Antibody-drug conjugates containing antibodies against human L1CAM and their use

Abstract

Disclosed herein are methods for treating solid tumors in a subject.

Description

[Technical Field]

[0001] Related applications This application claims priority to Republic of Korea Patent Application No. 10-2023-0032106, filed on 10 March 2023, and all contents of said application are incorporated herein by reference. [Background technology]

[0002] Cancer is a disease caused by abnormal and uncontrolled cell proliferation in the body's tissues, and is a result of uncontrolled cell proliferation in various tissues. Tumors in early-stage cancer can be removed by surgical and radiotherapy, while metastatic tumors are generally treated palliatively using chemotherapy. Most parenterally administered chemotherapeutic agents can induce undesirable adverse effects or serious toxicity as a result of systemic administration. Therefore, the focus of development is on developing novel chemotherapeutic agents that achieve improved efficacy and minimal toxicity / adverse effects through improved selective action of chemotherapeutic agents against tumor cells or immediately adjacent tissues.

[0003] Antibody-drug conjugates (ADCs) are a targeted technology in which a toxin or drug is bound to an antibody, which then binds to an antigen, releasing the toxin or drug into the cell and causing cell death in tumor cells. This technology has shown superior efficacy with antibody drugs and can substantially reduce the risk of adverse effects compared to conventional anticancer drugs because it allows for the specific delivery of the drug to targeted cancer cells while minimizing impact on healthy cells, and the drug is released only under specific conditions.

[0004] The basic structure of an antibody-drug conjugate is "antibody-linker-small molecule drug (toxin)". Here, the linker not only plays a functional role in linking the antibody and the drug, but also ensures that the drug is properly released after circulating in the body and reaching target cells through antibody-drug dissociation (for example, as a result of enzymatic hydrolysis), thereby demonstrating efficacy against the target cancer cells. In other words, the stability of the linker plays a very important role in the efficacy and systemic toxicity of the antibody-drug conjugate (Discovery Medicine 2010, 10(53):329-39).85-8 2018-08-14.

[0005] The use of monoclonal antibodies for cancer treatment has been highly successful. Monoclonal antibodies are suitable for targeted addressing of tumor tissue and tumor cells. Antibody-drug conjugates are a novel and promising option for the treatment of lymphoma and solid tumors, and more recently, immunomodulatory antibodies have shown significant success in clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering techniques, mechanisms of action and resistance, and the interaction between the immune system and cancer cells.

[0006] Antigens expressed on the surface of human cancer cells refer to a broad range of targets that are overexpressed, mutated, or selectively expressed compared to normal tissues. A key challenge is identifying suitable antigens for antibody-based therapies. These therapeutics exert their efficacy by mediating changes in antigen or receptor function (i.e., as stimulants or antagonists), modulating the immune system through Fc and T cell activation, and through the delivery of specific drugs that bind to antibodies targeting specific antigens. Molecular technologies that can alter the pharmacokinetics, function, size, and immune stimulation of antibodies are emerging as crucial elements in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of techniques for selecting optimized antibodies, including affinity and binding between the target antigen and the antibody, antibody structure selection, and therapeutic approaches (signaling or blockade of immune function).

[0007] From the aforementioned perspective, there is a continuous need for novel antibody-drug conjugates to treat diseases such as cancer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Discovery Medicine 2010,10(53):329-39).85-8 2018-08-14 [Overview of the Initiative]

[0009] The present invention provides, in particular, antibody-drug conjugates (ADCs) that target L1CAM (neuronal cell adhesion molecule L1), active metabolites of such ADCs, methods for preparing such ADCs, and the use of such ADCs in the treatment and / or prevention of diseases (e.g., proliferative and / or angiogenic diseases, e.g., cancer). More specifically, the present invention provides antibody-drug conjugates comprising an antibody or its antigen-binding fragment that binds to L1CAM, and pharmaceutical compositions comprising such conjugates. In one embodiment, this disclosure relates to formula I: [ka] (In the formula, Ab is as follows (a) to (f), that is (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It is an anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody or its antigen-binding fragment, Each B is independently an active agent, each G is independently a glucuronic acid moiety, or

Chem.

[0010] In another aspect, the Disclosure provides a pharmaceutical composition comprising a conjugate disclosed herein and a pharmaceutically acceptable excipient.

[0011] In yet another aspect, the Disclosure provides a method for treating or preventing hyperplasia, cancer, or angiogenic disease in a subject, comprising administering the subject a conjugate or a pharmaceutically acceptable salt thereof disclosed herein. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a schematic diagram showing the manufacturing method and structure of an exemplary antibody-drug conjugate (ADC1). [Figure 2] Figure 2 is a schematic diagram showing the manufacturing method and structure of an exemplary antibody-drug conjugate (ADC2). [Figure 3] Figure 3 is a schematic diagram showing the manufacturing method and structure of an example antibody-drug conjugate (ADC3). [Figure 4] Figure 4 is a graph showing the hydrophobicity of the antibody AFF4-CaaX, the prenylated antibody AFF4-CaaX, and the antibody-drug conjugates ADC1-ADC3, as measured using HIC-HPLC. [Figure 5] Figure 5 is a graph showing the molecular weights of antibody-drug conjugates ADC1 and ADC3 using intact mass. [Figure 6] Figure 6 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a breast cancer cell lineage JIMT-1 xenograft model. [Figure 7] Figure 7 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a colorectal cancer cell lineage SW480 xenograft model. [Figure 8] Figure 8 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in the MeWo xenograft model of melanoma cancer cells. [Figure 9] Figure 9 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a non-small cell lung cancer cell line Calu-6 xenograft model. [Figure 10] Figure 10 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in an ovarian cancer cell line SNU840 xenograft model. [Figure 11] Figure 11 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a pancreatic cancer cell line Patu8988s xenograft model. [Figure 12] Figure 12 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a small cell lung cancer cell line NCI-H146 xenograft model. [Figure 13] Figure 13 is a graph showing the tumor growth inhibitory effect of antibody-drug conjugates in a small cell lung cancer cell line NCI-H69 xenograft model. [Figure 14] Figure 14 is a graph showing the pharmacokinetics of antibody-drug conjugates. [Figure 15] Figure 15 is a graph showing the lack of acute toxicity of antibody-drug conjugates. [Modes for carrying out the invention]

[0013] Detailed description of the invention Human-derived L1CAM (neuronal cell adhesion molecule L1) is a 200-220 kDA transmembrane protein with 323 amino acids (1101 extracellular domains, 23 transmembrane domains, and 114 intracellular domains) and is widely known as a neuron cell adhesion molecule. L1CAM belongs to the L1 protein family, which plays a crucial role in neuronal transport, neurite growth, axon guidance, axon and dendrite enhancement, as well as myelination and synapse formation. L1CAM is expressed in sites where cancer stem cells are present within certain tumors and is widely known to play a causal role in biological responses closely related to cancer stem cells, such as endothelial-mesenchymal transition (EMT) and drug resistance. Furthermore, L1CAM is widely known to be abnormally expressed in various neuroblasts, solenomas, gynecological cancers (cervical cancer, ovarian cancer, uterine cancer, endometrial cancer), ovarian cancer, pancreatic cancer, lung cancer, colorectal cancer, bile duct cancer, and melanoma, and is directly associated with treatment resistance and poor prognosis of malignant tumors.

[0014] In certain embodiments, the present invention provides an antibody-linker-drug (e.g., toxin) system. The disclosed antibody binds to L1CAM, which is abnormally expressed in various cancer tissues. Furthermore, the antibody-linker-drug contains an effective self-immolative group to further enhance the effect of the antibody. This effective self-immolative group is stable in plasma and circulating blood and can readily release and exert efficacy of the drug in cancer cells. The linker containing the effective self-immolative group is stable in plasma and circulating blood and can readily release and exert efficacy of the drug in cancer cells (see U.S. Patent No. 9,919,057 incorporated herein by reference). As a result, disclosed herein is a novel antibody-drug conjugate (ADC) that effectively targets L1CAM in the treatment and / or prevention of proliferative and / or metastatic cancers. By applying a linker containing an effective self-sacrificing group that is stable in plasma and circulating blood but can readily release and exert efficacy of the drug in cancer cells to an anti-L1CAM antibody to further enhance the effect of the antibody, it is possible to provide a useful antibody-drug conjugate (ADC) that effectively targets L1CAM in the treatment and / or prevention of diseases (e.g., proliferative and / or metastatic cancer).

[0015] The antibody-drug conjugates disclosed herein enable effective, specific, and selective delivery of drugs to L1CAM-expressing cells by including anti-L1CAM antibodies that specifically and more strongly bind to the type of L1CAM primarily presented in tumors. By stably conjugating the antibody to the drug while maintaining in vivo stability (and particularly stability in serum and even circulating blood) while exhibiting the intended cytotoxicity, and by utilizing linker technology that includes self-sacrificing groups that allow for easy release of the drug within cancer cells to maximize efficacy, these conjugates demonstrate the effect of providing antibody-linker-drug systems that significantly reduce toxicity while safely delivering drugs and / or toxins to target cells and effectively exerting efficacy.

[0016] Accordingly, in certain embodiments, the present invention provides an antibody-drug conjugate targeting L1CAM, or a pharmaceutically acceptable salt thereof.

[0017] In certain embodiments, the present invention provides pharmaceutical compositions comprising an antibody-drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof.

[0018] In one embodiment, this disclosure relates to formula I: [ka] (In the formula, Ab is as follows (a) to (f), that is (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It is an anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody or its antigen-binding fragment, Each B is an active agent independently. Each G independently comprises a glucuronic acid portion, or [ka] And, R 3 is a hydrogen or carboxyl protecting group, Each R 4 These are independently hydrogen or hydroxyl protecting groups, R 1 and R 2 Each of them is independently hydrogen, C 1-8 Alkyl, or C 3-8 It is a cycloalkyl, Each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R''NR'-, -SONR'-, or -PO2NR'-, where C, S, or P is directly bonded to a phenyl ring, and NR' is bonded to L. R' and R'' are each independently hydrogen and C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono-, or di-C 1-8 Alkylamino, C 3-10 Heteroaryl or C 6-10 It is Ariel, Each Z is independently C 1-8 They are alkyl, halogen, cyano, or nitro, Each L is an independent linker, and the following (A) and / or (B), i.e. (A)(i) One or more unsaturated bonds, (ii) heteroarylenes (e.g., heteroarylenes in alkylene or heteroalkylene chains), or (iii) C 1-20 Alkyl substituents C including 1-50 Alkylenes or heteroalkylenes with 1 to 50 member rings, and / or (B) Formula II: [ka] at least one isoprenyl unit having the structure represented by Includes, n1 and n2 are integers independently selected from 1 to 20, and n3 is an integer between 0 and 3. We provide a conjugate having a structure represented by the given formula.

[0019] In yet another aspect, this disclosure relates to formula I: [ka] (In the formula, Ab is as follows (a) to (f), that is (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 16 (GYSITSDYX1WN), where X1 is A or T. (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 17 (YISYSGSX1SYX2PSLKS), where X2 is H or N. (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 18 (SX1SYX2YGFAY), where X1 is L or F and X2 is G, S or A. (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 19 (SASYRYX1), where X1 is T or I, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It is an anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody or its antigen-binding fragment, Each B is an active agent independently. Each G independently comprises a glucuronic acid portion, or [ka] And, R 3 is a hydrogen or carboxyl protecting group, Each R 4 These are independently hydrogen or hydroxyl protecting groups, R 1 and R 2 Each of them is independently hydrogen, C 1-8 Alkyl, or C 3-8 It is a cycloalkyl, Each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R''NR'-, -SONR'-, or -PO2NR'-, where C, S, or P is directly bonded to a phenyl ring, and NR' is bonded to L. R' and R'' are each independently hydrogen and C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8Alkoxy, C 1-8 Alkylthio, mono-, or di-C 1-8 Alkylamino, C 3-10 Heteroaryl or C 6-10 It is Ariel, Each Z is independently C 1-8 They are alkyl, halogen, cyano, or nitro, Each L is an independent linker, and the following (A) and / or (B), i.e. (A)(i) One or more unsaturated bonds, (ii) heteroarylenes (e.g., heteroarylenes in alkylene or heteroalkylene chains), or (iii) C 1-20 Alkyl substituents C including 1-50 Alkylenes or heteroalkylenes with 1 to 50 member rings, and / or (B) Formula II: [ka] at least one isoprenyl unit having the structure represented by Includes, n1 and n2 are integers independently selected from 1 to 20, and n3 is an integer between 0 and 3. We provide a conjugate having a structure represented by the given formula.

[0020] In some embodiments, the conjugate is (a) to (f) below, namely (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 8, (b) Heavy chain CDR2 containing the amino acid sequence of Sequence ID No. 17 (YISYSGSX1SYX2PSLKS), where X1 is Y and X2 is N. (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 18 (SX1SYX2YGFAY), where X1 is F and X2 is S. (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It contains an antibody or its antigen-binding fragment.

[0021] In some embodiments, the conjugate is (a) to (f) below, namely (a) Heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NOs. 1 and 8, (b) Heavy chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 9, 10, and 11, (c) Heavy chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 3, 12, 13, and 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 comprising amino acid sequences selected from SEQ ID NOs. 5 and 15, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It contains an antibody or its antigen-binding fragment.

[0022] In some embodiments, the conjugate is (i) to (x) below, i.e. (i)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (ii)(a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (iii)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (iv)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 1, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (v)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (vi)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 15, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (vii)(a) Heavy chain CDR1 containing the amino acid sequence of sequence number 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 15, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (viii)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 1, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 12, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (ix)(a) Heavy chain CDR1 containing the amino acid sequence of sequence number 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, or (x)(a) Heavy chain CDR1 containing the amino acid sequence of Sequence ID No. 1, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, It contains an antibody or its antigen-binding fragment.

[0023] In some embodiments, the conjugate is (a) to (f) below, namely (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 It contains an antibody or its antigen-binding fragment.

[0024] In some embodiments, the variable X1 or X2 included in one sequence number may be an amino acid sequence independently selected from X1 or X2 included in another sequence number. For example, the variable X1 or X2 included in sequence number 17 may each be an amino acid sequence independently selected from the variable X1 included in sequence number 16, the variable X1 or X2 included in sequence number 18, or the variable X1 included in sequence number 19.

[0025] In some embodiments, the antibody may contain mutations at the Fc site, specifically 1 to 10 mutations (mutations 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Each mutation may be introduced into one strand of the Fc site, or independently and symmetrically into two strands of the Fc site.

[0026] In some embodiments, the antibody may contain variants in the CL site and / or CH1, specifically 1 to 10 mutations (mutations 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0027] In various embodiments, a mutation refers to a deletion (deletion of one amino acid), an insertion (insertion of one amino acid), or a substitution (substitution of one amino acid with another different amino acid). In various embodiments involving multiple mutations, each amino acid difference may be selected independently of, for example, a deletion, insertion, or substitution.

[0028] In some embodiments, the antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 listed in Table 1. Specifically, the antibody and its antigen-binding fragment may contain H1 to H4 with respect to the heavy chain variable domain of the humanized antibody OV549.20. AFF1 to AFF10 may undergo further affinity maturation treatment in the humanized antibody.

[0029] [Table 1] TIFF2026510495000012.tif158161

[0030] In some embodiments, the antibody or its antigen-binding fragment may include a light chain variable region comprising the light chain CDR1, light chain CDR2, and light chain CDR3 amino acid sequences listed in Table 2. Specifically, the antibody and its antigen-binding fragment may include L1 or L2 with respect to the light chain variable domain of the humanized antibody of mouse antibody OV549.20. The antibody may be combined with the H1-H4 heavy chain variable domains and the L1 or L2 light chain variable domains shown in Table 1.

[0031] [Table 2] TIFF2026510495000014.tif90161

[0032] In some embodiments, the antibody or its antigen-binding fragment comprises one or more specific framework regions as described herein.

[0033] In some embodiments, the antibody or its antigen-binding fragment comprises one or more arbitrary framework sequences. Specifically, the framework region sequence may be a human framework sequence.

[0034] In this specification, the term “framework (FR) amino acid portion” refers to amino acids in the framework region of an immunoglobulin chain. In this specification, the term “framework” or “FR region” refers to amino acid portions in a variable region that is not part of the CDR. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. The framework region usually supports the structure of the antibody (without contact with the antigen) or supports binding to the antigen by direct contact with the antigen. Methods for producing antibodies having a CDR sequence are well known to those skilled in the art and may also include, for example, introducing an expression vector encoding a desired framework sequence into the nucleic acid sequence encoding the CDR.

[0035] In some embodiments, the heavy chain variable region FR1 to FR4 may be selected independently of the light chain variable region FR1 to FR4. Specifically, the FR of the heavy chain variable region may include the amino acid sequences shown in Table 3, and the FR of the light chain variable region may include the amino acid sequences shown in Table 4.

[0036] In some embodiments, the heavy chain variable region may include framework regions containing the amino acid sequences of SEQ ID NO: 39 and 41-46. Specifically, the heavy chain variable region of the antibody may include 1 to 4 framework regions (FRs) selected from the amino acid sequences of SEQ ID NO: 39 and 41-46. More specifically, the heavy chain variable region may include FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4. More specifically, FR1 of the heavy chain variable region may contain the amino acid sequence of SEQ ID NO: 39, FR2 of the heavy chain variable region may contain an amino acid sequence selected from SEQ ID NO: 41-44, FR3 of the heavy chain variable region may contain the amino acid sequence of SEQ ID NO: 45, and FR4 of the heavy chain variable region may contain the amino acid sequence of SEQ ID NO: 46.

[0037] [Table 3]

[0038] In some embodiments, the light chain variable region may include framework regions containing amino acid sequences selected from SEQ ID NOs. 51-56 or SEQ ID NO. 60. Specifically, the light chain variable region of the antibody may include 1-4 framework regions (FRs) selected from the amino acid sequences of SEQ ID NOs. 51-56 and SEQ ID NO. 60. More specifically, the light chain variable region may include FR1, CDRL1, FR2, CDRL2, FR3, CDRL3, and FR4. More specifically, FR1 of the light chain variable region may contain amino acid sequences selected from SEQ ID NOs. 51 and 55, FR2 of the light chain variable region may contain amino acid sequences selected from SEQ ID NOs. 48 or 52, FR3 of the light chain variable region may contain amino acid sequences selected from SEQ ID NOs. 53 and 56, and FR4 of the light chain variable region may contain amino acid sequences selected from SEQ ID NOs. 50, 54, or 60.

[0039] [Table 4]

[0040] In some embodiments, an antibody or its antigen-binding fragment that specifically binds to human L1CAM (neuronal cell adhesion molecule L1) may include a heavy chain variable region containing an amino acid sequence selected from SEQ ID NOs. 23-24, and may also include a light chain variable region containing an amino acid sequence selected from SEQ ID NOs. 20-22. More specifically, as shown in Table 5, the heavy chain variable region may contain an amino acid sequence selected from SEQ ID NOs. 23-24, and the light chain variable region may contain an amino acid sequence selected from SEQ ID NOs. 20-22.

[0041] [Table 5] TIFF2026510495000018.tif226162TIFF2026510495000019.tif226162TIFF2026510495000020.tif22616 2TIFF2026510495000021.tif226162TIFF2026510495000022.tif226162TIFF2026510495000023.tif66162

[0042] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may include a heavy chain containing an amino acid sequence selected from SEQ ID NOs. 58 and 37, and a light chain containing an amino acid sequence selected from SEQ ID NOs. 38 and 59. More specifically, as shown in Table 6, the heavy chain may contain an amino acid sequence selected from SEQ ID NOs. 37 and 58, and the light chain may contain an amino acid sequence selected from SEQ ID NOs. 38 or 59.

[0043] More specifically, the antibodies or antigen-binding fragments disclosed herein may include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30. The antibodies or antigen-binding fragments disclosed herein may also include a heavy chain containing the amino acid sequence of SEQ ID NO: 37.

[0044] More specifically, the antibodies or antigen-binding fragments disclosed herein may include LALA mutations in the heavy chain variable region and the heavy chain constant region, which include the amino acid sequence of SEQ ID NO: 30. The antibodies or antigen-binding fragments disclosed herein may also include a heavy chain, which includes the amino acid sequence of SEQ ID NO: 58. For example, an L236A / L237A substitution can be given as the LALA mutation.

[0045] More specifically, the antibodies or antigen-binding fragments disclosed herein may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 20. The antibodies or antigen-binding fragments disclosed herein may also include a heavy chain containing the amino acid sequence of SEQ ID NO: 38.

[0046] More specifically, the antibodies or antigen-binding fragments disclosed herein may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 20 and a CaaX peptide portion at the C-terminus of the light chain (GGGGGGGCVIM, SEQ ID NO: 7). The antibodies or antigen-binding fragments disclosed herein may also include a heavy chain containing the amino acid sequence of SEQ ID NO: 59.

[0047] In some embodiments, the antibody or its antigen-binding fragment may include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence exhibiting at least 80% sequence identity with SEQ ID NO: 30.

[0048] More specifically, the antibody or its antigen-binding fragment may include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 30.

[0049] More specifically, the antibody or its antigen-binding fragment may include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30, an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 30, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 30.

[0050] In some embodiments, the antibody or its antigen-binding fragment may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence exhibiting at least 80% sequence identity with SEQ ID NO: 20.

[0051] More specifically, the antibody or its antigen-binding fragment may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 20.

[0052] More specifically, the antibody or its antigen-binding fragment may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0053] In some embodiments, the antibody or its antigen-binding fragment may include a combination of a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, or a combination of a heavy chain variable region containing an amino acid sequence showing at least 80% sequence identity to SEQ ID NO: 30 and a light chain variable region containing an amino acid sequence showing at least 80% sequence identity to SEQ ID NO: 20, and the antibody or its antigen-binding fragment specifically binds to human L1CAM.

[0054] More specifically, the antibody or its antigen-binding fragment may include a combination of a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, or a combination of a heavy chain variable region containing an amino acid sequence showing at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 30 and a light chain variable region containing an amino acid sequence showing at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 20, and the antibody or its antigen-binding fragment specifically binds to human L1CAM.

[0055] More specifically, the antibody or its antigen-binding fragment exhibits sequence identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% relative to SEQ ID NO: 30. The antibody or its antigen-binding fragment may also include a combination of a heavy chain variable region containing an amino acid sequence and a light chain variable region containing an amino acid sequence exhibiting at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with respect to SEQ ID NO: 20, and the antibody or its antigen-binding fragment specifically binds to human L1CAM.

[0056] [Table 6] TIFF2026510495000025.tif227161TIFF2026510495000026.tif227161TIFF2026510495000027.tif96161

[0057] In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody.

[0058] In some embodiments, the humanized antibody is as follows (a) to (c), namely (a)(i) a variable domain framework region from the heavy chain of a human antibody or from the human consensus framework, and (ii) a variable domain framework region from the light chain of the human antibody or from the human consensus framework, (b)(i) at least one amino acid in the variable domain framework region from the heavy chain is substituted with a corresponding amino acid from the heavy chain of the mouse antibody or the mouse consensus framework, or (ii) the variable domain framework region from the light chain is substituted with a corresponding amino acid from the light chain of the mouse antibody or the mouse consensus framework, and (c)(i) The amino acid substitution in the heavy chain is the following amino acid substitution, i.e. At 34th place, T (human) was replaced with A (mouse). At 58th place, Y (human) was replaced with F (mouse). At position 61, N (human) was replaced with H (mouse), and At rank 103, S (human) was replaced with G (mouse). To be selected from It may include.

[0059] In some embodiments, the amino acid substitution in the heavy chain is the following amino acid substitution, i.e. At rank 238, A (human) was replaced with G (mouse). At position 241, replace D (human) with S (human), and / or At rank 335, E (human) was replaced with I (human). Selected from.

[0060] In some embodiments, the humanized antibody is (i) and (ii) below, namely (i) A variable heavy chain framework region from the heavy chain of a human antibody or the human consensus framework, wherein the variable heavy chain framework region contains one or more of the following mutations, namely T34A, Y58F, N61H, and S103G, and (ii) The light chain of the human antibody or the variable light chain framework region from the human consensus framework Includes.

[0061] In some embodiments, the humanized antibody contains an LALA mutation in the heavy chain constant region. In some embodiments, the LALA mutation includes an L236A / L237A substitution.

[0062] In some embodiments, the antibody or its antigen-binding fragment may be selected from monoclonal antibodies, domain antibodies (dAb), single-chain antibodies (scAb), Fab fragments, Fab' fragments, F(ab')2 fragments, scFab fragments, Fv fragments, dsFv fragments, single-chain variable fragments (scFv), scFv-Fc fragments, single-domain heavy chain antibodies, single-domain light chain antibodies, variant antibodies, multimeric antibodies, minibody antibodies, diabody antibodies, bispecific antibodies, and multispecific antibodies.

[0063] The antibody-drug conjugates disclosed herein enable effective, specific, and selective delivery of drugs to L1CAM-expressing cells by including anti-L1CAM antibodies that specifically and more strongly bind to the type of L1CAM primarily presented in tumors. By stably conjugating the antibody to the drug while maintaining in vivo stability (and particularly stability in serum and even circulating blood) while exhibiting the intended cytotoxicity, and by utilizing linker technology that includes self-sacrificing groups that allow for easy release of the drug within cancer cells to maximize efficacy, these conjugates demonstrate the effect of providing antibody-linker-drug systems that significantly reduce toxicity while safely delivering drugs and / or toxins to target cells and effectively exerting efficacy.

[0064] In certain embodiments, n1 is 1, 2, 3, or 4. In certain preferred embodiments, n1 is 1. In other preferred embodiments, n1 is 2.

[0065] In certain embodiments, n2 is 1, 2, 3, or 4. In certain preferred embodiments, n2 is 1.

[0066] In a particular preferred embodiment, each G is [ka] And R 3 and R 4 Each of these is H.

[0067] In a particular embodiment, R 1 and R 2 Each of them is hydrogen.

[0068] In a particular preferred embodiment, each W is independently -C(O)NR'-.

[0069] In certain embodiments, R' is H.

[0070] In certain embodiments, R'' is H.

[0071] In certain embodiments, Z is a halo (e.g., bromo) or cyano.

[0072] In a particular embodiment, n3 is 1. In a particular preferred embodiment, n3 is 0.

[0073] In certain embodiments, L is C 1-50 Contains alkylene. In other embodiments, L is C 1-50 Contains heteroalkylene. In certain embodiments, L contains unsaturated bonds (e.g., 1, 2, 3, 4, or 5 unsaturated bonds). In certain embodiments, L is C 1-20 Substitute with alkyl.

[0074] In a particular preferred embodiment, L is given by formula IIIa:

[0075] [ka] (In the formula, n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) It contains an isoprenyl group having the structure represented by .

[0076] In certain embodiments, L comprises a peptide. In certain embodiments, the peptide comprises at least one hydrophilic amino acid. In certain embodiments, the peptide comprises an amino acid having a side chain having a charged moiety (e.g., an amine, guanidine, or carboxyl moiety) at a neutral pH in aqueous solution. In certain embodiments, the peptide comprises an amino acid selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0077] In certain embodiments, the antibody comprises a cysteine ​​residue, and L is covalently linked to the antibody via a thioether bond by a sulfur atom in the cysteine ​​residue (e.g., by a sulfur atom in the side chain of the cysteine ​​residue). In certain embodiments, the C-terminus of the antibody comprises an amino acid motif recognized by an isoprenoid transferase. In certain embodiments, the isoprenoid transferase is farnesyl proteotransferase (FTase) or geranylgeranyl transferase (GGTase). In certain embodiments, the amino acid motif comprises a cysteine ​​residue, and L is covalently linked to the antibody via a thioether bond by a sulfur atom in the cysteine ​​residue (e.g., by a sulfur atom in the side chain of the cysteine ​​residue). In certain preferred embodiments, the amino acid motif has the following sequence: CYYX (In the formula, C is cysteine, Each Y is an aliphatic amino acid, and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine) has.

[0078] In certain embodiments, each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine. In certain embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. In certain preferred embodiments, the amino acid motif has the sequence GGGGGGGCVIM. In certain preferred embodiments, L comprises an oxime. In certain embodiments, the oxygen atom of the oxime is on the side of Y linked to the active agent, and the carbon atom of the oxime is on the side of L linked to Ab. In certain embodiments, the carbon atom of the oxime is on the side of Y linked to the active agent, and the oxygen atom of the oxime is on the side of L linked to Ab.

[0079] In certain preferred embodiments, L is of formula IVa or IVb:

Chemical formula

[0080] In certain embodiments, V is -O-. In certain embodiments, X is -O-. In certain embodiments, R 21 is H. In a particular embodiment, R 22 is H. In a particular embodiment, R 23 is H. In a particular embodiment, R 24 is H. In a particular embodiment, R 25 H is. In certain embodiments, r is 2. In certain embodiments, p is 2. In certain embodiments, q is 2, 5, or 11. In certain embodiments, w is 6 to 20.

[0081] In a particular preferred embodiment, L is

[0082] [ka] It comprises at least one polyethylene glycol unit represented by . In a particular embodiment, L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyethylene glycol units. In a more preferred embodiment, L comprises an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

[0083] In certain embodiments, L includes a portion formed by the reaction of an alkyne with an azide, or by the reaction of an aldehyde or ketone group with a hydrazine or hydroxylamine.

[0084] In a particular preferred embodiment, L is a function of the formula Va, Vb, Vc, Vd, or Ve: [ka] (L1 is a single bond or C 1-30 It is alkylene and R 11 is H or C 1-10 (It is alkyl.) Includes a unit having a structure represented by .

[0085] In a particular embodiment, L 1 It is a single bond. In certain embodiments, L is C 1-30 It is an alkylene. In certain embodiments, R 11 is H. In a particular embodiment, R 11 is C 1-10 It is an alkyl group (for example, methyl).

[0086] In certain preferred embodiments, L is (i) to (iiib) below, namely (i) A branched unit covalently bonded to Ab by a primary linker, (ii) A first branch that connects the first B to the branching unit, (iiia) A second branch that connects the second B to the branching unit, (iiib) A second branch containing the branch unit to which an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is attached. Includes.

[0087] In certain embodiments, L includes a second branch that conjugates a second active agent to the branched unit via a second cleavage group. In certain embodiments, L includes a second branch that includes the branched unit to which an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is conjugated. In certain embodiments, the branched unit is of formula VIa, VIb, VIc, or VId: [ka] (In the ceremony G 1 , G 2 , G 3 Each is connected independently. [Chemistry] or [Chemistry] and R 30 is H or alkyl, R 40 is H, alkyl or L5-CO2R 50 and R 50 is H or alkyl, and L 2 L 3 L 4 and L 5 are each independently a bond or alkylene) has a structure represented by

[0088] In certain embodiments, the branching unit is of formula VIe: [Chemistry] (where R 30 is H or alkyl) has a structure represented by

[0089] In certain embodiments, the branching unit is of formula VIf: [Chemistry] (where R <000—096>is H or alkyl) has a structure represented by

[0090] In certain embodiments, the conjugate comprises 1, 2, 3, or 4 branched linkers. In certain embodiments, each branched linker comprises at least 2 active agents. In certain embodiments, each branched linker comprises 2 active agents. In certain embodiments, the branching unit is lysine and the primary linker is attached to the C-terminus of the lysine.

[0091] In certain embodiments, the conjugate is cleavable in target cells (for example, the conjugate cleaves to release one or more active agents).

[0092] In a particular embodiment, the conjugate is as follows: [ka] (n11, n22, and n33 are each independent integers between 0 and 30, and (AA represents an amino acid group) Includes a structure represented by .

[0093] In certain embodiments, each active agent is independently a chemotherapeutic agent or toxin. In certain embodiments, each active agent is independently an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof. In certain embodiments, the active agent is independently an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, or an antiparasitic agent. In certain embodiments, the active agent is (a) to (q) below, i.e. (a) Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787 / ZK 222584, Oxaliplatin, 5-Fluorouracil, Leucovorin, Rapamycin, Lapatinib, Ronafarnib, Sorafenib, Gefitinib, AG1478, AG1571, Thiotepa, Cyclophosphamide, Busulfan, Improsulfan, Piposulfan, Benzodopa, Carbocon, Meturedopa, Uredopa, Ethyleneimine, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoramide, Trimethylolomelamine, Bratacin, Bratacinone, Camptothecin, Topotecan, Briostatin, Callystatin, CC-1065, Adzeresin, Calzelsin, Bizeresin, Cryptophycin 1, Cryptophycin 8, Drasta Chloramin, Duocalmycin, KW-2189, CB1-TM1, Erytherobin, Pancratistatin, Sarcodictyin, Spongestatin, Chlorambucil, Chlornafadin, Cholophosphamide, Estramustine, Ifosfamide, Mechloretamine, Melphalan, Novembichin, Phenesterine, Prednimustine, Trophosphamide, Uracil Mustard, Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimnustine, Calitiamycin, Calitiamycin Gamma 1, Calitiamycin Omega 1, Dynemicin, Dynemicin AA) Clodronate, esperamicin, neocardinostatin chromophore, alacinomysins, actinomycin, antimycin, azacerin, bleomycins, kactinomycin, carabicin, carninomycin, cardinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomarcelomycin Marcellomycin, Mitomycin C, Mycophenolic acid, Nogaramycin, Olibomycin derivatives, Peplomycin, Potfiromycin, Puromycin, Queramycin, Rhodorubicin, Streptomigrin, Streptozocin, Tubercidine, Ubenimex, Dinostatin, Zolubicin, 5-Fluorouracil, Denopterin, Methotrexate, Pteropterin, Trimethotrex Sart, fludarabine, 6-mercaptopurine, thiamiprine, tiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone, propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, acegraton, aldophosphamide glycosideGlycoside, aminolevulinic acid, enyluracil, amsacrine, bestrubsil, bisanthren, edatraxate, defofomine, demecoltin, diaziquan, elfornithine, eriptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, meitansine, anthamitocins, mitogluco Mitoxanthrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxanthrone, 2-ethylhydrazide, procarbazine, polysaccharide-K, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triadicone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A A) Loridine A, Anguidine, Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Gacytosine, Arabinoside, Cyclophosphamide, Thiotepa, Paclitaxel, Paclitaxel albumin-bound nanoparticle formulation, Docetaxel, Chlorambucil, Gemcitabine, 6-Thiogunine, Mercaptopurine, Cisplatin, Carboplatin, Vinblastine, Platinum, Etoposide, Ifosfamide, Mitoxantrone, Vincristine, Vinorelbine, Novantrone, Teniposide, Edatrexate, Daunomycin, Aminopterin, Xeloda, Iba Ndronate, CPT-11, SN-38, GI-147211C, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, jaimatecan, calenitecan, silatecan, raltothecan, exatecan, diflomotecan, belotecan, raltothecan, topoisomerase inhibitor RFS2000, topoisomerase inhibitor S39625, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids thereof, (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian duct inhibitors, mouse gonadotropin-related peptides, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythro Poietin, bone inducing factor, interferon, interferon-α, interferon-β, interferon-γ, colony-stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factor, LIF, kit ligand, or combination thereof. (c) Diphtheria toxin, botulinum toxin, tetanus toxin, decentritoxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolinobenzodiazepine, pyridinobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, lysine, AM Toxins, auristatin, tubulicin, geldanamycin, mytansinoids, calicheamycin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, drastatin, drastatin analogs, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocalmycin, engine antibiotics, esperamicin, epotilon, toxoids, or combinations thereof. (d) Affinity ligand, wherein the affinity ligand is a substrate, inhibitor, activator, neurotransmitter, radioisotope, or a combination thereof. (e) A radioactive label, 32P, 35S, a fluorescent dye, a high electron density reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to the target, or a combination thereof, (f) An immunomodulatory compound, an anticancer agent, an antiviral drug, an antibacterial drug, an antifungal drug, and an antiparasitic drug, or a combination 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, leuprorelin, goserelin, or troxacitabine, (j) An aromatase inhibitor, (k) A protein kinase inhibitor, (l) A lipid kinase inhibitor, (m) An antisense oligonucleotide, (n) A ribozyme, (o) Vaccines, (p) An anti-angiogenic drug, and (q) A topoisomerase I inhibitor is independently selected from.

[0094] In certain embodiments, at least one active agent is a pyrrolobenzodiazepine dimer. In certain embodiments, the active agent is a pyrrolobenzodiazepine dimer, and the pyrrolobenzodiazepine dimer is substituted with X at the N10 position and / or with X' at the N'10 position, where X or X' links the pyrrolobenzodiazepine dimer to the conjugate, X and X' are each independently -C(O)O- * or -C(O)- * and * is the binding site of the pyrrolobenzodiazepine dimer and the linker.

[0095] In certain embodiments, at least one active agent is a compound of formula X: [ka] (In the ceremony Each dotted line represents an arbitrary double bond. G' is a glucuronide group or a galactoside group. Z' is H, C 1-8 Alkyl, Halo, NO2, CN, [ka] Selected from, X' is -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR 32 -, -S(O)2NR 32 -,-P(O)R 33 NR 32 -, -S(O)NR 32 -, and -PO2NR 32 - Selected from, Xa' is a bonded, substituted, or unsubstituted C. 1-6 Alkylene, where C 1-6 Alkylene substituents are hydrogen, C 1-8 Alkyl, or C 3-8 It is a cycloalkyl, R X1 and R X1’ H, OH, =O, =CH2, CN, R m , OR m 、=CH-R m , =C(R m’ )2, OSO2-R m CO2R m COR m , Haro, Jiharo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenil, C 5-7 Ariel, C 3-6 Heteroaryl, or C 1-6 Alkyl or C 1-6 alkoxy-substituted C 5-7Each is independently selected from the arrows, R X2 , R X2’ R X3 , and R X3’ H, R m , OH, OR m , NR m 2, NO2, and Haro are each selected independently. R X4 and R X4’ H, R m , OH, OR m SH, SR m NH2, NHR m , NR m 2, Halo, and C 1-6 Each alkyl group is independently selected, R X5 and R X5’ H, R m , OH, OR m SH, SR m NH2, NHR m , NR m 2. -NR m R m’ NO2, -NR m C(O)R m ', -NR m C(O)OR m’ , -NR m C(O)NR m R m’ ,-S(O)R m -S(O)2R m -S(O)NR m R m’ -S(O)2NR m R m’ , -NR m S(O)R m’ , -NR m S(O)2R m’ ,-P(O)R m -P(O)2R m ,-P(O)NR m R m’ -P(O)2NR m R m’ , -NR m P(O)R m’ , -NR m P(O)2R m’, and are selected independently from Halo, Y and Y' are each independently O, S, or N(H), R X6 C is independent 3-12 Alkilen, C 3-12 Alkenylene or C 3-12 It is a heteroalkylene, R X6 It is either unsubstituted or -NH2, -NHR m ,-NHC(O)R m ,-NHC(O)CH2-[OCH2CH2] n31 -R XX , or -[CH2CH2O] n31 -R XX It has been replaced with, The R XX H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, Halogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 Aryl, mono, or di-C 1-8 It is an alkylamino compound, where each n31 is an integer from 1 to 6. R X7 and R X7’ H and C are independent of each other. 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, -C(O)R r , -C(O)OR s or -C(O)NR r R r’ And, R r , R r’ , and R s H and C are independent of each other. 1-7 Alkyl, C 2-7 Alkenil, C 2-7 Alkinyl, C 3-13 Cycloalkyl, 3-7 member heterocycloalkyl, C 5-10 It is an aryl or a 5-7 member heteroaryl. Each R m’ R m CO2R m COR m Selected independently from CHO, CO2H, and halo, Each R m H, OH, C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenil, C 2-12 Alkinyl, C 5-20 Ariel, C 5-20 Heteroaryl, C 3-6 A cycloalkyl group is independently selected from cycloalkyl groups, 3-7 membered heterocyclyl groups, 3-7 membered heterocycloalkyl groups, and 5-7 membered heteroaryl groups, where C 5-20 Ariel, C 5-20 Heteroaryl, C 3-6 One or more hydrogen atoms in cycloalkyls, 3-7 membered heterocyclyls, 3-7 membered heterocycloalkyls, and 5-7 membered heteroaryls are OH, =O, C 1-12 Alkyl, or C 1-12 It may be substituted with an alkoxy, and R 31 , R 36 , and R 39 H, C 1-8 Alkyl, C 2-6 Alkenil, C 1-6 Independently selected from alkoxy and alkyloxyalkyl groups, R 32 and R 33 Each of these is H, alkyl, alkoxy, or aralkyl, and n 32 (is 0, 1, 2, 3, or 4) It has a structure represented by the following:

[0096] In a particular preferred embodiment, G' is [ka] That is the case.

[0097] In a particular embodiment, R X1and R X1’ is =CH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenil, C 5-7 Ariel, C 3-6 Heteroaryl, or C 5-7 Each is independently selected from the arrows, and furthermore, here C 3-6 Heteroaryl or C 5-7 Ariel is C 1-6 Alkyl or C 1-6 It is substituted with alkoxy.

[0098] In a particular embodiment, R X2 is either H or OH.

[0099] In a particular embodiment, R X2’ is either H or OH.

[0100] In a particular embodiment, R X3 is either H or OH.

[0101] In a particular embodiment, R X3’ is either H or OH.

[0102] In a particular embodiment, R X5 H, OH, -S(O)R m S(O)2R m ,-P(O)R m , and -P(O)2R m And here R m H, OH, C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, or C 2-12 It is alkinyl.

[0103] In a particular embodiment, R X5’ H, OH, -S(O)R m S(O)2R m ,-P(O)R m , and -P(O)2R m And here R m H, OH, C1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, or C 2-12 It is alkinyl.

[0104] In a particular embodiment, R X4 is C 1-6 It is an alkoxy.

[0105] In a particular embodiment, R X4’ is C 1-6 It is an alkoxy.

[0106] In certain embodiments, Y is O.

[0107] In certain embodiments, Y' is O.

[0108] In a particular embodiment, R X6 is C 3-12 Alkilen, C 3-12 Alkenylene or C 3-12 It is a heteroalkylene and R X6 -NH2, -NHR m ,-NHC(O)R m ,-NHC(O)CH2-[OCH2CH2] n -R XX , or -[CH2CH2O] n -R XX It has been replaced with, R XX H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, Halogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 Aryl, mono, or di-C 1-8 It is an alkylamino compound, and n is an integer between 1 and 6.

[0109] In a particular embodiment, R X6 is C 3-12 It is alkylene.

[0110] In certain embodiments, X' is -C(O)-.

[0111] In certain embodiments, Z' is [ka] That is the case.

[0112] In a particular embodiment, R 36 It is an alkyloxyalkyl group.

[0113] In a particular embodiment, R 39 H is H.

[0114] In a particular embodiment, the conjugate is [ka] [ka] [ka] [ka] [ka] [ka] [ka] (In the formula, MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F, and (The dotted line represents the connection to the rest of the conjugate.) Includes.

[0115] In a particular embodiment, the conjugate is [ka]

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[0116] In certain preferred embodiments, the active agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0117] In a particular preferred embodiment, the conjugate is [ka] or a salt thereof, in the formula, [ka] This is the aforementioned antibody.

[0118] In a particular preferred embodiment, the conjugate is [ka] or a salt thereof, in the formula, [ka] This is the aforementioned anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody.

[0119] In a particular preferred embodiment, the conjugate is [ka] or a salt thereof, in the formula, [ka] This is the aforementioned anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody.

[0120] In another aspect, the Disclosure provides pharmaceutical compositions comprising conjugates and pharmaceutically acceptable excipients disclosed herein.

[0121] In yet another aspect, the Disclosure provides a method for treating or preventing hyperplasia, cancer, or angiogenic disease in a subject, comprising administering the subject a conjugate disclosed herein or a pharmaceutically acceptable salt thereof. In a particular preferred embodiment, the method treats cancer. In a particular embodiment, 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, or melanoma.

[0122] Pharmaceutical composition The compositions and methods of the present invention can be used to treat individuals in need of treatment. In certain embodiments, the individuals are mammals such as humans, or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered as a pharmaceutical composition comprising, for example, the compounds of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents, or vehicles such as glycols or glycerols, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive administration routes (i.e., routes such as injection or transplantation that avoid transport or diffusion across the epithelial barrier), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized reconstituters, powders, solutions, syrups, suppositories, and injections. The composition may also be present in transdermal delivery systems, such as skin patches. The composition may also be present in solutions suitable for topical administration, such as lotions, creams, or ointments.

[0123] A pharmaceutically acceptable carrier may include, for example, a physiologically acceptable agent that acts to stabilize, increase the solubility of, or increase the absorption of a compound, such as the compound of the present invention. Examples of such physiologically acceptable agents include sugars such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, which may incorporate, for example, the compound of the present invention. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to manufacture and administer.

[0124] In this specification, the term “pharmaceutically acceptable” is used to mean a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit-to-risk ratio.

[0125] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, and their derivatives; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) Examples include isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic, suitable substances used in pharmaceutical formulations.

[0126] Pharmaceutical compositions (preparations) can be administered to a target by any of many routes of administration, such as orally (e.g., aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue), absorbed through the oral mucosa (e.g., sublingually), subcutaneously, transdermally (e.g., as patches applied to the skin), and topically (e.g., as creams, ointments, or sprays applied to the skin). Compounds may also be formulated for inhalation. In certain embodiments, compounds may simply be dissolved or suspended in sterile water. Details of suitable administration routes and compositions thereof can be found, for example, in U.S. Patents 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited herein.

[0127] The formulation can be conveniently provided in unit dosage forms and can be prepared by any method well known in the field of pharmaceutical technology. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is the amount of the compound that produces the therapeutic effect. Generally, out of 100 percent, this amount is about 1 percent to about 99 percent of the active ingredient, preferably about 5 percent to about 70 percent, and most preferably about 10 percent to about 30 percent.

[0128] Methods for preparing these formulations or compositions include the step of combining an active compound, such as the compound of the present invention, with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and closely combining the compound of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then shaping the product as needed.

[0129] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavoring base, usually sucrose and acacia or tragacanth), lyophilized agents, powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or oil-in-water or water-in-oil emulsions, or licks or syrups, or lozenges (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or mouthwashes, each containing a predetermined amount of the compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, lick, or paste.

[0130] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient may be carried by one or more pharmaceutically acceptable carriers, e.g., sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, e.g., glycerol; (4) disintegrants, e.g., agar, calcium carbonate. Mix with any of the following: (5) um, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (6) dissolution retarders, e.g., paraffin; (7) absorption enhancers, e.g., quaternary ammonium compounds; (8) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (9) absorbents, e.g., kaolin and bentonite clay; (10) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (11) complexing agents, e.g., modified and unmodified cyclodextrins; and (12) colorants. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also include a buffer. Similar types of solid compositions can also be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc.

[0131] Tablets can be prepared by compression or molding, sometimes with one or more auxiliary components. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose sodium), a surfactant, or a dispersant. Molded tablets can be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent using a suitable machine.

[0132] Tablets and other solid dosage forms of pharmaceutical compositions, such as sugar-coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored and prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical field. They may also be formulated with other polymer matrices, liposomes, and / or microspheres, for example, using hydroxypropyl methylcellulose in various proportions to give a desired release profile, to provide sustained or controlled release of the active ingredient therein. They may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions may also optionally contain opacifying agents and may be compositions that optionally release the active ingredient(s) in a delayed manner, only in or preferentially in specific parts of the gastrointestinal tract. Examples of embedding compositions that can be used include polymer substances and waxes. The active ingredient may, where appropriate, be in the form of microencapsulated material accompanied by one or more of the above-mentioned excipients.

[0133] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized reconstituted agents, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain, for example, water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, and inert diluents commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof.

[0134] In addition to inert diluents, oral compositions may also contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavorings, colorants, fragrances and preservatives.

[0135] The suspension may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, as well as mixtures thereof.

[0136] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0137] In addition to the active compound, ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0138] In addition to the active compound, the powder and spray formulations may contain excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof. The spray formulations may additionally contain conventional propellants such as chlorofluorohydrocarbons, as well as volatile unsubstituted hydrocarbons such as butane and propane.

[0139] Transdermal patches offer the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. The rate of such flow can be controlled either by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0140] As used herein, the terms “parenteral administration” and “administered parenterally” typically refer to, but are not limited to, modes of administration other than enteral and local administration by injection, including intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration include one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or one or more active compounds in combination with a sterile powder, which may be reconstituted into a sterile injection solution or dispersion immediately before use, and may contain antioxidants, buffers, bacteriostatic agents, or solutes that make the formulation isotonic with the blood, suspension, or thickener of the target recipient.

[0141] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the particle size required in the case of dispersions, and using surfactants.

[0142] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. The inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbic acid, can ensure prevention of microbial action. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin can provide long-term absorption of the injectable pharmaceutical form.

[0143] In some cases, it is desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous substance. In this case, the rate of drug absorption depends on the rate of dissolution, which may then depend on the crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oily vehicle.

[0144] Depot formulations for injection are prepared by forming a microencapsulation matrix of the target compound in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot-injectable formulations can also be prepared by encapsulating the drug in liposomes or microemulsifies that are compatible with body tissues.

[0145] For use in the method of the present invention, the active compound can be provided by itself or, for example, in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition containing 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient.

[0146] The delivery method can also be provided by refillable or biodegradable devices. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein-based biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-biodegradable polymers, can be used to form grafts for sustained release of compounds at specific target sites.

[0147] The actual dosage level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient.

[0148] The selected dosage level depends on a variety of factors, including the activity of the specific compound or combination of compounds used, or their esters, salts, or amides; the route of administration; the time of administration; the elimination rate of the specific compound(s) used; the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific compound(s) used; the age, sex, weight, condition, overall health, and medical history of the patient being treated; and similar factors well known in the medical field.

[0149] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the therapeutically effective dose of the required pharmaceutical composition. For example, a physician or veterinarian can start administration of the pharmaceutical composition or compound at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. "Therapeutic dose" means a concentration of the compound sufficient to produce the desired therapeutic effect. It is generally understood that the effective dose of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective dose include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, other types of therapeutic agents administered with the compound of the present invention. A larger total dose may be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0150] Generally, the appropriate daily dose of the active compound used in the compositions and methods of the present invention is the minimum effective dose of the compound required to produce a therapeutic effect. Such an effective dose is generally determined by the factors described above.

[0151] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, or six or more subdoses, optionally administered separately in unit dosage forms, at appropriate intervals throughout the day. In certain embodiments of the present invention, the active compound may be administered twice or three times daily. In preferred embodiments, the active compound is administered once daily.

[0152] Patients receiving this treatment are any animal in need of treatment, such as primates, especially humans; as well as other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and common pets.

[0153] In certain embodiments, the compounds of the present invention may be used alone or in combination with other types of therapeutic agents.

[0154] This disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in compositions and methods of the present invention. In certain embodiments, salts intended for the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, salts intended for the present invention include, but are not limited to, L-arginine, venentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydravamin, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, salts intended for the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, the salts intended for use in the present invention are, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carboxylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, Examples include gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hypolic acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, L-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0155] pharmaceutically acceptable acid addition salts can also exist as various solvates with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvates may be derived from the solvent of crystallization, those inherent to the solvent of preparation or crystallization, or those associated with such solvents.

[0156] Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0157] Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium disulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0158] definition Unless otherwise specified herein, scientific and technical terms used in this application shall have meanings that are generally understood by those skilled in the art. In general, the nomenclature and techniques used in relation to 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.

[0159] The methods and techniques described herein are, unless otherwise indicated, typically carried out in accordance with prior art practices and as described in the various general and more specific references cited and discussed throughout this 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 ed.”, WH Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, WH Freeman & Co., NY (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0160] Chemical terms used herein, unless otherwise defined, are used in accordance with the customary usage of the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms," ​​Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0161] All of the foregoing, as well as any other publications, patents, and published patent applications referenced herein, are incorporated herein by reference. In the event of any conflict, this specification shall prevail, including its specific definitions.

[0162] The term "pharmaceutical" is used herein to refer to chemical compounds (e.g., organic or inorganic compounds, mixtures of compounds), biological macromolecules (e.g., nucleic acids, antibodies containing parts thereof, and humanized, chimeric and human antibodies, as well as monoclonal antibodies, proteins or parts thereof, e.g., peptides, lipids, carbohydrates), or extracts produced from biomaterials such as bacterial, plant, fungal, or animal (especially mammalian) cells or tissues. Pharmaceuticals include, for example, pharmaceuticals with known structures and pharmaceuticals with unknown structures.

[0163] As used herein, the term “conjugate” refers to a cell-binding substance covalently bound to one or more molecules of a cytotoxic compound. In this regard, the “cell-binding substance” is a molecule that has affinity for a biological target and may also be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and furthermore, the conjugate performs the function of directing the biologically active compound to the biological target. In certain embodiments of this disclosure, the conjugate may be designed to target tumor cells via a cell surface antigen. The antigen may be a cell surface antigen that is overexpressed or expressed in abnormal cell types. Specifically, the target antigen may be expressed only on proliferating cells (e.g., tumor cells). The target antigen may be selected based on different expressions, typically different expressions in proliferating tissue and normal tissue.

[0164] The terms “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, e.g., humans, primates, domestic animals (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0165] "Treating" a condition or patient means taking measures to obtain beneficial or desirable outcomes, including clinical outcomes. Beneficial or desirable clinical outcomes include, but are not limited to, reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; reduction of disease severity; stabilization of the disease state (i.e., no worsening); prevention of disease progression; delay or slowing of disease progression; improvement or mitigation of the disease state; and remission (whether partial or total). "Treatment" may also mean extending survival compared to the survival expected if no treatment were received.

[0166] The term “prevention” is recognized in the art and is well understood in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or any other medical condition, and includes the administration of a composition that reduces the frequency of symptoms of a medical condition in a subject compared to a subject not treated with the composition, or delays its onset. Thus, cancer prevention includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment by, for example, a statistically and / or clinically significant amount, compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population compared to an untreated control population.

[0167] "Administering" a substance, compound, or drug to a subject, or "administration," can be carried out using one of the various methods known to those skilled in the art. For example, a compound or drug can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, orally, sublingually, orally (by ingestion), intranasally (by inhalation), intrathecally, intracerebrally, and percutaneously (e.g., by absorption through cutaneous ducts). A compound or drug can also be appropriately introduced by refillable or biodegradable polymer devices or other devices, such as patches and pumps, or by formulations, which provide sustained release, sustained release, or controlled release of the compound or drug. Administration can also be carried out, for example, once, multiple times, and / or over one or more long periods.

[0168] Appropriate methods for administering a substance, compound, or drug to a subject also depend, for example, on the subject's age and / or physical condition, as well as the chemical and biological properties of the compound or drug (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or drug is administered orally to the subject, for example, by ingestion. In some embodiments, the orally administered compound or drug is in the form of a sustained-release formulation or a sustained-release formulation, or is administered using a device for such sustained-release or sustained-release.

[0169] As used herein, the term "co-administration" refers to any combination of two or more different therapeutic agents, where the second agent is administered while the previously administered therapeutic agent is still effective in the body (for example, both agents are effective simultaneously in the patient, which may include a synergistic effect between the two agents). For example, different therapeutic compounds may be administered in the same formulation, in separate formulations, simultaneously, or sequentially. Thus, an individual receiving such treatment may benefit from the combined effects of different therapeutic agents.

[0170] The "therapeutic effective dose" or "therapeutic effective amount" of a drug or medication is the amount of the drug or medication administered to a subject that produces the intended therapeutic effect. Complete therapeutic effect does not necessarily occur with a single dose, but may only occur after a series of doses. Therefore, a therapeutic effective dose may be administered in more than one dose. The exact effective dose required for a subject depends, for example, on the subject's size, health status, and age, as well as the nature and severity of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective dose for a given situation through routine experimentation.

[0171] As used herein, the terms “optional” or “optionally” (“optionally”) mean that the events or circumstances described below may or may not occur, and such descriptions encompass both cases in which the events or circumstances occur and cases in which they do not. For example, “optionally substituted alkyl” means both cases in which the alkyl can be substituted and cases in which the alkyl is not substituted.

[0172] Those skilled in the art will understand that substituents and substitution patterns for the compounds of the present invention can be selected by methods known in the art and by the methods described below to obtain chemically stable compounds that can be readily synthesized from readily available starting materials. When a substituent itself is substituted with two or more groups, these groups can reside on the same carbon or on different carbons, as long as a stable structure is consequently obtained.

[0173] As used herein, the term “optionally substituted” means that 1 to 6 hydrogen groups in a given structure are substituted with groups of a particular substituent, such substituents include, but are not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” means that 1 to 4 hydrogen groups in a given structure are substituted with the substituents described above. More preferably, 1 to 3 hydrogen groups are substituted with the substituents described above. It is understood that substituents may be further substituted.

[0174] In this invention, the aforementioned prefix (for example, C 1-12 , C 3-8 "Etc." refers to the number of ring atoms or the range of the number of ring atoms, regardless of whether the ring atoms are carbon atoms or heteroatoms.

[0175] As used herein, the term "alkyl" refers to a saturated aliphatic group, and for example, but is not limited to, C1-C11. 10 Linear alkyl groups or C1-C 10 Examples include branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 linear alkyl group or a C1-C6 branched alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 linear alkyl group or a C1-C4 branched alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. The "alkyl" group can be optionally substituted.

[0176] The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0177] The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group, which can be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0178] The term "acyloxy" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0179] The term "alkoxy" refers to an alkyl group to which oxygen is bonded. Typical alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[0180] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0181] The term "alkyl" refers to a saturated aliphatic group, and examples include linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, linear or branched alkyl groups have 30 or fewer carbon atoms in their skeleton (for example, C in the case of a linear group). 1-30 In the case of a branched chain, C 3-30 ), more preferably having 20 or fewer carbon atoms.

[0182] Furthermore, the term “alkyl” as used herein, in the examples and in the claims is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter of which refer to alkyl moieties having substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton, such alkyl moieties include haloalkyl groups (such as trifluoromethyl and 2,2,2-trifluoroethyl).

[0183] The term "alkenyl" refers to alkyl groups that have at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CHCH3), 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl.

[0184] The term "alkynyl" refers to an alkyl group having at least one carbon-carbon triple bond, and examples of unsaturated alkynyl groups include ethynyl and 2-propynyl.

[0185] "C x~y " or "C x~ C y The term "alkyl" means a group containing x to y carbon atoms in a chain, when used in combination with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. C0 alkyl indicates hydrogen when the group is at a terminal position and a bond when it is located internally. 1~6 Alkyl groups, for example, contain 1 to 6 carbon atoms in their chain.

[0186] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0187] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, which can be represented by the general formula alkylS-.

[0188] As used herein, the term "amide" refers to the following groups: [ka] In the formula, R 9 and R 10 Each of these independently represents either a hydrogen atom or a hydrocarbyl group, or R 9 and R 10Together with the N atoms to which they bond, they complete a heterocycle with 4 to 8 atoms in the ring structure.

[0189] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines and their salts, for example, [ka] This refers to the part that can be represented by, in the formula, R 9 , R 10 , and R 10 ' represents a hydrogen or a hydrocarbyl group, or R 9 and R 10 Together with the N atoms to which they bond, they complete a heterocycle with 4 to 8 atoms in the ring structure.

[0190] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0191] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0192] As used herein, the term “aryl” includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, where at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of aryl groups include benzene, naphthalene, phenanthrene, phenol, and aniline.

[0193] The term "carbamate" is recognized in the art and is based on the following principles. [ka] It refers to, and in the formula, R 9 and R 10 This independently represents a hydrogen atom or a hydrocarbyl group.

[0194] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0195] The term "carbocyclic ring" includes monocyclic rings with 5 to 7 members and bicyclic rings with 8 to 12 members. Each ring in a bicyclic carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. A carbocyclic ring includes a bicyclic molecule in which one, two, or three or more atoms are shared between two rings. The term "condensed carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring in a condensed carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. In exemplary embodiments, an aromatic ring, e.g., phenyl, may be condensed with a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of a carbocyclic ring, as long as the valence allows. Exemplary “carbocyclic rings” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octa-3-ene, naphthalene, and adamantane. Exemplary condensed carbocyclic rings include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hepta-3-ene. The “carbocyclic rings” may be substituted at any one or more positions that can support hydrogen atoms.

[0196] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0197] The term "carbonate" is recognized in this technical field and refers to the -OCO2- group.

[0198] As used herein, the term "carboxyl" refers to the group represented by the formula -CO2H.

[0199] The term "cycloalkyl" includes substituted or unsubstituted non-aromatic monocyclic structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term "cycloalkyl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which rings is cycloalkyl, and the cycloalkyl ring has substituents (e.g., R 100 A cyclic ring is added, for example, and the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, and tetrahydroquinoline.

[0200] The term "ester" as used herein means -C(O)OR 9 It refers to the base, and in the formula, R 9 This represents a hydrocarbyl group.

[0201] As used herein, the term "ether" refers to a hydrocarbyl group linked to another hydrocarbyl group via oxygen. Therefore, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers may be symmetric or asymmetric. Examples of ethers, but not limited to, include heterocyclic-O-heterocyclic and aryl-O-heterocyclic groups. Ethers also include "alkoxyalkyl" groups, which can be represented by the general formula: alkyl-O-alkyl.

[0202] The term "formyl" refers to -C(=O)H.

[0203] As used herein, the terms "halo" and "halogen" mean halogens, including chloro, fluoro, bromo, and iodine.

[0204] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to alkyl groups substituted with hetaralkyl groups.

[0205] The terms "heteroaryl" and "hetalil" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, wherein the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetalil" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0206] As used herein, the term “heteroatom” means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0207] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclic group.

[0208] The terms “heterocyclyl,” “heterocyclic,” and “heterocyclic formula” refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, wherein the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms “heterocyclyl” and “heterocyclic formula” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heterocyclic, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactone, and lactam.

[0209] As used herein, the term "hydrocarbyl" refers to a group bonded via a carbon atom that does not have an =O substituent or an =S substituent, and typically has at least one carbon-hydrogen bond and a primary carbon skeleton, but may optionally contain a heteroatom. Therefore, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyls for the purposes of this application, but substituents such as acetyl (having an =O substituent on the bonded carbon) and ethoxy (bonded via oxygen rather than carbon) are not considered hydrocarbyls. Examples of hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbosicle, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0210] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

[0211] The term “lower” means, when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, that the substituent has 10 or fewer atoms, preferably 6 or fewer. For example, “lower alkyl” refers to an alkyl group having 10 or fewer carbon atoms, preferably 6 or fewer. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are, respectively, lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, as described in the descriptions of hydroxyalkyl and aralkyl (in which case, for example, when counting the carbon atoms of the alkyl substituent, atoms in the aryl group are not counted).

[0212] The terms “polycyclyl,” “polycyclic,” and “polycyclic formula” refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, for example, the rings are “fused rings.” Each ring in the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring in the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms.

[0213] The term "sulfate" is recognized in the art and refers to the -OSO3H group or its pharmaceutically acceptable salts.

[0214] The term "sulfonamide" is recognized in the art and refers to a group represented by the following general formula: [ka] In the formula, R 9 and R 10 The '' independently represents hydrogen or hydrocarbyl.

[0215] The term "sulfoxide" is recognized in this technical field and refers to the -S(O)- group.

[0216] The term "sulfonate" is recognized in the art and refers to an SO3H group or a pharmaceutically acceptable salt thereof.

[0217] The term "sulfone" is recognized in this technical field and refers to the -S(O)2- group.

[0218] The term “substituted” refers to a portion of a skeleton having substituents that substitute hydrogens on one or more carbon atoms. “Substituted” or “substituted with…” will be understood to imply the implicit condition that such substitution results in a stable compound that does not undergo transformation, for example, spontaneously by rearrangement, cyclization, or elimination, depending on the substituted atom and the acceptable valence of the substituent. As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. In some embodiments, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of the organic compound. There may be one or more acceptable substituents for a given organic compound, and they may be the same or different. For the purposes of the present invention, heteroatoms such as nitrogen may have any acceptable substituents of the organic compounds described herein that satisfy hydrogen substitution and / or heteroatom valence. Substituents may include any substituents described herein, such as halogens, hydroxyls, carbonyls (carboxyls, alkoxycarbonyls, formyls, or acyls, etc.), thiocarbonyls (thioesters, thioacetates, or thioformates, etc.), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that, where appropriate, the substituted moieties on the hydrocarbon chain may be substituted themselves.

[0219] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0220] As used herein, the term "thioester" refers to -C(O)SR 9 Base or -SC(O)R 9 It refers to the base, In the formula, R 9 This represents hydrocarbyl.

[0221] As used herein, the term "thioether" is equivalent to an ether in which oxygen is replaced by sulfur.

[0222] The term "urea" is recognized in the art and can be represented by the following general formula: [ka] In the formula, R 9 and R 10 This independently represents hydrogen or hydrocarbyl.

[0223] As used herein, the term “modulate” includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0224] The term "pharmaceutically acceptable" is recognized in the art. In certain embodiments, this term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0225] "Pharmacologically acceptable salt" or "salt" is used herein to refer to an acid addition salt or basic addition salt that is suitable or suitable for the treatment of a patient.

[0226] As used herein, the term “pharmaceutically acceptable acid addition salt” means any non-toxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts, such as sodium monohydrogen orthophosphate and potassium bisulfate. Exemplary organic acids that form suitable salts include mono, di, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Salts of monobasic acids may be formed, or salts of dibasic acids may be formed, and such salts may exist in hydrated, solvated, or substantially anhydrous forms. Generally, acid addition salts of compounds of Formula I have higher solubility in water and various hydrophilic organic solvents and generally exhibit higher melting points compared to their free base forms. The selection of appropriate salts is known to those skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0227] As used herein, the term “pharmaceutically acceptable basic addition salt” means any non-toxic organic or inorganic base addition salt of any acid compound represented by formula I or any of its intermediates. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of suitable salts is known to those skilled in the art.

[0228] Many of the compounds useful in the methods and compositions of this disclosure have at least one chiral center in their structure. This stereocenter may be present in an R configuration or an S configuration, and the R and S notations are used in accordance with the rules set out in Pure Appl. Chem. (1976), 45, 11-30. This disclosure intends all stereoisomeric forms (including all possible mixtures of stereoisomers), such as enantiomers and diastereomers, of compounds, salts, prodrugs, or mixtures thereof. See, for example, WO 01 / 062726.

[0229] Furthermore, certain compounds containing an alkenyl group may exist as Z (tuzamen) or E (entgegen) isomers. In either case, this disclosure includes both mixtures and distinct individual isomers.

[0230] A “prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that, after administration, is metabolized (e.g., hydrolyzed or oxidized) in the host to form a compound of the Disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds having a biologically unstable or cleavable (protecting) group on the functional group of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramides as biologically unstable or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, which are incorporated herein by reference. The prodrugs of the Disclosure are metabolized to produce compounds of formula I. The Disclosure includes, to its extent, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of appropriate prodrugs are described, for example, in "Design of Prodrugs" by Ed. H. Bundgaard, Elsevier, 1985.

[0231] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filter, diluent, excipient, solvent, or encapsulating material, that is useful for preparing a drug for medical or therapeutic purposes.

[0232] As used herein, the terms “logarithm of solubility,” “LogS,” or “logS” are used in the art to quantify the water solubility of compounds. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is often associated with insufficient absorption. The LogS value is the unitless logarithm (base 10) of solubility measured in mol / liter.

[0233] The term "identity" refers to the similarity or relevance of two or more polypeptide or nucleic acid sequences, determined by aligning and comparing the sequences of those polypeptides or nucleic acids. Such sequence identity is typically expressed as an "identity percentage," which represents the proportion of identical amino acids or nucleotides between the compared molecules, calculated based on the smallest molecule among those compared. Methods for calculating identity between nucleic acids or polypeptides by aligning them are known in the art and may also be referred to herein.

[0234] The term "affinity" or "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment and the antigen, determined by the characteristics of the antigen, such as its size, shape, and / or charge, as well as the CDR sequence of the antibody or antigen-binding fragment. Methods for determining such affinity are known in the art and may also be referred to herein.

[0235] The antibodies or their antigen-binding fragments used in this invention have a dissociation constant (KD) of ≤ 10 -6 When the KD is M, it is said that the antibody "specifically binds" to a target such as an antigen. -8When it is M, it binds to the target with "high affinity".

[0236] As used in this invention, the term “antigen-binding fragment” of an antibody or immunoglobulin chain (light or heavy chain) refers to a portion of the antibody that contains several amino acid deficiencies compared to the full-length chain but is capable of specifically binding to a target antigen. These fragments may be biologically active in that they are capable of specifically binding to a target antigen or are competitive with other antibodies or antigen-binding fragments for binding to a particular epitope. In some embodiments, such fragments contain at least one CDR in the full-length light or heavy chain, and in some embodiments, they contain a short heavy chain and / or a light chain, or a portion thereof. These biologically active fragments may be produced by recombinant DNA technology or, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFV, scFV-Fc, diabody, minibody, scAb, and dAb, and these fragments may be derived from any mammal, such as humans, mice, rats, camelids, or rabbits. In this invention, a functional portion of an antibody, such as one or more CDRs, can be covalently bound to a second protein or small molecule compound and can be used as a targeted therapeutic agent against a specific target.

[0237] The "Fc" region is used to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and the mutant Fc region, and in this invention, includes two heavy chain fragments containing the CH2 and CH3 domains of the antibody. These two heavy chain fragments are linked to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The boundary of the Fc region of the immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The lysine at the C-terminus of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during the production or purification of the antibody, or by recombination of the nucleic acid encoding the heavy chain of the antibody. Accordingly, the composition of untreated antibodies may include an antibody population from which all K447 residues have been removed, an antibody population from which K447 residues have not been removed, and an antibody population having a mixture of the K447-removed antibodies and the unremoved antibodies. Suitable natural sequence Fc regions for use in the antibodies of this disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0238] The "Fab fragment" in this invention consists of one light chain and one heavy chain containing only a variable region and CH1. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. scFab is a Fab molecule consisting of two molecules linked by a flexible linker.

[0239] In the present invention, the "Fab' fragment" includes the region between the CH1 and CH2 domains of the heavy chain in addition to the Fab fragment, and this region forms an interchain disulfide bond between the two heavy chains of the two molecules of the Fab' fragment, thereby forming an F(ab')2 molecule.

[0240] As described above, the "F(ab')2 fragment" in the present invention comprises two light chains and two heavy chains including a variable region, CH1, and a portion of the constant region between the CH1 domain and the CH2 domain, with two interchain disulfide bonds forming between the heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are linked to each other by disulfide bonds.

[0241] In this invention, the "Fv region" is an antibody fragment that includes variable regions of the heavy and light chains but does not include a constant region. sdFv has its heavy and light chains linked by disulfide bonds. scFv is Fv linked by a flexible linker. scFv-Fc has its Fc linked to scFv. Minibody has its CH3 linked to scFv. Diabody contains two scFv molecules.

[0242] The “single-chain Fv” or “scFv” antibody fragments in the present invention comprise the VH and VL domains of an antibody, and these domains are located within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains, enabling the scFv to form a desired structure for antigen binding.

[0243] In the present invention, a "single-chain antibody" (scAb) is a single polypeptide chain containing one constant region of a heavy chain or a constant region of a light chain, wherein the heavy chain and the variable region of the light chain are linked by a flexible linker. For more information on single-chain antibodies, see, for example, U.S. Patent No. 5,260,203 disclosed herein for reference.

[0244] In the present invention, a "domain antibody" (dAb) is an immunologically functional immunoglobulin fragment containing only a variable region of the heavy chain or a variable region of the light chain. For example, in one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of these bivalent domain antibodies can target the same or different antigens.

[0245] As used herein, “complementarity-determining regions” (CDRs, i.e., CDR1, CDR2, and CDR3) refer to the amino acid portions of the antibody variable domain required for antigen binding. Antibodies typically contain six CDRs: three in the VH (H1 CDR, H2 CDR, H3 CDR) and three in the VL (L1 CDR, L2 CDR, L3 CDR). In naturally occurring antibodies, H3 and L3 exhibit the most diversity among the six CDRs, and H3, in particular, is thought to play a unique role in conferring excellent specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. For example, see Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0246] As used herein, the “framework region” (FR) is the variable domain portion excluding the CDR portion. Each variable domain typically has four FRs, which are identified as FR1, FR2, FR3, and FR4.

[0247] As used herein, a "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites in such a bivalent antibody may have the same antigen specificity, or they may be bispecific antibodies that bind to different antigens.

[0248] As used herein, a "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0249] As used herein, “chimeric antibody” means an antibody (immunoglobulin) in which a portion of its heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and in a fragment of such antibody (only if they exhibit the desired biological activity) (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). An example of a chimeric antibody of interest herein is a PRIMATIZED® antibody in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque monkey with the target antigen.

[0250] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing the minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody), in which case residues from the recipient's CDR are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having desired specificity, affinity, and / or capacity. In some cases, the FR residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity. Generally, a humanized antibody contains at least one, and typically two, variable domains substantially all of which, in the variable domain, all or substantially all hypervariable loops correspond to those of non-human immunoglobulin sequences, and all or substantially all FR regions are those of human immunoglobulin sequences, although the FR regions may contain one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, and immunogenicity. The number of these amino acid substitutions in the FR is typically 6 or less in the H chain and 3 or less in the L chain. The humanized antibody may further contain at least a portion of the immunoglobulin constant region (Fc), typically the Fc of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0251] A “human antibody” is a human antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. (Hoogenboom and Winter, J.Mol.Biol., 227:381(1991); Marks et al., J.Mol.Biol., 222:581(1991)). Similarly available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77(1985); and Boerner et al., J.Immunol., 147(1):86-95(1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals, such as immunized xenomouses (see, for example, U.S. Patents 6,075,181 and 6,150,584, for XENOMOUSE™ technology), which have been modified to produce such antibodies in response to antigen challenge but whose endogenous gene locus has been deactivated. See also Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006), for example, human antibodies produced by human B-cell hybridoma technology.

[0252] The "Human Consensus Framework" is a framework of the most commonly found amino acid residues when selecting human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. These subgroups are typically those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, with respect to VL, the subgroup could be subgroups κI, κII, κIII, or κIV as described by Kabat et al. above. Furthermore, with respect to VH, the subgroup could be subgroup I, subgroup II, or subgroup III as described by Kabat et al. above.

[0253] A "affinity-matured" antibody is an antibody that has one or more modifications in one or more CDRs, the modifications resulting in improved affinity of the antibody to an antigen compared to a parent antibody that does not possess those modifications. In one embodiment, the affinity-matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are prepared by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described, for example, by Barbas et al. Proc Nat.Acad.Sci.USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.Immunol.155:1994-2004 (1995); Jackson et al., J.Immunol.154(7):3310-9 (1995); and Hawkins et al., J.Mol.Biol.226:889-896 (1992).

[0254] Examples Although the present invention has been described in general terms up to this point, it will be easier to understand by referring to the following examples, which are included merely to illustrate specific aspects and embodiments of the present invention and are not intended to limit the present invention. [Examples]

[0255] Example 1: Production of an exemplary human L1CAM (neuronal cell adhesion molecule L1) specific antibody L1CAM (neuronal cell adhesion molecule L1) is widely expressed in tumors. The anti-L1CAM humanized antibody AFF4, which specifically binds to the exemplified L1CAM, was prepared by the method described in European Patent Application No. EP22196917.3 (the full contents of which are incorporated herein by reference). The amino acid sequence of the antibody AFF4 is shown in Tables 7 and 8 below.

[0256] [Table 7] TIFF2026510495000082.tif222160TIFF2026510495000083.tif62160

[0257] Furthermore, for ADC synthesis, AFF4-CaaX was prepared by introducing the LALA mutation (L236A / L237A) into the heavy chain constant region of the antibody AFF4, and introducing the CaaX peptide portion (GGGGGGGCVIM, SEQ ID NO: 7) into the C-terminus of the light chain. The prepared antibody AFF4-CaaX was used for ADC synthesis. The amino acid sequence of the antibody AFF4-CaaX is shown in Table 8 below.

[0258] [Table 8] TIFF2026510495000085.tif221162 [Examples]

[0259] Example 2: Synthesis of Exemplary Compounds of the Disclosure Preparation of Compound 1 [ka]

[0260] Compound 1 was prepared using the method disclosed in U.S. Patent No. 11,173,214 (the full contents of which are incorporated herein by reference). EI-MS m / z:[1 / 2M+H] + 1623.6,[1 / 3M+H] + 1082.8.

[0261] The structure of MMAE (monomethyl auristatin E) in compound 1 is as follows: [ka] That is the case.

[0262] Preparation of Compound 2 [ka] Compound 2 was prepared using the method disclosed in U.S. Patent No. 11,654,197 (the full contents of which are incorporated herein by reference). EI-MS m / z:[M+H] + 1698.2,[1 / 2M+H] + 849.6.

[0263] Preparation of compounds 4-7 [ka] [ka]

[0264] Preparation of compound 4 Compound 3 (PCT application no. PCT / KR2023 / 018766, the full contents of which are incorporated herein by reference) (1.5 g, 0.67 mmol) was dissolved in (24 mL) of dichloromethane. Next, 0.22 mL of pyridine (2.68 mmol) and 0.79 g of bis(pentafluorophenyl) carbonate (2.01 mmol) were added under a nitrogen atmosphere at room temperature, and the mixture was stirred for 16 hours. The reaction solution was diluted in 80 mL of ethyl acetate, rinsed with 50 mL x 2 saturated sodium bicarbonate aqueous solutions, then with 50 mL of brine, and dehydrated using anhydrous sodium sulfate. Compound 4 (2.06 g) was obtained by filtration and concentration under reduced pressure without further purification, and was subsequently used in the next process. EI-MS m / z:[M+H] + 2670.3,[1 / 2M+H] + 1335.7,[1 / 3M+H] + 890.8.

[0265] Preparation of compound 5 Compound 4 (100 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (3 mL), and then, under a nitrogen atmosphere with stirring for 3 hours at room temperature, exatecan mesylate (44 mg, 0.08 mmol), N,N-diisopropylethylamine (0.05 mL, 0.28 mmol), and 1-hydroxy-7-benzotriazole (HOAt, 2.55 mg, 0.02 mmol) were sequentially added. The reaction mixture was diluted with ethyl acetate (40 mL), washed sequentially with saturated ammonium chloride aqueous solution (30 mL), saturated sodium bicarbonate aqueous solution (30 mL), and brine (30 mL), and then dried over anhydrous sodium sulfate. Compound 5 (140 mg) was obtained after filtration and concentration under reduced pressure without further purification. EI-MS m / z:[1 / 2M+H] + 1587.5,[1 / 3M+H] + 1058.8.

[0266] Preparation of compound 6 Compound 5 (550 mg, 0.17 mmol) was dissolved in methanol (7 mL) and tetrahydrofuran (7 mL), and then a solution of lithium hydroxide (65.8 mg, 1.57 mmol) dissolved in distilled water (8 mL) was gradually added under a nitrogen atmosphere at -40°C. Next, this mixture was stirred for 2 hours so that the reaction temperature was gradually raised to 0°C. The reaction mixture was neutralized with acetic acid, concentrated under reduced pressure, purified by HPLC, and then freeze-dried to obtain compound 6 (236 mg, 44%) in the form of a yellow solid. EI-MS m / z:[1 / 2M+H] + 1446.7,[1 / 3M+H] + 964.9.

[0267] Preparation of compound 7 Compound 6 (236 mg, 0.08 mmol) was dissolved in acetonitrile (10 mL), and phosphoric acid (85%, 5 mL) was added at 0°C. After stirring at room temperature for 4 hours, the reaction mixture was purified by HPLC and subsequently freeze-dried to obtain compound 7 (138 mg, 61%), a yellow solid. EI-MS m / z:[1 / 2M+H] + 1340.5, [1 / 3M+H] + 894.1. [Examples]

[0268] Example 3: Synthesis of Exemplary ADCs of the Disclosure The example ADCs were prepared using the following two procedures. LCB14-0606 was prepared using the method disclosed in U.S. Patent No. 9,669,107 (the full contents of which are incorporated herein by reference). The structural formula of LCB14-0606 is as follows: [ka] That is the case.

[0269] Step 1: Preparation of prenylated antibodies using LCB14-0606 AFF4-CaaX was prepared according to Example 1. A mixture containing AFF4-CaaX for the prenylation reaction was prepared and reacted at 30°C for 16 hours. This reaction mixture contained a total of 24 μM of the antibody, 600 mg or 400 nM of FTase (Genscript), and 144 μM of LCB14-0606 in a buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 10 μM ZnCl2, 0.5 mM DTT). After the reaction, the prenylated antibody was desalted using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer. As a result, a total of 544 mg of prenylated antibody was produced.

[0270] Step 2: Drug Conjugation Methods Oxime bonding reaction of ADC1 (conjugation by oxime bond formation) The oxime bond formation reaction between the prenylated antibody and the linker-drug was carried out by mixing 100 μM sodium acetate buffer, pH 5.2, 10% DMSO, 350 mg of prenylated antibody (total 24 μM), and 10 equivalents of the linker-drug (compound 1 from Example 1) with stirring at 30°C and 500-600 rpm for 24 hours. After the reaction, excess low molecular weight compounds were removed using FPLC (AKTA purifier, GE Healthcare), and the protein fraction was recovered and concentrated. As a result, a total of 336 mg of ADC1 was obtained. The preparation procedure and structure are shown in Figure 1.

[0271] ADC2 oxime bonding reaction (conjugation by oxime bond formation) The oxime bond formation reaction between the prenylated antibody and the linker-drug was carried out by mixing 100 μM sodium acetate buffer, pH 5.2, 10% DMSO, 14.4 mg of prenylated antibody (total 24 μM), and 10 equivalents of the linker-drug (compound 2 from Example 2) with stirring at 30°C and 500-600 rpm for 6 hours. After the reaction, excess low molecular weight compounds were removed using FPLC (AKTA purifier, GE Healthcare), and the protein fraction was recovered and concentrated. As a result, a total of 13.5 mg of ADC2 was obtained. The preparation procedure and structure are shown in Figure 2.

[0272] ADC3 oxime bonding reaction (conjugation by oxime bond formation) The oxime bond formation reaction between the prenylated antibody and the linker-drug was carried out by mixing 100 μM sodium acetate buffer, pH 5.2, 10% DMSO, 10 mg of prenylated antibody (total 48 μM), and 4 equivalents of the linker-drug (compound 7 from Example 3) with stirring at 30°C and 500-600 rpm for 6 hours. After the reaction, excess low molecular weight compounds were removed using FPLC (AKTA purifier, GE Healthcare), and the protein fraction was recovered and concentrated. As a result, a total of 7.7 mg of ADC3 was obtained. Its preparation procedure and structure are shown in Figure 3.

[0273] To confirm the properties of the three ADCs prepared above, analysis was performed using a mobile phase prepared with ammonium sulfate, acetonitrile, and potassium phosphate, and an HPLC instrument equipped with an HIC column. The results are shown in Figure 4. Furthermore, the molecular weights of ADC1 and ADC3 were confirmed by intact mass analysis, and the results are shown in Figure 5. As shown in Figure 4, ADC1, ADC2, and ADC3 all exhibited different levels of hydrophobicity, with the order being ADC1 > ADC2 > ADC3. Furthermore, the purity at the same DAR (drug-antibody ratio) was confirmed to be 97% or higher.

[0274] As shown in Figure 5, the molecular weight of ADC1 was 153,535,27 Da, and the molecular weight of ADC3 was 152,404,38 Da.

[0275] Table 9 summarizes the ADCs fabricated using the two steps described above.

[0276] [Table 9] [Examples]

[0277] Example 4: Evaluation of cancer cell binding affinity of anti-L1CAM antibody The cell binding affinity of the AFF4-CaaX antibody prepared in Example 1 was confirmed using pancreatic cancer cell lines (PANC-1, PATU-8988s), breast cancer cell lines (JIMT-1, MDA-MB-231, SK-BR-3, HCC1395, MCF7), ovarian cancer cell lines (SK-OV-3, OVCAR-3, SNU-840, SNU-8), colorectal cancer cell lines (HCT116, SW480, SNU-1197), lung cancer cell lines (H69, PC-9, NCI-H460, Calu-6), melanoma cell lines (A2058, MeWo, G361), and neuroblastoma cell lines (SK-N-F1).

[0278] Specifically, the above-mentioned cancer cell line was cultured, and the cells were further divided into two groups: a control antibody-treated group and an anti-L1CAM antibody-treated group. Subsequently, nonspecific binding of the antibody was removed by incubating the cells in a phosphate-buffered saline solution containing 2% fetal bovine serum at 4°C for 1 hour. Next, the control antibody and anti-L1CAM antibody were incubated with each group of the cell line at 4°C for 1 hour to bind the antibodies to the L1CAM protein on the surface of the cancer cells. Subsequently, antibodies that did not bind to the cell surface were removed using a phosphate-buffered saline solution containing 2% fetal bovine serum. Subsequently, the cells were incubated with a fluorescently labeled secondary antibody that binds to the control antibody and the anti-L1CAM antibody at 4°C for 1 hour. After removing the secondary antibody that did not bind to the antibody using a phosphate-buffered saline solution, the fluorescence intensity of the cells was measured using a flow cytometer, and the fluorescence intensity of the cell line in the anti-L1CAM antibody group was calculated by comparing it with the fluorescence intensity of the cell line in the control group to confirm the binding affinity of the antibody to the cancer cell line. The results are shown in Tables 10-15 below.

[0279] [Table 10]

[0280] [Table 11]

[0281] [Table 12]

[0282] [Table 13]

[0283] [Table 14]

[0284] [Table 15]

[0285] As shown in Tables 10-15, the AFF4-CaaX antibody was able to strongly bind to all cancer cell lines. [Examples]

[0286] Example 5: In vitro cytotoxicity evaluation The cell proliferation inhibitory activity of the three ADCs prepared in Example 3 against cancer cell lines was measured.

[0287] Specifically, commercially available human breast cancer cell lines (JIMT-1, MDA-MB-231, SK-BR-3, HCC1395, MCF7), ovarian cancer cell lines (OVCAR-3, SNU-840, SNU-8), pancreatic cancer cell lines (PANC-1, PATU-8988s), lung cancer cell lines (NCI-H69, PC-9, NCI-H460, Calu-6), colorectal cancer cell lines (HCT116, SW480, SNU-1197), and melanoma cell lines (A2058, MeWo, G-361) were used as cancer cell lines, while commercially available human kidney cells (HK-2, HEK293T), skin cells (HaCaT), and liver cells (Fa2N-4) were used as control normal cell lines.

[0288] Furthermore, each cancer cell line was seeded in 96-well plates at a concentration of 500–9,000 cells / well and cultured for 24 hours. Subsequently, the cells were treated with ADC at concentrations of 0.256 pM–100 nM (5-fold serial dilution). After 144 hours, the number of viable cells was quantified using SRB (sulforhodamine B) dye or Cell Titer Glo, and the results are shown in Tables 16–18 below.

[0289] [Table 16]

[0290] [Table 17]

[0291] [Table 18]

[0292] As shown in Tables 16 and 17, the cytotoxicity assay results for ADC1 and ADC2 suggest that the pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC2) exhibits significantly stronger cytotoxicity compared to the auristatin-based antibody-drug conjugate (ADC1) in most cancer cell lines, with the exception of the colorectal cancer cell line HCT116 and the breast cancer cell lines HCC1395 and MCF7. Furthermore, as shown in Table 18, the cytotoxicity assay results for ADC1, 2, and 3 suggest that in the colorectal cancer cell line SW480 and the melanoma cell line MeWo, the cytotoxicity is in the order of ADC2 > ADC1 > ADC3.

[0293] Furthermore, the cytotoxicity results in normal cells are shown in Table 19.

[0294] [Table 19]

[0295] As shown in Table 19, ADCs exhibited weak cytotoxicity in normal cell lines. Specifically, ADC2 showed weaker cytotoxicity compared to ADC1 in normal cell lines, but ADC1 and ADC2 showed similar cytotoxicity in Fa2N-4 and HEK293T cells.

[0296] These results suggest that the exemplified ADCs exhibit weak cytotoxicity in normal cells but strong cytotoxicity in cancer cells. In particular, ADC2 showed weaker cytotoxicity in normal cells and stronger cytotoxicity in cancer cells compared to other ADCs. [Examples]

[0297] Example 6: Evaluation of in vivo anti-cancer efficacy The tumor growth inhibitory effect of the ADC prepared in Example 3 was analyzed using a tumor xenograft mouse model.

[0298] Specifically, breast cancer cell lines (JIMT-1), colorectal cancer cell lines (SW480), melanoma cell lines (MeWo), colorectal cancer cell lines (HT29), small cell lung cancer cell lines (NCI-H146, NCI-H69), ovarian cancer cell lines (SNU-840), pancreatic cancer cell lines (PATU-8988s), and non-small cell lung cancer cell lines (Calu-6) were used for cell culture, and 2,000,000 to 5,000,000 cells were mixed with 100 μL of PBS solution and 100 μL of Matrigel. Subsequently, these mixtures were subcutaneously transplanted into Balb / c nude mice to create a tumor xenograft mouse model. The tumor size was 100-300 mm. 3 When the tumor size was reached, ADC was administered intravenously according to the dosage and administration plan shown in Table 20. The tumor size was measured immediately before the first dose (day 1) and periodically for a certain period (up to 56 days), and the results are shown in Figures 6-13.

[0299] [Table 20]

[0300] As shown in Figure 6, ADCs demonstrated significant suppression of tumor growth in the JIMT-1 model of breast cancer cells. Specifically, the auristatin-based antibody-drug conjugate (ADC1) showed superior tumor growth suppression at 5 mg / kg compared to the control group, and the pyrrolobenzodiazepine-based antibody-drug conjugate (ADC2) showed tumor growth suppression at 1 mg / kg.

[0301] Furthermore, as shown in Figure 7, in the SW480 model of colorectal cancer cells, both ADC1 and ADC2 showed concentration-dependent tumor growth inhibition compared to the control group. Moreover, ADC2 showed significant tumor growth inhibition even at low doses.

[0302] Furthermore, as shown in Figure 8, in the MeWo model of melanoma cell lines, both ADC1 and ADC2 showed concentration-dependent suppression of tumor growth compared to the control group.

[0303] Furthermore, as shown in Figure 9, in the Calu-6 model of non-small cell lung cancer cells, ADC1 was 4 mg / kg (BIW). * 2) Dosage group and ADC3 4 mg / kg (QD * 1. BIW * 2) Both dose groups showed significant inhibition of tumor growth compared to the control group.

[0304] Furthermore, as shown in Figure 10, in the SNU840 model of ovarian cancer cells, 4 mg / kg of ADC1 and 8 mg / kg of ADC3 (QD * 1 and BIW * 2) Both groups showed significant suppression of tumor growth compared to the control group.

[0305] Furthermore, as shown in Figure 11, in the Patu8988s model of pancreatic cancer cells, 4 mg / kg of ADC1 showed significant suppression of tumor growth compared to the control group.

[0306] Furthermore, as shown in Figure 12, in the NCI-H146 model of small cell lung cancer cells, the 4 mg / kg ADC1 and 16 mg / kg ADC3 groups showed significant suppression of tumor growth compared to the control group.

[0307] Furthermore, as shown in Figure 13, in the NCI-H69 model of small cell lung cancer cells, ADC1 at 2 mg / kg and ADC3 at 16 mg / kg showed significant suppression of tumor growth compared to the control group. [Examples]

[0308] Example 7: Analysis of pharmacokinetics in a mouse model To analyze the pharmacokinetics of ADCs, ADC1 and ADC3 were administered intravenously to nude mice at single doses of 2 mg / kg and 8 mg / kg, respectively. Serum concentrations of ADCs and antibodies were measured using LC-MS by collecting blood samples at 0 minutes, 3 minutes, 3 hours, 1-, 2-, 3-, 7-, 9-, and 14-days after administration. Five nude mice were used at each time point.

[0309] Figure 14 shows the results of pharmacokinetic analyses of ADC1 and ADC3 in mouse models, indicating that ADC1 and ADC3 had superior half-lives. In particular, there was almost no difference in the half-lives of the ADCs and the antibody used as a control. [Examples]

[0310] Example 8: Single-dose acute toxicity study in rats To analyze the acute toxicity of ADCs, ADC1 (10 mg / kg, 20 mg / kg) and ADC3 (100 mg / kg, 150 mg / kg) were administered intravenously as a single dose into the tail vein of Sprague-Dawley rats (n=5), and the rats were observed for 4 weeks.

[0311] Figure 15 shows that administration of ADC1 or ADC3 to rats did not result in significant toxic symptoms, as measured by body weight change, at the highest concentrations, and no abnormal findings were observed at autopsy compared to the control group.

[0312] Built-in by reference All publications and patents referenced herein are incorporated herein by reference in their entirety, as if each individual publication or patent were explicitly and individually indicated to be incorporated by reference. Any conflict shall be governed by this application, including all definitions herein.

[0313] Equivalents While specific embodiments of the invention have been described, the above specification is illustrative and not limiting. Many modifications of the invention will be obvious to those skilled in the art upon consideration of this specification and the appended claims. The full scope of the invention should be determined by referring to the claims together with the full scope of their equivalents, and to this specification together with such modifications.

[0314] Sequence information

Claims

1. Formula I: 【Chemistry 1】 (In the formula, Ab is as follows (a) to (f), that is (a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 8, (b) Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, (c) Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, (d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, (e) Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and (f) Light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, This is an anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody or its antigen-binding fragment, Each B is an active agent independently. Each G independently comprises a glucuronic acid portion, or 【Chemistry 2】 And, R ３ is a hydrogen or carboxyl protecting group, Each R ４ These are independently hydrogen or hydroxyl protecting groups, R １ and R ２ Each is independently hydrogen, C １－８ Alkyl, or C ３－８ It is a cycloalkyl, W is independently -C(O)-, -C(O)NR'-, -C(O)O-, and -SO ２ NR'-, -P(O)R''NR'-, -SONR'- or -PO ２ NR', where C, S, or P is directly bonded to the phenyl ring, and NR' is bonded to L. R' and R'' are each independently hydrogen, C １－８ alkyl, C ３－８ cycloalkyl, C １－８ alkoxy, C １－８ alkylthio, mono- or di-C １－８ alkylamino, C ３－１０ heteroaryl or C ６－１０ aryl, and Each Z is independently C １－８ They are alkyl, halogen, cyano, or nitro, Each L is an independent linker, and the following (A) and / or (B), i.e. (A) (i) One or more unsaturated bonds, (ii) Heteroarylenes (e.g., heteroarylenes in alkylene or heteroalkylene chains), or (iii) C １－２０ Alkyl substituents C including １－５０ Alkylenes or heteroalkylenes with 1 to 50 member rings, and / or (B) Formula II: 【Transformation 3】 At least one isoprenyl unit having the structure represented by Includes, n1 and n2 are integers independently selected from 1 to 20, and n3 is an integer between 0 and 3. A conjugate having a structure represented by the symbol.

2. Ab is as follows (i) and (ii), that is (i) A variable heavy chain framework region from a human antibody heavy chain or human consensus framework, wherein the variable heavy chain framework region contains one or more of the following mutations: T34A, Y58F, N61H, and S103G, and (ii) The light chain of the human antibody or the variable light chain framework region from the human consensus framework. The conjugate according to claim 1, including the following:

3. The conjugate according to claim 1 or 2, wherein Ab contains a LALA mutation in the heavy chain constant region.

4. The conjugate according to claim 3, wherein the LALA mutations include L236A and L237A.

5. The conjugate according to any one of claims 1 to 4, wherein Ab is one selected from a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFab fragment, an Fv fragment, a dsFv fragment, a single-chain variable fragment (scFv), an 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.

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

7. The conjugate according to any one of claims 1 to 6, wherein n1 is 1, 2, 3, or 4.

8. The conjugate according to any one of claims 1 to 6, wherein n1 is 1 or 2.

9. The conjugate according to any one of claims 1 to 8, wherein n2 is 1, 2, 3, or 4.

10. The conjugate according to any one of claims 1 to 8, wherein n2 is 1.

11. Each G 【Chemistry 4】 And R ３ and R ４ The conjugate according to any one of claims 1 to 10, wherein each of them is H.

12. R １ and R ２ The conjugate according to any one of claims 1 to 11, wherein each of them is hydrogen.

13. The conjugate according to any one of claims 1 to 12, wherein each W is independently -C(O)NR'-.

14. The conjugate according to any one of claims 1 to 13, wherein R' is H.

15. The conjugate according to any one of claims 1 to 14, wherein R'' is H.

16. The conjugate according to any one of claims 1 to 15, wherein Z is a halo (e.g., bromo) or a cyano.

17. The conjugate according to any one of claims 1 to 16, wherein n3 is 1.

18. The conjugate according to any one of claims 1 to 17, wherein n3 is 0.

19. L is C １－５０ A conjugate according to any one of claims 1 to 18, comprising alkylene.

20. L is C １－５０ A conjugate according to any one of claims 1 to 19, comprising a heteroalkylene.

21. The conjugate according to any one of claims 1 to 20, wherein L comprises an unsaturated bond (e.g., 1, 2, 3, 4, or 5 unsaturated bonds).

22. L is C １－２０ A conjugate according to any one of claims 1 to 21, which is substituted with an alkyl group.

23. L is given by equation IIIa: 【Transformation 5】 (In the formula, n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) A conjugate according to any one of claims 1 to 22, comprising an isoprenyl group having the structure represented by .

24. The conjugate according to any one of claims 1 to 23, wherein L contains a peptide.

25. The conjugate according to claim 24, wherein the peptide comprises at least one hydrophilic amino acid.

26. The conjugate according to claim 24 or 25, wherein the peptide comprises an amino acid having a side chain having a charged portion (e.g., an amine, guanidine, or carboxyl portion) at a neutral pH in an aqueous solution.

27. The conjugate according to any one of claims 24 to 26, wherein the peptide comprises an amino acid selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

28. The conjugate according to any one of claims 1 to 27, wherein the antibody contains a cysteine ​​residue, and L is covalently linked to the antibody via a thioether bond by a sulfur atom in the cysteine ​​residue (for example, by a sulfur atom in the side chain of the cysteine ​​residue).

29. The conjugate according to any one of claims 1 to 28, wherein the C-terminus of the antibody includes an amino acid motif recognized by an isoprenoid transferase.

30. The conjugate according to claim 29, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

31. The conjugate according to claim 29 or 30, wherein the amino acid motif includes a cysteine ​​residue, and L is covalently linked to the antibody via a thioether bond by a sulfur atom in the cysteine ​​(for example, by a sulfur atom in the side chain of the cysteine ​​residue).

32. The aforementioned amino acid motif has the following sequence, namely CYYX (In the formula, C is cysteine, Each Y is an aliphatic amino acid, and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. The conjugate according to claim 31, including the above.

33. The conjugate according to claim 32, wherein each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine.

34. The conjugate according to claim 32, wherein the amino acid motif has the sequence CVIM or CVLL.

35. The conjugate according to any one of claims 29 to 34, wherein at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

36. The conjugate according to any one of claims 29 to 35, wherein the amino acid motif comprises the sequence GGGGGGGGCVIM.

37. The conjugate according to any one of claims 1 to 36, wherein L contains an oxime.

38. The conjugate according to claim 37, wherein the oxygen atom of the oxime is on the L side that is bonded to the active agent, and the carbon atom of the oxime is on the L side that is bonded to Ab.

39. The conjugate according to claim 37, wherein the carbon atom of the oxime is on the Y side that is bonded to the active agent, and the oxygen atom of the oxime is on the L side that is bonded to Ab.

40. L is expression IVa or IVb: 【Transformation 6】 (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 -, preferably -O-, X is -O-, C １－８ Alkylene, or -NR ２１ -, preferably -O-, R ２１ ~R ２５ H and C are independent of each other. １－６ Alkyl, C １－６ Alkyl C ６－２０ Aryl, or C １－６ Alkyl C ３－２０ It is a heteroaryl, Each r is an independent integer from 1 to 10, preferably 2. Each p is an independent integer between 0 and 12, preferably 2. q is an integer from 1 to 20, preferably 2, 5, or 11, and w is an integer between 1 and 20, preferably between 6 and 20. A conjugate according to any one of claims 1 to 39, comprising the structure represented by the above.

41. The conjugate according to claim 40, wherein V is -O-.

42. The conjugate according to claim 40 or 41, wherein X is -O-.

43. R ２１ The conjugate according to any one of claims 40 to 42, wherein H.

44. R ２２ The conjugate according to any one of claims 40 to 43, wherein H.

45. R ２３ The conjugate according to any one of claims 40 to 44, wherein H.

46. R ２４ The conjugate according to any one of claims 40 to 45, wherein H.

47. R ２５ The conjugate according to any one of claims 40 to 46, wherein H.

48. The conjugate according to any one of claims 40 to 47, wherein r is 2.

49. The conjugate according to any one of claims 40 to 48, wherein p is 2.

50. The conjugate according to any one of claims 40 to 49, wherein q is 2 or 5.

51. A conjugate according to any one of claims 40 to 50, wherein w is 6 to 20.

52. L, 【Transformation 7】 The conjugate according to any one of claims 40 to 51, comprising at least one polyethylene glycol unit represented by .

53. The conjugate according to claim 52, wherein L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyethylene glycol units.

54. The conjugate according to any one of claims 1 to 53, wherein L contains an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

55. The conjugate according to any one of claims 1 to 54, wherein L comprises a portion formed by the reaction of an alkyne with an azide, or by the reaction of an aldehyde or ketone group with a hydrazine or hydroxylamine.

56. L is an expression Va, Vb, Vc, Vd, or Ve: 【Transformation 8】 (L １ is a single bond or C １－３０ It is alkylene and R １１ is H or C １－１０ (It is alkyl.) A conjugate according to any one of claims 1 to 55, comprising a unit having a structure represented by [the specified formula].

57. L １ The conjugate according to claim 56, wherein the bond is a single bond.

58. L is C １－３０ The conjugate according to claim 56, wherein the conjugate is alkylene.

59. R １１ The conjugate according to any one of claims 56 to 58, wherein H.

60. R １１ C １－１０ The conjugate according to any one of claims 56 to 58, wherein the element is alkyl (for example, methyl).

61. L is as follows (i) to (iiib), that is (i) A branched unit covalently bonded to Ab by a first-class linker, (ii) A first branch that connects the first B to the branching unit, (iiia) A second branch that connects the second B to the branching unit, or (iiib) Second branch containing the branch unit to which an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is attached. A conjugate according to any one of claims 1 to 60, including the conjugate described in any one of claims 1 to 60.

62. The conjugate according to claim 61, wherein L includes a second branch that connects the second B to the branching unit via a second cleavage group.

63. The conjugate according to claim 61, wherein L comprises a second branch containing the branched unit to which an alkyl or heteroalkyl (e.g., polyethylene glycol monomer or polyethylene glycol oligomer) is bonded.

64. The aforementioned branching unit is given by formulas VIa, VIb, VIc, or VId: 【Chemistry 9】 (In the formula, G １ G ２ G ３ Each is connected independently. 【Chemistry 10】 or 【Chemistry 11】 And, R ３０ is H or alkyl, R ４０ is H, alkyl or L ５ -CO ２ R ５０ And, R ５０ is H or alkyl, and L ２ , L ３ , L ４ , and L ５ (Each is independently bonded or alkylene.) A conjugate according to any one of claims 61 to 63, having a structure represented by the above.

65. The aforementioned branching unit is given by equation VIe: 【Chemistry 12】 (R in the formula) ３０ (is H or alkyl) A conjugate according to any one of claims 61 to 63, having a structure represented by the above.

66. The aforementioned branching unit is given by equation Vif: 【Chemistry 13】 (R in the formula) ３０ (is H or alkyl) A conjugate according to any one of claims 61 to 63, having a structure represented by the above.

67. The conjugate according to any one of claims 1 to 66, wherein the conjugate includes one, two, three, or four branch linkers.

68. The conjugate according to claim 67, wherein each branched linker contains at least two active agents.

69. The conjugate according to claim 67, wherein each branched linker contains two active agents.

70. The conjugate according to any one of claims 61 to 69, wherein the branching unit is lysine and the primary linker is bonded to the C-terminus of lysine.

71. The conjugate according to any one of claims 1 to 70, wherein the conjugate is cleavable in target cells (for example, the conjugate cleaves to release one or more active agents).

72. The aforementioned conjugate is as follows: 【Chemistry 14】 (n11, n22, and n33 are each independent integers from 0 to 30, and (AA represents an amino acid group) A conjugate according to any one of claims 1 to 71, comprising the structure represented by the above.

73. The conjugate according to any one of claims 1 to 72, wherein each active agent is independently a chemotherapeutic agent or a toxin.

74. The conjugate according to any one of claims 1 to 72, wherein each active agent is independently an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

75. The conjugate according to any one of claims 1 to 72, wherein the active agent is independently an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, or an antiparasitic agent.

76. The active agent is (a) to (q) below, that is (a) Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787 / ZK222584, Oxaliplatin, 5-Fluorouracil, Leucovorin, Rapamycin, Lapatinib, Ronafarnib, Sorafenib, Gefitinib, AG1478, AG1571, Thiotepa, Cyclophosphamide, Busulfan, Improsulfan, Piposulfan, Benzodopa, Carbocon, Metsuredopa, Uredopa, Ethyleneimine, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Ethylenethiophosphoramide, Trimethylolmelamine, Bratacin, Bratacinone, Camptothecin, Topotecan, Briostatin, Calistatin, CC-1065, Adzeresin, Calzelsin, Bizeresin, Cryptophycin 1, Cryptophycin 8, Dorastatin, Duocalmycin, KW-2189, CB1-TM1, Erytherobin, Pancratistatin, Sarcodicin, Spongestatin, Chlorambucil, Chlornafadin, Colophosphamide, Estramustine, Ifosfamide, Mechloretamine, Melphalan, Nobuenvicin Fenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calitiamicin, calitiamicin gamma 1, calitiamicin omega 1, dynemycin, dynemycin A, clodronate, esperamicin, neocardinostatin chromophore, arasinomycins, actinomycin, antimycin, azaserin, bleomycins, kakuchinomycin, carabicin, carninomycin, cardinophilin, chromomycins, dactino Mycin, daunorubicin, detorubcin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomarcelomycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin derivatives, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptomygrin, streptozocin, tubercidine,Ubenimex, dinostatin, zolubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimethrexate, fludarabine, 6-mercaptopurine, thiamiprine, tiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone, propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, acegra Ton, Aldophosphamide Glycoside, Aminolevulinic Acid, Enyluracil, Amsacrin, Bestrabusil, Bisanthren, Edatraxate, Defofamine, Demecoltin, Diadiquan, Elfornithine, Erliptinium Acetate, Etoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Ronidymin, Maytansine, Ansamitocins, Mitoguazone, Mitoxanthrone, Mopidanmol, Nitraeline, Pentostatin, Fenamet, Pirarubicin, Rosoxanthrone, 2-Ethylhydrazide, Procarbazine, Polysaccharide-K, Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadicone, 2,2',2''-Trichlorotriethylamine, T-2 Toxin, Beraclin A, Loridine A, Anguidin, Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Gacitosine, Arabinoside, Cyclophosphamide, Thiotepa, Paclitaxel, Paclitaxel Albumin-Conjugated Nanoparticle Formulation, Docetaxel, Chlorambucil, Gemcitabine, 6-Thiogunine, Mercaptopurine, Cisplatin, Cal Boplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, SN-38, GI-147211C, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, jaimatecan, calenitecan, silatecan, raltothecan, exatecan,Diflomotecan, berotecan, raltotecan, topoisomerase inhibitor RFS2000, topoisomerase inhibitor S39625, difluoromethylornithine, retinoic acid, capecitabine, or their pharmaceutically acceptable salts, solvates, or acids. (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian duct inhibitors, mouse gonadotropin-related peptides, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythro Poetin, bone induction factor, interferon, interferon-α, interferon-β, interferon-γ, colony-stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factor, LIF, kit ligand, or combination thereof. (c) Diphtheria toxin, botulinum toxin, tetanus toxin, decentritoxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolinobenzodiazepine, pyridinobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, lysine, AM toxin, auristatin, tubulicin, geldanamycin, mytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dorastatin, dorastatin analog, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analog or derivative, duocalmycin, engine antibiotic, esperamicin, epothyron, toxoid, or combination thereof. (d) Affinity ligand, wherein the affinity ligand is a substrate, inhibitor, activator, neurotransmitter, radioisotope, or a combination thereof. (e) Radioactive labels, 32P, 35S, fluorescent dyes, high electron density reagents, enzymes, biotin, streptavidin, dioxygenin, haptens, immunogenic proteins, nucleic acid molecules having sequences complementary to the target, or combinations thereof. (f) Immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or combinations thereof, (g) Tamoxifen, raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole, (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine (j) Aromatase inhibitors, (k) Protein kinase inhibitors, (l) Lipid kinase inhibitors, (m) Antisense oligonucleotide, (n) Ribozyme, (o) Vaccines, (p) Anti-angiogenic drugs, and (q) Topoisomerase I inhibitors A conjugate according to any one of claims 1 to 72, independently selected from the above.

77. The conjugate according to any one of claims 1 to 74, wherein at least one active agent is a pyrrolobenzodiazepine dimer.

78. The active agent is a pyrrolobenzodiazepine dimer, The pyrrolobenzodiazepine dimer is substituted with X at the N10 position or with X' at the N'10 position. Here, X or X' is linked to the pyrrolobenzodiazepine dimer, X and X' are each independently -C(O)O- ＊ , or -C(O)- ＊ and ＊ This is the binding site between the pyrrolobenzodiazepine dimer and L. The conjugate according to any one of claims 1 to 74.

79. The aforementioned conjugate, 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】 【Chemistry 15-5】 【Chemistry 15-6】 【Chemistry 15-7】 (In the formula, MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F, and (The dotted line represents the connection to the rest of the conjugate.) A conjugate according to any one of claims 1 to 77, including the conjugate described in any one of claims 1 to 77.

80. The aforementioned conjugate, 【Chemistry 16-1】 【Chemistry 16-2】 【Chemistry 16-3】 【Chemistry 16-4】 【Chemistry 16-5】 【Chemistry 16-6】 【Chemistry 16-7】 【Chemistry 16-8】 【Chemistry 16-9】 【Chemistry 16-10】 【Chemistry 16-11】 【Chemistry 16-12】 [Chemistry 16-13] [Chemistry 16-14] 【Chemistry 16-15】 【Chemistry 16-16】 [Chemistry 16-17] [Chemistry 16-18] [Chemistry 16-19] 【Chemistry 16-20】 (In the formula, MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F, and (The dotted line represents the connection to the rest of the conjugate.) A conjugate according to any one of claims 1 to 77, including the conjugate described in any one of claims 1 to 77.

81. The conjugate according to any one of claims 1 to 97, wherein the active agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

82. The aforementioned conjugate, 【Chemistry 17】 or a salt thereof, in the formula, [Chemistry 18] The conjugate according to claim 1, wherein the antibody is the anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody.

83. The aforementioned conjugate, 【Chemistry 19】 or a salt thereof, in the formula, 【Chemistry 20】 The conjugate according to claim 1, wherein the antibody is the anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody.

84. The aforementioned conjugate, 【Chemistry 21】 or a salt thereof, in the formula, 【Chemistry 22】 The conjugate according to claim 1, wherein the antibody is the anti-human neuronal cell adhesion molecule L1 (L1CAM) antibody.

85. The conjugate according to any one of claims 82 to 84, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO:

61.

86. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 85 and a pharmaceutically acceptable excipient.

87. A method for treating or preventing hyperplasia, cancer, or neovascular disease in a subject, comprising administering a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 85 to the subject.

88. The method according to claim 87, wherein cancer is treated by the method described above.

89. The method according to claim 87 or 88, 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, or melanoma.

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