitution of S (human) for Y (mouse) at position 67; Substitution of G (human) for S (mouse) at position 64 and substitution of S (human) for Y (mouse) at position 67; a substitution of S (human) for Y (mouse) at position 67 and a substitution of T (human) for V (mouse) at position 72; or Substitution of S (human) for Y (mouse) at position 67 and substitution of T (human) for D (mouse) at position 85.

[0037] In certain embodiments, a humanized antibody comprises: (a) (i) a variable heavy chain framework region from a heavy chain of a human antibody or from a human consensus framework, wherein the variable heavy chain framework region comprises one or more of the following amino acid sequence changes: Y27F, T30S, V37L, M48I, G49A, I70L, and R72V; and (ii) a variable light chain framework region from a light chain of a human antibody or from a human consensus framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0038] Amino acid substitutions can increase affinity and enhance the stability of antibodies while maintaining their antigen-binding activity. Stabilization of therapeutic antibodies can result in improved serum half-life, lower dosage requirements, reduced side effects, and improved shelf life. Chimeric CH-2EF and CH-2G10 antibodies were constructed first, followed by early humanized versions: Hu-2EF-4, Hu-2G10-1, etc. These antibodies can exhibit negative characteristics such as chemical instability with aggregate formation and (for CH-2EF antibody) loss of solubility, as well as loss of affinity for Trop-2. Amino acid substitutions can generate humanized monoclonal antibodies with high affinity, including Hu-2EF-7, H, Hu-2G10-5, and Hu-2G10-6, which are not recognized by anti-mouse antibodies and specifically direct distinct regions of the extracellular domain of Trop2 and various forms of post-translational modifications.

[0039] In certain embodiments, human VH sequences homologous to the VH frameworks disclosed herein were searched in the GenBank database, and the VH sequence encoded by the human cDNA with NCBI accession number: X65888.1 was selected as the acceptor for humanization (X65888.1-VH). In certain embodiments, the CDR sequences of the VH disclosed herein were first transferred to the corresponding positions of X65888.1-VH. In certain embodiments, human amino acid residues were replaced with the corresponding mouse residues at framework positions 27, 30, 37, 48, 49, 67, 68, 70, and 72 (numbered from the first amino acid of the mature form), where the three-dimensional model of the variable region suggested significant contacts with the CDRs.

[0040] In mouse 2G10 VH, positions 67 and Lys and Ala at position 68, both located closer to the CDRs, were predicted to be important for the formation of the CDR structure, whereas the amino acid residues of the human acceptor X65888.1-VH are Arg at position 67 and Val at position 68, which are similar to the corresponding mouse residues in terms of molecular properties and structure. Therefore, to further reduce potential immunogenicity, a second humanized VH (VH2) was designed in which the amino acid residues of the human acceptor were maintained at positions 67 and 68, i.e., Arg and Val, respectively.

[0041] Based on GenBank homology search using the VL framework sequence, the human VK region encoded by the cDNA with NCBI accession number: AY043146.1 was selected as the acceptor for humanization. First, the CDR sequences of VL were transferred to the corresponding positions of AY043146.1VL. Then, human amino acid residues were replaced with the corresponding residues of mouse VL at positions 49 and 67, where the three-dimensional model of the variable region showed significant contact with the CDRs. Although Tyr at position 67 in mouse VL was predicted to be important for the formation of the CDR structure, further analysis of the three-dimensional model of the 2G10 variable region suggested that this Tyr at position 67 could be replaced with Ser, the residue located at the corresponding position in the human acceptor AY043146.1VL, without affecting antigen binding. Therefore, to further reduce potential immunogenicity, a second humanized VL was designed derived from VL1, in which Ser at position 67 in the human acceptor sequence was maintained.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof may be selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

[0043] The linkers described herein can be cleavable, non-cleavable, and hydrophilic or hydrophobic.

[0044] In certain embodiments, the cleavable linker is cleavable under intracellular or extracellular conditions, thereby releasing the active agent from the antibody construct-active agent conjugate in the intracellular environment.

[0045] The cleavable linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., lysosome, endosome, or caveolae). The cleavable linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In general, the peptidyl linker has a length of at least 2 amino acids or a length of at least 3 amino acids. Cleavage agents can include cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most common is a peptidyl linker that can be cleaved by an enzyme present in antigen-expressing cells. For example, a peptidyl linker (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker) that can be cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, can be used. Other examples of these linkers are described, for example, in U.S. Pat. No. 6,214,345. In addition, the peptidyl linker that can be cleaved by intracellular proteases can be, for example, a Val-Cit linker, a Phe-Lys linker (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker), or a Val-Ala linker. The Val-Cit linker or Val-Ala linker can contain a pentafluorophenyl group and can contain a succinimide group or a maleimide group. Alternatively, the Val-Cit or Val-Ala linker can contain a pentafluorophenyl group, and can contain a 4-aminobenzoic acid (PABA) group and a maleimide group, and can contain a PABA group and a succinimide group.

[0046] In addition, the cleavable linker can be easily hydrolyzed in a pH-sensitive manner, i.e., at a certain pH value. Generally, the pH-sensitive linker can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-asconamides, orthoesters, acetals, and ketals) that can be hydrolyzed in lysosomes can be used (see, for example, U.S. Pat. Nos. 5,122,368, 5,824,805, and 5,622,929, as well as Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123, and Neville et al., 1989, Biol. Chem. 264:14653-14661). These linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or pH 5.0, which is the approximate pH of lysosomes. Examples of hydrolyzable linkers include thioether linkers (eg, a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0047] The linker may also be cleavable under reducing conditions (e.g., disulfide linkers). For example, various disulfide linkers, including N-succinimidyl-5-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-thio)toluene)-(SMPT), and those that can be formed using SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931, and U.S. Pat. No. 4,880,935).

[0048] In addition, the linker can be a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12), a β-glucuronide linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832-40), or a β-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376-382).

[0049] The non-cleavable linker can be a maleimidocaproyl linker. The maleimidocaproyl linker can include N-maleimidomethylcyclohexane-1-carboxylate. The maleimidocaproyl linker can contain a succinimide group. The maleimidocaproyl linker can contain a pentafluorophenyl group. The linker can be a combination of a maleimido group and one or more polyethylene glycol molecules. The linker can be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. The linker can be a maleimido-PEG4 linker. The linker can be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. The linker can be a combination of a pentafluorophenyl group and a maleimidocaproyl linker containing one or more polyethylene glycol molecules. The linker can contain a maleimide linked to a polyethylene glycol molecule, the polyethylene glycol allowing for greater linker flexibility or allowing longer linkers to be used. The linker may be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker.

[0050] In certain embodiments, the linker may be a cleavable linker.

[0051] In certain embodiments, the linker can be a protease cleavable linker, an acid cleavable linker, a disulfide linker, a self-immolative or self-stabilizing linker, a malonate linker, a maleimidobenzoyl linker, a 3'-N-amide analog, a β-glucuronide linker, or a β-galactoside linker.

[0052] In certain embodiments, the protease-cleavable linker may comprise a thiol-reactive spacer or dipeptide, more specifically, the protease-cleavable linker may comprise a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, or a p-amino-benzyloxycarbonyl spacer.

[0053] In certain embodiments, the acid cleavable linker can be a hydrazine linker or a quaternary ammonium linker.

[0054] Exemplary antibody drug conjugates are disclosed in U.S. Pat. Nos. 10,583,197, 9,993,568, 9,951,072, 9,919,057, 9,669,107, 11,413,353, 11,173,214, 11,167,040, 10,980,890, 10,583,197, 10,383,949, 10,273,235, 10,183,997, and 10,118,965, the contents of each of which are incorporated herein by reference in their entirety.

[0055] In certain embodiments, the linker may have the structure of general formula II:

[0056] [ka] In the general formula II, The wavy symbol is a site that is linked to an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, * is the moiety that is linked to an active agent; G is a glucuronic acid moiety or

[0057] [ka] and R 3 is hydrogen or a carboxyl protecting group, and R 4 each is independently hydrogen or a hydroxyl protecting group; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl, W is -C(O)-, -C(O)NR'-, -C(O)O-, -SONR'-, -P(O)R''NR', -SONR'-, or -PONR'-, where C, S, or P is directly attached to the phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 is aryl, Z is independently hydrogen, C 1-8 alkyl, halogen, cyano, or nitro; n is 0 to 3; L is any one selected from the following A) or B): A) At least one of the following is satisfied: C 1-50 Alkylene or 1 to 50 membered heteroalkylene: (i) L contains one or more unsaturated bonds; (ii) two atoms in L are substituted with divalent substituents completing a heteroarylene; (iii) L is 1 to 50 membered heteroalkylene; (iv) the alkylene is at least one C 1-20 is substituted with alkyl, and B) comprises at least one isoprenyl derivative unit of the following general formula III, which can be recognized by an isoprenoid transferase:

[0058] [ka]

[0059] In certain embodiments, the conjugate may have a structure of general formula IIa: [General formula IIa]

[0060] [ka] During the ceremony, Each B' is an active agent; Each G is independently a glucuronic acid moiety or

[0061] [ka] and R 3 is hydrogen or a carboxyl protecting group; Each R 4 is independently hydrogen or a hydroxyl protecting group; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl, W is -C(O)-, -C(O)NR'-, -C(O)O-, -SONR'-, -P(O)R''NR', -SONR'-, or -PONR'-, where C, S, or P is directly attached to the phenyl ring of formula IIa; R′ and R″ are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20Heteroaryl, or C 6-20 is aryl, Each Z is independently hydrogen, C 1-8 alkyl, halogen, cyano, or nitro; n is 0, 1, 2, or 3; L, A) C 1-50 Alkylene or C 1-50 including heteroalkylene, (i) one or more unsaturated bonds (ii) contains heteroarylene, or (iii) at least one C 1-20 is substituted with alkyl, or B) at least one isoprenyl group having a structure represented by general formula III;

[0062] [ka] l and m are each independently 1 to 20.

[0063] The present disclosure also provides a conjugate represented by the following general formula IIa, or a pharma- ceutically acceptable salt or solvate thereof:

[0064] [ka] During the ceremony, the Ab is an anti-TROP2 (tumor-associated calcium signaling factor 2, also known as TACSTD2) antibody or an antigen-binding fragment thereof; Each B' is an active agent; Each G is independently a glucuronic acid moiety or

[0065] [ka] and R 3 is hydrogen or a carboxyl protecting group; Each R 4is independently hydrogen or a hydroxyl protecting group; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl, W is -C(O)-, -C(O)NR'-, -C(O)O-, -SONR'-, -P(O)R''NR', -SONR'-, or -PONR'-, where C, S, or P is directly attached to the phenyl ring of formula IIa; R′ and R″ are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 is aryl, Each Z is independently hydrogen, C 1-8 alkyl, halogen, cyano, or nitro; n is 0, 1, 2, or 3; L, A) C 1-50 Alkylene or C 1-50 including heteroalkylene, (i) one or more unsaturated bonds (ii) contains heteroarylene, or (iii) at least one C 1-20 is substituted with alkyl, or B) at least one isoprenyl group having a structure represented by general formula III;

[0066] [ka] l and m are each independently 1 to 20.

[0067] Additional conjugates of this structure, as well as detailed methods of preparation, are disclosed in U.S. Pat. Nos. 9,919,057, 9,993,568, 10,980,890, and 11,413,353, the contents of each of which are incorporated by reference in their entirety.

[0068] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; The light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: The amino acid sequence of SEQ ID NO: 15 or 20, A sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or The heavy chain variable region comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: Amino acid sequence of SEQ ID NO: 16; A sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or The light chain variable region comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0071] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0072] In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the humanized antibody comprises: (i) a variable heavy chain framework region from a heavy chain of a human antibody or from a human consensus framework, the variable heavy chain framework region comprising one or more of the following amino acid sequence changes: Y27F, T30S, V37L, M48I, G49A, I70L, and R72V; (ii) a variable light chain framework region from a light chain of a human antibody or from a human consensus framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

[0074] In certain preferred embodiments, each G is

[0075] [ka] It is.

[0076] In certain embodiments, R 1 and R 2 are each hydrogen.

[0077] In certain embodiments, R 3 is hydrogen.

[0078] In certain embodiments, each R 4 is a hydroxyl protecting group.

[0079] In certain embodiments, n is 0.

[0080] In certain preferred embodiments, each W is -C(O)NR'-, further comprising C attached directly to the phenyl ring of formula IIa and NR' attached to L.

[0081] In certain embodiments, each R 4 are independently hydrogen.

[0082] In certain embodiments, R 1 and R 2 are each hydrogen, n is 0, Each W is -C(O)NR'-, where C is directly attached to the phenyl ring of formula IIa and R' is hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 aryl, where NR′ is attached to L;

[0083] In certain embodiments, L comprises a nitrogen-containing 1-50 membered heteroalkylene.

[0084] In certain embodiments, L comprises a hydrophilic amino acid, hi certain embodiments, W comprises two or more atoms of a hydrophilic amino acid, and the nitrogen of W forms a peptide bond with the carbonyl of the hydrophilic amino acid.

[0085] In certain embodiments, L is a nitrogen-containing 1-50 membered heteroalkylene and the linker comprises two or more atoms of a hydrophilic amino acid; The nitrogen forms a peptide bond with the carbonyl of a hydrophilic amino acid.

[0086] In certain embodiments, L is covalently linked to the antibody by a thioether bond, wherein the thioether bond involves the sulfur atom of a cysteine ​​of the antibody.

[0087] In certain embodiments, the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C-terminus of the antibody, The thioether bond involves the sulfur atom of the cysteine ​​amino acid motif.

[0088] In certain embodiments, the amino acid motif has the sequence CYYX, further comprising: C is cysteine, Y is an aliphatic amino acid; X is selected from glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine; The thioether bond involves the sulfur atom of the cysteine ​​amino acid motif.

[0089] In certain embodiments, the amino acid motif has the sequence CYYX, further comprising: Y is selected from alanine, isoleucine, leucine, methionine, and valine.

[0090] In certain preferred embodiments, the amino acid motif has the sequence CVIM (SEQ ID NO:24) or CVLL (SEQ ID NO:25).

[0091] In certain embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is a glycine.

[0092] In certain preferred embodiments, L comprises the amino acid sequence of GGGGGGCVIM (SEQ ID NO:22) at the C-terminus.

[0093] In certain embodiments, L is C 1-50 Includes heteroalkylene.

[0094] In certain preferred embodiments, L comprises an oxime. In certain embodiments, the oxygen atom of the oxime is on the side of L that is linked to W, and the carbon atom of the oxime is on the side of L that is linked to Ab. In certain embodiments, the carbon atom of the oxime is on the side of L that is linked to W, and the oxygen atom of the oxime is on the side of L that is linked to Ab.

[0095] In certain embodiments, L is an oxime-containing C 1-50 is heteroalkylene, The oxygen atom of the oxime is on the side of L that is connected to W, The carbon atom of the oxime is on the side of L that is linked to Ab, or the carbon atom of the oxime is on the side of L that is connected to W, and The oxygen atom of the oxime is on the side of L that is linked to Ab.

[0096] In certain preferred embodiments, L comprises an oxime and at least one isoprenyl unit covalently attaches the oxime to Ab (e.g., at least one isoprenyl unit attaches the oxime directly or indirectly to Ab).

[0097] In certain embodiments, L comprises a connecting unit represented by general formula VIII or general formula IX: [General formula VIII] -(CH2) r (V(CH2) p ) q - [Chemical formula IX] -(CH2CH2X) w - V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 - and X is -O-, C 1-8 Alkylene, or -NR 21 - and R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl-C 3-20 is heteroaryl, r is 0 to 10; p is 0 to 10; q is 1 to 20; w is 1 to 20.

[0098] In certain embodiments, q is 1-10.

[0099] In certain embodiments, r is 1 or 2.

[0100] In certain embodiments, p is 1 or 2.

[0101] In certain embodiments, V is --O--.

[0102] In certain embodiments, q is 1 to 10; r and p are each 1 or 2; V is -O-.

[0103] In certain embodiments, X is --O--.

[0104] In certain embodiments, w is 1-10.

[0105] In certain embodiments, in X is -O-, w is 1 to 10.

[0106] In certain preferred embodiments, L is

[0107] [ka] where n40 is 1 to 10, preferably at least 2.

[0108] In certain embodiments, L comprises an oxime and at least one polyethylene glycol unit covalently attaches the oxime to the active agent.

[0109] In certain embodiments, L comprises a linking unit formed by reaction between an alkyne and an azide, or between an aldehyde or ketone group and a hydrazine or hydroxylamine.

[0110] In certain embodiments, L further comprises a linking unit represented by the following general formula IVa, IVb, IVc, IVd, or IVe:

[0111] [ka] During the ceremony, L1 and L2 each independently represent a single bond or C 1-30 is alkylene, R 11 is hydrogen or C 1-10 It is an alkyl.

[0112] In certain embodiments, L and L are each independently a single bond, C 11 Alkylene or C12 It is alkylene.

[0113] In certain embodiments, the isoprenoid transferase is a farnesyl protein transferase (FTase) or a geranylgeranyl transferase (GGTase).

[0114] In certain embodiments, L is branched and i) a branching unit covalently coupled to an antibody by a primary linker; ii) a first branch, wherein a first active agent is covalently coupled to the branch unit by a secondary linker and a cleavage group; and iii) a second branch, a) a second active agent is covalently coupled to the branching unit by a secondary linker and a cleavage group; or b) The polyethylene glycol moiety comprises a second branch that is covalently coupled to the branching unit.

[0115] In certain embodiments, the branching unit is

[0116] [ka] wherein: L 2 , L 3 , and L 4 each independently represents a bond or -C n H 2n - and n is 1 to 30; G 1 , G 2 , and G 3 each independently represents a bond,

[0117] [ka] represents R 30 is hydrogen or C 1-30 is alkyl, R 40 is hydrogen or L 5 -COOR6, L 5 is a bond or -C n’ H 2n’ - and n' is 1 to 10; R6 is hydrogen or C 1-30 It is an alkyl.

[0118] In certain preferred embodiments, the cleavable group is cleavable in the target cell to release one or more active agents.

[0119] In certain embodiments, at least one branched linker is covalently coupled to the Ab, At least two active agents are covalently coupled to the branched linker. In certain embodiments, one branched linker is coupled to the Ab. In certain embodiments, two branched linkers are coupled to the Ab. In certain embodiments, three branched linkers are coupled to the Ab. In certain embodiments, four branched linkers are coupled to the Ab. In certain embodiments, each branched linker is coupled to two active agents. In certain embodiments, the conjugate comprises at least two different active agents. In certain embodiments, at least one branched linker is coupled to two different active agents.

[0120] In certain embodiments, the branching unit is a nitrogen atom. In other embodiments, the branching unit is an amide and the first linker comprises an amide carbonyl. In yet other embodiments, the branching unit is an amide and the second linker comprises an amide carbonyl. In certain preferred embodiments, the branching unit is a lysine.

[0121] In certain embodiments, the conjugate has a structure represented by:

[0122] [ka] or a pharma- ceutically acceptable salt thereof, wherein: B' and B'' are each an active agent; n1 to n3 each independently represent 0 to 30; AA is an amino acid group.

[0123] In certain embodiments, the conjugate comprises a structure represented by the following:

[0124] [ka]

[0125] [ka] wherein B' and B'' are the same or different active agents; Pyrrolobenzodiazepine dimers, such as compounds of formula III: m and n each independently represent 0 to 30; or a pharma- ceutically acceptable salt thereof.

[0126] In certain embodiments, the conjugate comprises:

[0127] [ka] or a pharma- ceutically acceptable salt thereof, wherein the wave symbol represents the binding site of the antibody construct; * represents a binding site for an active agent, and n is 0-20.

[0128] In certain embodiments, the first linker of the antibody-drug conjugate comprises an alkylene having 1-100 carbon atoms, preferably 1-50 carbon atoms, or the alkylene comprises at least one unsaturated bond, the alkylene comprises at least one heteroarylene, and the carbon atoms of the alkylene are substituted with one or more heteroatoms selected from nitrogen (N), oxygen (O), and sulfur (S), or the alkylene is further substituted with one or more alkyls having 1-20 carbon atoms.

[0129] In certain embodiments, at least one carbon atom of the alkylene is replaced with nitrogen, and the first linker comprises at least two atoms of a hydrophilic amino acid, the nitrogen forming a peptide bond with the main chain carbonyl of the hydrophilic amino acid, which may be, for example, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

[0130] In certain embodiments, the branched linker of the antibody-active agent comprises an amino acid, preferably arginine, aspartic acid, glutamic acid, lysine, or ornithine, having a side chain with a moiety that carries a charge at neutral pH in aqueous solution. The amino acid may be located anywhere on the branched linker. For example, the oxime of the branched linker may be covalently attached to the polyethylene glycol unit of the branched linker. Alternatively or in addition, these amino acids may be present in the second linker, optionally within each second linker.

[0131] In certain embodiments, the conjugate has a structure represented by:

[0132] [ka] or a pharma- ceutically acceptable salt thereof, wherein B′ and B″ are each an active agent; n1 to n3 each independently represent 0 to 30; AA refers to an amino acid group.

[0133] In certain embodiments, AA refers to an amino acid group having one or more amino acids attached.

[0134] In certain embodiments, i) the linker comprises a peptide sequence of multiple amino acids, and ii) at least two active agents are covalently attached to the side chains of the amino acids.

[0135] In certain embodiments, the amino acid group is a group of 1-20 amino acids with main chain or side chain linkages.

[0136] In certain embodiments, the amino acid group is a group of 1-20 arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine residues linked in main chain or side chains.

[0137] In certain embodiments, the amino acid group is a group of 1-20 arginine, aspartic acid, glutamic acid, lysine, or ornithine residues linked in main chain or side chain links.

[0138] In certain embodiments, the amino acid group comprises between 1 and 20 amino acids, for example, at least one lysine.

[0139] In certain embodiments, the amino acid group comprises a lysine backbone or side chain linkage.

[0140] In certain embodiments, the conjugate comprises:

[0141] [ka] wherein MMAE is monomethylauristatin E.

[0142] Additional conjugates of this structure, as well as detailed methods of preparation, are disclosed in US Pat. Nos. 11,173,214 and 11,167,040, the contents of each of which are incorporated herein by reference in their entirety.

[0143] In certain embodiments, the term "active agent moiety" as used herein refers to a compound that includes an active agent and a covalent bond that attaches the active agent to a linker or portion thereof.

[0144] In the present disclosure, the active agent moiety may be directly attached to the linker, or one or more, particularly two or more, three or more, or four or more active agent moieties may be directly attached to the linker.

[0145] In certain embodiments, the active agents are each independently selected from a chemotherapeutic agent and a toxin.

[0146] In addition, the active agent may be an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof, and may be selected for use from the active agents listed below: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacin Non, camptothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novem Novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, anthromycin cin), azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin,Epirubicin, esorubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, thrombin Rimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, dromostanolone, propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, Bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenameth, pirarubicin, rosoxaxanthrone Inthrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, schizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracrine A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, paclitaxel,Albumin engineered nanoparticle formulations of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronic acid, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharma- ceutically acceptable salt, solvate, or acid thereof; (b) monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor factor, CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, polypeptide factor, LIF, Kit ligand, or combinations thereof; (c) diphtheria toxin, botulinum toxin, tetanus toxin, decentretoxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, indolinobenzodiazepines, pyridinobenzodiazepines, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, auristatin, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065, analogs or derivatives, duocarmycins, enediyne antibodies, esperamicins, epothilones, toxoids, or combinations thereof; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an active drug, a neurotransmitter, a radioisotope, or a combination thereof; (e) a radioactive label, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, dioxygenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination thereof; (f) an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, or toremifene; (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole; (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) aromatase inhibitors; (k) protein kinase inhibitors; (l) lipid kinase inhibitors; (m) antisense oligonucleotide; (n) ribozyme; (o) Vaccines; and (p) Antiangiogenic agents.

[0147] In certain embodiments, the active agent is

[0148] [ka]

[0149] [ka]

[0150] [ka]

[0151] [ka] and In the formula, y is 1 to 10, The wavy lines indicate the connections to the conjugates.

[0152] In certain embodiments, the active agent is a pyrrolobenzodiazepine dimer, the N10 position of the pyrrolobenzodiazepine dimer is substituted with X or the N'10 position is substituted with X', and X or X' connects the pyrrolobenzodiazepine dimer to a linker; X and X' are each independently -C(O)O- * or -C(O)- * and * refers to the binding site between the pyrrolobenzodiazepine dimer and the linker.

[0153] In certain preferred embodiments, the pyrrolobenzodiazepine dimer is represented by formula III:

[0154] [ka] During the ceremony, The wavy line indicates the point of attachment to the conjugate (e.g., the carbonyl of formula IIa attached to B'); The dotted line represents an optional double bond; R1 and R1 ’ are each independently H, OH, =O, =CH2, CN, or R m , OR m , =CH-R m’ =C(R m’ )2, O-SO2-R m , CO2R m , C.O.R. m , halo, and dihalo; R m’ But R m , CO2R m , C.O.R. m , CHO, COH, and halo; R m is substituted or unsubstituted C 1-12 Alkyl, substituted or unsubstituted C 2-12 Alkenyl, substituted or unsubstituted C 2-12 Alkynyl, substituted or unsubstituted C 5-20 Aryl, substituted or unsubstituted C 3-6 Heteroaryl, substituted or unsubstituted C 3-6 cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocyclyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and substituted or unsubstituted 5- to 7-membered heteroaryl; 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 When cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is substituted, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6Each hydrogen atom of the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl is independently selected from methoxy, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered R2, R3, R5, R2 ’ , R3 ’ , and R5 ’ However, each independently, H, R m , O.H., O.R. m , S.H., S.R. m , NH2, NHR m , N.R. m R m’ , NO2, Me3Sn, and halo; R4 and R4 ’ However, each independently, H, R m , O.H., O.R. m , S.H., S.R. m , NH2, NHR m , N.R. m R m’ , NO2, Me3Sn, halo, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n’ , -OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NRn R n’ , -S(O)NR n R n’ , -OS(O)NR n R n’ , -OS(O)2NR n R n’ , -NR n R n’ , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o’ , -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o’ , -NR n S(O)2NR o R o’ , -C(O)R n , -C(O)OR n , and -C(O)NR n R n’ Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-12 For aryl and 5- to 7-membered heteroaryl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-12 The hydrogen atoms of the aryl and the 5- to 7-membered heteroaryl are each independently represented by C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-12Aryl, 5-7 membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p’ , -OS(O)R p , -OS(O)2R p , -SR p , -S(O)R p , -S(O)2R p , -S(O)NR p R p’ , -S(O)NR p R p’ , -OS(O)NR p R p’ , -OS(O)2NR p R p’ , -NR p R p’ , -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q H, -NR p S(O)R q , -NR p S(O)2R q , -NR p S(O)NR q H, -NR p S(O)2NR q H, -C(O)R p , -C(O)OR p or -C(O)NR p R p may be replaced by R n , R n’ , R o , R o’ R p , R p’ , and R q However, each independently, H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 aryl, and 5- to 7-membered heteroaryl; X is selected from -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR-, -S(O)NR-, -P(O)R'NR-, -S(O)NR-, and -PONR-; Xa is a bond or a substituted or unsubstituted C 1-6 Alkylene, C 1-6 When alkylene is substituted, C 1-8 Alkyl or C 3-8 cycloalkyl substituted; R and R' are each independently H, OH, NH, ONH, NHNH, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 1-8 Alkoxy, substituted or unsubstituted C 1-8 Alkylthio, substituted or unsubstituted C 3-20 Heteroaryl, substituted or unsubstituted C 5-20 Aryl, or mono- or di-C 1-8 represents alkylamino, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, and C 5-20 When aryl is substituted, it can be OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-6 Alkyl, C 1-6 Alkoxy and C 6-12 aryl; Y and Y' are each independently selected from O, S, and N(H); R6 is a substituted or unsubstituted saturated or unsaturated C 3-12 A hydrocarbon chain, which may be interrupted by one or more heteroatoms, NMe, or substituted or unsubstituted aromatic rings, and the chain or aromatic ring may be -NH, -NR m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R or -[CH2CH2O] n-R may be substituted or unsubstituted, R m and R are the above R m and as defined for R, n is 1 to 12; R7 and R7 ’ each independently represents H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 Alkynyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r’ , -OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r’ , -S(O)NR r R r’ , -OS(O)NR r R r’ , -OS(O)2NR r R r’ , -NR r R r’ , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s’ , -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s’ , -NR r S(O)2NR s R s , -C(O)R r , -C(O)OR sor -C(O)NR r R r’ and C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 For aryl and 5- to 7-membered heteroaryl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 The hydrogen atoms of the aryl and the 5- to 7-membered heteroaryl are each independently represented by C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6-10 Aryl, 5-7 membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t’ , -OS(O)R t , -OS(O)2R t , -SR t , -S(O)R t , -S(O)2R t , -S(O)NR t R t’ , -S(O)NR t R t’ , -OS(O)NR t R t’ , -OS(O)2NR t R t’ , -NR t R t’ , -NR t C(O)R u , -NR t C(O)OR u , -NR t C(O)NR u R u’ , -NR t S(O)R u , -NR t S(O)2R u , -NR tS(O)NR u R u’ , -NR t S(O)2NR u R u’ , -C(O)R t , -C(O)OR t or -C(O)NR t R t’ may be replaced by R r , R r’ , R s , R s’ , R t , R t’ , R u , and R u ' are each independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5-10 aryl, and 5- to 7-membered heteroaryl; G is a glucuronide group or a galactoside group; Each Z is H, C 1-8 Alkyl, halo, NO2, CN,

[0155] [ka] is selected from R9, R 10 , and R 16 However, each independently, H, C 1-8 Alkyl, C 2-6 Alkenyl, C 1-6 selected from alkoxy, alkyloxyalkyl, and methyloxyethyl; n30 is 1 to 3.

[0156] In certain embodiments, the dotted line indicates R and R, or R 1` and R 7` This indicates the presence of a double bond between carbons that holds the

[0157] In certain embodiments, R is R m and Rm is a substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 5-7 Aryl, and substituted or unsubstituted C 3-6 Heteroaryl is selected from the group consisting of aryl, hetero ...

[0158] In certain embodiments, R2, R3, and R5 are each independently H or OH.

[0159] In certain embodiments, R4 is C 1-6 It is an alkoxy.

[0160] In certain embodiments, R4 is methoxy, ethoxy, or butoxy.

[0161] In certain embodiments, X is selected from -C(O)O-, -C(O)-, and -C(O)NR-; R is, independently at each occurrence, H, OH, N, CN, NO, SH, NH, ONH, NHNH, halo, substituted or unsubstituted C. 1-8 Alkyl, or substituted or unsubstituted C 1-8 Alkoxy, if substituted, C 1-8 Alkyl or C 1-8 The alkoxy is substituted with OH, N3, CN, NO2, SH, NH2, ONH2, NNH2, or halo.

[0162] In certain embodiments, X is -C(O)NR-.

[0163] In certain embodiments, R6 is a substituted or unsubstituted saturated or unsaturated C 3-8 is a hydrocarbon chain, One or more of the carbon atoms of the hydrocarbon chain are replaced by a heteroatom or a substituted or unsubstituted aromatic ring, the heteroatom being O, S, or N(H), the aromatic ring being benzene, pyridine, imidazole, or pyrazole, and the chain or aromatic ring being -NHC(O)CH2-[OCH2CH2] in any one or more positions of the hydrogen atoms on the chain or aromatic ring.n -R or -[CH2CH2O] n -R, n is 1 to 6.

[0164] In certain embodiments, n is 1-10.

[0165] In certain embodiments, Xa is a bond or C 1-3 It is alkylene.

[0166] In certain embodiments, Z is H,

[0167] [ka] Wherein R9, R 10 , and R 16 However, each independently, H, C 1-3 Alkyl, C 1-3 The alkyl group is selected from alkoxy and alkyloxymethyl.

[0168] In certain embodiments, R9 is methyloxyalkyl.

[0169] In certain embodiments, R 10 is methyloxyalkyl.

[0170] In certain embodiments, R 16 is methyloxyalkyl.

[0171] In certain embodiments, R, R 10 , or R 16 is -(CH2CH2O) m -(CH2) m2 CH3, and further, in the formula, m is 1 to 6, and m2 is 0 to 2.3.

[0172] In certain embodiments, R2 is H.

[0173] In certain embodiments, R3 is H.

[0174] In certain embodiments, R7 is H.

[0175] In certain embodiments, R4 is alkoxy (e.g., methoxy).

[0176] In certain embodiments, R5 is OH.

[0177] In certain embodiments, R1 is =CH2, CH3, or phenyl optionally substituted with methoxy.

[0178] In certain embodiments, Y is O.

[0179] In certain embodiments, R2' is H.

[0180] In certain embodiments, R3' is H.

[0181] In certain embodiments, R7' is H.

[0182] In certain embodiments, R4' is alkoxy (eg, methoxy).

[0183] In certain embodiments, R5' is OH.

[0184] In certain embodiments, R1' is =CH2, CH3, or phenyl optionally substituted with methoxy.

[0185] In certain embodiments, Y' is O.

[0186] In certain embodiments, X is -C(O)O-.

[0187] In certain embodiments, Xa is CH2.

[0188] In certain embodiments, G is a glucuronide group.

[0189] In certain embodiments, G is

[0190] [ka] It is.

[0191] In certain embodiments, n30 is 1.

[0192] In certain embodiments, Z is

[0193] [ka] It is.

[0194] In certain embodiments, R9 is H.

[0195] In certain embodiments, R 16 is alkyloxyalkyl (e.g., methoxyethyl).

[0196] In certain embodiments, Z is

[0197] [ka] It is.

[0198] In certain embodiments, R 10 is alkyl (e.g., methyl).

[0199] In certain embodiments, R6 is alkyl (eg, pentyl).

[0200] In certain embodiments, the conjugate has a structure selected from the following:

[0201] [ka]

[0202] [ka]

[0203] [ka]

[0204] [ka] where the dashed overlaid bond represents the point of attachment to L.

[0205] In certain embodiments, the conjugate has a structure selected from the following:

[0206] [ka]

[0207] [ka]

[0208] [ka]

[0209] [ka]

[0210] [ka]

[0211] [ka]

[0212] [ka]

[0213] [ka]

[0214] [ka]

[0215] [ka]

[0216] [ka]

[0217] [ka] Including, wherein MMAE is monomethylauristatin E; MMAF is monomethylauristatin F; The wavy lines are the points of attachment to the conjugates.

[0218] In certain aspects, the present disclosure also provides a pharmaceutical composition comprising the conjugate for the prevention or treatment of hyperproliferative, cancer, or angiogenic diseases.

[0219] In certain embodiments, the pharmaceutical composition further comprises a pharma- ceutically effective amount of a chemotherapeutic agent.

[0220] In certain embodiments, the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma.

[0221] The present disclosure also provides a pharmaceutical preparation comprising the conjugate.

[0222] Antibody or antigen-binding fragment thereof The present disclosure provides antibodies that bind to human TROP2 (tumor-associated calcium signaling factor 2, TACSTD2). The antibodies according to the present disclosure described herein are polypeptides that include one or more complementarity-determining areas or regions (CDRs).

[0223] In some embodiments, the CDRs are comprised in "framework" regions, which orient the CDRs such that they have appropriate antigen-binding properties.

[0224] In certain aspects, the present disclosure provides an antibody-drug conjugate comprising an anti-TROP2 antibody that binds to TROP2.In certain embodiments, the antibody disclosed herein can be used to bind to TROP expressed in tumors and deliver drugs to tumors.In certain embodiments, the antibody-drug conjugate disclosed herein has improved stability compared to antibody-drug conjugates known in the art.

[0225] In certain embodiments, the antibody includes, but is not limited to, a monoclonal antibody, a bispecific antibody, a diabody, a multispecific antibody, a multibody, a minibody, a domain antibody, an antibody mimetic (or synthetic antibody), a chimeric antibody, a humanized antibody, a human antibody, or an antibody fusion (or antibody conjugate), and fragments thereof, including the various forms of antibodies disclosed herein.

[0226] In certain embodiments, an antibody fragment of an antibody according to the present disclosure includes a Fab, Fab', F(ab')2, scFab, Fv, dsFv, scFv, scFv-Fc, minibody, diabody, scAb, or dAb.

[0227] In certain embodiments, an antibody according to the present disclosure may consist of only light chain or only heavy chain polypeptides comprising the variable regions shown in Tables 1-3.

[0228] CDR sequences that may be included in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof according to an embodiment of the present disclosure are shown in Tables 1 to 3, respectively.

[0229] Antibodies according to the present disclosure may share certain regions or sequences with other antibodies disclosed herein. In certain embodiments, the constant regions of the antibodies or antigen-binding fragments thereof may be shared. In certain embodiments, the Fc region may be shared. In certain embodiments, the frame of the variable regions may be shared.

[0230] The heavy and light chain variable regions according to the present disclosure may be linked to at least a portion of a human constant region. The choice of constant region may be determined in part by whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. In addition, human IgG1 and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa.

[0231] The variable regions of immunoglobulin chains generally have the same overall structure and comprise relatively conserved framework regions (FR) linked by three hypervariable regions called "complementarity determining regions" or CDRs. The CDRs of the variable regions from each chain comprising a heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a specific epitope of a target protein. These elements of naturally occurring light and heavy chain variable regions are typically comprised in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The position of the amino acid sequence corresponding to each variable region can be determined by Kabat (Kabat et al., (1983) USDept, of Health and Human Services, "Sequences of Proteins of Immunological Interest"), Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), or in a manner related to the OPAL library (Hye Young Yang et.al., 2009 Mol. Cells 27:225). The CDRs determined by each definition may overlap or be subsets that include the other when compared with each other. Those skilled in the art will be able to easily select CDR sequences according to the above definitions, taking into account the variable region sequences of the antibody.

[0232] In certain embodiments, the antibody according to the present disclosure is a humanized antibody. Humanized antibody refers to any antibody in which the constant regions of a non-human antibody are completely replaced with human forms of the constant regions, and at least a portion of the variable regions of the non-human antibody, except for the three loops of the outer amino acid sequence of each variable region that binds to the target structure, are completely or partially replaced with the corresponding portions of a human antibody.

[0233] Certain mutations can be introduced into the framework regions to enhance the stability of the antibody while maintaining the antigen-binding activity of the antibody. Stabilization of therapeutic antibodies can result in improved serum half-life, lower dosage requirements, reduced side effects, improved shelf life, and reduced shipping and storage costs.

[0234] In certain embodiments, a humanized antibody according to the present disclosure comprises: (a) comprises (i) a variable heavy chain framework region from a heavy chain of a human antibody or from a human consensus framework, and (ii) a variable light chain framework region from a light chain of a human antibody or from a human consensus framework.

[0235] In certain embodiments, at least one amino acid in the variable domain framework region from the heavy chain is replaced with the corresponding amino acid from the heavy chain of a murine antibody or a murine consensus framework, or the variable domain framework region from the light chain is replaced with the corresponding amino acid from the light chain of a murine antibody or a murine consensus framework.

[0236] In certain embodiments, the amino acid substitutions in the heavy chain are selected from the following amino acid substitutions: Substitution of Y (human) with F (mouse) at position 27; Substitution of T (human) with S (mouse) at position 30; Substitution of V (human) with L (mouse) at position 37; Substitution of M (human) with I (mouse) at position 48; Substitution of G (human) for A (mouse) at position 49; Substitution of I (human) with L (mouse) at position 70, and Substitution of R (human) with V (mouse) at position 72; (ii) the amino acid substitution in the light chain is an S (mouse) to Y (human) substitution at position 49;

[0237] In certain embodiments, the amino acid substitutions in the heavy chain may further include the following amino acid substitutions: Substitution of R (human) with K (mouse) at position 67, and / or Substitution of V (human) with A (mouse) at position 68.

[0238] In certain embodiments, the amino acid substitutions in the light chain may further include the following amino acid substitutions: Substitution of S (human) with Y (mouse) at position 67; substitution of I (human) for T (mouse) at position 2 and substitution of S (human) for Y (mouse) at position 67; substitution of T (human) for R (mouse) at position 22 and substitution of S (human) for Y (mouse) at position 67; Substitution of K (human) for E (mouse) at position 42 and substitution of S (human) for Y (mouse) at position 67; a substitution of G (human) for S (mouse) at position 64 and a substitution of S (human) for Y (mouse) at position 67; a substitution of S (human) for Y (mouse) at position 67 and a substitution of T (human) for V (mouse) at position 72; or Substitution of S (human) for Y (mouse) at position 67 and substitution of T (human) for D (mouse) at position 85.

[0239] In certain embodiments, a humanized antibody comprises: (a) (i) a variable heavy chain framework region from a heavy chain of a human antibody or from a human consensus framework, wherein the variable heavy chain framework region comprises one or more of the following amino acid sequence changes: Y27F, T30S, V37L, M48I, G49A, I70L, and R72V; and (ii) a variable light chain framework region from a light chain of a human antibody or from a human consensus framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0240] In certain embodiments, the heavy and light chain variable region sequences comprising the substitutions are shown, respectively, in Tables 1-3. In certain embodiments, the substitutions comprise substitutions in the FR regions shown in Tables 1-3.

[0241] In certain embodiments, the present disclosure discloses one or more amino acid sequences having substantial sequence identity to one or more amino acid sequences disclosed herein. Substantial identity means that the effects disclosed herein are maintained in the presence of sequence mutations. In certain embodiments, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity to the heavy chain variable region shown in Tables 1-3. In another embodiment, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity to the light chain variable region shown in Tables 1-3. For example, in the case of a variant exhibiting 90% identity, 95% identity, or 99% identity to the sequence of an antibody or antigen-binding fragment thereof according to the present disclosure, any mutations occur in the framework of the variable region, but not in the CDRs.

[0242] In certain embodiments, the nucleic acid encoding the antibody or fragment thereof according to the present disclosure is a nucleic acid encoding a full-length antibody comprising the CDRs, the variable regions comprising the CDRs, and the variable regions and constant regions disclosed herein. Once the amino acid sequence is determined, the nucleic acid sequence can be readily determined taking into account known reverse transcription programs, codon usage, and the like.

[0243] Antigen specificity and affinity for antibodies In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure have specificity and affinity for human TROP2 antigen suitable for use as antibody therapeutics / diagnostics. In certain embodiments, the affinity for aggregates may be KD<1,000 nM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM, e.g., KD<1,000 nM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM. -6 M~10 -12 It can be M.

[0244] Antibody production In the present disclosure, the non-human antibody may be derived, for example, from any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or a non-human primate (e.g., monkeys such as cynomolgus or rhesus monkeys) or ape (e.g., chimpanzee). The non-human antibody may be produced by immunizing the animal by using methods known in the art. For methods for producing humanized antibodies according to embodiments of the present disclosure, see US Patent Application No. 15 / 532598. Fully human antibodies may be produced by administering antigen to a transgenic animal containing human immunoglobulin loci, or by treating a phage display library expressing a human antibody repertoire with antigen and then selecting the target antibody. The antibody may be polyclonal or monoclonal, or may be synthesized in a cellular host through expression of recombinant DNA.

[0245] Monoclonal antibodies (mAbs) can be produced using conventional monoclonal antibody methods, such as standard somatic cell hybridization techniques (see Kohler and Milstein, 1975, Nature 256:495).

[0246] Methods for expressing antibodies The antibodies disclosed herein can be expressed in hybridoma cell lines or expression cell lines other than hybridomas. The expression constructs encoding the antibodies can be used to transform mammalian, insect, or microbial host cells. Constructs such as plasmids can be produced as described in the preceding text using any of a variety of known methods for introducing polynucleotides into host cells. The specific method can vary depending on the type of host cell. Methods for introducing heterologous polynucleotides into mammalian cells are widely known in the art and include, but are not limited to, dextran-mediated transfer, calcium phosphate precipitation, polybrene-mediated transfer, protoplast fusion, electrophoresis, encapsulation of the transferred polynucleotide using liposomes, mixing nucleic acid with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0247] Use of anti-human TROP2 antibody-drug conjugates for therapeutic purposes It is known that the expression of TROP2 in cancer is associated with poor prognosis of cancer patients and also affects cancer metastasis.For example, TROP2 is overexpressed in lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, etc. For anti-cancer antibody therapy, for example, anti-human TROP2 antibodies can be used in a form linked to various cytotoxic drugs via linkers to eliminate TROP2-overexpressing cancer cells, as described herein.Therefore, antibodies that bind to TROP2 can be used alone or in a form linked to anti-cancer chemotherapeutic agents, cytotoxic agents, or radioactive substances, and can be embodied as cell therapy agents such as CAR-T cells that target anti-cancer targets, and therefore can be used as targeted therapeutic agents directed to TROP2-expressing cells.

[0248] Treatment methods: pharmaceutical formulations and routes of administration The present disclosure also provides a method of treatment using the antibody-drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof. In certain embodiments, the antibody-drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, is provided to a patient. The antibody-drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, inhibits cancer cell metastasis by binding to human TROP2 expressed on the surface of the cancer cell. In certain embodiments, the antibody in the form of an antibody bound to a cytotoxic agent binds to human TROP2 expressed on the surface of the cancer cell, thereby specifically delivering the cytotoxic agent bound to the antibody to the cancer cell to induce the death of the cancer cell. In certain embodiments, the antibody in the form of an antibody specific for the same or another target binds to human TROP2 expressed on the surface of the cancer cell, thereby increasing the specificity of multiple antibodies to the cancer cell or inducing a connection between the cancer cell and other types of cells, such as immune cells, to induce the death of the cancer cell.

[0249] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, and a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Also provided is a method of treating a cancer patient, for example, by administering such a pharmaceutical composition. The term "patient" includes a human patient.

[0250] The pharmaceutical composition may include a pharma- ceutically acceptable carrier. Carrier is used to mean including excipient, diluent, or adjuvant. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, saline, buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

[0251] The pharmaceutical composition can be prepared as any formulation according to a general method.The composition can be formulated into an oral administration formulation (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral administration formulation (e.g., injection).In addition, the composition can be prepared as a systemic or local formulation.

[0252] The pharmaceutical composition may contain an effective amount of an antibody or an antigen-binding fragment thereof, an anti-cancer drug, or a combination thereof. The term "effective amount" refers to an amount sufficient to exhibit a preventive or therapeutic effect when administered to an individual in need of prevention or treatment. The effective amount may be appropriately selected according to the cell or individual selected by a person skilled in the art. The effective amount may be determined according to factors including the severity of the disease, the age, weight, health, and sex of the patient, the patient's sensitivity to the drug, the administration time, the administration route, the excretion rate, the duration of treatment, and the drug used in combination with or simultaneously with the composition used, as well as other factors well known in the medical field.

[0253] The dosage of the pharmaceutical composition may range, for example, from 10 μg / kg to about 30 mg / kg per adult, optionally from 0.1 mg / kg to about 30 mg / kg, or alternatively from 0.3 mg / kg to about 20 mg / kg. The pharmaceutical composition may be administered once daily, multiple times daily, once every 1 to 4 weeks, or 1 to 12 times per year.

[0254] The present disclosure will now be described in more detail with reference to examples and experimental examples.

[0255] The following examples and experimental examples are intended to aid in the understanding of the present disclosure and are not intended to limit the scope of the disclosure.

[0256] definition Unless otherwise defined in this disclosure, scientific and technical terms used herein have the meanings as commonly understood by those skilled in the art. Furthermore, unless otherwise specifically required by context, the singular includes the plural and the plural includes the singular. In general, the nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0257] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000), Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", WH Freeman & Co., New York (2000), Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", WH Freeman & Co., NY (1999), and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0258] Chemical terms used herein, unless otherwise defined herein, are used according to conventional usage in the art as exemplified in "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

[0259] All of the above, and any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including specific definitions, will control.

[0260] The term "agent" is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of compounds), a biological macromolecule (such as nucleic acids, antibodies, portions thereof, including humanized, chimeric and human antibodies and monoclonal antibodies, proteins or portions thereof, e.g., peptides, lipids, carbohydrates), or an extract made from biological material such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Agents include, for example, agents with known structures and agents with unknown structures.

[0261] "Patient," "subject," or "individual" are used interchangeably and refer to either a human or non-human animal. These terms include mammals such as humans, primates, farm animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice, rats).

[0262] "Administering" or "administration" of a substance, compound or agent to a subject can be performed using one of a variety of methods known in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through the skin tract). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations that provide sustained, sustained or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0263] The appropriate method of administering a substance, compound or agent to a subject also depends, for example, on the age and / or physical condition of the subject, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, the compound or agent is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a sustained or timed release formulation or is administered using a device for such sustained or timed release.

[0264] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents, such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective on a patient at the same time, and a synergistic effect of the two agents may be involved). For example, the different therapeutic compounds may be administered simultaneously or sequentially, in the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0265] As used herein, the term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" refers to cases where the alkyl can be substituted as well as cases where the alkyl is not substituted.

[0266] It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to result in chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art, as well as the methods described below. When a substituent is itself substituted with multiple groups, it is understood that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0267] As used herein, the term "optionally substituted" refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of one to four hydrogen radicals in a given structure with the above substituents. More preferably, one to three hydrogen radicals are replaced by the above substituents. It is understood that the substituents can be further substituted.

[0268] The term "conjugate" as used herein refers to a cell-binding agent that is covalently attached to one or more molecules of a cytotoxic compound. In this regard, the "cell-binding agent" is a molecule that has affinity for a biological target, and may be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and the binding agent functions to direct the biologically active compound to the biological target. In certain embodiments, the conjugate may be designed to target tumor cells through a cell surface antigen. The antigen may be a cell surface antigen that is overexpressed or expressed in abnormal cell types. In particular, the target antigen may be expressed only on proliferative cells (e.g., tumor cells). The target antigen may be selected based on its differential expression between proliferative and normal tissues in general. In the present disclosure, the antibody is attached to a linker.

[0269] In this disclosure, a "variant" of a polypeptide, e.g., an antigen-binding fragment, protein, or antibody, is a polypeptide that has insertions, deletions, additions, and / or substitutions of one or more amino acid residues compared to another polypeptide sequence, including fusion polypeptides. Protein variants also include those that have been modified by proteolytic enzymatic cleavage, phosphorylation, or other post-translational modifications, but that retain the biological activity, such as binding and specificity for ROR1, of the antibodies disclosed herein. A variant may have about 99% identity, about 98% identity, about 97% identity, about 96% identity, about 95% identity, about 94% identity, about 93% identity, about 92% identity, about 91% identity, about 90% identity, about 89% identity, about 88% identity, about 87% identity, about 86% identity, about 85% identity, about 84% identity, about 83% identity, about 82% identity, about 81% identity, or about 80% identity to the sequence of an antibody or antigen-binding fragment thereof according to the present disclosure. Percent identity (%) or homology can be calculated by methods known in the art.

[0270] In certain embodiments, the percent homology or identity can be calculated by 100×[(same position) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in the compared sequences A and B (Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0271] In the present disclosure, conservative amino acid substitution refers to a substitution that does not substantially affect the activity or antigenicity of a polypeptide. A polypeptide may contain one or more conservative substitutions. Non-limiting examples of which are shown in Table 3 below.

[0272] As used herein, the term "derivative" of a polypeptide refers to a polypeptide having a chemical modification at one or more residues through conjugation with other chemical moieties that differs from an insertion, deletion, addition, or substitution variant.

[0273] The term "naturally-occurring" as used herein with respect to polypeptides, nucleic acids, host cells, and the like, refers to materials that occur in nature.

[0274] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that need to be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps are not counted in the target sequence, since gaps are neither nucleotides nor amino acids. Similarly, gaps are not counted in the reference sequence, since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions where an identical amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Comparison of sequences and determination of the percent sequence identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from a variety of sources and are for alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website (on the World Wide Web at blast.ncbi.nlm.nih.gov). Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences and BLASTP is used to compare amino acid sequences.Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher which are part of the EMBOSS suite of bioinformatics programs, also available from the European Bioinformatics Institute (EBI) on the world wide web at ebi.ac.uk / Tools / psa.

[0275] As used herein, "homology" with respect to a peptide, polypeptide, or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art that are required to achieve maximum alignment over the entire length of the sequences being compared.

[0276] In the present disclosure, "affinity" is the strength of interaction between an antibody or an antigen-binding fragment thereof and an antigen, and is determined by the characteristics of the antigen, such as the size, shape, and / or charge of the antigen, and the CDR sequence of the antibody or an antigen-binding fragment thereof. Methods for determining affinity are known in the art, and the following may be used as a reference.

[0277] The antibody or antigen-binding fragment thereof has a dissociation constant (KD) of ≦10 -6 M. An antibody is said to "specifically bind" to its target, such as an antigen, if it has a KD of ≦1×10 -8 If M, then it specifically binds to the target with "high affinity."

[0278] As used in this disclosure, an "antigen-binding fragment" of an antibody or immunoglobulin chain (heavy or light chain) includes a portion of an antibody that lacks some amino acids compared to the full-length chain, but is capable of specifically binding to an antigen. The fragment can be considered biologically active in that it can specifically bind to a target antigen or can compete with other antibodies or antigen-binding fragments thereof to bind to a particular epitope. In certain embodiments, such a fragment includes at least one CDR present in a full-length light or heavy chain, and in some embodiments, a shortened heavy and / or light chain, or a portion thereof. The biologically active fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFv, scFv-Fc, diabodies, minibodies, These include, but are not limited to, cabs, and dAbs, and may be from any mammal, including but not limited to, human, mouse, rat, camelid, or rabbit. Functional portions of antibodies, such as one or more CDRs disclosed in this disclosure, may be covalently linked to a secondary protein or small compound, and thereby used as targeted therapeutics against specific targets.

[0279] In the present disclosure, the "Fc" region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, which are linked to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0280] In this disclosure, a "Fab fragment" consists of one light chain and one heavy chain that contains only the variable region and CH1. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. In an scFab, the Fabs of two molecules are linked by a flexible linker.

[0281] In this disclosure, a "Fab' fragment" includes the region between the CH1 and CH2 domains of the heavy chain in addition to the Fab fragment. Disulfide bonds can form between the two heavy chains of the Fab' fragment of the two molecules to form a F(ab')2 molecule.

[0282] In the present disclosure, the above-mentioned "F(ab')2 fragment" comprises two light chains and two heavy chains including a variable region CH1 and a part of the constant region between the CH1 domain and the CH2 domain, and an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are joined to each other by disulfide bonds between them.

[0283] In the present disclosure, an "Fv region" is a fragment of an antibody that contains the variable regions of the heavy and light chains but does not contain the constant regions. In sdFVs, the heavy and light chains are linked by disulfide bonds. In scFc, the Fv is linked by a flexible linker. In scFv-Fc, the Fc is linked to the scFV. In minibodies, the CH3 is linked to the scFV. Diabodies contain two molecules of scFV.

[0284] In the present disclosure, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In addition, the Fv polypeptide may contain a polypeptide linker between the Vh and VL domains that enables the scFv to form a target structure for antigen binding.

[0285] In the present disclosure, a "single-chain antibody (cab)" is a single polypeptide chain comprising one of the heavy or light chain constant regions, in which the heavy and light chain variable regions are linked by a flexible linker. For single-chain antibodies, see U.S. Patent No. 5,260,203, which is herein disclosed by reference.

[0286] In the present disclosure, a "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In certain embodiments, two or more VH regions are covalently linked via a peptide linker to form a bivalent domain antibody. The two VH regions of this bivalent domain antibody may target the same or different antigens.

[0287] In the present disclosure, "complementarity determining region" (CDR; (i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable domain of an antibody that are necessary for binding to an antigen. Each variable domain typically has three CDR domains identified as CDR1, CDR2, and CDR3.

[0288] In this disclosure, "framework regions" (FRs) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4.

[0289] In the present disclosure, a "bivalent antigen-binding protein" or a "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites comprised in a bivalent antibody may have the same antigen specificity, or the antigen-binding sites may be bispecific antibodies that bind to different antigens.

[0290] In the present disclosure, a "multispecific antigen binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0291] In this disclosure, "linker" refers to a compound that covalently attaches a cytotoxic compound to an antibody.

[0292] In this disclosure, "unsubstituted or substituted" is used to refer to a parent group which may be unsubstituted or substituted, and "substituted" refers to a parent group having at least one substituent, where a substituent refers to a chemical moiety covalently bonded or fused to the parent group.

[0293] In this disclosure, "halo" refers to fluorine, chlorine, bromine, iodine, and the like.

[0294] In this disclosure, "alkyl" is a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound. As used herein, the term "alkyl" refers to any group having a C1-C 10 Straight chain alkyl group or C1-C 10 It refers to saturated aliphatic groups including, but not limited to, branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. An "alkyl" group can be optionally substituted. Furthermore, the term "alkyl" refers to saturated aliphatic groups including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In a preferred embodiment, the straight or branched chain alkyl has 30 or less aryl groups in its backbone (e.g., C 1-30 , C for branched chains 3-30 ), more preferably having 20 or fewer carbon atoms. Examples of saturated alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, etc., examples of saturated linear alkyl include methyl, ethyl, n-propyl, n-pentyl (amyl), n-hexyl, n-heptyl, etc., examples of saturated branched cyclic alkyl include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, etc.

[0295] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0296] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0297] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0298] "C x~y " or "C x ~C y The term "alkyl" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. CO alkyl indicates a hydrogen in the group's terminal position, and if internal, indicates a bond. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0299] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0300] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0301] As used herein, the term "amide" refers to the group:

[0302] [ka] In the formula, R 9 and R 10 each independently represents a hydrogen or a hydrocarbyl group, or R 9 and R10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0303] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, salts thereof, such as the moiety that can be represented by:

[0304] [ka] In the formula, R 9 , R 10 , and R 10‘ each independently represents a hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0305] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0306] The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group.

[0307] The term "alkoxy" as used herein refers to -OR, where R is an alkyl group, examples of which include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, and the like.

[0308] The term "alkenyl" as used herein refers to an alkyl having at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CHCH3), 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl.

[0309] In this disclosure, the term "alkynyl" as used herein refers to an alkyl group having at least one carbon-carbon triple bond, and examples of unsaturated alkynyl groups can include ethynyl and 2-propynyl.

[0310] The term "carboxy" as used herein refers to -C(=O)OH.

[0311] The term "formyl" as used herein refers to -C(=O)H.

[0312] As used herein, the term "aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. For example, "C 5-7 The term "aryl" refers to a moiety having 5 to 7 ring atoms that is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and is defined as "C 5-10 The term "aryl" refers to a moiety having 5 to 10 ring atoms that is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. In this regard, the prefix (C 5-7 , C 5-10 ) refers to the number of ring atoms or range of numbers of ring atoms, whether they are carbon atoms or heteroatoms. For example, "C 5-6 The term "aryl" refers to an aryl group having 5 or 6 ring atoms. In this regard, the ring atoms may all be carbon atoms as in "carboaryl groups". Examples of carboaryl groups include, but are not limited to, those derived from benzene, naphthalene, azulene, anthracene, phenanthrene, naphthacene, and pyrene. Examples of aryl groups containing fused rings, at least one of which is aromatic, include, but are not limited to, those derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene. Alternatively, the ring atoms may contain one or more heteroatoms as in "heteroaryl groups".

[0313] The term "carbamate" is art-recognized and refers to a group

[0314] [ka] In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0315] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0316] The term "carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle can be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or more than two atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle can be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, can be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle, as long as valences permit. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any one or more positions capable of retaining a hydrogen atom.

[0317] The term "carbonate" is art-recognized and refers to a group -OCO 2- Refers to...

[0318] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0319] The term "cycloalkyl" as used herein refers to an alkyl group that is a cyclyl group, and refers to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon compound. Examples of cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane, and methylcyclohexane; or unsaturated monocyclic hydrocarbon compounds such as cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene, and methylcyclohexene; and saturated heterocyclic hydrocarbon compounds such as norcarane, norphenene, and norbornene.

[0320] As used herein, the term "ester" refers to an ester selected from the group -C(O)OR 9 In the formula, R 9 represents a hydrocarbyl group.

[0321] The term "ether" as used herein refers to a hydrocarbyl group bonded to another hydrocarbyl group via oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0322] The term "halo" or "halogen" as used herein refers to fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I).

[0323] The terms "hetarylalkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0324] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0325] The term "heterocyclic alkyl," as used herein, refers to an alkyl group substituted with a heterocyclic group.

[0326] The term "heteroaryl" as used herein refers to an aryl containing one or more heteroatoms, examples of which include pyridine, pyrimidine, benzothiophene, furyl, dioxolanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, and pyrimidinyl. More specifically, examples thereof include benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, benzodioxole, benzofuran, benzotriazole, benzothiofuran, benzothiazole, C9 having two condensed rings derived from benzothiazole, chromene, iso-chromene, chroman, iso-chroman, benzodioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, C10 having two condensed rings derived from pteridine, 10 , C with two condensed rings derived from benzodiazepines 11 C, which has three fused rings derived from carbazole, dibenzofuran, dibenzothiophene, carboline, pyrimidine, and pyridoindole 13, acridine, xanthene, thioxanthene, oxanthrene, phenoxathiin, phenazine, phenoxazine, phenothiazine, thianthrene, phenanthridine, phenanthroline, and C with three fused rings derived from phenazine 14 Examples include:

[0327] The term "heterocyclyl" as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound. Additionally, the term "heterocyclyl" or "heterocycle" as used herein refers to a monosaturated or partially unsaturated non-aromatic compound or non-aromatic polycyclic ring system containing at least one heteroatom (i.e., at least one cyclic heteroatom selected from oxygen, nitrogen, and sulfur) in the ring. Unless otherwise specified, a heterocyclyl group has from 5 to about 20 ring atoms, such as from 5 to 10 ring atoms, such as from 3 to 12 ring atoms. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring) having about 1 to 6 cyclic carbon atoms in the ring and about 1 to 3 cyclic heteroatoms selected from oxygen, nitrogen, and sulfur. The rings of multiple fused ring systems may be connected to each other through fused, spiro, and bridged bonds, so long as valence requirements are met. Examples of heterocycles include azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidinone, tetrahydrofuran, tetrahydrothiophene, dihydropyridine, tetrahydropyridine, quinuclidine, N-bromopyrrolidine, N-chloropiperidine, and the like.

[0328] The term "hydrocarbyl" as used herein refers to a group that is bonded through a carbon atom that does not have =O or =S substituents, typically has at least one carbon-hydrogen bond and a predominantly carbon backbone, but can optionally include heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has =O substituent on the bonded carbon) and ethoxy (which is bonded through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0329] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0330] The term "lower" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer atoms, preferably 6 or fewer atoms, in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are, for example, hydroxyalkyl and aralkyl in reference (where, for example, atoms in an aryl group are not counted when counting the carbon atoms of an alkyl substituent), respectively, lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents.

[0331] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings (e.g., the rings are "fused rings"). Each ring of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains from 3 to 10 atoms, preferably from 5 to 7 atoms, in the ring.

[0332] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharma- ceutically acceptable salt thereof.

[0333] The term "sulfonamide" is art recognized and refers to a group that can be represented by the general formula:

[0334] [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0335] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0336] The term "sulfonate" is art-recognized and refers to the group --SO.sub.3H, or a pharma- ceutically acceptable salt thereof.

[0337] The term "sulfone" is art-recognized and refers to the group -S(O)2-.

[0338] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the replacing atom and substituent, as well as the implicit proviso that the substitution results in a stable compound that does not spontaneously undergo transformation, such as, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain may themselves be substituted, if appropriate.

[0339] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0340] As used herein, the term "thioester" refers to the group -C(O)SR 9 Or -SC(O)R 9 In the formula, R 9 represents a hydrocarbyl.

[0341] The term "thioether" as used herein is equivalent to an ether where the oxygen is replaced with a sulfur.

[0342] The term "urea" is art-recognized and can be represented by the general formula:

[0343] [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0344] As used herein, the term "modulate" includes the inhibition or suppression of a function or activity (such as cell proliferation), as well as the enhancement of a function or activity.

[0345] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may exist in the R or S configuration, and the R and S designations are used according to the rules set forth in Pure Appl.Chem.(1976),45,11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs, or mixtures thereof, including all possible mixtures of stereoisomers. See, for example, WO 01 / 062726.

[0346] Furthermore, certain compounds containing alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers, and in each case the present disclosure includes both mixtures and the separate individual isomers.

[0347] "Prodrug" or "Pharmaceutically acceptable prodrug" refers to a compound that is metabolized in the host after administration, e.g., hydrolyzed or oxidized, to form a compound of the present disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on the functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to generate the active compound. Examples of prodrugs that use esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to generate a compound of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs”, Ed. H. Bundgaard, Elsevier, 1985.

[0348] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is useful in formulating a drug for medicinal or therapeutic use.

[0349] As used herein, the term "logarithm of solubility," "LogS," or "logS" is used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution properties. Low solubility often leads to poor absorption. The LogS value is the unit stripped logarithm (decimal) of solubility measured in mol / liter.

[0350] In this disclosure, the prefixes (e.g., C 1-12 , C 3-8etc.) refers to the number of ring atoms or range of numbers of ring atoms, whether they are carbon atoms or heteroatoms. For example, as used herein, "C 3-6 The term "heterocyclyl" refers to a heterocyclyl group having 3 to 6 ring atoms.

[0351] The term "prodrug", as used herein, refers to a compound that can be converted under physiological conditions in vivo (e.g., enzymatic oxidation, reduction and / or hydrolysis), directly or indirectly, into a pyrrolobenzodiazepine drug by the action of enzymes or gastric acid.

[0352] In the present disclosure, a "pharmaceutically acceptable salt" may be an acid addition salt formed with a pharmaceutically acceptable free acid, and an organic acid or an inorganic acid may be used as the free acid.

[0353] Examples of organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, meta-sulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid, and examples of inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0354] For example, if the compound has a functional group that can be anionic or inionic (e.g., -COOH can be -COO-), the compound can form a salt with a suitable cation. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3+ , and NR4 + ), but are not limited to these.

[0355] Some examples of suitable substituted ammonium ions are: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. Typical examples of quaternary ammonium ions include N(CH3)4 + Examples include:

[0356] The compound is or can be a cation (e.g., -NH2 becomes -NH3 + If the compound has a functional group that may be 0, 1 or 2, the compound may form a salt with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0357] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetioxybenzoic benzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, gluteptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, palmic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid and carboxymethyl cellulose.

[0358] The term "solvate," as used herein, refers to a molecular complex between a compound according to the present disclosure and a solvent molecule, examples of which include, but are not limited to, a compound according to the present disclosure bound to water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethyl acetate, acetic acid, ethanolamine, or mixtures thereof.

[0359] It may be convenient or desirable to prepare, purify, and / or handle an equivalent solvate of the active compound.The term "solvate" is used herein in its conventional sense to refer to a complex of a solute (e.g., an active compound or its salt) and a solvent.When the solvent is water, the solvate may be conveniently referred to as a hydrate, such as a monohydrate, a dihydrate, or a trihydrate.

[0360] The phrase "effective amount" or "therapeutically effective amount" as used herein refers to the amount required to achieve the desired therapeutic result (for dosage, and duration and means of administration). An effective dose is the minimum amount of activator required to provide a minimal therapeutic benefit to a subject, and is less than a toxic dose. For example, dosages may range from about 100 ng to about 100 mg / kg per patient, more typically from about 1 μg / kg to about 10 mg / kg. Where the active compound is a salt, ester, amide, prodrug, or the like, dosages are calculated based on that of the parent compound, and therefore the actual weight used will be increased proportionately. Pyrrolobenzodiazepine compounds according to the present disclosure may be formulated to contain 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of active ingredient per unit dosage form, but the present disclosure is not limited thereto. The active ingredient may be administered to obtain a peak plasma concentration of the active compound of about 0.05 μM to about 100 μM, about 1 μM to about 50 μM, or about 5 μM to about 30 μM. For example, a 0.1% to 5% w / v solution of the active ingredient in saline may be administered via intravenous injection.

[0361] The concentration of the active compound in the pharmaceutical composition can be determined by the absorption, inactivation, and excretion rate of the drug, as well as other factors known to those skilled in the art. The dosage can vary depending on the severity of the symptoms / disease. In addition, the dosage and method of administration for a particular patient can be adjusted according to the professional judgment of the person supervising administration, taking into account the severity of the patient's symptoms / disease, needs, age, drug response, etc., and the concentration ranges described herein are only examples and are not intended to limit the embodiments of the claimed compositions. In addition, the active ingredient can be administered once, or a much smaller dosage of the active ingredient can be administered in multiple doses.

[0362] The term "linker" as used herein refers to a compound that covalently attaches a cytotoxic compound to an antibody.

[0363] The terms "intracellularly cleaved" and "intracellular cleavage" as used herein refer to a metabolic process or reaction inside the cell to an antibody construct-active agent conjugate, whereby the covalent attachment, e.g., linker, between the active agent (B) and the antibody construct (Ab) is broken, thus causing the free drug or other metabolic product of the conjugate to dissociate from the antibody on the inside of the cell. EXAMPLES

[0364] The invention will now be generally described, which will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0365] Example 1: Generation of antibodies specific to human TROP2 (Tumor-associated calcium signaling factor 2, TACSTD2) Anti-TROP2 humanized antibodies 2G10-5 and 2G10-6, which bind more strongly and specifically to TROP2 (tumor-associated calcium signaling 2, TACSTD2), which is mainly expressed in tumors, were produced by the method described in U.S. Patent Application No. 15 / 532598. Additionally, the amino acid sequences of the antibodies are shown in Tables 1 and 2 below.

[0366] [Table 1]

[0367] [Table 2]

[0368] In addition, for ADC synthesis, an antibody clone 2G10-5-CaaX was constructed by condensing the CaaX peptide moiety (GGGGGGGCVIM; (SEQ ID NO:22) to the C-terminus of the light chain of antibody 2G10-5 (SEQ ID NO:18), and the antibody was produced through CHO cell-based transient expression. The produced antibody 2G10-5-CaaX was used for ADC synthesis. The amino acid sequence of the antibody is shown in Table 3.

[0369] [Table 3]

[0370] Example 2: Synthetic Preparation of Compounds 1, 2, 3, and 4 <2-1> Preparation of compound 1

[0371] [ka]

[0372] Compound 1 was prepared using the method described in Korean Patent Application No. 10-2018-7018068.

[0373] The structure of compound 1, monomethyl auristatin E (MMAE), is as follows:

[0374] [ka]

[0375] <2-2> Preparation of compound 2

[0376] [ka]

[0377] Compound 2 was prepared using the method described in Korean Patent Application No. 10-2018-0036895.

[0378] <2-3>Preparation of Compound 3

[0379]

Chem.

[0380] Compound 3 was prepared using the method described in Korean Patent Application No. 10-2020-0188314.

[0381] <2-4>Preparation of Compound 4

[0382]

Chem.

[0383] Compound 4 was prepared using the method described in Korean Patent Application No. 10-2018-0036895.

[0384] Example 3: Synthesis and Production of ADC The ADC was prepared through the following two steps. The commonly used LCB14-0606 and LCB14-0512 were prepared using the method described in Korean Patent Application No. 10-2013-7032628. The structural formulas of LCB14-0606 and LCB14-0512 are as follows:

[0385]

Chem.

[0386] Step 1: Production of Prenylated Antibody <Production of Prenylated Antibody Using LCB14-0606> The prenylation reaction mixture of antibody 2G10-5-CaaX of Example 1 was prepared, and the reaction was carried out at 30 °C for 16 hours. The reaction mixture consisted of a buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, and 0.5 mM DTT) containing 24 μM of the antibody, 600 nM of FTase (Genscript), and 144 μM of LCB14-0606. After the reaction was completed, the prenylated antibody was desalted using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.

[0387] <Production of prenylated antibody using LCB14-0512> The prenylation reaction mixture of antibody 2G10-5-CaaX of Example 1 was prepared, and the reaction was carried out at 30 °C for 2 hours. The reaction mixture consisted of a buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, and 0.1 mM DTT) containing 24 μM of the antibody, 600 nM of FTase (Genscript), and 200 μM of LCB14-0512. After the reaction was completed, the prenylated antibody was desalted using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.

[0388] Step 2: Drug conjugation method <Conjugation by oxime bond formation> The mixture for the oxime bond formation reaction between the prenylated antibody and the linker-drug was prepared by mixing 100 μM of Na-acetate buffer pH 5.2, 10% DMSO, 24 μM of the prenylated antibody, and 240 μM of the linker-drug (self-made, compound 1, 2, or 3 of Example 2), and gently stirred at 30 °C. After reacting for 6 hours or 24 hours, an FLPC (AKTA purifier, GE healthcare) process was performed to remove the excess small compound used, and the protein fraction was collected and concentrated.

[0389] <Conjugation by copper-free click reaction> For the production of ADCs using copper-free click coupling reaction between prenylated antibodies and linker-drugs, a reaction mixture was prepared by mixing PBS buffer (pH 7.4), 1% DMSO, 10 μM prenylated antibodies, and 100 μM linker-drug (self-produced, compound 4 in Example 2) and reacting at 25° C. for 6 hours, after which the excess small molecule compounds used were removed by FPLC (AKTA purifier, GE healthcare) or G25 Sepharose column process, and the protein fractions were collected and concentrated.

[0390] The ADCs produced through the above two steps are shown in Table 4 below and in Figures 1 to 4.

[0391] [Table 4]

[0392] <Experimental Example 1> In vitro cytotoxicity evaluation The cell proliferation inhibitory activity of the ADC produced according to Example 2 and the drug contained in the ADC alone against cancer cell lines was measured.

[0393] Specifically, commercially available human pancreatic cancer cell lines (BxPC-3, Capan-1, and PATU-8988s), breast cancer cell lines (JIMT-1 and MDA-MB-468), gastric cancer cell lines (NCI-N87, SNU-601, and SNU-620), colon cancer cell lines (HCT15, HT29, DLD-1, SW480, and COLO205), ovarian cancer cell lines (OVCAR-3), prostate cancer cell lines (PC-3 and LNCaP), and non-small cell lung cancer cell lines (NCI-H1781, HCC827, Calu-1, and Calu-3) were used as cancer cell lines. MMAE was used as the sole drug contained in the ADC prepared according to Example 3, and SG2057 (CAS#: 260417-62-7, MedKoo Biosciences) was used as the PBD dimer. Each cancer cell line was seeded at 500 cells / well to 5,000 cells / well in 96-well plates, cultured for 24 hours, and then treated with ADC1, ADC2, and ADC3 produced according to Example 3, and the drugs MMAE and SG2057 alone, each at concentrations from 0.256 pM to 100 nM (5-fold serial dilutions). After 144 hours, viable cell numbers were quantified using sulforhodamine B (SRB) dye or Cell titer glo.

[0394] As a result, as shown in Tables 5 to 7, it was confirmed that the pyrrolobenzodiazepine antibody-drug conjugates (ADC2 and ADC3) exhibited significantly stronger cytotoxicity in most cancer cell lines compared to the auristatin antibody-drug conjugate (ADC1).

[0395] [Table 5]

[0396] [Table 6]

[0397] [Table 7]

[0398] <Experimental Example 2> Analysis of tumor growth inhibition effect in vivo The tumor growth inhibitory potency of the ADCs produced according to Example 3 was analyzed in a tumor-implanted mouse model.

[0399] Specifically, pancreatic cancer cell lines (BxPC-3 and Capan-1), breast cancer cell lines (MDA-MB-468), gastric cancer cell lines (NCI-N87), colon cancer cell lines (HT29), and non-small cell lung cancer cell lines (HCC827 and NCI-H2170) were cultured at cell numbers of 1 × 10 6 ~7×10 6 The cultured cells were suspended in 100 μl of PBS, mixed with 100 μl of Matrigel, and then grafted into Balb / c nude mice so that the tumor size was 100 mm 3 ~200mm 3 When the tumor size reached 100 mg / kg / day, each of the ADCs produced according to Example 3 was administered intravenously according to the dosing regimen shown in Tables 8 to 10 below. The size of the tumor implanted in the mice was measured immediately before the first administration (Day 1), and then periodically for 56 days. In the control cases, Trodelvy (sacituzumab govitecan-hziy) or DS-1062BS (datopotamab deruxtecan) was administered.

[0400] [Table 8]

[0401] [Table 9]

[0402] [Table 10]

[0403] As a result, as shown in Figures 5A and 5B, when the auristatin antibody-drug conjugate (ADC1) using the oxime reaction was administered to a mouse model implanted with a pancreatic cancer cell line, both the 2 mg / kg and 4 mg / kg doses exhibited tumor growth inhibition efficacy in a dose-dependent manner compared to the control, as confirmed through the first experiment (Figure 5A). In a mouse model implanted with a breast cancer cell line, tumor growth inhibition efficacy was confirmed in both the group administered with the auristatin antibody-drug conjugate (ADC1) using the oxime reaction and the group administered with the pyrrolobenzodiazepine antibody-drug conjugate (ADC2 and ADC3).

[0404] Furthermore, as shown in Figures 6A to 6D, a second experiment confirmed that ADC1 had superior efficacy compared to the commercially available drug Trodelvy in all four cancer cell line transplanted mouse models (MDA-MB-468, BxPC-3, NCI-N87, and HCC827).

[0405] Furthermore, as shown in Figures 7A to 7E, it was confirmed through the third experiment that ADC1 had superior efficacy compared to the commercially available drug Trodelvy in all five cancer cell line transplant mouse models (BxPC-3, HCC827, Capan-1, NCI-H2170, and HT-29), and that ADC1 had efficacy equivalent to that of the competing drug DS-1062BS.

Claims

1. A conjugate represented by general formula I or a pharmaceutically acceptable salt thereof, [General formula I] A-[L-(B) ｌ ] ｍ During the ceremony, Ab is an anti-TROP2 (tumor-associated calcium signaling factor 2, also known as TACSTD2) antibody or its antigen-binding fragment, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:

6. The light chain variable region includes light chain CDR1 containing the amino acid sequence of SEQ ID NO: 9, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:

13. L is the linker, Each B independently contains an active agent. A conjugate or a pharmaceutically acceptable salt thereof, wherein l and m are each independently between 1 and 20.

2. Ab is a heavy chain variable region, The amino acid sequence of sequence number 15 or 20, A sequence having at least 90% sequence identity with the amino acid sequence of Sequence ID No. 15 or 20, while maintaining the heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 2, the heavy chain CDR2 containing the amino acid sequence of Sequence ID No. 4, and the heavy chain CDR3 containing the amino acid sequence of Sequence ID No. 6, or The conjugate according to claim 1, comprising a heavy chain variable region that includes a sequence having at least 95% sequence identity with respect to the amino acid sequence of sequence number 15 or 20, while maintaining the heavy chain CDR1 containing the amino acid sequence of sequence number 2, the heavy chain CDR2 containing the amino acid sequence of sequence number 4, and the heavy chain CDR3 containing the amino acid sequence of sequence number 6.

3. Ab is a light chain variable region, The amino acid sequence of sequence number 16, A sequence having at least 90% sequence identity with the amino acid sequence of Sequence ID No. 16, while maintaining the light chain CDR1 containing the amino acid sequence of Sequence ID No. 9, the light chain CDR2 containing the amino acid sequence of Sequence ID No. 11, and the light chain CDR3 containing the amino acid sequence of Sequence ID No. 13, or The conjugate according to claim 1, comprising a light chain variable region that includes a sequence having at least 95% sequence identity with respect to the amino acid sequence of sequence number 16, while maintaining the light chain CDR1 containing the amino acid sequence of sequence number 9, the light chain CDR2 containing the amino acid sequence of sequence number 11, and the light chain CDR3 containing the amino acid sequence of sequence number 13.

4. The conjugate according to claim 1, wherein Ab comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

16.

5. Ab is humanized, (i) A variable heavy chain framework region from the heavy chain of a human antibody or from the human consensus framework, wherein the variable heavy chain framework region includes one or more amino acid sequence changes: Y27F, T30S, V37L, M48I, G49A, I70L, and R72V, (ii) A variable light chain framework region from a human antibody light chain or from a human consensus framework, wherein the variable light chain framework region comprises a variable light chain framework region comprising an amino acid sequence change: Y49S, according to claim 1.

6. The conjugate according to claim 1, wherein Ab is selected from a monoclonal antibody, a single-chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single-chain variable fragment (scFv), a scFv-Fc fragment, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

7. The conjugate is represented by general formula IIa or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 During the ceremony, Each B' is an active agent independently. Each G independently comprises a glucuronic acid portion or 【Chemistry 2】 And, Each R ３ However, independently, it is a hydrogen or carboxyl protecting group, Each R ４ However, independently, they are hydrogen or hydroxyl protecting groups. R １ and R ２ However, each independently, hydrogen, C １－８ Alkyl, or C ３－８ It is a cycloalkyl, Each W is independently —C(O)—, —C(O)NR′—, —C(O)O—, —SO ２ NR′—, —P(O)R″NR′, —SONR′—, or —PONR′—, wherein said C, S, or P is directly bonded to the phenyl ring of Formula IIa, ２ NR′—, and R' and R'' are, independently, hydrogen and C. １－８ Alkyl, C ３－８ Cycloalkyl, C １－８ Alkoxy, C １－８ Alkylthio, mono-, or di-C １－８ Alkylamino, C ３－２０ Heteroaryl, or C ６－２０ It is Ariel, Each Z independently, C １－８ It is alkyl, halogen, cyano, or nitro, and n is 0, 1, 2, or 3. Each L acts independently, A) C １－５０ Alkylene or C １－５０ It contains heteroalkylenes, and (i) One or more unsaturated bonds, (ii) Heteroarylene, or (iii) at least one C １－２０ containing alkyl substituents, or B) comprising at least one isoprenyl group having a structure represented by general formula III, 【Transformation 3】 The conjugate according to claim 1, wherein l and m are each independently 1 to 20.

8. A conjugate represented by general formula IIa or a pharmaceutically acceptable salt thereof, 【Chemistry 4】 During the ceremony, Ab is an anti-TROP2 (tumor-associated calcium signaling factor 2, also known as TACSTD2) antibody or its antigen-binding fragment. Each B' is an active agent independently. Each G independently comprises a glucuronic acid portion or 【Transformation 5】 And, Each R ３ However, independently, it is a hydrogen or carboxyl protecting group, Each R ４ However, independently, they are hydrogen or hydroxyl protecting groups. R １ and R ２ However, each independently, hydrogen, C １－８ Alkyl, or C ３－８ It is a cycloalkyl, Each W independently corresponds to -C(O)-, -C(O)NR'-, -C(O)O-, and -SO ２ NR'-, -P(O)R''NR', -SONR'-, or -PO ２ It is NR'-, and the C, S, or P is directly bonded to the phenyl ring of formula IIa, R' and R'' are, independently, hydrogen and C. １－８ Alkyl, C ３－８ Cycloalkyl, C １－８ Alkoxy, C １－８ Alkylthio, mono-, or di-C １－８ Alkylamino, C ３－２０ Heteroaryl, or C ６－２０ It is Ariel, Each Z independently, C １－８ It is alkyl, halogen, cyano, or nitro, and n is 0, 1, 2, or 3. Each L acts independently, A) C １－５０ Alkylene or C １－５０ It contains heteroalkylenes, and (i) One or more unsaturated bonds, (ii) Heteroarylene, or (iii) at least one C １－２０ containing alkyl substituents, or B) comprising at least one isoprenyl group having a structure represented by general formula III, 【Transformation 6】 A conjugate or a pharmaceutically acceptable salt thereof, wherein l and m are each independently between 1 and 20.

9. Ab includes a heavy chain variable region and a light chain variable region, The heavy chain variable region includes heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:

6. The conjugate according to claim 8, wherein the light chain variable region includes a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO:

13.

10. Ab is a heavy chain variable region, The amino acid sequence of sequence number 15 or 20, A sequence having at least 90% sequence identity with the amino acid sequence of Sequence ID No. 15 or 20, while maintaining the heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 2, the heavy chain CDR2 containing the amino acid sequence of Sequence ID No. 4, and the heavy chain CDR3 containing the amino acid sequence of Sequence ID No. 6, or The conjugate according to claim 9, comprising a heavy chain variable region that includes a sequence having at least 95% sequence identity with the amino acid sequence of sequence number 15 or 20, while maintaining the heavy chain CDR1 containing the amino acid sequence of sequence number 2, the heavy chain CDR2 containing the amino acid sequence of sequence number 4, and the heavy chain CDR3 containing the amino acid sequence of sequence number 6.

11. Ab is a light chain variable region, The amino acid sequence of sequence number 16, A sequence having at least 90% sequence identity with the amino acid sequence of Sequence ID No. 16, while maintaining the light chain CDR1 containing the amino acid sequence of Sequence ID No. 9, the light chain CDR2 containing the amino acid sequence of Sequence ID No. 11, and the light chain CDR3 containing the amino acid sequence of Sequence ID No. 13, or The conjugate according to claim 9, comprising a light chain variable region that includes a sequence having at least 95% sequence identity with respect to the amino acid sequence of sequence number 16, while maintaining the light chain CDR1 containing the amino acid sequence of sequence number 9, the light chain CDR2 containing the amino acid sequence of sequence number 11, and the light chain CDR3 containing the amino acid sequence of sequence number 13.

12. The conjugate according to claim 9, wherein Ab comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

16.

13. Ab is humanized, and (i) A variable heavy chain framework region from the heavy chain of a human antibody or from the human consensus framework, wherein the variable heavy chain framework region includes one or more amino acid sequence changes: Y27F, T30S, V37L, M48I, G49A, I70L, and R72V, (ii) A variable light chain framework region from a human antibody light chain or from a human consensus framework, wherein the variable light chain framework region comprises a variable light chain framework region comprising an amino acid sequence change: Y49S, according to claim 9.

14. The conjugate according to claim 9, wherein Ab is selected from a monoclonal antibody, a single-chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single-chain variable fragment (scFv), a scFv-Fc fragment, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

15. Each G, 【Transformation 7】 And R3 and R4 are each hydrogen. The conjugate according to claim 7 or 8.

16. R １ and R ２ However, each is hydrogen, n is 0, Each W is -C(O)NR'-, where C is directly bonded to the phenyl ring of formula IIa, and R' is hydrogen, C １－８ Alkyl, C ３－８ Cycloalkyl, C １－８ Alkoxy, C １－８ Alkylthio, mono-, or di-C １－８ Alkylamino, C ３－２０ Heteroaryl, or C ６－２０ The conjugate according to claim 7 or 8, wherein NR' is bonded to L.

17. L is a nitrogen-containing heteroalkylene with 1 to 50 members. L contains two or more atoms of hydrophilic amino acids, The conjugate according to claim 7 or 8, wherein the nitrogen forms a peptide bond with the carbonyl of the hydrophilic amino acid.

18. The conjugate according to claim 7 or 8, wherein L is covalently bonded to Ab by a thioether bond, and the thioether bond contains a sulfur atom of the cysteine ​​of Ab.

19. Ab comprises an amino acid motif at the C-terminus of Ab that is recognizable by an isoprenoid transferase, The conjugate according to claim 18, wherein the thioether bond contains a sulfur atom of the cysteine ​​of the amino acid motif, and the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

20. (i) The amino acid motif has a CYYX sequence, in the formula, C is cysteine, Each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine. X is selected from glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine. (ii) The conjugate according to claim 19, wherein the amino acid motif has a CVIM (SEQ ID NO: 24) or CVLL sequence (SEQ ID NO: 25).

21. The conjugate according to claim 19, wherein at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

22. The conjugate according to claim 1 or 8, wherein Ab contains the amino acid sequence GGGGGGGGCVIM (SEQ ID NO: 22) at its C-terminus.

23. L is C １－５０ The conjugate according to claim 1 or 8, comprising a heteroalkylene.

24. The conjugate according to claim 1 or 8, wherein L contains an oxime.

25. L contains C １－５０ It is a heteroalkylene, The oxygen atom of the oxime is on the side of L that is bonded to W, and The carbon atoms of the oxime are on the side of L that is linked to Ab, or The carbon atoms of the oxime are on the side of L that is bonded to W, and The conjugate according to claim 7 or 8, wherein the oxygen atom of the oxime is on the side of L that is linked to Ab.

26. The conjugate according to claim 1 or 8, wherein L contains an oxime, and at least one isoprenyl unit covalently bonds the oxime to Ab.

27. L further includes a unit of connection represented by general formula VIII or general formula IX, [General formula VIII] -(CH) ２ ) ｒ (V(CH) ２ ) ｐ ) ｑ - [Chemical formula IX] -(CH ２ CH ２ X) ｗ - V is a single bond, -O-, -S-, -NR ２１ -, -C(O)NR ２２ -, -NR ２３ C(O)-, -NR ２４ SO ２ -, or -SO ２ NR ２５ - and X is -O-, C １－８ Alkylene, or -NR ２１ - and R ２１ ~R ２５ However, each independently, hydrogen, C １－６ Alkyl, C １－６ Alkyl C ６－２０ Aryl, or C １－６ Alkyl-C ３－２０ It is a heteroaryl, r is between 0 and 10, p is between 0 and 10, q is between 1 and 20, The conjugate according to claim 1 or 8, wherein w is 1 to 20.

28. q is between 1 and 10, r and p are each 1 or 2, The conjugate according to claim 27, wherein V is -O-.

29. X is -O-, The conjugate according to claim 27, wherein w is 1 to 10.

30. L, 【Transformation 8】 The conjugate according to claim 27, comprising, in the formula, n40 being 1 to 10.

31. The conjugate according to claim 27, wherein L contains an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to an active agent.

32. The conjugate according to claim 27, wherein L comprises a bonding unit formed by a reaction between an alkyne and an azide, or between an aldehyde or ketone group and a hydrazine or hydroxylamine.

33. L includes a bonding unit represented by the general formula IVa, IVb, IVC, IVd, or IVe, 【Chemistry 9】 During the ceremony, L １ However, single bond or C １－３０ It is alkylene, R １１ However, hydrogen or C １－１０ The conjugate according to claim 27, wherein it is alkyl.

34. L is branched, and i) A branch unit covalently coupled to Ab by a primary linker, ii) A first branch, wherein a first active agent is covalently coupled to the branch unit by a secondary linker and a cleaving group, and iii) The second branch, a) The second active agent is covalently coupled to the branched unit by a secondary linker and a cleaving group, or b) The conjugate according to claim 1 or 8, comprising a second branch in which a polyethylene glycol portion is covalently coupled to the branch unit.

35. The aforementioned branching unit is 【Chemistry 10】 It has a structure represented by the formula, in which, L ２ , L ３ , and L ４ However, each can be used independently, or combined or -C ｎ H ２ｎ - and n is between 1 and 30. G １ G ２ , and G ３ However, each is independent, and combined, 【Chemistry 11】 This represents, R ３０ However, hydrogen or C １－３０ It is alkyl, R ４０ is hydrogen or L ５ -COOR ６ and L ５ However, bond or -C ｎ’ H ２ｎ’ - and n' is between 1 and 10, R ６ is hydrogen or C １－３０ alkyl, the conjugate according to claim 34.

36. The conjugate according to claim 34, wherein at least two active agents are covalently coupled to the branch L.

37. The conjugate according to claim 36, wherein one, two, three, or four branches L are coupled to Ab.

38. The conjugate according to claim 34, wherein each L is coupled to one or more active agents.

39. The conjugate according to claim 34, wherein the branching unit contains lysine.

40. The aforementioned conjugate has the following structure: 【Chemistry 12】 or comprising a pharmaceutically acceptable salt thereof, in the formula, B' and B'' are each independently active agents. n1 to n3 are each independently between 0 and 30. The conjugate according to claim 1 or 8, wherein AA is an amino acid group.

41. The aforementioned conjugate has the following structure: 【Chemistry 13】 【Chemistry 14】 or containing pharmaceutically acceptable salts thereof, The conjugate according to claim 1 or 8, wherein B' and B'' are each independently an active agent, and m and n each independently refer to 0 to 30.

42. The conjugate according to claim 1 or 8, wherein the active agent is a chemotherapeutic agent or a toxin.

43. The conjugate according to claim 1 or 8, wherein the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

44. The aforementioned active agent, (a) Erlotinib, Bortezomib, Fulvestrant, Sunitinib, Letrozole, Imatinib Mesylate, PTK787 / ZK222584, Oxaliplatin, 5-Fluorouracil, Leucovorin, Rapamycin, Lapatinib, Ronafarnib, Sorafenib, Gefitinib, AG1478, AG1571, Thiotepa, Cyclophosphamide, Busulfan, Improsulfan, Piposulfan, Benzodepa, Carbocon, Metsuredepa, Uredepa, Ethyleneimine, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoramide, Trimethylolmelamine, Bratacin, Bratacinone, Camptothecin Topotecan, bryostatin, calistatin, adzeresin, carzeresin, bizeresin, cryptophycin 1, cryptophycin 8, duocalmycin, KW-2189, CB1-TM1, eloiterobin, pancratistatin, sarcodicin, spongistatin, chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, melphalan, nobembicin, fenestrine, prednimustine, trophosphamide, uracil mustard, carmustine, chlor Rozotocin, Fotemustine, Lomustine, Nimustine, Ranimnustine, Calichemycin, Calichemycin Gamma 1, Calichemycin Omega 1, Dynemycin, Dynemycin A, Clodronate, Esperamicin, Neocardinostatin chromophore, Akurasinomycin, Actinomycin, Antimycin, Azaserin, Bleomycin, Carabicin, Carninomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, 6-Diazo-5-Oxo-L-Norleucine, Doxorubicin, Morphorino - Doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptomygrin, streptozocin, tubercidine, ubenimex, dinostatin, zolbicin, denopterin, methotrexate, pteropterin,Trimethrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone, propionate, epithiostanol, mepiciostane, testactone, aminoglutethimide, mitotane, trilostane, folinic acid, acegraton, aldofol Sphamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, demecolsin, diaziquan, elfornithine, eriptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, ronidynin, mytansin, ansamitosin, mitogluzone, mitoxantrone, mopidammol, pentostatin, fenamet, pirarubicin, losoxantrone, 2-ethylhydride Radid, Procarbazine, Polysaccharide-K, Lazoxane, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadiquan, 2,2',2''-Trichlorotriethylamine, T-2 Toxin, Beraclin A, Loridine A, Anguidin, Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Arabinoside, Paclitaxel, Paclitaxel Albumin-Modified Nanoparticle Formulation, Docetax Cell, gemicitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornitine, retinolic acid, capecitabine, or pharmaceutically acceptable salts, solvates, or acids thereof; (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian duct inhibitors, mouse gonadotropin-related peptides, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoiesis Ethin, bone induction factor, interferon, interferon-α, interferon-β, interferon-γ, colony-stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factor, LIF, kit ligand, or combination thereof; (c) Diphtheria toxin, botulinum toxin, tetanus toxin, central migration toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolinobenzodiazepine, pyridinobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, lysine, AM toxin, auristatin, tubulcin, geldanamycin, meitansinoid, calicheamicin, SG2285, drastatin, drastatin analogs, cryptophycin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, engine antibodies, epotilon, toxoids, or combinations thereof; (d) affinity ligands, wherein the affinity ligand is a substrate, inhibitor, active agent, neurotransmitter, radioisotope, or combination thereof; (e) Radioactive labels, 32P, 35S, fluorescent dyes, high electron density reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules having sequences complementary to the target, or combinations thereof; (f) Immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, or combinations thereof; (g) Tamoxifen, raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, LY117018, onapristone, or toremifene; (h) 4(5)-imidazole, megestrol acetate, exemestane, or anastrozole; (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) Aromatase inhibitors; (k) Protein kinase inhibitors; (l) Lipid kinase inhibitors; (m) Antisense oligonucleotide; (n) Ribozyme; (o) vaccine; and (p) The conjugate according to claim 1 or 8, selected from anti-angiogenic agents.

45. The active agent is a pyrrolobenzodiazepine dimer. The N10 position of the pyrrolobenzodiazepine dimer is substituted with X, or the N'10 position is substituted with X', and X or X' links the pyrrolobenzodiazepine dimer to the linker. X and X' are each independently -C(O)O- ＊ or -C(O)- ＊ And, ＊ The conjugate according to claim 1 or 8, wherein the conjugate refers to the binding site between the pyrrolobenzodiazepine dimer and the linker.

46. The aforementioned active agent is represented by: 【Chemistry 15】 During the ceremony, The dotted line represents an optional double bond. R １ and R １ ’ However, each is independent of H, OH, =O, =CH ２ , CN, R ｍ , OR ｍ _=CH-R ｍ’ = C(R ｍ’ ) ２ O-SO ２ -R ｍ CO ２ R ｍ COR ｍ Selected from, Halo, and Jihalo, R ｍ’ However, R ｍ CO ２ R ｍ COR ｍ , CHO, CO ２ H and Halo are selected, R ｍ However, substitution or non-substitution C １－１２ Alkyl, substituted, or unsubstituted C ２－１２ Alkenyl, substituted, or unsubstituted C ２－１２ Alkynyl, substituted, or unsubstituted C ５－２０ Aryl, substituted, or unsubstituted C ３－６ Heteroaryl, substituted, or unsubstituted C ３－６ Selected from cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocyclyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and substituted or unsubstituted 5- to 7-membered heteroaryl, the C １－１２ Alkyl, C ２－１２ Alkenil, C ２－１２ Alkinyl, C ５－２０ Ariel, C ５－２０ Heteroaryl, C ３－６ When a cycloalkyl, 3-7 membered heterocyclyl, 3-7 membered heterocycloalkyl, or 5-7 membered heteroaryl is substituted, the C １－１２ Alkyl, C ２－１２ Alkenil, C ２－１２ Alkinyl, C ５－２０ Ariel, C ５－２０ Heteroaryl, C ３－６ Each hydrogen atom of a cycloalkyl, 3-7 membered heterocyclyl, 3-7 membered heterocycloalkyl, or 5-7 membered heteroaryl is independently methoxy, C １－１２ Alkyl, C ２－１２ Alkenil, C ２－１２ Alkinyl, C ５－２０ Ariel, C ５－２０ Heteroaryl, C ３－６ It may be substituted with a cycloalkyl, a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 5- to 7-membered heteroaryl. R ２ , R ３ , R ５ , R ２ ’ , R ３ ’ , and R ５ ’ However, H and R are independent of each other. ｍ OH, OR ｍ SH, SR ｍ NH ２ NHR ｍ , NR ｍ R ｍ’ NO ２ Me ３ Selected from Sn and Halo, R ４ and R ４ ’ However, H and R are independent of each other. ｍ OH, OR ｍ SH, SR ｍ NH ２ NHR ｍ , NR ｍ R ｍ’ NO ２ Me ３ Sn, halo, substituted or unsubstituted C １－６ Alkyl, substituted, or unsubstituted C １－６ alkoxy, substituted, or unsubstituted C ２－６ Alkenyl, substituted, or unsubstituted C ２－６ Alkynyl, substituted, or unsubstituted C ３－６ Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C ５－１２ Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -CN, -NCO, -OR ｎ , -OC(O)R ｎ , -OC(O)NR ｎ R ｎ’ , -OS(O)R ｎ , -OS(O) ２ R ｎ , -SR ｎ , -S(O)R ｎ , -S(O) ２ R ｎ , -S(O)NR ｎ R ｎ’ , -S(O) ２ NR ｎ R ｎ’ , -OS(O)NR ｎ R ｎ’ , -OS(O) ２ NR ｎ R ｎ’ , -NR ｎ R ｎ’ , -NR ｎ C(O)R ｏ , -NR ｎ C(O)OR ｏ , -NR ｎ C(O)NR ｏ R ｏ’ , -NR ｎ S(O)R ｏ , -NR ｎ S(O) ２ R ｏ , -NR ｎ S(O)NR ｏ R ｏ’ , -NR ｎ S(O) ２ NR ｏ R ｏ’ , -C(O)R ｎ , -C(O)OR ｎ , and -C(O)NR ｎ R ｎ’ Selected from, the C １－６ Alkyl, C １－６ Alkoxy, C ２－６ Alkenil, C ２－６ Alkinyl, C ３－６ Cycloalkyl, 3-7 member heterocycloalkyl, C ５－１２ The hydrogen atoms of aryl and 5- to 7-membered heteroaryl groups are each independently C １－６ Alkyl, C １－６ Alkoxy, C ２－６ Alkenil, C ２－６ Alkinyl, C ３－６ Cycloalkyl, 3-7 member heterocycloalkyl, C ５－１２ Aryl, 5-7 member heteroaryl, -OR ｐ , -OC(O)R ｐ , -OC(O)NR ｐ R ｐ’ , -OS(O)R ｐ , -OS(O) ２ R ｐ , -SR ｐ , -S(O)R ｐ , -S(O) ２ R ｐ , -S(O)NR ｐ R ｐ’ , -S(O) ２ NR ｐ R ｐ’ , -OS(O)NR ｐ R ｐ’ , -OS(O) ２ NR ｐ R ｐ’ , -NR ｐ R ｐ’ , -NR ｐ C(O)R ｑ , -NR ｐ C(O)OR ｑ , -NR ｐ C(O)NR ｑ H, -NR ｐ S(O)R ｑ , -NR ｐ S(O) ２ R ｑ , -NR ｐ S(O)NR ｑ H, -NR ｐ S(O) ２ NR ｑ H, -C(O)R ｐ , -C(O)OR ｐ , or -C(O)NR ｐ R ｐ It can be replaced by, R ｎ , R ｎ’ , R ｏ , R ｏ’ R ｐ , R ｐ’ , and R ｑ However, H and C are independent of each other. １－７ Alkyl, C ２－７ Alkenil, C ２－７ Alkinyl, C ３－１３ Cycloalkyl, 3-7 member heterocycloalkyl, C ６－１０ Selected from aryls and 5- to 7-membered heteroaryls, X is -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR-, -S(O) ２ NR-, -P(O)R'NR-, -S(O)NR-, and -PO ２ Selected from NR-, Xa is bonded, substituted, or unsubstituted C. １－６ It is alkylene, and the C １－６ If alkylene is substituted, C １－８ Alkyl, or C ３－８ Substituted with cycloalkyl groups, R and R' are each independently H, OH, substituted or unsubstituted C １－８ The C represents alkyl, １－８ If alkyl is substituted, OH, N ３ , CN, NO ２ SH, NH ２ , ONH ２ NHNH ２ Hello, C １－６ Alkyl, C １－６ Alkoxy and C ６－１２ Substituted with a substituent selected from aryls, Y and Y' are each independently selected from O, S, and N(H), R ６ However, saturated or unsaturated C, whether substituted or unsubstituted. ３－１２ A hydrocarbon chain, wherein the chain may be interrupted by one or more heteroatoms, NMe, or substituted or unsubstituted aromatic rings, and the chain or aromatic ring has -NH, -NR at any one or more positions of hydrogen atoms on the chain or aromatic ring. ｍ ,-NHC(O)R ｍ , -NHC(O)CH ２ - [OCH ２ CH ２ ] ｎ -R, or -[CH ２ CH ２ O] ｎ - It can be substituted with R, or it is not substituted, and n is between 1 and 12. R ７ and R ７ ’ However, each independently, H, substituted or unsubstituted C １－６ Alkyl, substituted, or unsubstituted C ２－６ Alkenyl, substituted, or unsubstituted C ２－６ Alkynyl, substituted or unsubstituted C ３－６ Cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C ６－１０ Aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -OR ｒ , -OC(O)R ｒ , -OC(O)NR ｒ R ｒ’ , -OS(O)R ｒ , -OS(O) ２ R ｒ , -SR ｒ , -S(O)R ｒ , -S(O) ２ R ｒ , -S(O)NR ｒ R ｒ’ , -S(O) ２ NR ｒ R ｒ’ , -OS(O)NR ｒ R ｒ’ , -OS(O) ２ NR ｒ R ｒ’ , -NR ｒ R ｒ’ , -NR ｒ C(O)R ｓ , -NR ｒ C(O)OR ｓ , -NR ｒ C(O)NR ｓ R ｓ’ , -NR ｒ S(O)R ｓ , -NR ｒ S(O) ２ R ｓ , -NR ｒ S(O)NR ｓ R ｓ’ , -NR ｒ S(O) ２ NR ｓ R ｓ , -C(O)R ｒ , -C(O)OR ｓ , or -C(O)NR ｒ R ｒ’ And the above C １－６ Alkyl, C ２－６ Alkenil, C ２－６ Alkinyl, C ３－６ Cycloalkyl, 3-7 member heterocycloalkyl, C ６－１０ The hydrogen atoms of aryl and 5- to 7-membered heteroaryl groups are each independently C １－６ Alkyl, C ２－６ Alkenil, C ２－６ Alkinyl, C ３－６ Cycloalkyl, 3-7 member heterocycloalkyl, C ６－１０ Aryl, 5-7 member heteroaryl, -OR ｔ , -OC(O)R ｔ , -OC(O)NR ｔ R ｔ’ , -OS(O)R ｔ , -OS(O) ２ R ｔ , -SR ｔ , -S(O)R ｔ , -S(O) ２ R ｔ , -S(O)NR ｔ R ｔ’ , -S(O) ２ NR ｔ R ｔ’ , -OS(O)NR ｔ R ｔ’ , -OS(O) ２ NR ｔ R ｔ’ , -NR ｔ R ｔ’ , -NR ｔ C(O)R ｕ , -NR ｔ C(O)OR ｕ , -NR ｔ C(O)NR ｕ R ｕ’ , -NR ｔ S(O)R ｕ , -NR ｔ S(O) ２ R ｕ , -NR ｔ S(O)NR ｕ R ｕ’ , -NR ｔ S(O) ２ NR ｕ R ｕ’ , -C(O)R ｔ , -C(O)OR ｔ , or -C(O)NR ｔ R ｔ’ It can be replaced by, R ｒ , R ｒ’ , R ｓ , R ｓ’ , R ｔ , R ｔ’ , R ｕ , and R ｕ 'However, H and C are independent of each other. １－７ Alkyl, C ２－７ Alkenil, C ２－７ Alkinyl, C ３－１３ Cycloalkyl, 3-7 member heterocycloalkyl, C ５－１０ Selected from aryls and 5- to 7-membered heteroaryls, G is a glucuronide group or a galactoside group, Each Z is H, C １－８ Alkyl, Halo, NO ２ , CN, 【Chemistry 16】 Selected from, R ９ , R １０ , and R １６ However, H and C are independent of each other. １－８ Alkyl, C ２－６ Alkenil, C １－６ Selected from alkoxy, alkyloxyalkyl, and methyloxyethyl, The conjugate according to claim 1 or 8, wherein n30 is 1 to 3.

47. (i) The conjugate has a structure selected from the following: 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 ; or (ii) The structure of the conjugate is selected from the following: 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 And, 【Chemistry 31】 Includes, In the formula, MMAE is monomethyl auristatin E. MMAF stands for monomethyl auristatin F. The conjugate according to any one of claims 1 to 6.

48. The conjugate is as follows: 【Chemistry 32】 or a pharmaceutically acceptable salt thereof During the ceremony, 【Transformation 33】 Ab is Ab, The conjugate according to claim 1, wherein Ab comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

16.

49. A pharmaceutical composition comprising the conjugate according to claim 1, 8, or 48 and a pharmaceutically acceptable excipient.

50. A pharmaceutical composition for the prevention or treatment of hyperplasia, cancer, or angiogenic disease, comprising the conjugate described in claim 1, 8, or 48.

51. A pharmaceutical composition for the prevention or treatment of cancer, wherein the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma, according to claim 50.

52. Use of the conjugate according to claim 1, 8, or 48 in the manufacture of a pharmaceutical product for the prevention or treatment of hyperplasia, cancer, or angiogenic disease.

53. The use according to claim 52, wherein the pharmaceutical agent is for the prevention or treatment of cancer, and the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma.

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