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

Abstract

The present disclosure relates to antibody-drug conjugates (ADCs) targeting human TROP2 (Tumor-Associated Calcium Signal Transducer 2, TACSTD2) and uses thereof, and more particularly, the present invention relates to: ADCs comprising antibodies or antigen-binding fragments thereof that bind to human TROP2; and the use of the ADC for the manufacture of a medicament for the treatment and / or treatment of a disease, more particularly a hyperproliferative and / or angiogenic disease, such as a cancer disease.

Description

[0001] Related Applications

[0002] This application claims priority to Korean Patent Application No. 10-2022-0043049, filed on April 6, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Cancer refers to a disease caused by an abnormal growth mass resulting from spontaneous overgrowth of body tissues and is the result of uncontrolled cell growth in various tissues. Early stage tumors can be removed by surgery and radiation therapy, and metastatic tumors are generally treated with chemotherapy.

[0004] Most parenterally administered chemotherapeutic agents may induce unwanted side effects and even severe toxicity due to systemic administration. Therefore, research and development emphasis is placed on developing therapies that can achieve improved efficacy and / or reduced minimal toxicity / side effects, for example, by selectively applying these chemotherapeutic agents in tumor cells or adjacent tissues. Summary of the invention

[0005] In certain aspects, the present invention provides an antibody-drug conjugate comprising an anti-TROP2 antibody that binds to TROP2. In certain embodiments, the antibodies disclosed herein bind to TROP expressed in a tumor and can be used to deliver a drug to a tumor. In certain embodiments, the antibody drug conjugate disclosed herein has improved stability compared to antibody drug conjugates known in the art.

[0006] In certain aspects, the present invention provides a conjugate having a structure represented by Formula I or a pharmaceutically acceptable salt thereof:

[0007] [General Formula I]

[0008] Ab-[L-(B) 1 ] m

[0009] in,

[0010] Ab is an anti-TROP2 (tumor-associated calcium signal transducer 2, also known as TACSTD2) antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein

[0011] 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; and

[0012] 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;

[0013] L is a linker;

[0014] B is the active agent moiety, and

[0015] 1 and m are each independently an integer selected from 1 to 20. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 FIG. 5 is a schematic diagram illustrating a method for preparing an antibody-drug conjugate ADC1 according to one embodiment of the present invention.

[0017] Figure 2 is a schematic diagram illustrating a method for preparing an antibody-drug conjugate ADC2 according to one embodiment of the present invention.

[0018] Figure 3 FIG. 5 is a schematic diagram illustrating a method for preparing an antibody-drug conjugate ADC3 according to one embodiment of the present invention.

[0019] Figure 4 is a schematic diagram illustrating a method for preparing antibody-drug conjugate ADC4 according to one embodiment of the present invention.

[0020] Figure 5A and Figure 5B Figure 2 shows the results of the first in vivo experiment in a mouse model transplanted with pancreatic cancer cell line BxPC-3 ( Figure 5A ) and a mouse model transplanted with the breast cancer cell line MDA-MB-468 ( Figure 5B ) confirmed the results of the tumor growth inhibitory efficacy of the antibody-drug conjugate according to one embodiment of the present invention.

[0021] FIG. 6A to FIG. 6D The second in vivo experiment shows that the breast cancer cell line MDA-MB-468 was transplanted into the mouse model ( Fig. 6A ), a mouse model transplanted with the pancreatic cancer cell line BxPC-3 ( Figure 6B ), a mouse model transplanted with gastric cancer cell line NCI-N87 ( Figure 6C ) and a mouse model transplanted with the non-small cell lung cancer cell line HCC827 ( Fig.6D ) confirmed the results of the tumor growth inhibitory efficacy of the antibody-drug conjugate according to one embodiment of the present invention.

[0022] 7A to 7EFigure 2 shows the expression of BxPC-3 pancreatic cancer cell line in a mouse model ( Fig. 7A ), a mouse model transplanted with the non-small cell lung cancer cell line HCC827 ( Figure 7B ), a mouse model transplanted with the pancreatic cancer cell line Capan-1 ( Figure 7C ), a mouse model transplanted with the non-small cell lung cancer cell line NCI-H2170 ( Fig.7D ) and a mouse model transplanted with the colon cancer cell line HT29 ( Fig. 7E ) confirmed the results of the tumor growth inhibitory efficacy of the antibody-drug conjugate according to one embodiment of the present invention. DETAILED DESCRIPTION

[0023] Antibody-drug conjugate (ADC) is a targeted technology for binding toxins or drugs to antibodies that bind to antigens, thereby releasing toxins in cells to cause the death of cancer cells and their analogs. ADC is able to precisely deliver drugs to target cancer cells while having minimal impact on healthy cells, and is released only under specific conditions, and therefore has excellent efficacy compared to antibody therapeutics themselves, and can significantly reduce the risk of side effects compared to existing anticancer agents.

[0024] 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 connecting the antibody to the drug, but in some cases also ensures that the drug is released from the antibody at the appropriate time, such as after reaching the target cell. That is, the stability of the linker plays an extremely important role in the efficacy and safety (e.g., systemic toxicity) of the antibody-drug conjugate (Discovery Medicine 2010, 10(53): 329-39).

[0025] The use of monoclonal antibodies for cancer therapy has achieved great success. For example, monoclonal antibodies are suitable for targeted addressing of tumor tissues and tumor cells. Antibody-drug conjugates have become a novel and effective option for the treatment of lymphomas and solid cancers, and immunomodulatory antibodies have also recently achieved great success in clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering techniques and their effects, resistance mechanisms and the interaction between the immune system and cancer cells.

[0026] Antigens expressed on the surface of human cancer cells are defined as a wide range of targets that are overexpressed, mutated, and selectively expressed compared to normal tissues. The main challenge is to identify antigens suitable for antibody-based therapies. These therapeutic agents mediate changes in antigen or receptor function (i.e., acting as stimulants or antagonists), regulate the immune system via Fc and T cell activation, and demonstrate efficacy via delivery of specific drugs combined with antibodies targeting specific antigens. Molecular techniques that can change antibody pharmacokinetics, action functions, size, and immune stimulation are becoming key factors in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of methods for selecting optimized antibodies, including affinity and combination of target antigens and antibodies, selection of antibody structures, and therapeutic methods (signaling blocking or immune function).

[0027] Human TROP2 (tumor-associated calcium signal transducer 2, TACSTD2) is a 35-46 kDa transmembrane calcium signal transducer glycoprotein with 323 amino acids (274 extracellular domains, 23 transmembrane domains and 26 intracellular domains), and belongs to the epithelial cell adhesion molecule (EpCAM) family, which is an important component in cell signaling, proliferation and differentiation. TROP2 expression is associated with tumor induction or tumor suppression in various carcinomas, but it is known that TROP2 generally acts as a tumor inducer.

[0028] Cleavage of the region between R87 and T88 in the 274 extracellular domain induces a structural rearrangement of TROP2, resulting in changes in biological activity. Although this cleavage does not occur in normal tissues, TROP2 cleavage occurs in most cancers, including skin cancer, ovarian cancer, colon cancer, and breast cancer, and thus TROP2 acts as a tumor inducer.

[0029] In certain aspects, the present invention provides an antibody-linker-drug (e.g., toxin) system that, by using a linker that optionally comprises a self-immolative group: is more stable in circulation (e.g., in plasma), allows the drug to be easily released in cancer cells to maximize efficacy, and allows the drug and / or toxin to stably reach the target cells.

[0030] Therefore, in certain embodiments, the present invention provides antibody-drug conjugates targeting human TROP2 (tumor-associated calcium signal transducer 2, TACSTD2) and pharmaceutically acceptable salts thereof.

[0031] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof.

[0032] In certain aspects, the present invention provides a conjugate represented by the following general formula I or a pharmaceutically acceptable salt or solvate thereof:

[0033] [General Formula I]

[0034] Ab-[L-(B) 1 ] m

[0035] in,

[0036] Ab is an anti-TROP2 (tumor-associated calcium signal transducer 2, also known as TACSTD2) antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein

[0037] 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; and

[0038] 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;

[0039] L is a linker;

[0040] B is an active agent moiety; and

[0041] In certain embodiments, the antibody or antigen-binding fragment thereof may include 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.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof may include 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.

[0043] In certain embodiments, the antibody or antigen-binding fragment thereof may include a heavy chain variable region consisting of: an amino acid sequence of SEQ ID NO: 15 or 20; or 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 an 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.

[0044] In certain embodiments, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising: an amino acid sequence of SEQ ID NO: 15 or 20; or a sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 15 or 20; a sequence having at least 95% sequence identity to an 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.

[0045] In certain embodiments, the antibody or antigen-binding fragment thereof may include a heavy chain variable region consisting of: an amino acid sequence of SEQ ID NO: 15 or 20; a sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 15 or 20; or a sequence having at least 95% sequence identity to an 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.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof may include 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.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof may include 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.

[0048] In certain embodiments, the antibody or antigen-binding fragment thereof may include a light chain variable region consisting of: an 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.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof may include a light chain variable region comprising: the 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 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.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof may include a light chain variable region consisting of: an 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 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.

[0051] 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.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof consists of: 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.

[0053] 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 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. The light chain variable region comprises a light chain CDR3 having the amino acid sequence of NO: 13, and specifically binds to human TROP2.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region consisting of 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 consisting of an amino acid sequence consisting of an amino acid sequence of SEQ ID NO: 15 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 an amino acid sequence consisting of 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: The light chain variable region comprises a light chain CDR3 having the amino acid sequence of NO: 13, and specifically binds to human TROP2.

[0055] 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 comprises an amino acid sequence having 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, or at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 15, while maintaining the amino acid sequence of SEQ ID NO: 16. NO:9, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:11, and a heavy chain variable region comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, and an amino acid sequence having 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, or at least 99.5% 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:11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, and an amino acid sequence having 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, or at least 99.5% sequence identity to the amino acid sequence of SEQ ID The invention also provides a light chain variable region comprising 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.

[0056] 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 comprises an amino acid sequence having 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, or at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 15, while maintaining the amino acid sequence of SEQ ID NO: 16. NO:9, a light chain CDR1 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 heavy chain variable region consisting of an amino acid sequence having 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, or at least 99.5% sequence identity with the amino acid sequence of SEQ ID NO:16, while maintaining 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 consisting of an amino acid sequence having 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, or at least 99.5% sequence identity with the amino acid sequence of The invention also provides a light chain variable region comprising 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.

[0057] In certain embodiments, the antibody is a humanized antibody.

[0058] In certain embodiments, a humanized antibody comprises: (a) (i) a variable heavy chain framework region from a human antibody heavy chain or from a human common framework; and (ii) a variable light chain framework region from a human antibody light chain or from a human common framework. In some embodiments, at least one amino acid in the variable domain framework region from the heavy chain is substituted with a corresponding amino acid from a mouse antibody heavy chain or a mouse common framework; or the variable domain framework region from the light chain is substituted with a corresponding amino acid from a mouse antibody light chain or a mouse common framework. In some embodiments, the amino acid substitutions in the heavy chain are selected from the following amino acid substitutions:

[0059] F (mouse) substituted for Y (human) at position 27;

[0060] S (mouse) substituted for T (human) at position 30;

[0061] L (mouse) substituted for V (human) at position 37;

[0062] I (mouse) substituted for M (human) at position 48;

[0063] A (mouse) substituted for G (human) at position 49;

[0064] L (mouse) replaces I (human) at position 70; and

[0065] V (mouse) substituted for R (human) at position 72, and

[0066] (ii) The amino acid substitution of the light chain was Y (human) replaced by S (mouse) at position 49.

[0067] In certain embodiments, the amino acid substitutions of the heavy chain may further include the following amino acid substitutions:

[0068] K (mouse) replaces R (human) at position 67; and / or

[0069] At position 68, A (mouse) replaced V (human).

[0070] In certain embodiments, the amino acid substitutions of the light chain may further include the following amino acid substitutions:

[0071] Y (mouse) substituted for S (human) at position 67;

[0072] T (mouse) substituted for I (human) at position 2 and Y (mouse) substituted for S (human) at position 67;

[0073] R (mouse) substituted for T (human) at position 22 and Y (mouse) substituted for S (human) at position 67;

[0074] E (mouse) substituted for K (human) at position 42 and Y (mouse) substituted for S (human) at position 67;

[0075] S (mouse) substituted for G (human) at position 64 and Y (mouse) substituted for S (human) at position 67;

[0076] Substitution of Y (mouse) for S (human) at position 67 and of V (mouse) for T (human) at position 72; or

[0077] At position 67, Y (mouse) replaced S (human) and at position 85, D (mouse) replaced T (human).

[0078] In certain embodiments, the humanized antibody comprises: (a)(i) a variable heavy chain framework region from a human antibody heavy chain or from a human common 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 human antibody light chain or from a human common framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0079] Amino acid substitutions can increase affinity and enhance the stability of antibodies while maintaining their antigen-binding activity. Stabilization of therapeutic antibodies can lead to improved serum half-life, reduced dosage requirements, reduced side effects, and improved storage life. Chimeric CH-2EF and CH-2G10 antibodies were first constructed, followed by early humanized versions: Hu-2EF-4, Hu-2G10-1, etc. These antibodies may exhibit undesirable characteristics, such as chemical instability, resulting in aggregate formation and loss of solubility (for CH-2EF antibodies), and loss of affinity for Trop-2. Amino acid substitutions can produce 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, especially for different regions of the extracellular domain of Trop2 and various forms of post-translational modifications.

[0080] In certain embodiments, the Genbank database is searched for human VH sequences homologous to the VH framework disclosed herein, and the VH sequence encoded by the human cDNA with NCBI accession number: X65888.1 is selected as the humanized receptor (X65888.1-VH). In certain embodiments, the CDR sequences of the VH disclosed herein are first transferred to the corresponding positions of X65888.1-VH. In certain embodiments, at framework positions 27, 30, 37, 48, 49, 67, 68, 70 and 72 (numbered from the first amino acid of the mature form) (i.e., positions where the three-dimensional model of the variable region indicates significant contact with the CDR), human amino acid residues are replaced by corresponding mouse residues.

[0081] Although Lys at position 67 and Ala at position 68 (both adjacent to CDR) in mouse 2G10 VH are predicted to be critical for the formation of CDR structure, the amino acid residues in 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 molecular properties and structure. Therefore, in order 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, namely Arg and Val, respectively.

[0082] Based on the GenBank homology search with the VL framework sequence, the human VK region encoded by the cDNA with NCBI deposit number: AY043146.1 was selected as the humanized receptor. The CDR sequence of VL was first transferred to the corresponding position of AY043146.1VL. Then, at positions 49 and 67 (i.e., the three-dimensional model of the variable region showed significant contact with the CDR), the human amino acid residues were replaced by the corresponding residues of the mouse VL. Although the Tyr at position 67 in the mouse VL is predicted to be crucial to the formation of the CDR structure, further analysis of the three-dimensional model of the 2G10 variable region showed that the Tyr at this position 67 was replaced by Ser (the residue at the corresponding position in the human receptor AY043146.1VL) without affecting the possibility of antigen binding. Therefore, in order to further reduce the potential immunogenicity, a second humanized VL (derived from VL1) was designed, in which Ser at position 67 in the human receptor sequence was maintained.

[0083] In certain embodiments, the antibody or antigen-binding fragment thereof can 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, a 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 mini antibody, a bifunctional antibody, a bispecific antibody, and a multispecific antibody.

[0084] The linkers described herein can be cleavable, non-cleavable and hydrophilic or hydrophobic.

[0085] In certain embodiments, the cleavable linker can be cleaved under intracellular or extracellular conditions, thereby releasing the active agent from the antibody construct-active agent conjugate in the intracellular environment.

[0086] The cleavable linker can be cleaved by a cleaving agent present in the intracellular environment (e.g., lysosomes, endosomes, or caveolae). The cleavable linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease, including but not limited to a lysosomal or endosomal protease. In general, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. The cleaving agent 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, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most common are peptidyl linkers that are cleaved by enzymes present in cells expressing the antigen. For example, a peptidyl linker that is cleaved by the thiol-dependent protease cathepsin-B, which is abundantly expressed in cancer tissues (e.g., Phe-Leu or Gly-Phe-Leu-Gly linkers) 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 use of a Val-Cit linker to synthesize doxorubicin) or a Val-Ala linker. The Val-Cit linker or the Val-Ala linker can contain a pentafluorophenyl group, and can contain a succinimide group or a maleimide group. Alternatively, the Val-Cit linker or the Val-Ala linker can contain a pentafluorophenyl group, can contain a 4-aminobenzoic acid (PABA) group and a maleimide group, and can contain a PABA group and a succinimide group.

[0087] In addition, the cleavable linker can be pH-sensitive, i.e., susceptible to hydrolysis at a specific pH. In general, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, and ketals) that can be hydrolyzed in lysosomes can be used (see, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, and 5,622,929; and 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 (e.g., in blood), but are unstable at pH 5.5 (which is close to the pH of lysosomes) or at less than pH 5.0. Examples of hydrolyzable linkers include thioether linkers (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0088] Furthermore, the linker can be cleaved 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-α-methyl-α-(2-pyridyl-thio) toluene)-(SMPT), as well as disulfide linkers 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).

[0089] 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).

[0090] The non-cleavable linker can be a maleimidocaproyl linker. The maleimidocaproyl linker can include N-maleimidocaproyl methylcyclohexane-1-carboxylate. The maleimidocaproyl linker can contain a succinimidyl group. The maleimidocaproyl linker can contain a pentafluorophenyl group. The linker can be a combination of a maleimidocaproyl 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 maleimidocaproyl-PEG4 linker. The linker can be a combination of a maleimidocaproyl linker containing a succinimidyl 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 may contain a maleimide attached to a polyethylene glycol molecule, wherein the polyethylene glycol makes the linker more flexible or enables the use of a longer linker. The linker may be a (maleimidocaproyl)-(valine-citrulline)-(p-aminobenzyloxycarbonyl) linker.

[0091] In certain embodiments, the linker can be a cleavable linker.

[0092] In certain embodiments, the linker can be a protease cleavable linker, an acid cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, a maleimidobenzoyl linker, a 3′-N-amide analog, a β-glucuronide linker, or a β-galactoside linker.

[0093] In certain embodiments, the protease cleavable linker may include a thiol-reactive spacer or a dipeptide, and more specifically, the protease cleavable linker may include a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, or a p-amino-benzyloxycarbonyl spacer.

[0094] In certain embodiments, the acid-cleavable linker can be a hydrazine linker or a quaternary ammonium linker.

[0095] Exemplary antibody drug conjugates are disclosed in US 10,583,197, US 9,993,568, US 9,951,072, US 9,919,057, US 9,669,107, US 11,413,353, US 11,173,214, US 11,167,040, US 10,980,890, US 10,583,197, US 10,383,949, US 10,273,235, US 10,183,997, and US 10,118,965, the contents of each of which are incorporated herein by reference in their entirety.

[0096] In certain embodiments, the linker can have the structure of Formula II.

[0097] [General Formula II]

[0098]

[0099] Wherein: In the general formula II,

[0100] The wavy symbol is the linker site for an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, and

[0101] * is the linking site to the active agent;

[0102] G is a glucuronic acid moiety or R 3 is hydrogen or a carboxyl protecting group, and each R 4 are independently hydrogen or a hydroxy protecting group;

[0103] R 1 and R 2 are independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl;

[0104] W is -C(O)-, -C(O)NR′-, -C(O)O-, -SO 2 NR′-, -P(O)R″NR′, -SONR′- or -PQ 2 NR′-, wherein C, S or P is directly bonded to the benzene ring; and R′ and R″ are each independently hydrogen, C1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl;

[0105] Z is independently hydrogen, C 1-8 Alkyl, halogen, cyano or nitro;

[0106] n is 0 to 3; and

[0107] L is any one selected from the following A) or B):

[0108] A)C 1-50 Alkylene or 1 to 50-membered heteroalkylene, satisfying at least one of the following:

[0109] (i) L includes one or more unsaturated bonds;

[0110] (ii) two atoms in L are substituted with a divalent substituent to form a heteroarylene group;

[0111] (iii) L is a 1- to 50-membered heteroalkylene group;

[0112] (iv) The alkylene group is replaced by at least one C 1-20 Alkyl substitution; and

[0113] B) comprises at least one isoprenyl derivative unit of the following general formula III, which can be recognized by an isoprenoid transferase:

[0114] [Formula III]

[0115]

[0116] In certain embodiments, the conjugate may have the structure of Formula IIa.

[0117] [General Formula IIa]

[0118]

[0119] in

[0120] Each B′ is an active agent;

[0121] Each G is independently a glucuronic acid moiety or

[0122] R 3 is hydrogen or a carboxyl protecting group;

[0123] Each R4 are independently hydrogen or a hydroxy protecting group;

[0124] R 1 and R 2 are independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl;

[0125] W is -c(O)-, -c(O)NR′-, -c(O)O-, -SO 2 NR′-, -P(O)R″NR′, -SONR′- or -PO 2 NR′-, wherein C, S or P is directly bonded to the benzene ring of formula IIa;

[0126] R' and R" are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono-C 1- 8-Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl;

[0127] Each Z is independently hydrogen, C 1-8 Alkyl, halogen, cyano or nitro;

[0128] n is 0, 1, 2, or 3;

[0129] L contains:

[0130] A)C 1-50 Alkylene or C 1-50 Heteroalkylene and comprising

[0131] (i) one or more unsaturated bonds

[0132] (ii) heteroarylene; or

[0133] (iii) by at least one C 1-20 Alkyl substitution; or

[0134] B) at least one isoprenyl group having a structure represented by the general formula III:

[0135] [General Formula III]

[0136] and

[0137] 1 and m are each independently 1 to 20.

[0138] The present invention also provides a conjugate represented by the following general formula IIa or a pharmaceutically acceptable salt or solvate thereof:

[0139] [General Formula IIa]

[0140]

[0141] in

[0142] Ab is against TROP2 (tumor-associated calcium signal transducer 2, also known as TACSTD2)

[0143] Antibodies or antigen-binding fragments thereof;

[0144] Each B′ is an active agent;

[0145] Each G is independently a glucuronic acid moiety or

[0146] R 3 is hydrogen or a carboxyl protecting group;

[0147] Each R 4 are independently hydrogen or a hydroxy protecting group;

[0148] R 1 and R 2 are independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl;

[0149] W is -C(O)-, -C(O)NR′-, -C(O)O-, -SO 2 NR′-, -P(O)R″NR′, -SONR′- or -PO 2 NR′-, wherein C, S or P is directly bonded to the benzene ring of formula IIa;

[0150] R' and R" are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl;

[0151] Each Z is independently hydrogen, C 1-8 Alkyl, halogen, cyano or nitro;

[0152] n is 0, 1, 2, or 3;

[0153] L contains:

[0154] A)C 1-50 Alkylene or C 1-50 Heteroalkylene and comprising

[0155] (i) one or more unsaturated bonds

[0156] (ii) heteroarylene; or

[0157] (iii) by at least one C 1-20 Alkyl substitution; or

[0158] B) at least one isoprenyl group having a structure represented by the general formula III:

[0159] [General Formula III]

[0160] and

[0161] 1 and m are each independently 1 to 20.

[0162] Other conjugates related to this structure and detailed methods of preparation are disclosed in US 9,919,057, US 9,993,568, US 10,980,890 and US 11,413,353; the contents of each of which are incorporated herein by reference in their entirety.

[0163] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0164] 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; and

[0165] 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.

[0166] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising:

[0167] The amino acid sequence of SEQ ID NO: 15 or 20;

[0168] having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining a sequence comprising a light chain CDR1 of the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 of the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 of the amino acid sequence of SEQ ID NO: 13; or

[0169] Having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0170] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising:

[0171] The amino acid sequence of SEQ ID NO: 16;

[0172] having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining a sequence of 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

[0173] Having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:16, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0174] 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.

[0175] In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the humanized antibody comprises:

[0176] (i) a variable heavy chain framework region from a human antibody heavy chain or from a human common 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

[0177] (ii) a variable light chain framework region from a human antibody light chain or from a human common framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0178] 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, a 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.

[0179] In certain preferred embodiments, each G is

[0180] In certain embodiments, R 1 and R 2 Each is hydrogen.

[0181] In certain embodiments, R 3 It's hydrogen.

[0182] In certain embodiments, each R 4 It is a hydroxyl protecting group.

[0183] In certain embodiments, n is 0.

[0184] In certain preferred embodiments, each W is -C(O)NR'-, further wherein C is directly bonded to the phenyl ring of Formula IIa and NR' is bonded to L.

[0185] In certain embodiments, each R4 is independently hydrogen.

[0186] In certain embodiments, R 1 and R 2 Each is hydrogen;

[0187] n is 0; and

[0188] Each W is -C(O)NR'-, c is directly bonded to the benzene 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl, wherein NR′ is bonded to L.

[0189] In certain embodiments, L comprises a nitrogen-containing 1- to 50-membered heteroalkylene group.

[0190] In certain embodiments, L comprises a hydrophilic amino acid. In 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 group of the hydrophilic amino acid.

[0191] In certain embodiments, L is a nitrogen-containing 1- to 50-membered heteroalkylene, the linker comprises two or more atoms of a hydrophilic amino acid, and

[0192] The nitrogen forms a peptide bond with the carbonyl group of the hydrophilic amino acid.

[0193] In certain embodiments, L is covalently bonded to the antibody via a thioether bond and the thioether bond comprises a sulfur atom of a cysteine ​​of the antibody.

[0194] In certain embodiments, the antibody comprises an amino acid motif at the C-terminus of the antibody that is recognized by an isoprenoid transferase, and

[0195] The thioether bond involves the sulfur atom of the cysteine ​​of the amino acid motif.

[0196] In certain embodiments, the amino acid motif has a CYYX sequence, further wherein:

[0197] c is cysteine;

[0198] Y is an aliphatic amino acid;

[0199] X is selected from the group consisting of glutamine, glutamic acid, serine, cysteine, methionine, alanine and leucine; and

[0200] The thioether bond involves the sulfur atom of the cysteine ​​of the amino acid motif.

[0201] In certain embodiments, the amino acid motif has a CYYX sequence, further wherein:

[0202] Y is selected from the group consisting of alanine, isoleucine, leucine, methionine and valine.

[0203] In certain preferred embodiments, the amino acid motif has a CVIM (SEQ ID NO: 24) or CVLL sequence (SEQ ID NO: 25).

[0204] In certain embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

[0205] In certain preferred embodiments, L comprises the amino acid sequence of GGGGGGGCVIM at the C-terminus (SEQ ID NO: 22).

[0206] In certain embodiments, L comprises C 1-50 Heteroalkylene.

[0207] In certain preferred embodiments, L comprises an oxime. In certain embodiments, the oxygen atom of the oxime is on the side of L attached to W and the carbon atom of the oxime is on the side of L attached to Ab. In certain embodiments, the carbon atom of the oxime is on the side of L attached to W and the oxygen atom of the oxime is on the side of L attached to Ab.

[0208] In certain embodiments, L is a C containing oxime 1-50 Heteroalkylene,

[0209] The oxygen atom of the oxime is on the side where L is connected to W,

[0210] The carbon atom of the oxime is on the side where L is attached to Ab, or

[0211] The carbon atom of the oxime is on the side where L is connected to W, and

[0212] The oxygen atom of the oxime is attached to the side of L to Ab.

[0213] In certain preferred embodiments, L comprises an oxime, and at least one prenyl unit covalently bonds the oxime to Ab (eg, at least one prenyl unit directly or indirectly bonds the oxime to Ab).

[0214] In certain embodiments, L comprises a linker unit represented by Formula VIII or Formula IX:

[0215] [General Formula VIII]

[0216] -(CH 2 ) r (V(CH 2 ) p ) q -

[0217] [Formula IX]

[0218] -(CH 2 CH 2 X) w -

[0219] 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 -;

[0220] X is -O-, C 1-8 Alkylene or -NR 21 -;

[0221] R 21 To R25 are independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl-C 3-20 heteroaryl;

[0222] r is 0 to 10;

[0223] p is 0 to 10;

[0224] q is 1 to 20; and

[0225] w is 1 to 20.

[0226] In certain embodiments, q is 1-10.

[0227] In certain embodiments, r is 1 or 2.

[0228] In certain embodiments, p is 1 or 2.

[0229] In certain embodiments, V is -O-.

[0230] In certain embodiments, q is 1 to 10;

[0231] r and p are each 1 or 2; and

[0232] V is -O-.

[0233] In certain embodiments, Z is -O-.

[0234] In certain embodiments, w is 1-10.

[0235] In certain embodiments, wherein:

[0236] Z is -O-; and

[0237] w is 1 to 10.

[0238] In certain preferred embodiments, L comprises wherein n40 is 1 to 10, preferably at least 2.

[0239] In certain embodiments, L comprises an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

[0240] In certain embodiments, L comprises a bonding unit formed by the reaction between an alkyne and an azide group or between an aldehyde or ketone group and hydrazine or hydroxylamine.

[0241] In certain embodiments, L further comprises a binding unit represented by the following general formula IVa, IVb, IVc, IVd or IVe:

[0242] [General Formula IVa]

[0243]

[0244] [General Formula IVb]

[0245]

[0246] [General Formula IVc]

[0247]

[0248] [General Formula IVd]

[0249]

[0250] [General Formula IVe]

[0251]

[0252] in

[0253] L 1 and L 2 Each is independently a single bond or C 1-30 an alkylene group; and

[0254] R 11 is hydrogen or C 1-10 alkyl.

[0255] In certain embodiments, L 1 and L 2 Each is independently a single bond, C 11 Alkylene or C 12 Alkylene.

[0256] In certain embodiments, the isoprenoid transferase is farnesyl protein transferase (Ftase) or geranylgeranyl transferase (GGTase).

[0257] In certain embodiments, L is branched and comprises:

[0258] i) a branching unit covalently coupled to the antibody via a primary linker;

[0259] ii) a first branch, wherein a first active agent is covalently coupled to the branch unit via a secondary linker and a cleavable group; and

[0260] iii) the second branch, in which:

[0261] a) a second active agent is covalently coupled to the branching unit via a secondary linker and a cleavable group;

[0262] or

[0263] b) The polyethylene glycol moiety is covalently coupled to the branching unit.

[0264] In certain embodiments, the branching unit has The structure represented by:

[0265] L 2 , L 3 and L 4 Each independently is a bond or -C n H 2n -,

[0266] n is 1 to 30;

[0267] G 1 , G 2 and G 3 Each independently represents a key,

[0268] R 30 is hydrogen or C 1-30 alkyl;

[0269] R 40 is hydrogen or L 5 -COOR 6 ;and

[0270] L 5 For key or -C n′ H 2n′ -;

[0271] n′ is 1 to 10; and

[0272] R 6 is hydrogen or C 1-30 alkyl.

[0273] In certain preferred embodiments, the cleavable group is capable of being cleaved in a target cell and is capable of releasing one or more active agents.

[0274] In certain embodiments, at least one branched linker is covalently coupled to Ab; and

[0275] At least two active agents are covalently coupled to the branch linker. In certain embodiments, one branch linker is coupled to Ab. In certain embodiments, two branch linkers are coupled to Ab. In certain embodiments, three branch linkers are coupled to Ab. In certain embodiments, four branch linkers are coupled to Ab. In certain embodiments, each branch 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 branch linker is coupled to two different active agents.

[0276] In certain embodiments, the branching unit is a nitrogen atom. In other embodiments, the branching unit is an amide and the primary linker comprises the carbonyl group of the amide. In other embodiments, the branching unit is an amide and the secondary linker comprises the carbonyl group of the amide. In certain preferred embodiments, the branching unit is lysine.

[0277] In certain embodiments, the conjugate comprises the structure represented by:

[0278]

[0279] or a pharmaceutically acceptable salt thereof; wherein

[0280] B′ and B″ are each active agents;

[0281] n1 to n3 are each independently 0 to 30; and

[0282] AA is the amino acid group.

[0283] In certain embodiments, the conjugate comprises a structure represented by:

[0284]

[0285]

[0286] wherein B′ and B″ refer to active agents that are the same or different from each other;

[0287] A pyrrolobenzodiazepine dimer, such as a compound of formula III; and

[0288] m and n each independently mean 0 to 30,

[0289] or a pharmaceutically acceptable salt thereof.

[0290] In certain embodiments, the conjugate comprises the structure represented by:

[0291]

[0292] or a pharmaceutically acceptable salt thereof, wherein the wavy symbol represents the binding site of the antibody construct, * represents the binding site of the active agent, and n is 0 to 20.

[0293] In certain embodiments, the first linker of the antibody-drug conjugate comprises an alkylene group having 1 to 100 carbon atoms, preferably 1 to 50 carbon atoms, or the alkylene group includes at least one unsaturated bond, the alkylene group comprises at least one heteroarylene group, the carbon atoms of the alkylene group are substituted with one or more heteroatoms selected from nitrogen (N), oxygen (O) and sulfur (S), or the alkylene group is further substituted with one or more alkyl groups having 1 to 20 carbon atoms.

[0294] In certain embodiments, at least one carbon atom of the alkylene group is substituted with nitrogen, the first linker comprises at least two atoms of a hydrophilic amino acid, and the nitrogen forms a peptide bond together with the main chain carbonyl of the hydrophilic amino acid. The hydrophilic amino acid can be, for example, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

[0295] In certain embodiments, the branch linker of the antibody active agent comprises an amino acid with a side chain having a portion that carries a charge at neutral pH in an aqueous solution, preferably arginine, aspartic acid, glutamic acid, lysine or ornithine. The amino acid may be located at any position on the branch linker. For example, the oxime of the branch linker may be covalently bonded to the polyethylene glycol unit of the branch linker. Alternatively or additionally, these amino acids may be present in the second linker, optionally in each second linker.

[0296] In certain embodiments, the conjugate comprises the structure represented by:

[0297]

[0298] or a pharmaceutically acceptable salt thereof;

[0299] wherein B′ and B″ are each an active agent;

[0300] n1 to n3 each independently refer to 0 to 30; and

[0301] AA refers to the amino acid group.

[0302] In certain embodiments, AA refers to an amino acid group to which one or more amino acids are bonded.

[0303] In certain embodiments, i) the linker comprises a peptide sequence of multiple amino acids; and ii) at least two active agents are covalently bonded to the side chains of the amino acids.

[0304] In certain embodiments, the amino acid group is a group formed by main chain linkage or side chain linkage of 1 to 20 amino acids.

[0305] In certain embodiments, the amino acid group is a group formed by main-chain or side-chain linkages of 1 to 20 arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine residues.

[0306] In certain embodiments, the amino acid group is a group formed by backbone or side chain linkages of 1 to 20 arginine, aspartic acid, glutamic acid, lysine, or ornithine residues.

[0307] In certain embodiments, the amino acid group includes 1 to 20 amino acids, such as at least one lysine.

[0308] In certain embodiments, the amino acid group comprises a backbone or side chain linkage of lysine.

[0309] In certain embodiments, the conjugate comprises the structure represented by:

[0310]

[0311] Among them, MMAE is monomethyl auristatin E.

[0312] Other conjugates related to this structure and detailed methods of preparation are disclosed in US 11,173,214 and US 11,167,040; the contents of each of which are incorporated herein by reference in their entirety.

[0313] In certain embodiments, the term "active agent moiety" as used herein refers to a compound comprising an active agent and a covalent bond that binds the active agent to a linker or a portion thereof.

[0314] In the present invention, the active agent moiety may be directly bound to the linker, and one or more, particularly two or more, three or more, or four or more active agent moieties may be directly bound to the linker.

[0315] In certain embodiments, the active agents are each independently selected from a chemotherapeutic agent and a toxin.

[0316] Additionally, the active agent may be an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof, and may be selected from the following active agents for use:

[0317] (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, A G1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine, hexamethylmelamine, triethylmelamine, triethylphosphoramide, triethylthiophosphoramide, trihydroxymethylmelamine, bullatacin, bullatacinone, camptothecin, ecin), topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcosamine Sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustardmustard), carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, anthramycin, azaserine ine), bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, lipid doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acidacid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin icin), 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmof ur), cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone, aminoglutethimid e), mitotane, trilostane, aldehyde folate, acetylglucosyl ester, aldehyde phosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomithine, elliptinium acetateacetate), etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, raZoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid acid), triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin AA), roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine ne), 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid thereof;

[0318] (b) Monokine, lymphoid mediators, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin , erythropoietin, osteoinductive 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, tumor necrosis factor, polypeptide factor, LIF, kit ligand or a combination thereof;

[0319] (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolinylbenzodiazepine, pyridobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin namycin), maytansinoid, calicheamicin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, dolestacin, dolestacin analogs, auristatin, nostoc, camptothecin, rhizotomycin, rhizotomycin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotics, esperamicin, epothilone, toxoids, or combinations thereof;

[0320] (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an active agent, a neurotransmitter, a radioisotope or a combination thereof;

[0321] (e) radioactive labels, 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, dioxigenin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to a target, or a combination thereof;

[0322] (f) immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, or combinations thereof;

[0323] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone or toremifene;

[0324] (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole;

[0325] (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxacitabine;

[0326] (j) aromatase inhibitors;

[0327] (k) protein kinase inhibitors;

[0328] (l) lipid kinase inhibitors;

[0329] (m) antisense oligonucleotides;

[0330] (n) ribonucleases;

[0331] (o) vaccines; and

[0332] (p) Anti-angiogenic agents.

[0333] In certain embodiments, the active agent is

[0334]

[0335]

[0336]

[0337]

[0338] wherein Y is 1 to 10; and

[0339] The wavy line indicates the connection to the conjugate.

[0340] In certain embodiments, the active agent is a pyrrolobenzodiazepine dimer;

[0341] The pyrrolobenzodiazepine dimer is substituted with X at the N10 position, or is substituted with X′ at the N′10 position, wherein X or X′ connects the pyrrolobenzodiazepine dimer to a linker;

[0342] X and X' are each independently -C(O)O-* or -C(O)-*; and

[0343] * is the binding site between the pyrrolobenzodiazepine dimer and the linker.

[0344] In certain preferred embodiments, the pyrrolobenzodiazepine dimer is represented by Formula III:

[0345]

[0346] in

[0347] The wavy line indicates the point of attachment to the conjugate (e.g., the carbonyl group of Formula IIa is attached to B′);

[0348] Dashed lines represent optional double bonds;

[0349] R 1 and R 1 ' are each independently selected from 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 , halogen and dihalogen;

[0350] R m′ Selected from R m , CO 2 R m , COR m , CHO, CO 2 H and halogen;

[0351] R m is selected from 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, wherein when the C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 When the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl or 5- to 7-membered heteroaryl is substituted, the C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6The corresponding hydrogen atoms in the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl or 5- to 7-membered heteroaryl may each be independently replaced by a 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 heteroaryl;

[0352] R 2 , R 3 , R 5 , R 2 ′、R 3 ′ and R 5 ' are each independently selected from H, R m , OH, OR m , SH, SR m NH 2 、NHR m NR m R m′ 、NO 2 、Me 3 Sn and halogen;

[0353] R 4 and R 4 ' are each independently selected from H, R m , OH, OR m , SH, SR m NH 2 、NHR m NR m R m′ 、NO 2 、Me 3 Sn, halogen, 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 Rn 、-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′ , where C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 The hydrogen atoms in the aryl and 5- to 7-membered heteroaryl groups may be replaced independently by C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered 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 Replacement, when the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl and 5- to 7-membered heteroaryl;

[0354] R n , R n′ , R o , R o′ , R p , R p′ and R q Each independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl and 5- to 7-membered heteroaryl;

[0355] X is selected from -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 NR-;

[0356] Xa is a bond or substituted or unsubstituted C 1-6 Alkylene, where C 1-6 Alkylene, when substituted, is replaced by C 1-8 Alkyl or C 3-8 Cycloalkyl substitution;

[0357] R and R' each independently represent H, OH, NH 2 ,ONH 2 , NHNH 2 , 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, wherein the C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl and C 5-20 Aryl, when substituted, is selected from OH, N 3 、CN、NO 2 , SH, NH 2 ,ONH 2 , NHNH 2 , halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 6-12 Substitution of aryl groups by substituents;

[0358] Y and Y' are each independently selected from O, S and N(H);

[0359] R 6 is substituted or unsubstituted saturated or unsaturated C 3-12A 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 may be replaced by -NH, -NR at any one or more hydrogen atom positions 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 -R substituted or unsubstituted, wherein R m and R are as described above for R m and R, and n is 1 to 12; and

[0360] R 7 and R 7 ' are 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 、-NRr 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′ , where C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 The hydrogen atoms in the aryl and 5- to 7-membered heteroaryl groups may be replaced independently by C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered 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)ORu 、-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′ Replacement, when the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl and 5- to 7-membered heteroaryl;

[0361] R r , R r′ , R s , R s′ , R t , R t′ , R u and R u′ Each independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl and 5- to 7-membered heteroaryl;

[0362] G is a glucuronide group or a galactoside group;

[0363] Each Z is selected from H, C 1-8 Alkyl, halogen, NO 2 、CN、

[0364] R 9 , R 10 and R 16 Each independently selected from H, C 1-8 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, alkoxyalkyl, and methoxyethyl; and

[0365] n30 is 1 to 3.

[0366] In certain embodiments, the dashed line indicates that the 1 and R 7 or R 1` and R 7` There are double bonds between the carbons.

[0367] In certain embodiments, R 1 YesR m And R m is selected from 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.

[0368] In certain embodiments, R 2 , R 3 and R 5 Each is independently H or OH.

[0369] In certain embodiments, R 4 It is C 1-6 Alkoxy.

[0370] In certain embodiments, R 4 It is methoxy, ethoxy or butoxy.

[0371] In certain embodiments, X is selected from -C(O)O-, -C(O)-, and -C(O)NR-; and R each independently represents H, OH, N 3 、CN、NO 2 , SH, NH 2 ,ONH 2 , NHNH 2 , halogen, substituted or unsubstituted C 1-8 Alkyl or substituted or unsubstituted C 1-8 Alkoxy, where C 1-8 Alkyl or C 1-8 When alkoxy is substituted, it is replaced by OH, N 3 、CN、NO 2 , SH, NH 2 ,ONH 2 , NNH 2 or halogen substituted.

[0372] In certain embodiments, X is -C(O)NR-.

[0373] In certain embodiments, R 6 is substituted or unsubstituted saturated or unsaturated C3-8 Hydrocarbon chain, where

[0374] One or more of the carbon atoms of the hydrocarbon chain are replaced by a heteroatom or a substituted or unsubstituted aromatic ring, wherein the heteroatom is O, S or N(H) and the aromatic ring is benzene, pyridine, imidazole or pyrazole, and the chain or aromatic ring may be replaced at any one or more hydrogen atom positions on the chain or aromatic ring by -NHC(O)CH 2 -[OCH 2 CH 2 ] n -R or -[CH 2 CH 2 O] n -R is replaced; and

[0375] n is 1 to 6.

[0376] In certain embodiments, n is 1-10.

[0377] In certain embodiments, Xa is a bond or C 1-3 Alkylene.

[0378] In certain embodiments, z is H, Where R 9 , R 10 and R 16 Each independently selected from H, C 1-3 Alkyl, C 1-3 Alkoxy and alkoxymethyl.

[0379] In certain embodiments, R 9 It is a methoxyalkyl group.

[0380] In certain embodiments, R 10 It is a methoxyalkyl group.

[0381] In certain embodiments, R 16 It is a methoxyalkyl group.

[0382] In certain embodiments, R 9 , R 10 or R 16 Yes-(CH 2 CH 2 O) m -(CH 2 ) m2 CH 3 , further wherein m is 1 to 6 and m2 is 0 to 2.3.

[0383] In certain embodiments, R 2 It's H.

[0384] In certain embodiments, R3 It's H.

[0385] In certain embodiments, R 7 It's H.

[0386] In certain embodiments, R 4 is an alkoxy group (eg, methoxy).

[0387] In certain embodiments, R 5 It's OH.

[0388] In certain embodiments, R 1 Yes = CH 2 , CH 3 or phenyl, optionally substituted with methoxy.

[0389] In certain embodiments, Y is O.

[0390] In certain embodiments, R 2 ′ is H.

[0391] In certain embodiments, R 3 ′ is H.

[0392] In certain embodiments, R 7 ′ is H.

[0393] In certain embodiments, R 4 ' is an alkoxy group (eg, methoxy group).

[0394] In certain embodiments, R 5 ′ is OH.

[0395] In certain embodiments, R 1 ' is = CH 2 , CH 3 or phenyl, optionally substituted with methoxy.

[0396] In certain embodiments, Y′ is O.

[0397] In certain embodiments, X is -C(O)O-.

[0398] In certain embodiments, Xa is CH 2 .

[0399] In certain embodiments, G is a glucuronide group.

[0400] In certain embodiments, G is

[0401] In certain embodiments, n30 is 1.

[0402] In certain embodiments, Z is

[0403] In certain embodiments, R 9 It's H.

[0404] In certain embodiments, R 16 is an alkoxyalkyl group (eg, methoxyethyl).

[0405] In certain embodiments, Z is

[0406] In certain embodiments, R 10 is an alkyl group (eg, methyl).

[0407] In certain embodiments, R 6 is an alkyl group (eg, pentyl).

[0408] In certain embodiments, the conjugate comprises a structure selected from:

[0409]

[0410]

[0411] The bond that overlaps with the dashed line represents the point of attachment to L.

[0412] In certain embodiments, the conjugate comprises a structure selected from:

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427] Where MMAE is monomethyl auristatin E;

[0428] MMAF is monomethylauristatin F; and

[0429] The wavy line is the point of attachment to the conjugate.

[0430] In certain aspects, the present invention also provides pharmaceutical compositions, including conjugates, for preventing or treating hyperproliferative, cancer or angiogenic diseases.

[0431] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically effective amount of a chemotherapeutic agent.

[0432] 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.

[0433] The present invention also provides a pharmaceutical preparation comprising the conjugate.

[0434] Antibodies or antigen-binding fragments thereof

[0435] The present invention provides antibodies that bind to human TROP2 (tumor-associated calcium signal transducer 2, TACSTD2).As described herein, the antibodies according to the present invention are polypeptides comprising one or more complementarity determining regions or regions (CDRs).

[0436] In some embodiments, the CDRs are included in a "framework" region, and the framework orients the CDRs so that the CDRs can have appropriate antigen binding properties.

[0437] In certain aspects, the present invention provides an antibody-drug conjugate comprising an anti-TROP2 antibody that binds to TROP2. In certain embodiments, the antibodies disclosed herein bind to TROP expressed in a tumor and can be used to deliver a drug to a tumor. In certain embodiments, the antibody drug conjugate disclosed herein has improved stability compared to antibody drug conjugates known in the art.

[0438] In certain embodiments, antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, bifunctional antibodies, multispecific antibodies, multifunctional antibodies, miniantibodies, domain antibodies, antibody mimetics (or synthetic antibodies), chimeric antibodies, humanized antibodies, human antibodies or antibody fusions (or antibody conjugates), and fragments thereof, and include various forms of the antibodies disclosed herein.

[0439] In certain embodiments, antibody fragments of antibodies according to the invention include Fab, Fab'; F(ab')2, scFab, Fv, dsFv, scFV, scFv-Fc, minibodies, diabodies, scAbs or dAbs.

[0440] In certain embodiments, the antibodies of the present invention may be composed of polypeptides having only light chains or polypeptides having only heavy chains, wherein the light chains or heavy chains include the variable regions shown in Tables 1 to 3.

[0441] The CDR sequences that may be included in the heavy chain and light chain variable regions of the antibody or antigen-binding fragment thereof according to one embodiment of the present invention are shown in Tables 1 to 3, respectively.

[0442] Antibodies according to the present invention share certain regions or sequences with other antibodies disclosed herein. In certain embodiments, the constant region of the antibody or its antigen binding fragment may be shared. In certain embodiments, the Fc region may be shared. In a certain embodiment, the framework of the variable region may be shared.

[0443] The heavy chain variable region and light chain variable region according to the present invention can be connected to at least a portion of a human constant region. The selection of the constant region can be determined in part by whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not have such cytotoxicity. In addition, human IgG1 and IgG3 induce cell-mediated effector functions that are stronger than human IgG2 and IgG4. The light chain constant region can be λ or κ.

[0444] The variable regions of immunoglobulin chains generally have the same overall structure and include relatively conserved framework regions (FRs) connected by three hypervariable regions, which are called "complementarity determining regions or regions or domains" or complementarity determining regions (CDRs). The CDRs derived from the variable regions of each chain (including the heavy chain / light chain pair) are generally aligned by the framework regions to form a structure that specifically binds to a specific antigenic determinant of the target protein. From N-terminus to C-terminus, these factors of naturally occurring light and heavy chain variable regions are generally included in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The positions corresponding to the amino acid sequences of each variable region can be determined by Kabat (Kabat et al., (1983), U.S. Department 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 associated with the OPAL library (Hye Young Yang et al., 2009, Mol. Cells 27: 225). When compared to each other, the CDRs determined by each definition may be overlapping subsets or subsets that include each other. Based on the variable region sequences of the antibody, a person generally familiar with the art will be able to easily select the CDR sequences as defined above.

[0445] In certain embodiments, the antibody according to the present invention is a humanized antibody. A humanized antibody refers to any antibody in which the constant region of a non-human antibody is completely replaced by the constant region of a human form, and at least a portion of the variable region of the non-human antibody (except for the three loops of the amino acid sequence outside each variable region that is bound to the target structure) is completely or partially replaced by the corresponding portion of a human antibody.

[0446] Certain mutations can be introduced into the framework regions to enhance the stability of the antibody while maintaining its antigen binding activity. Stabilization of therapeutic antibodies can result in improved serum half-life, reduced dosage requirements, reduced side effects, improved shelf life, and reduced shipping and storage costs.

[0447] In certain embodiments, a humanized antibody according to the invention comprises:

[0448] (a) (i) a variable heavy chain framework region from a human antibody heavy chain or from a human common framework; and (ii) a variable light chain framework region from a human antibody light chain or from a human common framework.

[0449] In certain embodiments, at least one amino acid in the variable domain framework region from the heavy chain is substituted with the corresponding amino acid from a mouse antibody heavy chain or a mouse common framework; or the variable domain framework region from the light chain is substituted with the corresponding amino acid from a mouse antibody light chain or a mouse common framework.

[0450] In certain embodiments, the amino acid substitutions in the heavy chain are selected from the following amino acid substitutions:

[0451] F (mouse) substituted for Y (human) at position 27;

[0452] S (mouse) substituted for T (human) at position 30;

[0453] L (mouse) substituted for V (human) at position 37;

[0454] I (mouse) substituted for M (human) at position 48;

[0455] A (mouse) substituted for G (human) at position 49;

[0456] L (mouse) replaces I (human) at position 70; and

[0457] V (mouse) substituted for R (human) at position 72, and

[0458] (ii) The amino acid substitution of the light chain is Y (human) replaced by S (mouse) at position 49.

[0459] In certain embodiments, the amino acid substitutions of the heavy chain may further include the following amino acid substitutions:

[0460] K (mouse) replaces R (human) at position 67; and / or

[0461] At position 68, A (mouse) replaced V (human).

[0462] In certain embodiments, the amino acid substitutions of the light chain may further include the following amino acid substitutions:

[0463] Y (mouse) substituted for S (human) at position 67;

[0464] T (mouse) substituted for I (human) at position 2 and Y (mouse) substituted for S (human) at position 67;

[0465] R (mouse) substituted for T (human) at position 22 and Y (mouse) substituted for S (human) at position 67;

[0466] E (mouse) substituted for K (human) at position 42 and Y (mouse) substituted for S (human) at position 67;

[0467] S (mouse) substituted for G (human) at position 64 and Y (mouse) substituted for S (human) at position 67;

[0468] Substitution of Y (mouse) for S (human) at position 67 and of V (mouse) for T (human) at position 72; or

[0469] At position 67, Y (mouse) replaced S (human) and at position 85, D (mouse) replaced T (human).

[0470] In certain embodiments, the humanized antibody comprises: (a)(i) a variable heavy chain framework region from a human antibody heavy chain or from a human common 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 human antibody light chain or from a human common framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

[0471] In certain embodiments, the heavy and light chain variable region sequences comprising substitutions are shown in Tables 1 to 3, respectively. In certain embodiments, the substitutions comprise substitutions of the FR regions shown in Tables 1 to 3.

[0472] In certain embodiments, the present invention discloses one or more amino acid sequences having significant sequence identity with one or more amino acid sequences disclosed herein. Significant identity means that the effects disclosed herein can be maintained in the presence of sequence changes. In certain embodiments, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity with the heavy chain variable region shown in Tables 1 to 3. In another embodiment, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity with the light chain variable region shown in Tables 1 to 3. For example, with respect to variants that exhibit 90% identity, 95% identity, or 99% identity with the sequence of an antibody or antigen-binding fragment thereof according to the present invention, any mutation occurs in the framework of the variable region rather than in the CDR.

[0473] In certain embodiments, the nucleic acid encoding the antibody of the present invention or its fragment is a nucleic acid encoding a full-length antibody, which includes the CDR disclosed herein, the variable region including the CDR, and the variable region and constant region. Once the amino acid sequence is determined, the nucleic acid sequence can be easily determined according to known reverse transcription procedures, codon usage, etc.

[0474] Antigen specificity and affinity of antibodies

[0475] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the present invention are specific for human TROP2 antigen and have an affinity 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, and may be, for example, 10 -6 M to 10 -12 M.

[0476] Antibody production

[0477] In the present invention, non-human antibodies can be derived from, for example, any animal producing antibodies, such as mice, rats, rabbits, goats, donkeys or non-human primates (e.g., monkeys, such as cynomolgus macaques or rhesus monkeys) or apes (e.g., chimpanzees). Non-human antibodies can be produced by immunizing animals using methods known in the art. According to one embodiment of the present invention, a method for producing humanized antibodies is referred to U.S. Patent No. 15 / 532598. Full human antibodies can be produced in the following manner: antigens are administered to transformed animals including human immunoglobulin loci, or by treating phage display libraries expressing human antibody libraries with antigens, and then selecting target antibodies. Antibodies can be polyclonal or monoclonal antibodies, or can be synthesized in a cell host via expressing recombinant DNA.

[0478] Monoclonal antibodies (mAbs) can be produced using conventional monoclonal antibody methods, such as standard somatic cell hybridization techniques known in the literature (see: Kohler and Milstein, 1975, Nature 256:495).

[0479] Methods for expressing antibodies

[0480] The antibodies disclosed herein can be expressed in a fusion tumor cell line or an expression cell line other than a fusion tumor. The expression construct encoding the antibody can be used to transform mammalian, insect or microbial host cells. As described in the foregoing description, any of the various known methods for introducing polynucleotides into host cells can be used to produce constructs, such as plasmids. The specific method can vary according to the type of host cell. The method for introducing heterogeneous polynucleotides into mammalian cells is well known in the art, and includes but is not limited to, for example, polydextrose-mediated transfer, calcium phosphate precipitation, polycondensation-mediated transfer, protoplast fusion, electrophoresis, encapsulation of transfer polynucleotides using liposomes, mixing of nucleic acids with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0481] Use of anti-human TROP2 antibody-drug conjugates for therapeutic purposes

[0482] TROP2 expression in cancer is associated with poor prognosis in cancer patients, and is known to also affect 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 and other cancers. For example, in anti-cancer antibody therapy, anti-human TROP2 antibodies as described herein can be used in the form of being connected to various cytotoxic agents via a linker to remove cancer cells that overexpress TROP2. Therefore, the antibody combined with TROP2 can be used alone or in the form of being combined with anti-cancer chemotherapeutics, cytotoxic agents or radioactive substances, or can be embodied as a cell therapy agent (such as CAR-T cells) to target anti-cancer targets, and therefore can be used as a targeted therapeutic agent for cells expressing TROP2.

[0483] Treatment options: drug formulations and routes of administration

[0484] One embodiment of the present invention also provides a method of treating using an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided to a patient. Antibody-drug conjugates or a pharmaceutically acceptable salt or solvate thereof inhibit cancer cell metastasis by binding to human TROP2 expressed on the surface of cancer cells. In certain embodiments, antibodies are bound to human TROP2 expressed on the surface of cancer cells in the form of a cytotoxic agent, so that the cytotoxic agent bound to the antibody is specifically delivered to cancer cells to induce cancer cell death. In certain embodiments, antibodies are bound to human TROP2 expressed on the surface of cancer cells in the form of antibodies specific to the same target or another target, thereby increasing the specificity of multiple antibodies to cancer cells or inducing the connection between cancer cells and other types of cells (e.g., immune cells) to induce cancer cell death.

[0485] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. In addition, for example, a method for treating a cancer patient by administering such a pharmaceutical composition is provided. The term "patient" includes a human patient.

[0486] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The meaning of carrier includes excipients, diluents or adjuvants. Carriers may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, normal saline, buffer (e.g., PBS), methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. Compositions may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives or combinations thereof.

[0487] The pharmaceutical composition can be prepared as any formulation according to general methods. The composition can be formulated into a formulation for oral administration (e.g., powder, tablet, capsule, syrup, pill or granule) or a formulation for parenteral administration (e.g., injection). In addition, the composition can be prepared as a systemic formulation or a local formulation.

[0488] The pharmaceutical composition may include an effective amount of an antibody or its antigen binding fragment, an anticancer agent or a combination thereof. The term "effective amount" refers to an amount sufficient to produce a preventive or therapeutic effect when an individual who needs to prevent or treat is administered. Those generally familiar with the art may appropriately select an effective amount based on the selected cell or individual. The effective amount may be determined based on factors including the severity of the disease, the patient's age, body weight, health status and sex, the patient's sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the treatment period, and the drug used in combination with the composition used or used simultaneously, and other factors known in the medical field.

[0489] The dosage range of the pharmaceutical composition can be, for example, 10 gg / kg to about 30 mg / kg per adult, optionally 0.1 mg / kg to about 30 mg / kg, or 0.3 mg / kg to about 20 mg / kg. The pharmaceutical composition can be administered once a day, multiple times a day, once every 1 to 4 weeks, or once to 12 times a year.

[0490] Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

[0491] The following examples and experimental examples are intended to help understand the present invention and are not intended to limit the scope of the present invention.

[0492] definition

[0493] Unless otherwise defined in the present invention, the scientific and technical terms used herein have the meanings as generally understood by those skilled in the art. In addition, unless the context specifically requires otherwise, the singular includes the plural and the plural includes the singular. In general, the nomenclature and its techniques used in conjunction with 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.

[0494] Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. 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 Edition", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis 7th Edition", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology 6th Edition", Sinauer Associates, Inc., Sunderland, MA (2000).

[0495] Unless otherwise defined herein, chemical terms used herein are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms,” Parker, S., ed., McGraw-Hill, San Francisco, CA. (1985).

[0496] All of the above and any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In the event of a conflict, the present specification, including its specific definitions, will control.

[0497] The term "agent" is used herein to refer to a chemical compound (e.g., an organic or inorganic compound, a mixture of compounds), a biological macromolecule (e.g., nucleic acids, antibodies (including portions thereof), and humanized antibodies, chimeric antibodies, human antibodies, and monoclonal antibodies, proteins, or portions thereof, such as peptides, lipids, carbohydrates), or an extract made from biological materials (e.g., bacteria, plants, fungi, or animal (especially mammalian) cells or tissues). Agents include, for example, agents of known structure and agents of unknown structure.

[0498] "Patient", "subject" or "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (such as canines, felines, etc.), and rodents (such as mice and rats).

[0499] A substance, compound, or agent may be "administered" or "administered" to a subject using one of a variety of methods known to those familiar with the art. For example, the compound or agent may be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinal, intracranially, and transdermally (by absorption, e.g., through a skin duct). The compound or agent may also be suitably introduced by a rechargeable or biodegradable polymer device or other device, such as a patch and pump or formulation that provides for extended, slowed, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0500] The appropriate method of administering a substance, compound, or agent to an individual will also depend on, for example, the age and / or physical condition of the individual 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 orally to an individual, for example, by ingestion. In some embodiments, the orally administered compound or agent is in the form of an extended release or sustained release formulation, or is administered using a device for such sustained release or extended release.

[0501] As used herein, the phrase "administered in combination" refers to any form of administration of two or more different therapeutic agents that allows the second agent to be administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are effective in the patient's body at the same time, which can include a synergistic effect of the two agents). For example, different therapeutic compounds can be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0502] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted as well as instances where the alkyl group is not substituted.

[0503] It should be 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 produce chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and the methods given below. If a substituted radical is substituted with more than one group, it should be understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure results.

[0504] As used herein, the term "optionally substituted" means that one to six hydrogen radicals in a given structure are replaced by free radicals of specified substituents, including but not limited to hydroxy, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silicon, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH 2 -O-alkyl, -OP(O)(O-alkyl) 2 or -CH 2 -OP(O)(O-alkyl) 2 Preferably, "optionally substituted" means that one to four hydrogen radicals in a given structure are replaced by the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents mentioned above. It should be understood that the substituents may be further substituted.

[0505] The term "conjugate" as used herein refers to a cell binding agent covalently bonded to one or more molecules of a cytotoxic compound. In this regard, a "cell binding agent" is a molecule with affinity for a biological target, and can be, for example, an antibody, particularly a monoclonal antibody or an antibody fragment, and the binding agent is used to direct the biologically active compound to the biological target. In certain embodiments, the conjugate can be designed to target tumor cells via cell surface antigens. The antigen can be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen can be expressed only on proliferating cells (e.g., tumor cells). The target antigen can be selected based on differential expression (usually between proliferating tissues and normal tissues). In the present invention, the antibody is bound to a linker.

[0506] In the present invention, a "variant" of a polypeptide (e.g., an antigen-binding fragment, a protein, or an antibody) is a polypeptide in which insertions, deletions, additions, and / or substitutions occur at one or more amino acid residues compared to other polypeptide sequences, and includes fusion polypeptides. Protein variants also include variants that are modified by protein enzymatic cleavage, phosphorylation, or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding and specificity for ROR1. The 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 with the sequence of the antibody or antigen-binding fragment thereof of the present invention. The percent identity (%) or homology can be calculated by methods known in the art.

[0507] In certain embodiments, the homology or identity percentage can be calculated by 100×[(same positions) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in the sequences A and B to be compared (Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0508] In the present invention, conservative amino acid substitutions refer to substitutions that do not substantially affect the activity or antigenicity of the polypeptide. A polypeptide may include one or more conservative substitutions. Non-limiting examples thereof are shown in Table 3 below.

[0509] As used herein, the term "derivative" of a polypeptide refers to a polypeptide that is chemically modified at one or more residues via conjugation to other chemical moieties, as distinguished from insertion, deletion, addition or substitution variants.

[0510] As used herein, the term "naturally occurring" with respect to polypeptides, nucleic acids, host cells, and the like, refers to a material that occurs in nature.

[0511] The term "percentage of sequence identity" between two polypeptide or polynucleotide sequences refers to the number of consistent matching positions shared by the sequences over the comparison window, taking into account the additions or deletions (i.e., spaces) that must be introduced to optimally align the two sequences. A matching position is any position where a consistent nucleotide or amino acid is present in the target and reference sequences. The spaces presented in the target sequence are not counted because the spaces are not nucleotides or amino acids. Similarly, the spaces presented in the reference sequence are not counted because the target sequence nucleotides or amino acids are counted instead of the nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated in the following manner: the number of positions where consistent amino acid residues or nucleic acid bases are present in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the percentage of sequence identity. The comparison of sequences and the determination of the percentage of sequence identity between the two sequences can be achieved using readily available software programs. Suitable software programs are available from various sources and are used to align protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is b12seq, which is part of the BLAST software suite available from the US government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov on the World Wide Web). B12seq uses the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher (part of the EMBOSS suite of bioinformatics programs) and are also available from the European Bioinformatics Institute (EBI) at ebi.ac.uk / Tools / psa on the World Wide Web.

[0512] As used herein, "homology" with respect to peptide, polypeptide or antibody sequences refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM(DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0513] In the present invention, "affinity" is the interaction force between an antibody or its antigen-binding fragment and an antigen, and is determined based on the characteristics of the antigen, such as the size, shape and / or charge of the antigen, and the CDR sequence of the antibody or its antigen-binding fragment. Methods for determining affinity are known in the art, and the following can be used as a reference.

[0514] When the dissociation constant (KD) is ≤10 -6 M, an antibody or antigen-binding fragment thereof is said to "specifically bind" to its target (e.g., antigen). -8 When M, the antibody specifically binds to the target with "high affinity".

[0515] As used in the present invention, an "antigen-binding fragment" of a chain (heavy or light chain) of an antibody or immunoglobulin includes a portion of an antibody that lacks some amino acids compared to the full-length chain but can specifically bind to an antigen. This fragment can be considered to have biological activity because the fragment can specifically bind to a target antigen or can compete with other antibodies or their antigen-binding fragments for binding to a specific antigenic determinant. In certain embodiments, such fragments include at least one CDR present in a full-length light or heavy chain, and in some embodiments, include a short chain heavy chain and / or light chain or a portion thereof. This biologically active fragment can be produced by recombinant DNA technology or can be produced, for example, by enzymatic or chemical cleavage of a complete antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFV, scFv-Fc, bifunctional antibodies, mini antibodies, 73cab and dAb, and can be derived from any mammal, including, but not limited to, humans, mice, rats, camels or rabbits. The functional portion of the antibody (eg, one or more CDRs disclosed in the present invention) can be linked to a secondary protein or a small compound through a covalent bond and thus used as a targeted therapeutic agent for a specific target.

[0516] In the present invention, the "Fc" region includes two heavy chain fragments, which include the CH2 and CH3 domains of an antibody. The two heavy chain fragments are connected to each other through two or more disulfide bonds and the hydrophobic interaction of the CH3 domain.

[0517] In the present invention, "Fab fragment" consists of one light chain and one heavy chain, which only includes the variable region and CH1. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. In scFab, two Fab molecules are connected by a flexible linker.

[0518] In the present invention, "Fab' fragment" includes the Fab fragment and further includes the region between the CH1 and CH2 domains of the heavy chain. A disulfide bond can be formed between the two heavy chains of two molecules of the Fab' fragment to form a F(ab')2 molecule.

[0519] In the present invention, as described above, the "F(ab')2 fragment" includes two light chains and two heavy chains, which include a variable region CH1 and a portion of the constant region between the CH1 and CH2 domains, wherein an interchain disulfide bond is formed between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments, and the two Fab' fragments are bonded to each other via a disulfide bond therebetween.

[0520] In the present invention, "Fv region" is a fragment of an antibody, which includes each variable region of the heavy chain and the light chain, but does not include the constant region. In sdFV, the heavy chain and the light chain are connected by a disulfide bond. In scFc, Fv is connected by a flexible linker. In scFv-Fc, Fc is connected to scFV. In mini antibodies, CH3 is connected to scFV. Bifunctional antibodies include two molecules of scFv.

[0521] In the present invention, "single-chain Fv" or "scFv" antibody fragments include the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain (which enables the scFV to form a target structure for antigen binding) and the VL domain.

[0522] In the present invention, "short chain antibody (74cab)" is a single polypeptide chain, which includes a heavy chain constant region or a light chain variable region, wherein the heavy chain and the light chain variable region are connected by a flexible linker. For short chain antibodies, reference can be made to U.S. Patent No. 5,260,203, and short chain antibodies are disclosed herein by reference.

[0523] In the present invention, "domain antibodies (dAbs)" are immunologically functional immunoglobulin fragments that include only the heavy chain variable region or the light chain variable region. 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 can target the same or different antigens.

[0524] In the present invention, "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 usually has three CDR domains, designated CDR1, CDR2 and CDR3.

[0525] In the present invention, "framework regions" (FR) are variable domain residues other than CDR residues. Each variable domain usually has four FRs, designated FR1, FR2, FR3 and FR4.

[0526] In the present invention, a "bivalent antigen binding protein" or "bivalent antibody" includes two antigen binding sites. The two antigen binding sites included in the bivalent antibody may have the same antigen specificity, or may be a bispecific antibody in which the antigen binding sites bind to different antigens.

[0527] In the present invention, a "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or antigenic determinants.

[0528] In the present invention, "linker" refers to a compound that covalently bonds a cytotoxic compound to an antibody.

[0529] In the present invention, "unsubstituted or substituted" is used to refer to a parent group that can be unsubstituted or substituted, "substituted" refers to a parent group with at least one substituent, and a substituent refers to a chemical moiety covalently bonded to the parent group or fused to the parent group.

[0530] In the present invention, "halo" refers to fluoro, chloro, bromo, iodo and the like.

[0531] In the present invention, "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 a saturated aliphatic group, including but not limited to C 1 -C 10 Straight chain alkyl or C 1 -C 10 Preferably, "alkyl" refers to a C 1 -C 6 Straight chain alkyl or C 1 -C 6 Most preferably, "alkyl" refers to a C 1 -C 4 Straight chain alkyl or C 1 -C 4Branched chain alkyl. 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, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl, etc. "Alkyl" groups may be optionally substituted. In addition, the term "alkyl" refers to saturated aliphatic groups, including straight chain alkyl, branched chain alkyl, cycloalkyl (alicyclic) groups, cycloalkyl substituted by alkyl, and alkyl substituted by cycloalkyl. In preferred embodiments, straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., straight chain C 1-30 、Branch chain C 3-30 ), and more preferably 20 or less carbon atoms. Examples of saturated alkyl groups may include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, etc., examples of saturated straight-chain alkyl groups may include methyl, ethyl, n-propyl, n-pentyl (pentyl), n-hexyl, n-heptyl, etc., and examples of saturated branched-chain cycloalkyl groups may include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, etc.

[0532] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0533] The term "amido" is art-recognized and refers to an amine group substituted with an acyl group, and may be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0534] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0535] Term "C x-y ″ or ″C x -C y "" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is intended to include groups containing from x to y carbons in the chain. 0 Alkyl represents hydrogen when the group is in the terminal position and represents a bond when it is internal. 1-6 Alkyl groups contain from one to six carbon atoms in the chain.

[0536] The term "alkylamino" as used herein refers to an amine group substituted with at least one alkyl group.

[0537] The term "alkylthio" as used herein refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0538] As used herein, the term "amido" refers to a group

[0539]

[0540] Where R 9 and R 10 Each independently represents hydrogen or a hydrocarbon group, or R 9 and R 10 Together with the N atom to which it is attached it forms a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0541] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, such as the moiety represented by

[0542]

[0543] Where R 9 , R 10 and R 10′ Each independently represents hydrogen or a hydrocarbon group, or R 9 and R 10 Together with the N atom to which it is attached it forms a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0544] The term "aminoalkyl" as used herein refers to an alkyl group substituted with an amine group.

[0545] The term "aralkyl" as used herein refers to an alkyl group substituted with an aryl group.

[0546] The term "alkoxy" as used herein refers to -OR wherein R is an alkyl group, and examples thereof may include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy and the like.

[0547] As used herein, the term "alkenyl" refers to an alkyl group having at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups may include ethenyl (vinyl, -CH=CH 2 ), 1-propenyl (-CH=CHCH 3 ), 2-propenyl, isopropenyl, butenyl, pentenyl and hexenyl.

[0548] In the present invention, the term "alkynyl" as used herein refers to an alkyl group having at least one carbon-carbon triple bond, and examples of the unsaturated alkynyl group may include ethynyl and 2-propynyl.

[0549] The term "carboxy" as used herein refers to -C(=O)OH.

[0550] The term "formyl" as used herein refers to -C(=O)H.

[0551] 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. 5-7 "Aryl" refers to a moiety having 5 to 7 ring atoms, which is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and the term "C 5-10 "Aryl" refers to a moiety having 5 to 10 ring atoms which is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. In this context, the prefix (C 5-7 , C 5-10 , etc.) refers to the number of ring atoms or a range of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, the term "C 5-6 "Aryl" refers to an aromatic group having 5 or 6 ring atoms. In this regard, the ring atoms may all be carbon atoms, such as a "carboaryl". Examples of carboaryl groups include, but are not limited to, carboaryl groups derived from benzene, naphthalene, indene, anthracene, phenanthrene, fused tetraphenyl and pyrene. Examples of aromatic groups containing fused rings (at least one of which is aromatic) include, but are not limited to, groups derived from indane, indene, isoindene, tetrahydronaphthalene, acenaphthene, fluorene, petroleum, acephenanthrene and aceanthrene. Alternatively, the ring atoms may contain one or more heteroatoms, such as a "heteroaryl".

[0552] The term "carbamate" is art-recognized and refers to a group

[0553]

[0554] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0555] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0556] The term "carbocycle" includes 5-7 membered monocyclic rings and 8-12 membered bicyclic rings. Each ring of the bicyclic carbocycle can be selected from saturated, unsaturated and aromatic rings. Carbocycles include bicyclic molecules in which one, two or three or more 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 another ring. Each ring of the 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, such as cyclohexane, cyclopentane or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings is included in the definition of carbocycle when the valence permits. 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 decahydronaphthalene, 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. "Carbocycle" may be substituted at any position or positions capable of having a hydrogen atom.

[0557] The term "carbonate" is art-recognized and refers to the group -OCO 2 -.

[0558] As used herein, the term "carboxyl" refers to a group consisting of the formula -CO 2 The group represented by H.

[0559] The term "cycloalkyl" as used herein refers to an alkyl group as a cyclic 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, cycloalkyl groups 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, norpinene, and norcamphene.

[0560] As used herein, the term "ester" refers to the group -c(O)OR 9 , where R 9 It represents a hydrocarbon group.

[0561] The term "ether" as used herein refers to a hydrocarbyl group connected to another hydrocarbyl group via an oxygen. Thus, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. An ether group may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

[0562]

[0046] The term "halo" or "halogen" as used herein refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0563] As used herein, the term "hetaralkyl" or "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group.

[0564] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0565] The term "heterocyclylalkyl" as used herein refers to an alkyl group substituted with a heterocyclyl group.

[0566] The term "heteroaryl" as used herein refers to an aryl group containing one or more heteroatoms, and examples thereof may include pyridine; pyrimidine; benzothiophene; furanyl; dioxolanyl; pyrrolyl; oxazolyl; pyridyl; pyridazinyl and pyrimidinyl. More specifically, examples thereof may include benzofuran; isobenzofuran; indole; isoindole; indolizine; indoline; isoindoline; purine (adenine or guanine); benzimidazole; indazole; benzoxazole; benzisoxazole; benzo; oxolene; benzofuran; benzotriazole dibenzothiofuran; benzothiazole; a C having two fused rings derived from benzothiazole, chromene, isochromene, chroman, isochroman, benzodioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, pyrazine, naphthyridine 9 ; C having two fused rings derived from pteridine 10 ; C having two fused rings derived from benzodiazepine, carbazole, dibenzofuran, dibenzothiophene, carboline, pyrimidine 11 ; C having three fused rings derived from pyridoindole, acridine, xanthene, thioxanthene, dibenzo-p-dioxin, phenoxathioin, phenoxazine, phenoxazine, phenathiazine, thiophene, phenanthridine, phenanthroline 13 ; and C having three fused rings derived from phenazine 14 .

[0567] As used herein, the term "heterocyclyl" refers to a monovalent moiety obtained by removing hydrogen atoms from the ring atoms of a heterocyclic compound. In addition, the terms "heterocyclyl" or "heterocycle" as used herein refer to a monosaturated or partially unsaturated non-aromatic compound or a non-aromatic polycyclic system, wherein the ring includes at least one heteroatom (i.e., at least one ring heteroatom selected from oxygen, nitrogen and sulfur). Unless otherwise specified, a heterocyclyl has 5 to about 20 ring atoms, such as 3 to 12 ring atoms, such as 5 to 10 ring atoms. Therefore, the term includes a monosaturated or partially unsaturated ring (e.g., 3, 4, 5, 6 or 7-membered ring) having about 1 to 6 ring carbon atoms and about 1 to 3 ring heteroatoms selected from oxygen, nitrogen and sulfur in the ring. The rings of the polycondensed ring system can be connected to each other via fusion, spiro and cross-linking bonds, as long as the valence requirements are met. Examples of heterocycles include azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidone, tetrahydrofuran, tetrahydrothiophene, dihydropyridine, tetrahydropyridine, quinuclidine, N-bromopyrrolidine, N-chloropiperidine, and the like.

[0568] As used herein, the term "hydrocarbyl" refers to a group that is bonded via a carbon atom, does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl, but substituents such as acetyl (which has a =O substituent on the attached carbon) and ethoxy (which is attached via oxygen rather than carbon) are not hydrocarbyl. Hydrocarbyl includes, but is not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0569] The term "hydroxyalkyl" as used herein refers to an alkyl group substituted with a hydroxy group.

[0570] The term "lower carbon" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy is intended to include groups having ten or less, preferably six or less atoms in the substituent. "Lower alkyl" refers, for example, to an alkyl group containing ten or less, preferably six or less carbon atoms. In certain embodiments, an acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituent as defined herein is a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, respectively, whether alone or in combination with other substituents, such as in the described hydroxyalkyl and aralkyl groups (in which case, for example, when counting the carbon atoms in the alkyl substituent, the atoms in the aryl group are not counted).

[0571] The terms "polycyclic group", "polycyclic" 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 shared by two adjacent rings, e.g., such rings are "fused rings". Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10, preferably 5 to 7 atoms in the ring.

[0572] The term "sulfate" is art-recognized and refers to the group -OSO 3 H or a pharmaceutically acceptable salt thereof.

[0573] The term "sulfonamido" is art-recognized and refers to a group represented by the general formula

[0574]

[0575] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0576] The term "sulfoxide" is art-recognized and refers to the group -S(O)-.

[0577] The term "sulfonate" is art-recognized and refers to the group -SO 3 H or a pharmaceutically acceptable salt thereof.

[0578] The term "sulfone" is art-recognized and refers to the group -S(O) 2 -.

[0579] The term "substituted" refers to a substituent having a hydrogen on one or more carbons of the main chain that replaces the moiety. It should be understood that "substituted" or "substituted" includes implicit restrictions, i.e., such substitutions are consistent with the allowed valences of the substituted atom and the substituent, and the substitution produces a stable compound, such as it will not spontaneously transform, for example, by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include non-cyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For suitable organic compounds, permissible substituents may be one or more and the same or different. For purposes of the present invention, for example, a heteroatom of nitrogen may have a hydrogen substituent and / or any permissible substituent of an organic compound described herein that satisfies the heteroatom valence. Substituents may include any of the substituents described herein, such as halogen, hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfonamide, sulfonamide, sulfonyl, heterocyclic, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted where appropriate.

[0580] As used herein, the term "sulfanyl" refers to an alkyl group substituted with a thiol group.

[0581] As used herein, the term "thioester" refers to the group -C(O)SR 9 or -SC(O)R 9 , where R 9 It represents a hydrocarbon group.

[0582] As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

[0583] The term "urea" is art-recognized and can be represented by the general formula

[0584]

[0585] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0586] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (eg, cell proliferation) as well as enhancing a function or activity.

[0587] A variety of compounds suitable for use in the methods and compositions of the present invention have at least one stereocenter in their structure. This stereocenter may be present in the R or S configuration, and the R and S symbols are used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present invention encompasses all stereoisomeric forms (including all possible mixtures of stereoisomers) of, for example, enantiomeric and diastereomeric forms of compounds, salts, prodrugs, or mixtures thereof. See, for example, WO 01 / 062726.

[0588] Furthermore, certain compounds containing alkenyl groups may exist as either Z (same side) or E (different side) isomers. In each case, the present invention includes both the mixture and the separate individual isomers.

[0589] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in the host to form a compound of the invention (e.g., a compound of formula I) after administration. Typical examples of prodrugs include compounds having biologically labile or cleavable (protecting) groups on the functional moiety of the active compound. Prodrugs include compounds that can undergo oxidation, reduction, amination, deamination, hydroxylation, dehydroxylation, hydrolysis, dehydrolysis, alkylation, dealkylation, acylation, deacylation, phosphorylation, or dephosphorylation to produce the active compound. Examples of prodrugs using esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present invention are metabolized to produce compounds of formula I. The scope of the present invention includes 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.

[0590] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material which is suitable for formulating a medicament for medical or therapeutic uses.

[0591] As used herein, the terms "Log of solubility", "LogS" or "logS' are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is generally associated with poor absorption. The LogS value is the unit exfoliation logarithm (base 10) of the solubility measured in moles / liter.

[0592] In the present invention, the prefix (such as C 1-12 , C3-8 , etc.) refers to the number of ring atoms or the number range of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, as used herein, the term "C 3-6 "Heterocyclyl" refers to a heterocyclic group having 3 to 6 ring atoms.

[0593] As used herein, the term "prodrug" refers to a compound that can be directly or indirectly converted into a pyrrolobenzodiazepine drug by the action of an enzyme or gastric acid under physiological conditions (eg, enzymatic oxidation, reduction and / or hydrolysis) in vivo.

[0594] In the present invention, the "pharmaceutically acceptable salt" may be an acid addition salt formed from a pharmaceutically acceptable free acid, and an organic acid or an inorganic acid may be used as the free acid.

[0595] 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, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid. Again, examples of inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0596] For example, when the compound has an anion or a functional group that may be an anion (e.g., -COOH may be -COO-), the compound may form a salt with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (e.g., Na + and K + ), alkaline earth metal cations (such as Ca 2+ and Mg 2+ ) and other cations (such as Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g. NH 3 R + NH 2 R 2 + 、NHR 3 + and NR 4 + ).

[0597] Some examples of suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. Examples of typical quaternary ammonium ions include N(CH 3 ) 4 + .

[0598] When the compound has a cationic or potentially cationic functional group (e.g. -NH 2 It can be -NH 3 + ), the compound can 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.

[0599] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic 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, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfonamide, 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 carboxymethylcellulose.

[0600] The term "solvate" as used herein refers to a molecular complex between a compound according to the present invention and a solvent molecule, and examples of such solvent molecules include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or a compound according to the present invention bound to a mixed solvent.

[0601] It may be convenient or desirable to prepare, purify and / or dispose of equivalent solvates 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 a salt thereof) and a solvent. When the solvent is water, the solvate may be appropriately referred to as a hydrate, such as a monohydrate, a dihydrate, or a trihydrate.

[0602] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount required to achieve the target therapeutic result (for the number of administrations and the period and mode of administration). The effective dose is the minimum active dose necessary for the subject to obtain the minimum therapeutic effect, and is less than the toxic dose. For example, the dose per patient may be in the range of about 100 ng to about 100 mg / kg, more usually in the range of about 1 μg / kg to about 10 mg / kg. When the active compound is a salt, ester, amide, prodrug, etc., the dose is calculated based on the parent compound, and therefore the actual weight used is increased proportionally. The pyrrolobenzodiazepine compound according to the present invention can be formulated to include 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 invention is not limited thereto. The active ingredient can be administered so that the peak plasma concentration of the active compound reaches 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 w / v% to 5 w / v% solution of the active ingredient in saline may be administered via intravenous injection.

[0603] The concentration of the active compound in the pharmaceutical composition can be determined by the absorption, inactivation and excretion rate of the drug and other factors generally known to those familiar with the art. The dosage can vary depending on the severity of the symptoms / disease. In addition, the dosage and administration method of a particular patient can be adjusted according to the professional judgment of the medication supervisor, taking into account the severity of the patient's symptoms / disease, necessity, age, drug response, etc., and the concentration ranges described herein are only exemplary and do not wish to limit the embodiments of the desired composition. In addition, the active ingredient can be administered once, or smaller doses of the active ingredient can be administered in multiple doses.

[0604] The term "linker" as used herein refers to a compound that covalently bonds a cytotoxic compound to an antibody.

[0605] As used herein, the term "intracellularly cleaved" or "intracellular cleavage" refers to a metabolic process or reaction of an antibody construct-active agent conjugate within a cell whereby the covalent attachment (e.g., linker) between the active agent (B) and the antibody construct (Ab) is broken, thereby causing the free drug or other metabolites of the conjugate to separate from the antibody within the cell.

[0606] Example

[0607] Having generally described the invention, the invention 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 limit the invention.

[0608] Example 1: Antibodies specific for human TROP2 (tumor-associated calcium signal transducer 2, TACSTD2) Production

[0609] Anti-TROP2 humanized antibodies 2G10-5 and 2G10-6 were produced by the method described in U.S. Pat. No. 15 / 532598, which more strongly and specifically bind to TROP2 (tumor-associated calcium signal transducer 2, TACSTD2) mainly expressed in tumors. In addition, the amino acid sequences of the antibodies are shown in Tables 1 and 2 below.

[0610] [Table 1]

[0611]

[0612]

[0613] [Table 2]

[0614]

[0615]

[0616] In addition, for ADC synthesis, the antibody strain 2G10-5-CaaX was constructed by fusing the CaaX peptide portion (GGGGGGGCVIM; SEQ ID NO: 22) to the C-terminus of the light chain (SEQ ID NO: 18) of the antibody 2G10-5, and the antibody was produced via transient expression based on CHO cells. The produced antibody 2G10-5-CaaX was used for ADC synthesis. The amino acid sequence of the antibody is shown in Table 3.

[0617] [Table 3]

[0618]

[0619]

[0620] Example 2: Synthesis of Compound 1, Compound 2, Compound 3 and Compound 4

[0621] <2-1> Preparation of Compound 1

[0622]

[0623] Compound 1 was prepared using the method described in Korean Patent Application No. 10-2018-7018068.

[0624] The structure of monomethyl auristatin E (MMAE) in compound 1 is as follows:

[0625]

[0626] <2-2> Preparation of Compound 2

[0627]

[0628] Compound 2 was prepared using the method described in Korean Patent Application No. 10-2018-0036895.

[0629] <2-3> Preparation of Compound 3

[0630]

[0631] Compound 3 was prepared using the method described in Korean Patent Application No. 10-2020-0188314.

[0632] <2-4> Preparation of Compound 4

[0633]

[0634] Compound 4 was prepared using the method described in Korean Patent Application No. 10-2018-0036895.

[0635] Example 3: Synthesis and preparation of ADC

[0636] ADC is prepared via the following two steps, and the commonly used LCB14-0606 and LCB14-0512 are 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:

[0637]

[0638] Step 1: Production of polyprenylated antibodies

[0639] <Production of polyprenylated antibodies using LCB14-0606>

[0640] The polyisoprenylation reaction mixture of the antibody 2G10-5-CaaX of Example 1 was prepared and the reaction was allowed to proceed at 30° C. for 16 hours. The reaction mixture consisted of 24 μM antibody, 600 nM farnesyl transferase (Genscript), and a buffer solution (50 mM Triss-HCl (pH 7.4), 5 mM MgCl 2 , 10 μM ZnCl 2 After the reaction was completed, the polyisoprenylated antibody was desalted using a G25 sepharose column (AKTA purifier, GE healthcare) equilibrated with a PBS buffer solution.

[0641] <Production of polyprenylated antibodies using LCB14-0512≥

[0642] The polyisoprenylation reaction mixture of the antibody 2G10-5-CaaX of Example 1 was prepared and the reaction was allowed to proceed at 30° C. for 2 hours. The reaction mixture consisted of 24 μM antibody, 600 nM farnesyl transferase (Genscript), and a buffer solution (50 mM Triss-HCl (pH 7.4), 5 mM MgCl 2 , 10 μM ZnCl 2 After the reaction was completed, the polyisoprenylated antibody was desalted using a G25 sepharose column (AKTA purifier, GE healthcare) equilibrated with a PBS buffer solution.

[0643] Step 2: Drug combination method

[0644] <Bonding via oxime bond formation>

[0645] A mixture for the oxime bond formation reaction between the polyisoprenylated antibody and the linker-drug was prepared by mixing 100 μM Na-acetate buffer solution pH 5.2, 10% DMSO, 24 μM polyisoprenylated antibody and 240 μM linker-drug (in-house, compound 1, 2 or 3 of Example 2), and gently stirring at 30° C. After reacting for 6 hours or 24 hours, the FLPC (AKTA purifier, GE healthcare) process was performed to remove excess small compounds used, and the protein fractions were collected and concentrated.

[0646] <Conjugation by copper-free click reaction>

[0647] In order to prepare ADC using the copper-free click coupling reaction between the polyisoprenylated antibody and the linker-drug, PBS buffer solution (pH 7.4), 1% DMSO, 10 μM polyisoprenylated antibody and 100 μM linker-drug (in-house, compound 4 in Example 2) were mixed to obtain a reaction mixture, and after reacting at 25° C. for 6 hours, excess small molecule compounds were removed via FPLC (AKTA purifier, GE healthcare) or G25 agarose gel column process, and the protein eluate fractions were collected and concentrated.

[0648] The ADCs generated by the above two steps are shown in Tables 4 and Figures 1 to 4 middle.

[0649] [Table 4]

[0650]

[0651]

[0652] <Experimental Example 1> In vitro cytotoxicity evaluation

[0653] The cell proliferation inhibitory activity of the ADC produced according to <Example 2> and the drug included in the ADC alone on cancer cell lines was measured.

[0654] Specifically, 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) commercially available as cancer cell lines were used. MMAE in the form of a single drug included in the ADC prepared according to Example 3 and SG2057 (cAS#: 260417-62-7, MedKooBiosciences) as a PBD dimer were used. 500 cells / well to 5,000 cells / well of each cancer cell line were seeded into a 96-well plate and cultured for 24 hours, and then each ADC1, ADC2 and ADC3 produced according to Example 3 and individual drugs MMAE and SG2057 were treated at concentrations ranging from 0.256 pM to 100 nM (5-fold serial dilutions). After 144 hours, the number of viable cells was quantified using sulforhodamine B (SRB) dye or Cell titer glo.

[0655] Therefore, as shown in Tables 5 to 7, it was confirmed that the pyrrolobenzodiazepine-based antibody-drug conjugates (ADC2 and ADC3) exhibited stronger cytotoxicity in most cancer cell lines compared to the auristatin-based antibody-drug conjugate (ADC1).

[0656] [Table 5]

[0657]

[0658] [Table 6]

[0659]

[0660] [Table 7]

[0661]

[0662] <Experimental Example 2> Analysis of in vivo tumor growth inhibition efficacy

[0663] The tumor growth inhibitory efficacy of the ADC produced according to Example 3 was analyzed in a mouse model of transplanted tumors.

[0664] 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. The cultured cells were suspended in 100 μl PBS so that the cell number became 1×10 6 Up to 7×10 6 , mixed with 100 μl of Matrigel, and then transplanted into Balb / c-nude mice. When the tumor size reached 100 mm 3 Up to 200mm 3 Each ADC prepared according to Example 3 was intravenously administered according to the dosing regimen shown in Tables 8 to 10 below. The size of the tumor implanted in the mouse was measured just before the first administration (Day 1), and then measured regularly for 56 days. As a control, Trodelvy (sacituzumab govitecan-hziy) or DS-1062BS (Datopotamab deruxtecan) was administered.

[0665] [Table 8]

[0666] First time (QD×1) MDA-MB-468 BxPC-3 ADC1 2.4mpk 2.4mpk ADC2 0.2, 0.3, 0.4 mpk - ADC3 1.4mpk -

[0667] [Table 9]

[0668]

[0669] [Table 10]

[0670]

[0671] Therefore, if Figure 5A and Figure 5B As shown in FIG, the first experiment confirmed that when an auristatin-based antibody-drug conjugate (ADC1) using an oxime reaction was administered to a mouse model transplanted with a pancreatic cancer cell line, both 2 mg / kg and 4 mg / kg showed tumor growth inhibition efficacy in a dose-dependent manner compared with the control group ( Figure 5AIn a mouse model transplanted with breast cancer cell lines, both the group administered with an auristatin-based antibody-drug conjugate (ADC1) using an oxime reaction and the group administered with pyrrolobenzodiazepine-based antibody-drug conjugates (ADC2 and ADC3) demonstrated tumor growth inhibitory efficacy.

[0672] like FIG. 6A to FIG. 6D As shown, and also confirmed by a second experiment, ADC1 has excellent efficacy in all four mouse models of transplanted cancer cell lines (MDA-MB-468, BxPC-3, NCI-N87 and HCC827) compared with the marketed drug Trodelvy.

[0673] like 7A to 7E As shown, the third experiment also confirmed that ADC1 had excellent efficacy in all five mouse models of transplanted cancer cell lines (BxPC-3, HCC827, Capan-1, NCI-H2170 and HT-29) compared with the marketed drug Trodelvy, and the efficacy of ADC1 was comparable to that of the competing drug DS-1062BS.

Claims

1. A conjugate having a structure represented by general formula I or a pharmaceutically acceptable salt thereof: [General Formula I] Ab-[L-(B)1]m in, Ab is an anti-TROP2 (tumor-associated calcium signal transducer 2, also known as TACSTD2) antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein 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; and 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 agent; and l and m are each independently 1 to 20.

2. The conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising: The amino acid sequence of SEQ ID NO: 15 or 20; having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 4 and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6; or having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 or 20, while maintaining the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, The heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:4 and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

3. The conjugate according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising: The amino acid sequence of SEQ ID NO: 16; having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or Having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:16, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

13. 4 . The conjugate of claim 1 , wherein 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.

5. The conjugate according to any one of claims 1 to 4, wherein the antibody is a humanized antibody.

6. The conjugate according to any one of claims 1 to 5, wherein the humanized antibody comprises: (i) a variable heavy chain framework region from a human antibody heavy chain or from a human common 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 human antibody light chain or from a human common framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

7. The conjugate according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is 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, a 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 bifunctional antibody, a bispecific antibody, and a multispecific antibody.

8. The conjugate according to any one of claims 1 to 7, wherein L is cleavable.

9. The conjugate according to any one of claims 1 to 8, wherein the conjugate has a structure represented by general formula IIa or a pharmaceutically acceptable salt thereof: [General Formula IIa] in Each B′ is an active agent; Each G is independently a glucuronic acid moiety or R 3 is hydrogen or a carboxyl protecting group; Each R 4 are independently hydrogen or a hydroxy protecting group; R 1 and R 2 are independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl; W is -C(O)-, -C(O)NR′-, -C(O)O-, -SO2NR′-, -P(O)R″NR′, -SONR′-, or -PO2NR′-, wherein C, S, or P is directly bonded to the benzene 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl; Each Z is independently hydrogen, C 1-8 Alkyl, halogen, cyano or nitro; n is 0, 1, 2, or 3; L contains: A)C 1-50 Alkylene or C 1-50 Heteroalkylene, and comprising (i) one or more unsaturated bonds (ii) heteroarylene; or (iii) by at least one C 1-20 Alkyl substitution; or B) at least one isoprenyl group having a structure represented by the general formula III: [General Formula III] and l and m are each independently 1 to 20.

10. A conjugate having a structure represented by the general formula IIa or a pharmaceutically acceptable salt thereof: [Formula IIa] in Ab is an anti-TROP2 (tumor-associated calcium signal transducer 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 R 3 is hydrogen or a carboxyl protecting group; Each R 4 are independently hydrogen or a hydroxy protecting group; R 1 and R 2 are independently hydrogen, C 1-8 Alkyl or c 3- 8 cycloalkyl; W is -C(O)-, -C(O)NR′-, -C(O)O-, -SO2NR′-, -P(O)R″NR′, -SONR′-, or -PO2NR′-, wherein C, S, or P is directly bonded to the benzene 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl; Each Z is independently hydrogen, C 1-8 Alkyl, halogen, cyano or nitro; n is 0, 1, 2, or 3; L contains: A)C 1-50 Alkylene or C 1-50 Heteroalkylene, and comprising (i) one or more unsaturated bonds (ii) heteroarylene; or (iii) by at least one C 1-20 Alkyl substitution; or B) at least one isoprenyl group having a structure represented by the general formula III: [General Formula III] and 1 and m are each independently 1 to 20.

11. The conjugate of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: 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; and 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.

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

13.

13. The conjugate according to claim 11 or 12, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising: The amino acid sequence of SEQ ID NO: 16; having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or Having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:16, while maintaining the sequence of the light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO:11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

13. 14 . The conjugate of claim 11 , wherein 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.

15. The conjugate according to any one of claims 11 to 14, wherein the antibody is a humanized antibody.

16. The conjugate according to any one of claims 11 to 15, wherein the humanized antibody comprises: (i) a variable heavy chain framework region from a human antibody heavy chain or from a human common 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 human antibody light chain or from a human common framework, wherein the variable light chain framework region comprises the following amino acid sequence change: Y49S.

17. The conjugate of any one of claims 11 to 16, wherein the antibody or antigen-binding fragment thereof is 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, a 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 bifunctional antibody, a bispecific antibody, and a multispecific antibody.

18. The conjugate according to any one of claims 9 to 17, wherein each G is 19. The conjugate according to any one of claims 9 to 18, wherein R 1 and R 2 Each is hydrogen.

20. The conjugate according to any one of claims 9 to 19, wherein R 3 It's hydrogen.

21. The conjugate according to any one of claims 9 to 20, wherein each R 4 It is a hydroxyl protecting group.

22. The conjugate according to any one of claims 9 to 21, wherein n is 0.

23. The conjugate according to any one of claims 9 to 22, wherein each W is -C(O)NR'-, further wherein C is directly bonded to the phenyl ring of formula IIa and NR' is bonded to L.

24. The conjugate according to any one of claims 9 to 23, wherein each R 4 are independently hydrogen.

25. The conjugate according to any one of claims 9 to 18, wherein: R 1 and R 2 Each is hydrogen; n is 0; and Each W is -C(O)NR'-, C is directly bonded to the benzene 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 Aryl, wherein NR′ is bonded to L.

26. The conjugate of any one of claims 9 to 25, wherein L comprises a nitrogen-containing 1 to 50-membered heteroalkylene group.

27. The conjugate of any one of claims 9 to 26, wherein L comprises a hydrophilic amino acid.

28. The conjugate of claim 27, wherein W comprises two or more atoms of the hydrophilic amino acid, and the nitrogen of W forms a peptide bond with the carbonyl group of the hydrophilic amino acid.

29. The conjugate according to any one of claims 9 to 28, wherein L is a nitrogen-containing 1- to 50-membered heteroalkylene group, The linker comprises two or more atoms of hydrophilic amino acids, and The nitrogen forms a peptide bond with the carbonyl group of the hydrophilic amino acid.

30. The conjugate according to any one of claims 9 to 29, wherein L is covalently bonded to the antibody via a thioether bond and the thioether bond comprises a sulfur atom of a cysteine ​​of the antibody.

31. The conjugate of claim 30, wherein: The antibody comprises an amino acid motif capable of being recognized by an isoprenoid transferase at the C-terminus of the antibody, and The thioether bond comprises the sulfur atom of the cysteine ​​of the amino acid motif.

32. The conjugate of claim 31, wherein the amino acid motif has a CYYX sequence, and further wherein: C is cysteine; Y is an aliphatic amino acid; X is selected from the group consisting of glutamine, glutamic acid, serine, cysteine, methionine, alanine and leucine; and The thioether bond comprises the sulfur atom of the cysteine ​​of the amino acid motif.

33. The conjugate of claim 31, wherein the amino acid motif has a CYYX sequence, and further wherein: Y is selected from the group consisting of alanine, isoleucine, leucine, methionine and valine. The conjugate of claim 31 , wherein the amino acid motif has a CVIM (SEQ ID NO: 24) or CVLL sequence (SEQ ID NO: 25).

35. The conjugate of any one of claims 31 to 34, wherein at least one of the first 1 to 20 amino acids of the amino acid motif is glycine.

36. The conjugate of any one of claims 1 to 35, wherein L comprises the amino acid sequence of GGGGGGGCVIM (SEQ ID NO: 22) at the C-terminus.

37. The conjugate according to any one of claims 1 to 36, wherein L comprises C 1-50 Heteroalkylene.

38. The conjugate of any one of claims 1 to 37, wherein L comprises an oxime.

39. The conjugate of claim 38, wherein the oxygen atom of the oxime is at the side where L is attached to W and the carbon atom of the oxime is at the side where L is attached to Ab.

40. The conjugate of claim 38, wherein the carbon atom of the oxime is on the side where L is attached to W and the oxygen atom of the oxime is on the side where L is attached to Ab.

41. The conjugate according to any one of claims 1 to 36, wherein L is C containing oxime 1-50 Heteroalkylene, The oxygen atom of the oxime is on the side where L is connected to W, The carbon atom of the oxime is at the side where L is attached to Ab, or The carbon atom of the oxime is on the side where L is connected to W, and The oxygen atom of the oxime is at the side where L is attached to Ab.

42. The conjugate of any one of claims 1 to 41, wherein L comprises an oxime, and at least one prenyl unit covalently bonds the oxime to Ab (eg, at least one prenyl unit directly or indirectly bonds the oxime to Ab).

43. The conjugate according to any one of claims 1 to 42, wherein L further comprises a linking unit represented by Formula VIII or Formula IX: [General Formula VIII] -(CH2) r (V(CH2) p ) q - [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 -; X is -O-, C 1-8 Alkylene or -NR 21 -; R 21 To R 25 are independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl-C 3-20 heteroaryl; r is 0 to 10; p is 0 to 10; q is 1 to 20; and w is 1 to 20. The conjugate of claim 43 , wherein q is 1 to 10.

45. The conjugate of claim 43 or 44, wherein r is 1 or 2.

46. ​​The conjugate of any one of claims 43 to 45, wherein p is 1 or 2.

47. The conjugate of any one of claims 43 to 46, wherein V is -O-.

48. The conjugate of claim 43, wherein: q is 1 to 10; r and p are each 1 or 2; and V is -O-.

49. The conjugate of any one of claims 43 to 48, wherein X is -O-.

50. The conjugate of any one of claims 43 to 48, wherein w is 1 to 10.

51. The conjugate according to any one of claims 43 to 48, wherein: Said X is -O-; and w is 1 to 10.

52. The conjugate of any one of claims 43 to 51, wherein L comprises wherein n40 is 1 to 10, preferably at least 2.

53. The conjugate of any one of claims 43 to 52, wherein L comprises an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

54. The conjugate according to any one of claims 43 to 53, wherein L comprises a bonding unit formed by the reaction between an alkyne and an azide group or between an aldehyde or ketone group and hydrazine or hydroxylamine.

55. The conjugate according to any one of claims 43 to 54, wherein L further comprises a binding unit represented by the following general formula IVa, IVb, IVc, IVd or IVe: [General Formula IVa] [General Formula IVb] [General Formula IVc] [General Formula IVd] [General Formula IVe] in L1 and L2 are each independently a single bond or C 1-30 an alkylene group; and R 11 is hydrogen or C 1-10 alkyl.

56. The conjugate of claim 55, wherein L1 and L2 are each independently a single bond, C 11 Alkylene or C 12 Alkylene.

57. The conjugate of any one of claims 3 to 56, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

58. The conjugate of any one of claims 1 to 57, wherein L is branched and comprises: i) a branching unit covalently coupled to the antibody via a primary linker; ii) a first branch, wherein a first active agent is covalently coupled to the branch unit via a secondary linker and a cleavable group; and iii) the second branch, in which: a) a second active agent is covalently coupled to the branching unit via a secondary linker and a cleavable group; or b) A polyethylene glycol moiety is covalently coupled to the branching unit.

59. The conjugate of claim 58, wherein the branching unit has The structure represented by L 2 , L 3 and L 4 Each independently is a bond or -C n H 2n -, n is 1 to 30; G 1 , G 2 and G 3 Each independently represents a key, R 30 is hydrogen or C 1-30 alkyl; R 40 is hydrogen or L 5 -COOR6; and L 5 is a key or -C n′ H 2n′ -; n′ is 1 to 10; and R6 is hydrogen or C 1-30 alkyl.

60. The conjugate of claim 58 or 59, wherein the cleavage group is capable of being cleaved in a target cell and is capable of releasing one or more active agents.

61. The conjugate according to any one of claims 58 to 60, wherein: At least one branched linker is covalently coupled to Ab; and At least two active agents are covalently coupled to the branched linker.

62. The conjugate of claim 61, wherein one branched linker is coupled to Ab.

63. The conjugate of claim 61, wherein two branched linkers are coupled to Ab.

64. The conjugate of claim 61, wherein three branched linkers are coupled to Ab.

65. The conjugate of claim 61, wherein four branched linkers are coupled to Ab.

66. The conjugate of any one of claims 59 to 65, wherein each branched linker is coupled to two active agents.

67. The conjugate of any one of claims 59 to 66, wherein the conjugate comprises at least two different active agents.

68. The conjugate of any one of claims 59 to 67, wherein at least one branched linker is coupled to two different active agents.

69. The conjugate of any one of claims 59 to 68, wherein the branching unit is a nitrogen atom.

70. The conjugate of any one of claims 59 to 68, wherein the branching unit is an amide and the primary linker comprises the carbonyl group of the amide.

71. The conjugate of any one of claims 59 to 69, wherein the branching unit is an amide and the secondary linker comprises the carbonyl group of the amide.

72. The conjugate of any one of claims 59 to 71, wherein the branching unit is lysine.

73. The conjugate of any one of claims 1 to 72, wherein the conjugate comprises a structure represented by: or a pharmaceutically acceptable salt thereof; wherein B′ and B″ are each active agents; n1 to n3 are each independently 0 to 30; and AA is the amino acid group.

74. The conjugate of any one of claims 1 to 73, wherein the conjugate comprises a structure represented by: or a pharmaceutically acceptable salt thereof; wherein B′ and B″ refer to active agents that are the same or different from each other; The pyrrolobenzodiazepine dimer, for example, a compound of formula III; and m and n each independently mean 0 to 30, or a pharmaceutically acceptable salt thereof.

75. The conjugate of any one of claims 1 to 74, wherein the active agent is a chemotherapeutic agent or a toxin.

76. The conjugate of any one of claims 1 to 75, 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.

77. The conjugate of any one of claims 1 to 73, wherein the active agent is selected from: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, mitramycin meturedopa, uredopa, ethylenimine, hexamethylmelamine, triethylenmelamine, triethylenphosphoramide, triethylenthiophosphoramide, trihydroxymethylmelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1 1), Nostoc 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustardmustard), carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore chromophore), aclacinomysins, actinomycin, antrmycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, lipid doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C C) mycophenolic acidacid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimesacil trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, aldehyde folate, acetylglucosyl ester, aldehyde phosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomithine, elliptinium acetateacetate), etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, raZoXane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid acid), triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin A, roridin AA), anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, boplatin), vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid thereof; (b) Monokine, lymphoid mediators, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, platelet production cytokines, erythropoietin, osteoinductive factors, interferons, interferons-α, interferons-β, interferons-γ, colony stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins (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 factors, LIF, kit ligand or a combination thereof; (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolinylbenzodiazepine, pyridobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin geldanamycin, maytansinoid, calicheamicin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, doleacin, doleacin analogs, auristatin, nostoc, camptothecin, rhizotomycin, rhizotomycin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotics, esperamicin, epothilone, toxoids, or combinations thereof; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an active agent, a neurotransmitter, a radioisotope or a combination thereof; (e) radioactive labels, 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, dioxigenin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to a target, or a combination thereof; (f) immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, or combinations thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, 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 oligonucleotides; (n) ribonucleases; (o) vaccines; and (p) Anti-angiogenic agents.

78. The conjugate of any one of claims 1 to 77, wherein: The active agent is a pyrrolobenzodiazepine dimer; The pyrrolobenzodiazepine dimer is substituted with X at the N10 position, or is substituted with X′ at the N′10 position, wherein X or X′ connects the pyrrolobenzodiazepine dimer to the 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.

79. The conjugate of claim 78, wherein the pyrrolobenzodiazepine dimer is represented by Formula III: in The wavy line indicates the point of attachment to the conjugate (e.g., the carbonyl of Formula IIa is attached to B′); the dashed line indicates an optional double bond; R1 and R1′ are each independently selected from H, OH, ═O, ═CH2, CN, R m , OR m , =CH-R m′ =C(R m′ 2. O-SO2-R m 、CO2R m , COR m , halogen and dihalogen; R m′ Selected from R m 、CO2R m , COR m , CHO, CO2H and halogen; R m is selected from 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, wherein when the c 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 When the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl or 5- to 7-membered heteroaryl is substituted, the C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 The corresponding hydrogen atoms in the cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl or 5- to 7-membered heteroaryl may each be independently replaced by a 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 heteroaryl; R2, R3, R5, R2′, R3′ and R5′ are each independently selected from H, R m , OH, OR m , SH, SR m 、NH2、NHR m NR m R m′ , NO2, Me3Sn and halogen; R4 and R4′ are each independently selected from H, R m , OH, OR m , SH, SR m 、NH2、NHR m NR m R m′ , NO2, Me3Sn, halogen, 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)NR n R n′ 、-S(O)2NR 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′ , wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 The hydrogen atoms in the aryl and 5- to 7-membered heteroaryl groups may be replaced independently by C 1-6 Alkyl, c 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 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)2NR 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 Replacement, when the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl and 5- to 7-membered heteroaryl; R n , R n′ , R o , R o′ , R p , R p′ and R q Each independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl and 5- to 7-membered heteroaryl; - Xa is a bond or substituted or unsubstituted C 1-6 Alkylene, where c 1-6 Alkylene, when substituted, is replaced by C 1-8 Alkyl or C 3-8 Cycloalkyl substitution; R and R' independently represent H, OH, NH2, ONH2, NHNH2, 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-C 1-8 Alkylamino or di-C 1-8 Alkylamino, wherein the C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl and C 5-20 Aryl, when substituted, is selected from OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-6 Alkyl, c 1-6 Alkoxy and C 6-12 Substitution of aryl groups by substituents; 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, wherein the chain may be interrupted by one or more heteroatoms, NMe or substituted or unsubstituted aromatic rings, and the chain or aromatic ring may be replaced at any one or more hydrogen atom positions on the chain or aromatic ring by -NH, -NR m 、-NHC(O)R m 、-NHC(O)CH2-[OCH2CH2] n -R or -[CH2CH2O] n -R substituted or unsubstituted, wherein R m and R are as described above for R m and R, and n is 1 to 12; and R7 and R7′ are 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)2R r 、-SR r 、-S(O)R r 、-S(O)2R r 、-S(O)NR r R r′ 、-S(O)2NR 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 s or -c(O)NR r R r′ , wherein the c 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 The hydrogen atoms in the aryl group and the 5- to 7-membered heteroaryl group may each be independently replaced by c 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 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)2NR 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 t S(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′ Replacement, when the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl and 5- to 7-membered heteroaryl; R r , R r′ , R s , R s′ , R t , R t′ , R u and R u′ Each independently selected from H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 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 selected from H, C 1-8 Alkyl, halogen, NO2, CN, R9, R 10 and R 16 Each independently selected from H, C 1-8 Alkyl, C 2-6 Alkenyl, c 1-6 Alkoxy, alkoxyalkyl, and methoxyethyl; and n30 is 1 to 3.

80. The conjugate of claim 79, wherein Y is O.

81. The conjugate of claim 79 or 80, wherein Y' is O.

82. The conjugate of any one of claims 79 to 81, wherein the dotted line represents a conjugate carrying R1 and R7 or R 1` and R 7` There are double bonds between the carbons.

83. The conjugate of any one of claims 79 to 82, wherein R1 is R m And R m Selected from 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.

84. The conjugate of any one of claims 79 to 83, wherein R2, R3 and R5 are each independently H or OH.

85. The conjugate of any one of claims 79 to 84, wherein R4 is C 1-6 Alkoxy.

86. The conjugate of any one of claims 79 to 84, wherein R4 is methoxy, ethoxy or butoxy.

87. The conjugate of any one of claims 79 to 86, wherein X is selected from -C(O)O-, -C(O)-, and -C(O)NR-; and R each independently represents H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, substituted or unsubstituted C 1-8 Alkyl or substituted or unsubstituted C 1-8 Alkoxy, where C 1-8 Alkyl or C 1-8 Alkoxy groups, when substituted, are substituted with OH, N3, CN, NO2, SH, NH2, ONH2, NNH2 or halo.

88. The conjugate of any one of claims 79 to 87, wherein X is -C(O)NR-.

89. The conjugate of any one of claims 79 to 88, wherein R6 is a substituted or unsubstituted saturated or unsaturated C 3-8 Hydrocarbon chain, where One or more of the carbon atoms of the hydrocarbon chain are replaced by a heteroatom or a substituted or unsubstituted aromatic ring, wherein the heteroatom is O, S or N(H) and the aromatic ring is benzene, pyridine, imidazole or pyrazole, and the chain or aromatic ring may be replaced at any one or more hydrogen atom positions on the chain or aromatic ring by -NHC(O)CH2-[OCH2CH2] n -R or -[CH2CH2O] n -R is replaced; and n is 1 to 6.

90. The conjugate of any one of claims 79 to 89, wherein n is 1 to 10.

91. The conjugate of any one of claims 79 to 90, wherein Xa is a bond or C 1-3 Alkylene.

92. The conjugate of any one of claims 99 to 110, wherein Z is H, Among them, R9, R 10 and R 16 Each independently selected from H, C 1-3 Alkyl, C 1-3 Alkoxy and alkoxymethyl.

93. The conjugate of claim 92, wherein R9 is methoxyalkyl.

94. The conjugate according to claim 92 or 93, wherein R 10 It is a methoxyalkyl group.

95. The conjugate of any one of claims 92 to 94, wherein R 16 It is a methoxyalkyl group.

96. The conjugate of any one of claims 92 to 95, wherein R9, R 10 or R 16 Yes -(CH2CH2O) m -(CH2) m2 CH3, further wherein m is 1 to 6 and m2 is 0 to 2.

97. The conjugate of any one of claims 92 to 95, wherein m is 1.

98. The conjugate of any one of claims 92 to 95, wherein m2 is 1.

99. The conjugate of claim 79, wherein R2 is H.

100. The conjugate of claim 99, wherein R3 is H.

101. The conjugate of claim 99 or 100, wherein R7 is H.

102. The conjugate of any one of claims 99 to 101, wherein R4 is alkoxy (eg, methoxy).

103. The conjugate of any one of claims 99 to 102, wherein R5 is OH.

104. The conjugate of any one of claims 99 to 103, wherein R1 is =CH2, CH3 or phenyl, optionally substituted with methoxy.

105. The conjugate of any one of claims 99 to 104, wherein Y is O.

106. The conjugate of any one of claims 99 to 105, wherein R2' is H.

107. The conjugate of any one of claims 99 to 106, wherein R3' is H.

108. The conjugate of any one of claims 99 to 107, wherein R7' is H.

109. The conjugate according to any one of claims 99 to 108, wherein R4' is alkoxy (eg methoxy).

110. The conjugate according to any one of claims 99 to 109, wherein R5' is OH.

111. The conjugate according to any one of claims 99 to 110, wherein R1' is =CH2, CH3 or phenyl, optionally substituted with methoxy.

112. The conjugate of any one of claims 99 to 111, wherein Y' is O.

113. The conjugate of any one of claims 99 to 112, wherein X is -C(O)O-.

114. The conjugate of any one of claims 99 to 113, wherein Xa is CH2.

115. The conjugate of any one of claims 99 to 114, wherein G is a glucuronide group.

116. The conjugate of claim 115, wherein G is 117. The conjugate of any one of claims 99 to 116, wherein n30 is 1.

118. The conjugate of any one of claims 99 to 117, wherein Z is 119. The conjugate of claim 118, wherein R9 is H.

120. The conjugate according to claim 118 or 119, wherein R 16 is an alkoxyalkyl group (eg, methoxyethyl).

121. The conjugate of any one of claims 99 to 117, wherein Z is 122. The conjugate of claim 121, wherein R 10 is an alkyl group (eg, methyl).

123. The conjugate of claim 121, wherein R6 is alkyl (eg, pentyl).

124. The conjugate of any one of claims 1 to 123, wherein the conjugate comprises a structure selected from: And the bond overlapping with the wavy line indicates the point of connection to L.

125. The conjugate of any one of claims 1 to 124, wherein the conjugate comprises a structure selected from: Where MMAE is monomethyl auristatin E; MMAF is monomethylauristatin F; and The wavy line indicates the point of attachment to the conjugate.

126. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 125 and a pharmaceutically acceptable excipient.

127. A pharmaceutical composition for preventing or treating hyperproliferation, cancer or angiogenic diseases, comprising the conjugate according to any one of claims 1 to 125.

128. The pharmaceutical composition of claim 127, further comprising a pharmaceutically effective amount of a chemotherapeutic agent.

129. The pharmaceutical composition of claim 127 or 128, 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.

130. A pharmaceutical preparation comprising the conjugate according to any one of claims 1 to 125 and a pharmaceutically acceptable carrier, wherein the pharmaceutical preparation is selected from injections, tablets, pills, powders, granules, capsules, dragees, suspensions, internal liquids, emulsions, syrups, emulsions, lyophilized preparations and suppositories.

131. A method of treating cancer or angiogenic disease in an individual in need thereof, the method comprising administering to the individual a conjugate according to any one of claims 1 to 125, or a pharmaceutically acceptable salt thereof.

132. The method of claim 131, wherein the method treats cancer.

133. The method of claim 131 or 132, wherein the cancer is 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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