Antibody-drug conjugates comprising anti-human L1CAM antibodies and uses thereof

Abstract

Methods of treating solid cancer in an individual are disclosed.

Description

[0001] Related Applications

[0002] This application claims priority to Korean Application No. 10-2023-0032106, filed on March 10, 2023, the contents of which are incorporated herein by reference in their entirety. BACKGROUND

[0003] Cancer is a disease caused by abnormal and uncontrolled cell growth in body tissue, and is a result of uncontrolled cell growth in various tissues. Early-stage cancer tumors can be removed by surgical and radiation treatment measures, and metastatic tumors are generally treated palliatively using chemotherapy. Most of the chemotherapeutic agents that are not administered orally can induce undesirable adverse effects or severe toxicity due to systemic administration. Accordingly, the focus of development has been on developing novel chemotherapeutic agents that achieve improved efficacy and minimal toxicity / adverse effects by improved and selective action of chemotherapeutic agents on tumor cells or immediately adjacent tissues.

[0004] An antibody-drug conjugate (ADC) is a targeting technology in which a toxin or a drug is bonded to an antibody, which in turn binds to an antigen, the toxin or the drug being released into a cell to cause cell death of a tumor cell. The technology has efficacy superior to that of an antibody-drug, and can substantially reduce the risk of adverse effects compared to conventional anticancer agents, because it specifically delivers a drug to a target cancer cell with minimal impact on healthy cells, and releases the drug only under specific conditions.

[0005] The basic structure of an antibody-drug conjugate is "antibody-linker-small molecule drug (toxin)". Here, the linker needs to not only play a functional role in connecting the antibody and the drug, but also needs to ensure that the drug is properly released by antibody-drug dissociation (e.g., due to enzymatic hydrolysis) after circulating through the body and reaching the target cell and exhibits efficacy against the target cancer cell. 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).

[0006] The use of monoclonal antibodies for cancer treatment has achieved remarkable success. Monoclonal antibodies are suitable for target-oriented addressing of tumor tissues and tumor cells. Antibody-drug conjugates have become a novel and potent option for treating lymphoma and solid cancer, and recently, immunomodulatory antibodies have achieved 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 interactions between the immune system and cancer cells.

[0007] Antigens expressed on the surface of human cancer cells refer to a wide range of targets that are overexpressed, mutated and selectively expressed compared to normal tissue. The key issue is to identify the proper antigens for antibody-based therapies. These therapeutics mediate changes in antigen or receptor function (i.e. as stimulators or antagonists), modulate the immune system through Fc and T cell activation, and exhibit efficacy by delivering specific drugs conjugated to antibodies targeting specific antigens. Molecular technologies that can alter antibody pharmacokinetics, function, size, and immunostimulation are becoming key elements in the development of novel antibody-based therapies. Evidence from therapeutic antibody clinical trials in cancer patients highlights the importance of methods for selecting optimal antibodies, including target antigen and antibody affinity and binding, selection of antibody structure, and therapeutic approach (blockade of signaling or immune function).

[0008] In view of the foregoing, there remains a need for novel antibody-drug conjugates to treat diseases, such as cancer. SUMMARY

[0009] The present invention provides, inter alia, antibody-drug conjugates (ADCs) targeting L1CAM (Nerve Cell Adhesion Molecule L1), active metabolites of these ADCs, methods for preparing these ADCs, uses of these ADCs in the treatment and / or prevention of diseases, such as proliferative and / or angiogenic diseases, e.g. cancer. More particularly, the present invention provides antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof that binds to L1CAM and pharmaceutical compositions comprising said conjugates.

[0010] In one aspect, the present disclosure provides a conjugate having a structure represented by Formula I:

[0011] [Formula I]

[0012]

[0013] wherein

[0014] Ab is an anti-human Nerve Cell Adhesion Molecule L1 (L1CAM) antibody or antigen-binding fragment thereof comprising:

[0015] (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0016] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0017] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0018] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0019] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0020] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6,

[0021] each B is independently an active agent;

[0022] each G is independently a glucuronic acid moiety or

[0023] R 3 is hydrogen or a carboxyl protecting group;

[0024] each R 4 is independently hydrogen or a hydroxyl protecting group;

[0025] R 1 and R 2 are each independently hydrogen, C 1-8 alkyl, or C 3-8 cycloalkyl;

[0026] each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'-, or -PO2NR'-, wherein C, S, or P is directly bound to the phenyl ring and NR' is bound to L;

[0027] each R' and R" is independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono C 1-8 alkylamino, or di C 1-8 alkylamino, C 3-10 heteroaryl, or C 6-10 aryl;

[0028] each Z is independently C 1-8 alkyl, halogen, cyano, or nitro;

[0029] each L is independently a linker and comprises:

[0030] A) C 1-50 alkylene or 1 to 50 membered heteroalkylene, comprising:

[0031] (i) one or more unsaturated bonds;

[0032] (ii) heteroarylene (e.g., in an alkylene or heteroalkylene chain); or

[0033] (iii) C 1-20alkyl substituents; and / or

[0034] B) at least one isoprenyl unit having a structure represented by Formula II:

[0035] [Formula II]

[0036]

[0037] n1and n2are each independently an integer selected from 1 to 20; and

[0038] n3is an integer from 0 to 3.

[0039] In another aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate disclosed herein and a pharmaceutically acceptable excipient.

[0040] In yet another aspect, the present disclosure provides a method of treating or preventing a proliferative, cancerous, or angiogenic disease in an individual, comprising administering to the individual a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Schematic diagram showing the method of production and structure of an exemplary antibody-drug conjugate (ADC1).

[0042] Figure 2 Schematic diagram showing the method of production and structure of an exemplary antibody-drug conjugate (ADC2).

[0043] Figure 3 Schematic diagram showing the method of production and structure of an exemplary antibody-drug conjugate (ADC3).

[0044] Figure 4 Graph showing the hydrophobicity profiles of antibody AFF4-CaaX, prenylated antibody AFF4-CaaX, and antibody-drug conjugates ADC1-ADC3 by HIC-HPLC.

[0045] Figure 5 Graph showing the molecular weights of antibody-drug conjugates ADC1 and ADC3 by intact mass.

[0046] Figure 6 Graph showing the tumor growth inhibition effect of antibody-drug conjugates in a breast cancer cell line JIMT-1 xenograft model.

[0047] Figure 7 Graph showing the tumor growth inhibition effect of antibody-drug conjugates in a colorectal cancer cell line SW480 xenograft model.

[0048] Figure 8 Graph showing tumor growth inhibition effect of antibody-drug conjugate in a melanoma cancer cell line MeWo xenograft model.

[0049] Figure 9 Graph showing tumor growth inhibition effect of antibody-drug conjugate in a non-small cell lung cancer cell line Calu-6 xenograft model.

[0050] Figure 10 Graph showing tumor growth inhibition effect of antibody-drug conjugate in an ovarian cancer cell line SNU840 xenograft model.

[0051] Figure 11 Graph showing tumor growth inhibition effect of antibody-drug conjugate in a pancreatic cancer cell line Patu8988s xenograft model.

[0052] Figure 12 Graph showing tumor growth inhibition effect of antibody-drug conjugate in a small cell lung cancer cell line NCI-H146 xenograft model.

[0053] Figure 13 Graph showing tumor growth inhibition effect of antibody-drug conjugate in a small cell lung cancer cell line NCI-H69 xenograft model.

[0054] Figure 14 Graph showing pharmacokinetics of antibody-drug conjugate.

[0055] Figure 15 Graph showing no acute toxicity of antibody-drug conjugate. DETAILED DESCRIPTION

[0056] Human L1CAM (neural cell adhesion molecule L1) is a 200 to 220 kDA transmembrane protein with 323 amino acids (1101 extracellular regions, 23 transmembrane regions and 114 intracellular regions) and is widely known as a neuronal cell adhesion molecule. L1CAM belongs to the L1 protein family and plays an important role in neuronal transmission, neurite outgrowth, axon guidance, axon and dendrite promotion, myelination and synapse formation. L1CAM is expressed in areas where cancer stem cells are present in specific tumors and is well known to play a causal role in biological reactions closely related to cancer stem cells (such as endothelial-mesenchymal transition (EMT) and drug resistance). In addition, L1CAM is abnormally expressed in malignant tumors, such as various neuroblastomas, endometriomas, 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 well known to be directly related to treatment resistance and poor prognosis of malignant tumors.

[0057] In certain aspects, the present invention provides an antibody-linker-drug (e.g., toxin) system. The disclosed antibodies bind to L1CAM, which is abnormally expressed in various cancer tissues. In addition, the antibody-linker-drug includes an effective self-immolative group to further enhance the effect of the antibody. The effective self-immolative group is stable in plasma and circulation, and allows the drug to be easily released and exhibits efficacy in cancer cells. The linker including the effective self-immolative group is stable in plasma and circulation, and allows the drug to be easily released and exhibits efficacy in cancer cells (see U.S. Patent No. 9,919,057, which is incorporated herein by reference). Therefore, 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 comprising an effective self-immolative group to an anti-L1CAM antibody that is stable in plasma and circulation but still allows for easy release of the drug and exhibits efficacy in cancer cells, the effect of the antibody can be further enhanced. This provides a beneficial antibody-drug conjugate (ADC) that effectively targets L1CAM in the treatment and / or prevention of diseases (e.g., proliferative and / or metastatic cancers).

[0058] By including an anti-LlCAM antibody that binds more potently and specifically to the LlCAM type predominantly exhibited in tumors, the antibody-drug conjugates disclosed herein are capable of delivering a drug effectively, specifically, and selectively to cells expressing LlCAM. By having an antibody with a stable linkage to a drug to maintain stability in vivo, especially in serum, even in circulation, while exhibiting the intended cytotoxicity, and by employing a linker technology that includes a self-immolative group that allows the drug to be released readily within the cancer cell to maximize efficacy, these conjugates exhibit the effect of providing an antibody-linker-drug system that allows the drug and / or toxin to reach the target cell safely and effectively exhibit efficacy, while having significantly reduced toxicity.

[0059] Thus, in certain embodiments, the present application provides an antibody-drug conjugate targeting LlCAM or a pharmaceutically acceptable salt thereof.

[0060] In certain embodiments, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof.

[0061] In one aspect, the present disclosure provides a conjugate having a structure represented by Formula I:

[0062] [Formula I]

[0063]

[0064] wherein

[0065] Ab is an anti-human neural cell adhesion molecule Ll (LlCAM) antibody or antigen binding fragment thereof comprising:

[0066] (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0067] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0068] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0069] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0070] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0071] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6,

[0072] each B is independently an active agent;

[0073] each G is independently a glucuronic acid moiety or

[0074] R 3 is hydrogen or a carboxyl protecting group;

[0075] each R 4 is independently hydrogen or a hydroxyl protecting group;

[0076] R 1 and R 2 are each independently hydrogen, C 1-8 alkyl or C 3-8 cycloalkyl;

[0077] each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'- or -PO2NR'-, wherein C, S or P is directly bound to the phenyl ring and NR' is bound to L;

[0078] each R' and R" is independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono C 1-8 alkylamino or di C 1-8 alkylamino, C 3-10 heteroaryl or C 6-10 aryl;

[0079] each Z is independently C 1-8 alkyl, halogen, cyano or nitro;

[0080] each L is independently a linker and comprises:

[0081] A) C 1-50 alkylene or 1 to 50 membered heteroalkylene, comprising:

[0082] (i) one or more unsaturated bonds;

[0083] (ii) heteroarylene (e.g., in an alkylene or heteroalkylene chain); or

[0084] (iii) C 1-20 alkyl substituents; and / or

[0085] B) at least one isoprenyl unit having a structure represented by Formula II:

[0086] [Formula II]

[0087]

[0088] each of n1 and n2 is independently an integer selected from 1 to 20; and

[0089] n3 is an integer from 0 to 3.

[0090] In yet another aspect, the present disclosure provides a conjugate having a structure represented by Formula I:

[0091] [Formula I]

[0092]

[0093] wherein

[0094] Ab is an anti-human neural cell adhesion molecule Ll (LlCAM) antibody or antigen binding fragment thereof, comprising:

[0095] (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16 (GYSITSDYXiWN); wherein Xi is A or T;

[0096] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17 (YISYSGXiSYX2PSLKS); wherein X2 is H or N;

[0097] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18 (SX1SYX2YGFAY); wherein Xi is L or F, and X2 is G, S, or A;

[0098] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0099] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19 (SASYRYXi); wherein Xi is T or I; and

[0100] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6;

[0101] each B is independently an active agent;

[0102] each G is independently a glucuronic acid moiety or

[0103] R 3 is hydrogen or a carboxyl protecting group;

[0104] each R 4 is independently hydrogen or a hydroxyl protecting group;

[0105] R 1 and R 2 are each independently hydrogen, C 1-8 alkyl, or C 3-8cycloalkyl;

[0106] each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'-, or -PO2NR'-, wherein C, S, or P is directly bound to the phenyl ring and NR' is bound to L;

[0107] each R' and R" is independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono C 1-8 alkylamino or di C 1-8 alkylamino, C 3-10 heteroaryl or C 6-10 aryl;

[0108] each Z is independently C 1-8 alkyl, halogen, cyano, or nitro;

[0109] each L is independently a linker and includes:

[0110] A) C 1-50 alkylene or 1- to 50-membered heteroalkylene, including:

[0111] (i) one or more unsaturated bonds;

[0112] (ii) heteroarylene (e.g., in an alkylene or heteroalkylene chain); or

[0113] (iii) C 1-20 alkyl substituents; and / or

[0114] B) at least one isoprenyl unit having a structure represented by Formula II:

[0115] [Formula II]

[0116]

[0117] each of n1and n2is independently an integer selected from 1 to 20; and

[0118] n3is an integer from 0 to 3.

[0119] In some embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof comprising:

[0120] (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0121] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17 (YISYSGX1SYX2PSLKS); wherein X1is Y, and X2is N;

[0122] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18 (SX1SYX2YGFAY); wherein X1is F, and X2is S;

[0123] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0124] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0125] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0126] In some embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:

[0127] (a) a heavy chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 8;

[0128] (b) a heavy chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, and 11;

[0129] (c) a heavy chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 13, and 14;

[0130] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0131] (e) a light chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 15; and

[0132] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0133] In some embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:

[0134] (i) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0135] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0136] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0137] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4;

[0138] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and

[0139] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; or

[0140] (ii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0141] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9;

[0142] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0143] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4;

[0144] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and

[0145] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; or

[0146] (iii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:8;

[0147] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9;

[0148] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0149] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4;

[0150] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and

[0151] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6; or

[0152] (iv) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0153] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0154] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0155] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0156] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0157] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0158] (v) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0159] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0160] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0161] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0162] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0163] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0164] (vi) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0165] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9;

[0166] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0167] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0168] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and

[0169] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0170] (vii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0171] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9;

[0172] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0173] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0174] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and

[0175] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0176] (viii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0177] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0178] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12;

[0179] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0180] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0181] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0182] (ix) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0183] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11;

[0184] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3;

[0185] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0186] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0187] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0188] (x) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0189] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2;

[0190] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3;

[0191] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0192] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0193] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0194] In some embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof comprising:

[0195] (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0196] (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0197] (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

[0198] (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0199] (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0200] (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0201] In some embodiments, a variable X1 or X2 contained in one SEQ ID NO can be an amino acid sequence independently selected from X1 or X2 contained in another SEQ ID NO. For example, the variable X1 or X2 contained in SEQ ID NO: 17 can each be an amino acid sequence independently selected from the variable X1 contained in SEQ ID NO: 16, the variable X1 or X2 contained in SEQ ID NO: 18, or the variable X1 contained in SEQ ID NO: 19.

[0202] In some embodiments, the antibody can comprise mutations at the Fc site, in particular, 1 to 10 mutations (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations). Each mutation can be introduced into one chain of the Fc site, or independently and symmetrically into both chains of the Fc site.

[0203] In some embodiments, the antibody can comprise mutations at the CL site and / or CH1, in particular, 1 to 10 mutations (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations).

[0204] In various embodiments, a mutation refers to a deletion (deletion of one amino acid), an insertion (insertion of one amino acid), or a substitution (one amino acid is substituted by another different amino acid). In various embodiments comprising multiple mutations, each amino acid difference can be independently selected from, for example, a deletion, an insertion, or a substitution.

[0205] In some embodiments, the antibody or antigen-binding fragment thereof can include a heavy chain variable region comprising the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 amino acid sequences listed in Table 1. In particular, the antibody and antigen-binding fragment thereof can include H1 to H4 of the heavy chain variable domain of the humanized antibody of mouse antibody OV549.20. AFF1 to AFF10 can have further advancement in the affinity maturation process in the humanized antibody.

[0206] Table 1. Heavy chain CDR sequences

[0207]

[0208] In some embodiments, the antibody or antigen-binding fragment thereof can 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. In particular, the antibody and antigen-binding fragment thereof can include L1 or L2 of the light chain variable domain of the humanized antibody of mouse antibody OV549.20. The antibody can be combined with the H1 to H4 heavy chain variable domains and L1 or L2 light chain variable domains shown in Table 1.

[0209] Table 2. Light chain CDR sequences

[0210]

[0211] In some embodiments, the antibody or antigen-binding fragment thereof includes one or more specific framework regions described herein.

[0212] In some embodiments, the antibody or antigen-binding fragment thereof includes one or more types of arbitrary framework sequences. In particular, the framework region sequences can be human framework sequences.

[0213] In the present specification, the term "framework (FR) amino acid moiety" refers to an amino acid in the framework region of an immunoglobulin chain. In the present specification, the term "framework" or "FR region" refers to an amino acid moiety in a variable region that is not part of a CDR. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. Framework regions generally support the structure of the antibody (not contacting the antigen) or directly contacting the antigen to support binding to the antigen. For example, methods of producing antibodies having CDR sequences are well known to those skilled in the art and can include introducing an expression vector encoding the desired framework sequences of the CDRs.

[0214] In some embodiments, the FR1 to FR4 of the heavy chain variable region can be independently selected from the FR1 to FR4 of the light chain variable region. In particular, the FR of the heavy chain variable region can comprise the amino acid sequences of Table 3, and the FR of the light chain variable region can comprise the amino acid sequences of Table 4.

[0215] In some embodiments, the heavy chain variable region can comprise a framework region comprising the amino acid sequence of SEQ ID NO: 39 and 41-46. In particular, the heavy chain variable region of the antibody can comprise 1 to 4 framework regions (FRs) selected from the amino acid sequences of SEQ ID NO: 39 and 41-46. More particularly, the heavy chain variable region can comprise FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4. More particularly, the FR1 of the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 39; the FR2 of the heavy chain variable region can comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 41 to 44; the FR 3 of the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 45; and the FR 4 of the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 46.

[0216] Table 3. VH framework region sequences

[0217]

[0218] In some embodiments, the light chain variable region can include a framework region comprising an amino acid sequence selected from SEQ ID NOs: 51-56 or SEQ ID NO: 60. In particular, the light chain variable region of the antibody can include 1 to 4 framework regions (FRs) selected from the amino acid sequences of SEQ ID NOs: 51-56 and SEQ ID NO: 60. More particularly, the light chain variable region can include a FR1, a CDRL1, a FR2, a CDRL2, a FR3, a CDRL3, and a FR4. More particularly, the FR1 of the light chain variable region can include an amino acid sequence selected from SEQ ID NOs: 51 and 55; the FR2 of the light chain variable region can include an amino acid sequence selected from SEQ ID NOs: 48 or 52; the FR3 of the light chain variable region can include an amino acid sequence selected from SEQ ID NOs: 53 and 56; and the FR4 of the light chain variable region can include an amino acid sequence selected from SEQ ID NOs: 50, 54, or 60.

[0219] Table 4. VL framework region sequences

[0220]

[0221] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human L1CAM (neural cell adhesion molecule L1) can include a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 23-34, and can include a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 20-22. More particularly, as shown in Table 5, the heavy chain variable region can include an amino acid sequence selected from SEQ ID NOs: 23-34, and the light chain variable region can include an amino acid sequence selected from SEQ ID NOs: 20-22.

[0222] Table 5. VH and VL sequences

[0223]

[0224]

[0225]

[0226]

[0227] The underlined sequences in the above table indicate CDR sequences.

[0228] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 58 and 37, and can include a light chain comprising an amino acid sequence selected from SEQ ID NO: 38 and 59. More particularly, as shown in Table 6, the heavy chain can include an amino acid sequence selected from SEQ ID NO: 37 and 58, and the light chain can include an amino acid sequence selected from SEQ ID NO: 38 or 59.

[0229] More particularly, the antibody or antigen-binding fragment thereof disclosed herein includes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain comprising the amino acid sequence of SEQ ID NO: 37.

[0230] More particularly, the antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30 and LALA mutations in the heavy chain constant region. The antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain comprising the amino acid sequence of SEQ ID NO: 58. For example, the LALA mutations can comprise substitutions L236A / L237A.

[0231] More particularly, the antibody or antigen-binding fragment thereof disclosed herein can include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. The antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain comprising the amino acid sequence of SEQ ID NO: 38.

[0232] More particularly, the antibody or antigen-binding fragment thereof disclosed herein can include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a CaaX peptide moiety (GGGGGGGGCVIM, SEQ ID NO: 7) at the C-terminus of the light chain. The antibody or antigen-binding fragment thereof disclosed herein can include a heavy chain comprising the amino acid sequence of SEQ ID NO: 59.

[0233] In some embodiments, the antibody or antigen-binding fragment thereof can include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 30.

[0234] More particularly, the antibody or antigen-binding fragment thereof can include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to SEQ ID NO: 30.

[0235] More particularly, the antibody or antigen-binding fragment thereof can include a heavy chain variable region including the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 90% or more sequence identity to SEQ ID NO: 30, or an amino acid sequence having at least 95% or more sequence identity to SEQ ID NO: 30.

[0236] In some embodiments, the antibody or antigen-binding fragment thereof can include a light chain variable region including the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 20.

[0237] More particularly, the antibody or antigen-binding fragment thereof can include a light chain variable region including the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to SEQ ID NO: 20.

[0238] Even more particularly, the antibody or antigen-binding fragment thereof can include a light chain variable region including the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% or more sequence identity to SEQ ID NO: 20, or an amino acid sequence having at least 95% or more sequence identity to SEQ ID NO: 20.

[0239] In some embodiments, the antibody or antigen-binding fragment thereof can include a combination of a heavy chain variable region including the amino acid sequence of SEQ ID NO: 30 and a light chain variable region including the amino acid sequence of SEQ ID NO: 20; or a combination of a heavy chain variable region including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 30 and a light chain variable region including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 20; and the antibody or antigen-binding fragment thereof specifically binds to human L1CAM.

[0240] More particularly, the antibody or antigen-binding fragment thereof can include a combination of a heavy chain variable region including the amino acid sequence of SEQ ID NO: 30 and a light chain variable region including the amino acid sequence of SEQ ID NO: 20; or a combination of a heavy chain variable region including an amino acid sequence having at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to SEQ ID NO: 30 and a light chain variable region including an amino acid sequence having at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to SEQ ID NO: 20; and the antibody or antigen-binding fragment thereof specifically binds to human L1CAM.

[0241] More particularly, the antibody or antigen-binding fragment thereof can comprise a combination of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; or a combination of a heavy chain variable region comprising an amino acid sequence having at least 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more sequence identity to SEQ ID NO: 30, and a light chain variable region comprising an amino acid sequence having at least 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more sequence identity to SEQ ID NO: 20; and the antibody or antigen-binding fragment thereof specifically binds to human L1CAM.

[0242] Table 6. Heavy and light chain amino acid sequences

[0243]

[0244]

[0245] The underlined sequences in the above table represent CDR sequences.

[0246] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody.

[0247] In some embodiments, the humanized antibody can comprise:

[0248] (a) (i) a variable domain framework region from a heavy chain of a human antibody or a human consensus framework; and (ii) a variable domain framework region from a light chain of a human antibody or a human consensus framework,

[0249] (b) (i) at least one amino acid from the variable domain framework region of the heavy chain is substituted with a corresponding amino acid from a heavy chain of a mouse antibody or a mouse consensus framework; or (ii) the variable domain framework region from the light chain is substituted with a corresponding amino acid from a light chain of a mouse antibody or a mouse consensus framework, and

[0250] (c) (i) the amino acid substitutions in the heavy chain are selected from the following amino acid substitutions:

[0251] T (human) at position 34 is substituted to A (mouse);

[0252] Y (human) at position 58 is substituted to F (mouse);

[0253] N (human) at position 61 is substituted to H (mouse); and

[0254] S (human) at position 103 is substituted to G (mouse).

[0255] In some embodiments, the amino acid substitutions in the heavy chain are selected from the following amino acid substitutions:

[0256] A (human) at position 238 is substituted to G (mouse);

[0257] D (human) at position 241 is substituted to S (human); and / or

[0258] E (human) at position 335 is substituted to I (human).

[0259] In some embodiments, the humanized antibody comprises:

[0260] (i) a variable heavy chain framework region from a heavy chain of a human antibody or a human consensus framework, wherein the variable heavy chain framework region comprises one or more of the following mutations: T34A, Y58F, N61H, and S103G; and

[0261] (ii) a variable light chain framework region from a light chain of the human antibody or the human consensus framework.

[0262] In some embodiments, the humanized antibody comprises LALA mutations in the heavy chain constant region. In some embodiments, the LALA mutations comprise substitutions L236A / L237A.

[0263] In some embodiments, the antibody or antigen-binding fragment thereof can be selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, a Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

[0264] By including an anti-LlCAM antibody that binds more potently and specifically to the LlCAM type predominantly exhibited in tumors, the antibody-drug conjugates disclosed herein are capable of effectively, specifically, and selectively delivering a drug to cells expressing LlCAM. By having an antibody with a stable linkage to a drug to maintain stability in vivo, especially in serum, even in circulation, while exhibiting the intended cytotoxicity, and by employing a linker technology that includes a self-immolative group that allows the drug to be released readily within the cancer cell to maximize efficacy, these conjugates exhibit the effect of providing an antibody-linker-drug system that allows the drug and / or toxin to reach the target cell safely and effectively exhibit efficacy, while having significantly reduced toxicity.

[0265] In certain embodiments, n1 is 1, 2, 3, or 4. In certain preferred embodiments, n1 is 1. In other preferred embodiments, n1 is 2.

[0266] In certain embodiments, n2 is 1, 2, 3, or 4. In certain preferred embodiments, n2 is 1.

[0267] In certain preferred embodiments, each G is and R 3 and R 4 are each H.

[0268] In certain embodiments, R 1 and R 2 are each hydrogen.

[0269] In certain preferred embodiments, each W is independently -C(O)NR'-

[0270] In certain embodiments, R' is H.

[0271] In certain embodiments, R" is H.

[0272] In certain embodiments, Z is halo (e.g., bromo) or cyano.

[0273] In certain embodiments, n3 is 1. In certain preferred embodiments, n3 is 0.

[0274] In certain embodiments, L includes C 1-50 alkylene. In other embodiments, L includes C 1-50 heteroalkylene. In certain embodiments, L includes an unsaturated bond (e.g., 1, 2, 3, 4, or 5 unsaturated bonds). In certain embodiments, L is substituted with C 1-20 alkyl.

[0275] In certain preferred embodiments, L includes isoprenyl having a structure represented by Formula Ilia:

[0276]

[0277] wherein n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0278] 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 with a moiety that carries a charge in a neutral aqueous pH solution (e.g., an amine, guanidine, or carboxyl moiety). In certain embodiments, the peptide comprises an amino acid selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[0279] In certain embodiments, the antibody comprises a cysteine residue and L is covalently attached to the antibody via a thioether linkage through a sulfur atom in the cysteine residue (e.g., through 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 a prenyl transferase. In certain embodiments, the prenyl transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). In certain embodiments, the amino acid motif comprises a cysteine residue and L is covalently attached to the antibody via a thioether linkage through a sulfur atom in the cysteine residue (e.g., through a sulfur atom in the side chain of the cysteine residue). In certain preferred embodiments, the amino acid motif has the following sequence:

[0280] CYYX

[0281] wherein:

[0282] C is cysteine;

[0283] each Y is an aliphatic amino acid; and

[0284] X is selected from the group consisting of glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine.

[0285] In certain embodiments, each Y is independently selected from the group consisting of 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.

[0286] In certain preferred embodiments, L comprises an oxime. In certain embodiments, the oxygen atom of the oxime is on the side of Y that is attached to the active agent, and the carbon atom of the oxime is on the side of L that is attached to Ab. In certain embodiments, the carbon atom of the oxime is on the side of Y that is attached to the active agent, and the oxygen atom of the oxime is on the side of L that is attached to Ab.

[0287] In certain preferred embodiments, L comprises a structure represented by Formula IVa or IVb:

[0288] -(CH2) r (V(CH2) p ) q -

[0289] Formula IVa,

[0290] -(CH2CH2X) w -

[0291] Formula IVb;

[0292] V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2-, or -SO2NR 25 -, preferably -O-;

[0293] X is -O-, C 1-8 alkylene, or -NR 21 -, preferably -O-;

[0294] R 21 to R 25 are each independently H, C 1-6 alkyl, C 1-6 alkyl C 6-20 aryl, or C 1-6 alkyl C 3-20 heteroaryl;

[0295] each r is independently an integer from 1 to 10, preferably 2;

[0296] each p is independently an integer from 0 to 12, preferably 2;

[0297] q is an integer from 1 to 20, preferably 2, 5, or 11; and

[0298] w is an integer from 1 to 20, preferably 6 to 20.

[0299] In certain embodiments, V is -O-. In certain embodiments, X is -O-. In certain embodiments, R 21 is H. In certain embodiments, R22 H. In certain embodiments, R 23 H. In certain embodiments, R 24 H. In certain embodiments, R 25 H. 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.

[0300] In certain preferred embodiments, L comprises at least one polyethylene glycol unit represented by In certain embodiments, L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyethylene glycol units. In yet another preferred embodiment, L comprises an oxime and at least one polyethylene glycol unit covalently binds the oxime to the active agent.

[0301] In certain embodiments, L comprises a moiety formed from the reaction of an alkyne with an azide or the reaction of an aldehyde or ketone group with a hydrazine or hydroxylamine.

[0302] In certain preferred embodiments, L comprises a unit having a structure represented by formula Va, Vb, Vc, Vd, or Ve:

[0303]

[0304] L 1 is a single bond or C 1-30 alkylene; and

[0305] R 11 is H or C 1-10 alkyl.

[0306] In certain embodiments, L 1 is a single bond. In certain embodiments, L is C 1-30 alkylene. In certain embodiments, R 11 is H. In certain embodiments, R 11 is C 1-10 alkyl (e.g., methyl).

[0307] In certain preferred embodiments, L comprises:

[0308] i) a branching unit covalently coupled to Ab by a primary linker;

[0309] ii) a first B coupled to a first branch of the branching unit; and

[0310] iiia) a second B coupled to a second branch of the branching unit; or

[0311] iiib) coupling a second branch of the branch unit comprising an alkyl or heteroalkyl group (e.g., a polyethylene glycol monomer or polyethylene glycol oligomer).

[0312] In certain embodiments, L comprises coupling a second active agent to a second branch of the branch unit via a second cleavage group. In certain embodiments, L comprises coupling a second branch of the branch unit comprising an alkyl or heteroalkyl group (e.g., a polyethylene glycol monomer or polyethylene glycol oligomer). In certain embodiments, the branch unit has a structure represented by Formulae Via, VIb, VIc, or VId:

[0313]

[0314] wherein

[0315] G 1 , G 2 , G 3 each independently is a bond,

[0316] R 30 is H or alkyl;

[0317] R 40 is H, alkyl, or L5-CO2R 50 ;

[0318] R 50 is H or alkyl; and

[0319] L 2 , L 3 , L 4 , and L 5 each independently is a bond or alkylene.

[0320] In certain embodiments, the branch unit has a structure represented by Formulae VIe:

[0321]

[0322] wherein R 30 is H or alkyl.

[0323] In certain embodiments, the branch unit has a structure represented by Formulae VIf:

[0324]

[0325] wherein R 30 is H or alkyl.

[0326] In certain embodiments, the conjugate comprises 1, 2, 3, or 4 branched linkers. In certain embodiments, each branched linker comprises at least two active agents. In certain embodiments, each branched linker comprises two active agents. In certain embodiments, the branched unit is lysine and the primary linker is bound to the C-terminus of the lysine.

[0327] In certain embodiments, the conjugate can be cleaved in a target cell (e.g., the conjugate is cleaved to release one or more active agents).

[0328] In certain embodiments, the conjugate comprises a structure represented by:

[0329]

[0330] n11, n22, and n33 are each independently an integer from 0 to 30; and

[0331] AA represents an amino acid group.

[0332] In certain embodiments, each active agent is independently a chemotherapeutic agent or a 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 independently selected from the following agents:

[0333] (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1,1) ) Cytostatic / cytotoxic agents: cytarabine, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, etoposide, teniposide, topotecan, irinotecan, sobuzoxane, 5-fluorouracil, floxuridine, tegafur, carmofur, doxifuridine, palafoxur, gemcitabine, 6-mercaptopurine, thioguanine, pentostatin, cladribine, 2-chlorodeoxyadenosine, nelarabine, cladribine, clofarabine, fludarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabinemustard), carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, alacinomysin, actinomycin, antrmycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, Marcelo marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, plicamycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like.acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, ro dorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone, enalapril, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epirubicin, esorubicin, glufosfamide, halicon, hexamethylmelamine, hydroxyurea, ixabarbine, lentinan, lonidamide, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide ("CTX"), dacarbazine, fluorouracil ("5-FU"), doxifluridine, gemcitabine ("GMIC"), 6- thioguanine, mercaptopurine ("6-MP"), methotrexate ("MTX"), cisplatin, carboplatin, oxaliplatin, procarbazine, psoralen, melphalan, mitomycin, mylotarg, nitorestat, ranimustine, ranimustine, temozolomide, tomudex, urethane, vindesine, dacarbazine, mannosulfan, mitomycin, mytomycin, nitrosurea, platinum compounds, procarbazine, taxol, teniposide, triaziquone, 2,2,2-trichloroethylhydroxylamine, urofollitropin, uramustine, vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinzolidine, and the like.acetate), etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, lo soxantrone, 2-ethylhydrazide, procarbazine, polysaccharide k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, canarypine, cactinomycin, carabicin, cararnine, aclarubicin, CC-1065, chloranmbucil, cytochalasin B, dacarbazine, daunorubicin, 6-diazo-5-oxo-l-norleucine, elaiplomycin, elliptinium acetate, epirubicin, esorubicin, grebleomycin, hydroxyitraconazole, izonici acid, mitoxantrone, nitoxantrone, nitrinex, peplomycin, potfiromycin, puromycin, quelamycin, swinomycin, zinostatin, zorubicin, ibronucleic acid, olomucine, temiserte, triptorelin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carzinophilin, chromomycin A3, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine, adriamycin, mycophenolic acid, nogalamycin, oltipraz, peplomycin, potfiromycin, puromycin, quelamycin, swinomycin, zorubicin, ibronucleic acid, olomucine, temiserte, triptorelin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carzinophilin, chromomycin A3, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine,A) anguidine, ethyl carbamate, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, SN-38, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxy camptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan, topoisomerase inhibitor RFS2000, topoisomerase inhibitor S39625, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid thereof;

[0334] (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-alpha, tumor necrosis factor-beta, mullerian inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factors, interferons, interferon-alpha, interferon-beta, interferon-gamma, colony stimulating factors (CSFs), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins (ILs), IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factors, LIF, kit ligand, or combinations thereof;

[0335] (c) diphtheria toxin, botulinum toxin, tetanus toxin, decentritoxin, cholera toxin, amanitin, alpha-amanitin, pyrrolobenzo-diazepine, pyrrolobenzo-diazepine derivative, indolinobenzo-diazepine, pyridinobenzo-diazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, cyanobacterium, camptothecin, rhizoxin, rhizoxin derivative, CC-1065, CC-1065 analog or derivative, duocarmycin, enediyne antibiotic, esperamycin, epothilone, toxoid, or combinations thereof;

[0336] (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an activator, a neurotransmitter, a radioisotope, or combinations thereof;

[0337] (e) radiolabels,32P,35S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, dioxigenin, haptens, immunogenic proteins, nucleic acid molecules with sequences complementary to a target, or combinations thereof;

[0338] (f) immunomodulatory compounds, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents, and anti-parasitic agents, or combinations thereof;

[0339] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;

[0340] (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

[0341] (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

[0342] (j) an aromatase inhibitor;

[0343] (k) a protein kinase inhibitor;

[0344] (l) a lipid kinase inhibitor;

[0345] (m) an antisense oligonucleotide;

[0346] (n) a ribozyme;

[0347] (o) a vaccine;

[0348] (p) an anti-angiogenic agent; and

[0349] (q) a topoisomerase I inhibitor.

[0350] In certain embodiments, the at least one active agent is a pyrrolobenzodiazepine dimer. In certain embodiments, the active agent is a pyrrolobenzodiazepine dimer;

[0351] The pyrrolobenzodiazepine dimer is substituted with X at position N10 or with X' at position N'10, wherein X or X' connects the pyrrolobenzodiazepine dimer to the conjugate;

[0352] X and X' are each independently -C(O)O-* or -C(O)-*; and

[0353] * indicates the binding site between the pyrrolobenzodiazepine dimer and the linker.

[0354] In certain embodiments, at least one active agent has a structure represented by Formula X:

[0355] [Formula X]

[0356]

[0357] in

[0358] Each dashed line represents an optionally present double bond;

[0359] G' is a glucuronide group or a galactoside group;

[0360] Z ' Selected from H, C 1-8 Alkyl, halogen, NO2, CN,

[0361] X' is selected from -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 -;

[0362] Xa' is a bond or a substituted or unsubstituted C 1-6 Alkylene, where C 1-6 The alkylene substituents are hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl;

[0363] R X1 and R X1' Each independently selected from C 1-6 Alkyl or C 1-6 Alkoxy-substituted H, OH, =O, =CH2, CN, R m , OR m , =CH-R m , =C(R m ')2, OSO2-R m, CO2R m , COR m , halo, dihalo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 5-7 aryl, C 3-6 heteroaryl or C 5-7 aryl;

[0364] R X2 , R X2 , R X3 and R X3 are each independently selected from the group consisting of H, R m , OH, OR m , NR m 2, NO2and halo;

[0365] R X4 and R X4' are each independently selected from the group consisting of H, R m , OH, OR m , SH, SR m , NH2, NHR m , NR m 2 halo and C 1-6 alkyl;

[0366] R X5 and R X5' are each independently selected from the group consisting of 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)2Rm 、-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 halogen;

[0367] Y and Y' are each independently O, S or N(H);

[0368] R X6 Independently C 3-12 Alkylene, C 3-12 Alkenylene or C 3-12 heteroalkylene;

[0369] R X6 Unsubstituted or substituted with: -NH2, -NHR m 、-NHC(O)R m 、-NHC(O)CH2-[OCH2CH2] n31 -R XX or -[CH2CH2O] n31 -R XX ;

[0370] 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, or single C 1-8 Alkylamino or di-C 1-8 Alkylamino, where each n 31 is an integer from 1 to 6;

[0371] R X7 and R X7' Each independently is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -C(O)R r 、-C(O)OR s or -C(O)NR r R r ';

[0372] R r 、R r ' and R seach independently H, C 1-7 alkyl, C 2-7 alkenyl, C 2-7 alkynyl, C 3-13 cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 aryl or 5- to 7-membered heteroaryl;

[0373] each R m' is independently selected from R m , CO2R m , COR m , CHO, CO2H, and halo, and

[0374] each R m is independently selected from H, OH, C 1-12 alkyl, C 1-12 alkoxy, C 2-12 alkenyl, C 2-12 alkynyl, C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl, wherein C 5-20 aryl, C 5-20 heteroaryl, C 3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl, one or more hydrogen atoms of which can be substituted with OH, =O, C 1-12 alkyl or C 1-12 alkoxy; and

[0375] R 31 , R 36 and R 39 each is independently selected from H, C 1-8 alkyl, C 2-6 alkenyl, C 1-6 alkoxy and alkoxyalkyl;

[0376] R 32 and R 33 each is H, alkyl, alkoxy, or aralkyl; and

[0377] n 32 is 0, 1, 2, 3, or 4.

[0378] In certain preferred embodiments, G' is

[0379] In certain embodiments, R X1 and R X1 ' each is independently selected from =CH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C5-7 aryl, C 3-6 heteroaryl or C 5-7 aryl; further wherein C 3-6 heteroaryl or C 5-7 aryl is substituted with C 1-6 alkyl or C 1-6 alkoxy.

[0380] In certain embodiments, R X2 is H or OH.

[0381] In certain embodiments, R X2 is H or OH.

[0382] In certain embodiments, R X3 is H or OH.

[0383] In certain embodiments, R X3' is H or OH.

[0384] In certain embodiments, R X5 is H, OH, -S(O)R m , S(O)2R m , -P(O)R m , and -P(O)2R m , wherein R m is H, OH, C 1-12 alkyl, C 1-12 alkoxy, C 2-12 alkenyl, or C 2-12 alkynyl.

[0385] In certain embodiments, R X5' is H, OH, -S(O)R m , S(O)2R m , -P(O)R m , and -P(O)2R m , wherein R m is H, OH, C 1-12 alkyl, C 1-12 alkoxy, C 2-12 alkenyl, or C 2-12 alkynyl.

[0386] In certain embodiments, R X4 is C 1-6 alkoxy.

[0387] In certain embodiments, R X4' is C 1-6 alkoxy.

[0388] In certain embodiments, Y is O.

[0389] In certain embodiments, Y' is O.

[0390] In certain embodiments, R X6 is C 3-12 alkylene, C 3-12 alkenylene, or C 3-12 heteroalkylene, and R X6 is substituted with -NH2, -NHR m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R XX , or -[CH2CH2O] n -R XX ;

[0391] R XX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, C 3-20 heteroaryl, C 5-20 aryl, or mono C 1-8 alkylamino or di C 1-8 alkylamino; and

[0392] n is an integer from 1 to 6.

[0393] In certain embodiments, R X6 is C 3-12 alkylene.

[0394] In certain embodiments, X' is -C(O)-.

[0395] In certain embodiments, Z ' is

[0396] In certain embodiments, R 36 is alkoxyalkyl.

[0397] In certain embodiments, R 39 is H.

[0398] In certain embodiments, the conjugate comprises

[0399]

[0400]

[0401]

[0402]

[0403]

[0404] wherein

[0405] MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F; and the dashed line indicates attachment to the remainder of the conjugate.

[0406] In certain embodiments, the conjugate comprises

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422] wherein

[0423] MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F; and

[0424] the dashed line indicates attachment to the remainder of the conjugate.

[0425] In certain preferred embodiments, the active agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0426] In certain preferred embodiments, the conjugate is:

[0427]

[0428] or a salt thereof, wherein:

[0429] is an antibody.

[0430] In certain preferred embodiments, the conjugate is:

[0431]

[0432] or a salt thereof, wherein:

[0433] is an anti-human neural cell adhesion molecule LI (LI CAM) antibody.

[0434] In certain preferred embodiments, the conjugate is:

[0435]

[0436] or a salt thereof, wherein:

[0437] is an anti-human neural cell adhesion molecule LI (LI CAM) antibody.

[0438] In another aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate disclosed herein and a pharmaceutically acceptable excipient.

[0439] In yet another aspect, the present disclosure provides a method of treating or preventing a proliferative, cancerous, or angiogenic disease in an individual comprising administering to the individual a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof. In certain preferred embodiments, the method treats a cancer. In certain embodiments, 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.

[0440] Pharmaceutical Compositions

[0441] The compositions and methods of the present application can be used to treat an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered in the form of a pharmaceutical composition that includes, for example, a compound of the present application 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, glycerol, oils (such as olive oil), or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, especially for invasive administration routes (i.e., routes that avoid transport or diffusion through an epithelial barrier, such as injection or implantation), the aqueous solutions are pyrogen-free, or substantially pyrogen-free. Excipients can be chosen, for example, to effect delayed release of a medicament or selective targeting of one or more cells, tissues, or organs. The pharmaceutical composition can be in unit dosage form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilizates for reconstitution, powders, solutions, syrups, suppositories, injectables, and the like. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be in a solution suitable for topical administration, such as a cream, ointment, or salve.

[0442] The pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize the compound (such as a compound of the present application), to increase solubility or to enhance absorption. These physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextrans; 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 (including a physiologically acceptable agent) depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) can also be a liposome or other polymer matrix, in which, for example, a compound of the present application can be incorporated. Liposomes (e.g., which include phospholipids or other lipids) are nontoxic, physiologically acceptable, and metabolizable delivery vehicles that can be relatively simple to make and administer.

[0443] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0444] 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 encapsulating material. Each carrier must "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (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 laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0445] The pharmaceutical compositions (formulations) can be administered to an individual by any of a variety of routes of administration including, for example, oral (e.g., as a drench, in tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes applied to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). The compounds can also be formulated for inhalation. In certain embodiments, the compounds can be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for such can be found in, for example, U.S. Pat. Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, and patents cited therein.

[0446] The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30% by weight. Typically, such a mixture will contain at least about 5% of the active compound.

[0447] Methods of preparing these formulations or compositions include the step of bringing into association active compound (e.g., a compound of the present application) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound of the present application with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0448] Formulations of the present application suitable for oral administration can be in the form of capsules (including sprinkle capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophilized forms, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present application as an active ingredient. Compositions or compounds can also be administered as a bolus, electuary or paste.

[0449] For preparing a solid dosage form for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers (e.g., sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acids; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.

[0450] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Binders such as gelatin or hydroxypropylmethyl cellulose, lubricants such as magnesium stearate or stearic acid, inert diluents such as lactose or milk sugar, as well as

[0451] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can also be formulated so as to provide a sustained or controlled release of the active ingredient therein using, for example, different proportions of hydroxypropylmethyl cellulose, other polymer matrices, liposomes and / or microspheres. They can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved in sterile water or some other sterile injectable medium just prior to use. These compositions also can optionally contain opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient(s) can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0452] Liquid dosage forms suitable for oral administration include pharmaceutically- acceptable emulsions, lyophilized formulations for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylen glycol, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, baby oil, olive oil, castor oil and sesame seed oil, glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0453] In addition to inert diluents, the oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and coloring agents, and preservatives.

[0454] Suspensions, in addition to the active compound, can contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0455] Dosage forms for the topical or transdermal administration of an active compound include powders, sprays, ointments, pastes, creams, lotions, jellies, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants, as appropriate.

[0456] Ointments, pastes, creams and gels, in addition to the active compound, can contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0457] In addition to the active compounds, the powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0458] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present application to the body. Such dosage forms can be made by dissolving or dispensing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0459] The phrase "parenteral administration" and "administered parenterally" as used herein, mean modes of administration other than oral and topical administration, and include, but are not limited to, injection, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which can contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0460] Examples of suitable aqueous and nonaqueous carriers which can be employed in the pharmaceutical compositions of the application include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0461] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0462] In some cases, in order to prolong the effect of a drug, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution that, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0463] Injectable depot forms are prepared by forming microencapsule matrices of the compound of the application using biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0464] For use in the methods of the application, the active compounds can be administered alone, or in combination with pharmaceutically acceptable carriers or diluents to provide a pharmaceutical composition in a form appropriate for individual dosing, such as tablets, capsules, injections and the like. The active compound itself, or in some embodiments, the combination of active compounds, is the agent which exerts the therapeutic effect.

[0465] Introduction methods can also be provided by reloadable or biodegradable devices. Various slow-release polymeric devices for the controlled delivery of drugs, including protein biopharmaceuticals, have been developed and tested in vivo in recent years. A variety of biocompatible polymers, including hydrogels, including biodegradable and non-degradable polymers, can be used to form implants that release a compound at a particular target site for an extended period of time.

[0466] Actual dosage levels of the active ingredients in the pharmaceutical compositions can vary so as to administer an amount of the active ingredient to a patient that is effective to treat the particular disease, condition, or disorder, without being toxic to the patient.

[0467] The selected dosage level will depend on a variety of factors including the activity of the particular compound or combination of compounds or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0468] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" means an amount of a compound sufficient to exert a. therapeutic effect. In general, it is understood that the effective amount of a compound will vary according to the weight, sex, age and medical history of the individual. Other factors influencing the effective amount can include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and another type of therapeutic agent being administered, if necessary, with a compound of the present application. A larger total dosage can be delivered by multiple administration of the agent. Methods of determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th Ed., 1814-1882, incorporated herein by reference).

[0469] In general, a suitable daily dose of an active compound used in the compositions and methods of the application will be that amount of the compound that is the lowest dose effective to produce the therapeutic effect. Generally, this will be in the range of 0.01 to 100 mg per kilogram body weight per day, preferably 0.1 to 50 mg per kilogram body weight per day, and most preferably 0.5 to 10 mg per kilogram body weight per day. The actual dosage employed can be varied depending upon the requirements of the patient and the severity of the condition being treated.

[0470] If desired, the effective daily dose of the active compound can optionally be divided into single or multiple sub-doses and administered at appropriate intervals throughout the day. In certain embodiments of the application, the active compound can be administered twice or thrice daily. In preferred embodiments, the active compound will be administered once daily.

[0471] The patient to be treated by such a method is any animal in need of such treatment, including primates, especially humans; and other mammals such as equines, bovines, porcines, ovines, felines and canines; and generally poultry and pets.

[0472] In certain embodiments, the compounds of the present application can be administered alone or in combination with another type of therapeutic agent.

[0473] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, salts contemplated by the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, salts contemplated by the present invention include, but are not limited to, L-arginate, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 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 contemplated by the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In certain embodiments, salts contemplated by the present invention include, but are not limited to, acid salts of 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), carbonic acid, cinnamic acid, citric acid, cyclamahydroxypropyl sulfonic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric 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 undecylenic acid.

[0474] Pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates are also possible. Such solvates can arise from the solvent of crystallization, be inherent in the solvent of preparation or crystallization, or be added to such solvents.

[0475] Humectants, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0476] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0477] Definitions

[0478] Unless otherwise defined herein, scientific and technical terms used in this application have the meanings that would be given to them by one of ordinary skill in the art. Generally, the nomenclature used herein and the techniques described herein follow those set forth in the standard reference works in the fields of chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry, as well as those set forth in various general and more specific references that are cited herein.

[0479] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited herein. See, e.g., “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th Ed.”, W.H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th Ed.”, W.H. Freeman & Co., New York (1999); and Gilbert et al., “Developmental Biology, 6th Ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0480] Unless otherwise defined herein, chemical terms as used herein are used according to conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", by Parker, S., ed., McGraw-Hill, San Francisco, C.A. (1985).

[0481] All of the above cited references and any other publications, patents and published patent applications referred to in this application are hereby incorporated by reference herein. In case of conflict between the present specification and any incorporated reference, the present specification shall control.

[0482] The term "agent" is used herein to denote a compound (e.g., an organic or inorganic compound, a mixture of compounds), a biological macromolecule (e.g., a nucleic acid, an antibody (including portions thereof) and humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate) or an extract made from biological materials (e.g., bacteria, plants, fungi or animal (particularly mammalian) cells or tissues). Agents include, for example, agents whose structure is known and agents whose structure is unknown.

[0483] The term "conjugate" as used herein refers to a cell-binding agent covalently bonded to one or more molecules of a cytotoxic compound. In this regard, a "cell-binding agent" is a molecule that has affinity for a biological target and can be, for example, an antibody, particularly a monoclonal antibody or antibody fragment, and the function of the binding agent is to direct a biologically active compound to the biological target. In certain embodiments of the disclosure, the conjugate can be designed to target tumor cells through a cell surface antigen. The antigen can be a cell surface antigen that is overexpressed or expressed in abnormal cell types. In particular, the target antigen can be expressed only on proliferating cells, such as tumor cells. The target antigen can be selected based on differential expression, typically between proliferating and normal tissue.

[0484] "Patient," "subject," or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, farm animals (including cows, pigs, etc.), pets (e.g., dogs, cats, etc.) and rodents (e.g., mice and rats).

[0485] "Treat," "treatment," or "treating" a condition or a patient refer to taking steps to obtain a beneficial or desired result, including clinical results. Beneficial or desired clinical results can include, but are not limited to, reducing or ameliorating one or more symptoms or conditions, diminishing the extent of a disease, stabilizing the disease state (i.e., not worsening), preventing the spread of disease, delaying or slowing the progression of a disease, ameliorating or improving the disease state, and remission, whether partial or total, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0486] The term "prevent," is art-recognized and, as used in reference to a condition such as local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure, or any other medical condition, is well understood in the art and includes administration of a composition that reduces the frequency or delays the onset of a symptom of a medical condition in an individual relative to an individual who does not receive the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to an untreated control population; and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0487] An individual can be "administered" a substance, compound, or agent using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin conduit). A compound or agent can also be suitably introduced by a refillable or biodegradable polymeric device or other device (e.g., patches and pumps or formulations that result in prolonged, slowed, or controlled release of the compound or agent). Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0488] A suitable method of administering a substance, compound, or agent to an individual will also depend on, for example, the age and / or physical condition of the individual, as well as the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, a compound or agent is administered orally to an individual, e.g., by ingestion. In some embodiments, an orally administered compound or agent is in the form of an extended release or slow release formulation, or is administered using a device for such slow or extended release.

[0489] As used herein, the term "administered in combination" refers to administration of two or more different therapeutic agents in any form wherein the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are simultaneously effective in the patient, which can include a synergistic effect of the two agents). For example, the different therapeutic compounds can be administered in the same formulation or in separate formulations, simultaneously or sequentially. Thus, an individual receiving such therapy can benefit from the combined effect of the different therapeutic agents.

[0490] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or pharmaceutical agent is an amount that, when administered to an individual, will have the intended therapeutic effect. Full therapeutic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations. The precise effective amount for an individual will depend upon, e.g., the size, health, and age of the individual, and the nature and extent of the condition being treated, e.g., cancer or MDS. One of ordinary skill in the art can determine the effective amount for a given situation by routine experimentation, taking into account the guidance provided herein.

[0491] As used herein, the term "optionally present" or "optionally" means that the event or circumstance described subsequently can or can not occur, and the description includes both the event or circumstance occurring and the event or circumstance not occurring. For example, "optionally substituted alkyl" refers to both the case where the alkyl is substituted and the case where the alkyl is not substituted.

[0492] It is understood that the substituents and substitution patterns on the compounds of the present application can be selected by one of ordinary skill in the art to generate chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and those methods given below. If a substituent is itself substituted with more than one group, it is understood that these multiple groups can be on the same carbon or different carbons as long as a stable structure results.

[0493] As used herein, the term "optionally substituted" refers to one to six hydrogen groups in a given structure being replaced by a designated substituent group, including but not limited to: hydroxy, hydroxyalkyl, alkoxy, halo, 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" refers to one to four hydrogen groups in a given structure being replaced by a substituent as mentioned above. More preferably, one to three hydrogen groups are replaced by a substituent as mentioned above. It is understood that the substituents can be further substituted.

[0494] In the present application, the prefix (e.g., C 1-12 , C 3-8"number of ring atoms" or "range of ring atoms" refers to the number of ring atoms or the range of ring atoms, regardless of whether the ring atoms are carbon atoms or heteroatoms.

[0495] As used herein, the term "alkyl" refers to, but is not limited to, C1-C 10 straight-chain alkyl or C1-C 10 saturated aliphatic groups. Preferably, "alkyl" refers to C1-C6straight-chain alkyl or C1-C6branched-chain alkyl. Most preferably, "alkyl" refers to C1-C4straight-chain alkyl or C1-C4branched-chain alkyl. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl, and the like. The "alkyl" group can be optionally substituted.

[0496] The term "acyl" is art-recognized and refers to a radical represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0497] The term "amido" is art-recognized and refers to an amino group substituted by an acyl group, and can be represented by, for example, the formula hydrocarbylC(O)NH-.

[0498] The term "acyloxy" is art-recognized and refers to a radical represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)O-.

[0499] The term "alkoxy" refers to an alkyl group with an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propyloxy, t-butyloxy, and the like.

[0500] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0501] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl, branched-chain alkyl, cyclic alkyl (alicyclic) groups, cyclic alkyl substituted alkyl, and alkyl substituted cyclic alkyl groups. In preferred embodiments, straight-chain or branched-chain alkyl groups have 30 or fewer carbon atoms in their backbone (e.g., C1-C30alkyl), and more preferably 20 or fewer. 1-30 branched-chain C 3-30 , and more preferably 20 or fewer.

[0502] Further, as used throughout the specification, examples, and claims, the term "alkyl" is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter referring to alkyl moieties having a substituent replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, and the like.

[0503] The term "alkenyl" refers to an alkyl group having at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups are ethenyl (vinyl, -CH=CH2), 1- propenyl (-CH=CHCH3), 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl, and the like.

[0504] 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, and the like.

[0505] The term "C x-y " or "C x -C y " when used in conjunction with a chemical moiety, such as an acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups with from x to y carbons in the chain. Co alkyl indicates a hydrogen if the group is at the terminal position, and if internal, indicates a bond. 1-6 An alkyl group contains one to six carbon atoms in the chain.

[0506] The term "alkylamino" as used herein refers to an amino group substituted with at least one alkyl group.

[0507] The term "alkylthio" as used herein refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0508] The term "acylamino" as used herein refers to a group

[0509]

[0510] wherein R 9 and R 10 each independently represent hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocyclic ring having from 4 to 8 atoms in the ring structure.

[0511] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, for example, moieties that can be represented by:

[0512]

[0513] wherein R 9 , R 10 and R 10 each independently represent hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocyclic ring having from 4 to 8 atoms in the ring structure.

[0514] The term "aminoalkyl" as used herein refers to an alkyl group substituted with an amino group.

[0515] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0516] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein 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 rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is aromatic, for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0517] The term "carbamate" is art-recognized and refers to the group:

[0518]

[0519] wherein R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0520] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0521] The term "carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring of a bicyclic carbocycle can be selected from saturated, unsaturated, and aromatic rings. A carbocycle includes bicyclic molecules in which two rings share one, two, or three or more atoms between the rings. The term "fused carbocycle" refers to bicyclic carbocycles in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle can be selected from saturated, unsaturated, and aromatic rings. In exemplary embodiments, an aromatic ring (e.g., phenyl) can be fused to a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane, or cyclohexene). Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle when valence permits. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any position or positions that can carry a hydrogen atom.

[0522] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0523] The term "carbonate" is art-recognized and refers to the group -OCO2-.

[0524] As used herein, the term "carboxyl" refers to a group represented by the formula -CO2H.

[0525] The term "cycloalkyl" includes substituted or unsubstituted non-aromatic monocyclic ring 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 rings, wherein two or more carbons are shared between two adjacent rings, wherein at least one ring is a cycloalkyl and a substituent (e.g., R 100 ) is attached to the cycloalkyl ring, for example, other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, benzodioxane, tetrahydroquinoline, and the like.

[0526] As used herein, the term "ester" refers to a group -C(O)OR 9 , wherein R 9 represents a hydrocarbyl group.

[0527] As used herein, the term "ether" refers to a hydrocarbyl group attached to another hydrocarbyl group through an oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocyclyl-O-heterocyclyl and aryl-O-heterocyclyl. An ether includes an "alkyloxyalkyl" group, which can be represented by the general formula alkyl-O-alkyl.

[0528] The term "formyl" refers to -C(=O)H.

[0529] As used herein, the terms "halo" and "halogen" mean halogen and include chlorine, fluorine, bromine, and iodine.

[0530] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a heteroaryl group.

[0531] The term "heteroaryl" (heteroaryl and hetaryl) includes substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structure includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more rings, wherein two or more carbons are shared between two adjacent rings, wherein at least one ring is a heteroaromatic ring, for example, other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0532] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferably, the heteroatom is nitrogen, oxygen, and sulfur.

[0533] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group.

[0534] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structure includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocycle" also include polycyclic ring systems having two or more rings, where two or more carbons are common to two adjacent rings, where at least one ring is a heterocycle, for example other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like.

[0535] As used herein, the term "hydrocarbyl" refers to groups which are bonded through carbon atoms, which groups do not have =0 or =S substituents and which groups generally have at least one carbon-hydrogen bond and a predominantly carbon backbone, but which groups can optionally include heteroatoms. Thus, for purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl, but substituents such as acetyl (which has a =0 substituent on the attached carbon) and ethoxy (which is attached through oxygen, not carbon) are not considered to be hydrocarbyl. Hydrocarbyl includes, but is not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0536] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

[0537] The term "lower" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups in which there are ten or fewer, preferably six or fewer, atoms present in the substituent. "Lower alkyl," for example, refers to alkyl groups containing ten or fewer, preferably six or fewer, carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents, for example, in the hydroxyalkyl and aralkyl groups (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0538] The terms "polycyclyl," "polycyclic," and "polycyclic ring" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl), where two or more atoms are common to two adjacent rings, for example, the rings are "fused." The rings of a polycyclic group can be substituted or unsubstituted. In certain embodiments, the rings of a polycyclic group contain 3 to 10, preferably 5 to 7, atoms in the ring.

[0539] The term "sulfate" is art-recognized and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0540] The term "sulfonamido" is art-recognized and refers to a group of the formula:

[0541]

[0542] wherein R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0543] The term "sulfoxide" is art-recognized and refers to a group -S(O)-.

[0544] The term "sulfonate" is art-recognized and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0545] The term "sulfone" is art-recognized and refers to a group -S(O)2-.

[0546] The term "substituted" means that the moiety has a substituent(s) that replaces a hydrogen on one or more carbons of the backbone. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atoms and the substituents, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, it is contemplated that the term "substituted" encompasses all permissible substituents of organic compounds. In some embodiments, permissible substituents include acyclic and cyclic, branched and unbranched carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For appropriate organic compounds, the permissible substituents can be one or more and the same or different at each occurrence. For purposes of this application, the heteroatoms of heteroatom-containing

[0547] As used herein, the term "sulfanyl" refers to an alkyl group substituted with a thiol group.

[0548] As used herein, the term "thioester" refers to the group -C(O)SR 9 or -SC(O)R9 ,

[0549] wherein R 9 represents a hydrocarbyl group.

[0550] As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

[0551] The term "urea" is art-recognized and can be represented by the general formula:

[0552]

[0553] wherein R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0554] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (e.g., cell proliferation) as well as enhancing a function or activity.

[0555] The term "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms that are, within the scope of sound medical judgment, compatible with human and animal tissue, suitable for use in contact with the tissues without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0556] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to either an acid addition salt or a base addition salt that is suitable for use in the treatment of patients or compatible with such treatments.

[0557] As used herein, the term "pharmaceutically acceptable acid addition salt" means any non-toxic organic or inorganic salt of a base compound represented by Formula I. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, among others, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di- and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono- or di-acid salts can be formed, and the salts can exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts of the compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally exhibit a higher melting point than their free base forms. The selection of an appropriate salt is known to those of skill in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, can be used, for example, in the isolation or purification of a compound of Formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0558] As used herein, the term "pharmaceutically acceptable base addition salt" means any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any intermediate thereof. Illustrative inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and pyridine, or ammonia. The choice of suitable salt is known to those skilled in the art.

[0559] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereogenic center in their structure. This stereogenic center can exist in the R or S configuration, the R and S symbols being used in accordance with the conventions described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms (including all possible mixtures of stereoisomers) of, for example, enantiomeric and diastereomeric forms of the compounds, salts, prodrugs, or mixtures thereof. See, e.g., WO 01 / 062726.

[0560] In addition, certain compounds containing alkenyl groups can exist as Z (syn) or E (anti) isomers. In each instance, the present disclosure includes both mixtures and individual isomers.

[0561] A "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host after administration to form a compound of the present disclosure (e.g., a compound of Formula I). Typical examples of prodrugs include compounds having biologically labile or cleavable (protecting) groups on functional moieties 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 yield the active compound. Examples of prodrugs using esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to yield the compound of Formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, e.g., in "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985.

[0562] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which is suitable for use in modulating a medicament for medical or therapeutic uses.

[0563] The term "log of solubility," "LogS" or "logS" as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly influences its absorption and distribution characteristics. Low solubility is often associated with poor absorption. The LogS value is the unit log (base 10) of the solubility measured in moles / liter.

[0564] The term "identity" refers to the similarity or relatedness of two or more polypeptide or nucleic acid sequences as determined by aligning and comparing the sequences of two or more polypeptides or nucleic acids. Such inter-sequence identity is typically expressed as a "percent identity," representing the proportion of identical amino acids or nucleotides between the compared molecules, calculated based on the smallest of the compared molecules. Methods for aligning nucleic acids or polypeptides to calculate their identity are known in the art and can also be mentioned herein.

[0565] The term "affinity" or "avidity" refers to the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen as determined by the features of the antigen (e.g., size, shape, and / or charge) and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining these affinities are known in the art and can also be mentioned herein.

[0566] An antibody or antigen-binding fragment thereof used in the present application is said to "specifically bind" to a target, such as an antigen, when the dissociation constant (KD) is < 10 -6 M. An antibody "binds with high affinity" to a target when the KD is < 1 x 10 -8 M. An antibody "binds with high affinity" to a target.

[0567] The term "antigen-binding fragment" of an antibody or immunoglobulin chain (light or heavy) as used herein refers to a portion of an antibody that comprises some amino acid deletions compared to the full-length chain, but that comprises an antibody portion that can specifically bind to a target antigen. These fragments can have a biological activity in that they are capable of specifically binding to a target antigen, or they compete with other antibodies or antigen-binding fragments for binding to a particular epitope. In some embodiments, these fragments include at least one CDR in a full-length light or heavy chain, and in some embodiments include a short heavy and / or light chain or portions thereof. These biologically active fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFv, scFv-Fc, diabodies, minibodies, scAb, and dAbs, and can be derived from any mammal, including human, mouse, rat, camel, or rabbit. In the present invention, a functional portion of an antibody (e.g., one or more CDRs) can be covalently linked to a second protein or small molecule compound, and used as a targeted therapeutic agent for a particular target.

[0568] The "Fc" region is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions, and in the present invention includes two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. These two heavy chain fragments are linked to each other by two or more disulfide bonds and hydrophobic interactions with the CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus. The C-terminal lysine (residue 447 according to EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody or by engineering the nucleic acid encoding the antibody heavy chain recombinantly. Thus, the composition of intact antibodies can include a population of antibodies that have all K447 residues removed, a population of antibodies that have no K447 residues removed, and a population of antibodies that have a mixture of antibodies with and without K447 residues. Native sequence Fc regions suitable for use in antibodies of the present disclosure include human IgGl, IgG2, IgG3, and IgG4.

[0569] A "Fab fragment" in the present invention consists of one light chain and one heavy chain that contains only the variable and CH1 domains. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules. A scFab is a Fab of two molecules connected by a flexible linker.

[0570] A "Fab' fragment" in the present invention comprises the region between the CH1 and CH2 domains of the heavy chain that is not present in the Fab fragment, which forms an interchain disulfide bond between the two heavy chains of the two molecules of the Fab' fragment, thereby forming a F(ab')2 molecule.

[0571] As described above, the "F(ab')2 fragment" in the present application comprises two light chains and two heavy chains, which comprise a variable region, CH1 and a portion of the constant region between CH1 and CH2 domains, which forms 2 interchain disulfide bonds between the heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are bound to each other through the disulfide bond therebetween.

[0572] The "Fv region" in the present application is an antibody fragment comprising the variable regions of the heavy and light chains, but not comprising the constant regions. The heavy and light chains in the sdFV are connected by a disulfide bond. The scFv is an Fv connected by a flexible linker. The Fc in the scFv-Fc is connected to the scFv. The CH3 in the minibody is connected to the scFv. The bivalent antibody contains two molecules of scFv.

[0573] The "single chain Fv" or "scFv" antibody fragment in the present application includes the VH and VL domains of an antibody, and these domains are in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding.

[0574] The "single chain antibody" (scAb) in the present application is a single polypeptide chain containing one heavy chain constant region or a light chain constant region, in which the heavy chain and light chain variable regions are connected by a flexible linker. For single chain antibodies, see, for example, U.S. Patent No. 5,260,203, which is incorporated by reference in the present application.

[0575] The "domain antibody" (dAb) in the present application is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. 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.

[0576] As used herein, "complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the portions of an antibody variable domain that are required for antigen binding. Generally, an antibody includes six CDRs; three in the VH (H1 CDR, H2 CDR, H3 CDR), and three in the VL (L1 CDR, L2 CDR, L3 CDR). In native antibodies, H3 and L3 exhibit the most diversity of the six CDRs, and H3 in particular is believed to play a unique role in conferring good specificity to an antibody. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). Indeed, naturally occurring camelid antibodies consisting of heavy chains only have functionality and stability in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0577] As used herein, "framework region" (FR) is the portion of a variable domain other than the CDR portion. Each variable domain generally has four FRs, identified as FR1, FR2, FR3, and FR4.

[0578] As used herein, a "bivalent antigen binding protein" or "bivalent antibody" comprises two antigen binding sites. The two antigen binding sites comprised in such bivalent antibodies can have the same antigen specificity, or can be a bispecific antibody that each binds to a different antigen.

[0579] As used herein, a "multispecific antigen binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0580] As used herein, "chimeric antibody" refers to an antibody (immunoglobulin) in which some portion of the heavy and / or light chain is identical with, or homologous to, the corresponding sequence in an antibody derived from a specific species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81 :6851-55 (1984)). Chimeric antibodies of interest herein include antibodies, wherein the antigen binding region of the antibody is derived from an antibody produced by immunizing a macaque monkey with, for example, an antigen of interest.

[0581] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, humanized antibodies can include residues not found in the recipient antibody or donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions can contain one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs is typically no more than six in the H chain, and no more than three in the L chain. A humanized antibody optionally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., 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, e.g., 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. Pat. Nos. 6,982,321 and 7,087,409.

[0582] A "human antibody" is one in which the amino acid sequence corresponding to the variable regions of the antibody is derived from human germline sequences. By "human antibody" is meant an antibody that is not a humanized antibody comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art. For example, it is now possible to produce human antibodies directly in transgenic animals or in cell lines in which human germline immunoglobulin sequences have been introduced. See, e.g., Taylor, M. D. et al., "Humanization of Antibodies Derived from Inbred Mice and Rats," Trends in Biotechnology 22, No. 4 (April 2004), pp. 174-180. For a review of the methods available for humanizing antibodies see, e.g., Almagro and Fransson, "Therapeutic Antibodies: Historical Perspectives and Future Prospects," J. Biomed. Biotechnol. 2011: 628156 (2011), which is incorporated by reference in its entirety.

[0583] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in the position of a selected human immunoglobulin VL or VH framework sequence. Generally, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include: for the VL, subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Further, for the VH, subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0584] An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alterations. In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by V H domain and V L domain shuffling. Random mutagenesis of CDR and / or framework residues are described by, for example, (Barbas) et al. Proc. Natl. 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).

[0585] Examples

[0586] Having now generally described the application, the same will be better understood by reference to the following Example, which is included merely for purposes of illustration of certain aspects and embodiments of the present application, and is not intended to limit the application.

[0587] Example 1: Generation of Exemplary Human L1CAM (Neural Cell Adhesion Molecule L1) Specific Antibodies

[0588] L1CAM (Nerve Cell Adhesion Molecule L1) is widely expressed in tumors. An exemplary anti-L1CAM humanized antibody AFF4 that specifically binds to L1CAM was generated by the methods described in European Patent Application No. EP22196917.3, the specification of which is incorporated herein by reference in its entirety. The amino acid sequence of antibody AFF4 is shown below in Tables 7 and 8.

[0589] Table 7. AFF4 amino acid sequence

[0590]

[0591] Further, for ADC synthesis, AFF4-CaaX was generated by introducing LALA mutations (L236A / L237A) into the heavy chain constant region of antibody AFF4 and introducing a CaaX peptide moiety (GGGGGGGGCVIM, SEQ ID NO: 7) to the C-terminus of the light chain. The generated antibody AFF4-CaaX was used for ADC synthesis. The amino acid sequence of antibody AFF4-CaaX is shown below in Table 8.

[0592] Table 8. AFF4-CaaX amino acid sequence

[0593]

[0594] Example 2: Synthesis of exemplary compounds of the disclosure

[0595] Production of Compound 1

[0596]

[0597] Compound 1 was generated using the methods disclosed in U.S. Patent No. 11,173,214, the specification of which is incorporated herein by reference in its entirety.

[0598] EI-MS m / z: [1 / 2 M+H] + 1623.6, [1 / 3 M+H] + 1082.8.

[0599] The structure of MMAE (monomethyl auristatin E) in Compound 1 is as follows:

[0600]

[0601] Production of Compound 2

[0602]

[0603] Compound 2 was produced using the method disclosed in U.S. Patent No. 11,654,197, the specification of which is incorporated herein by reference in its entirety.

[0604] EI-MS m / z: [M+H] + 1698.2, [1 / 2 M+H] + 849.6.

[0605] Production of Compounds 4 to 7

[0606]

[0607]

[0608] Production of Compound 4

[0609] Compound 3 (PCT / KR2023 / 018766, the entirety of which is incorporated herein by reference) (1.5 g, 0.67 mmol) was dissolved in dichloromethane (24 mL). Next, 0.22 mL of pyridine (2.68 mmol) and 0.79 g of bis(pentafluorophenyl)carbonate (2.01 mmol) were added at room temperature under a nitrogen atmosphere and stirred for 16 hours. The reaction solution was diluted with 80 mL of ethyl acetate, washed with 50 mL x 2 of saturated aqueous sodium bicarbonate solution, then washed with 50 mL of brine, and dehydrated using anhydrous sodium sulfate. Compound 4 (2.06 g) was obtained after filtration and concentration under reduced pressure, without further purification, and was then used in the next process.

[0610] EI-MS m / z: [M+H] + 2670.3, [1 / 2 M+H] + 1335.7, [1 / 3 M+H] + 890.8.

[0611] Production of Compound 5

[0612] Compound 4 (100 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (3 mL) before adding, under stirring, exetecan 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) sequentially at room temperature under nitrogen atmosphere for 3 hours. The reaction mixture was diluted with ethyl acetate (40 mL) and washed sequentially with saturated aqueous ammonium chloride (30 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL) before drying over anhydrous sodium sulfate. Compound 5 (140 mg) was obtained after filtration and concentration under reduced pressure without further purification.

[0613] EI-MS m / z: [1 / 2 M+H] + 1587.5, [1 / 3 M+H] + 1058.8.

[0614] Production of Compound 6

[0615] Compound 5 (550 mg, 0.17 mmol) was dissolved in methanol (7 mL), tetrahydrofuran (7 mL) before adding a solution of lithium hydroxide (65.8 mg, 1.57 mmol) dissolved in distilled water (8 mL) gradually at -40 °C under nitrogen atmosphere. The mixture was then stirred for 2 hours as the reaction temperature gradually increased to 0 °C. The reaction mixture was neutralized using acetic acid, concentrated under reduced pressure, purified via HPLC before being freeze-dried to obtain compound 6 (236 mg, 44%) in the form of a yellow solid.

[0616] EI-MS m / z: [1 / 2 M+H] + 1446.7, [1 / 3 M+H] + 964.9.

[0617] Production of Compound 7

[0618] 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 for 4 hours at room temperature, the reaction mixture was purified by HPLC before being freeze-dried to obtain compound 7 (138 mg, 61%) as a yellow solid.

[0619] EI-MS m / z: [1 / 2 M+H] + 1340.5, [1 / 3 M+H] + 894.1.

[0620] Example 3: Synthesis of Exemplary ADCs of the Disclosure

[0621] An exemplary ADC was produced using the following two steps. LCB14-0606 was produced using the method disclosed in U.S. Patent No. 9,669,107, the specification of which is incorporated herein by reference in its entirety. The structural formula of LCB14-0606 is as follows:

[0622]

[0623] Step 1: Production of prenylated antibody with LCB14-0606

[0624] AFF4-CaaX was prepared according to Example 1. The mixture for the prenylation reaction including AFF4-CaaX was prepared and reacted for 16 hours at 30°C. The reaction mixture contained a total of 24 mM antibody, 600 mg or 400 nM FTase (Genscript), and 144 mM LCB14-0606 in a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 mM ZnCl2, 0.5 mM DTT). After the reaction, the prenylated antibody was desalted using a G25 Sephadex gel column (AKTA purifier, GE healthcare) equilibrated with PBS buffer. As a result, a total of 544 mg of prenylated antibody was produced.

[0625] Step 2: Drug conjugation method

[0626] Oxime bond reaction of ADC 1 (conjugation through oxime bond formation)

[0627] The oxime bond formation reaction between the prenylated antibody and the linker-drug (Compound 1 of Example 1) was performed by mixing 100 mM sodium acetate buffer (pH 5.2), 10% DMSO, 350 mg of prenylated antibody (a total of 24 mM), and 10 equivalents of the linker-drug at 30°C under stirring at 500 to 600 rpm for 24 hours. After the reaction, the excess low molecular weight compounds were removed by FPLC (AKTA purifier, GE healthcare), and the protein fraction was collected and concentrated. As a result, a total of 336 mg of ADC 1 was obtained. The production process and its structure are shown in Figure 1 .

[0628] Oxime bond reaction of ADC 2 (conjugation through oxime bond formation)

[0629] The oxime bond formation reaction between the prenylated antibody and the linker-drug was performed by mixing 100 mM sodium acetate buffer (pH 5.2), 10% DMSO, 14.4 mg of the prenylated antibody (total 24 mM) and 10 equivalents of the linker-drug (compound 2 of Example 2) at 30°C under stirring at 500 to 600 rpm for 6 hours. After the reaction, the excess low molecular weight compounds were removed by FPLC (AKTA purifier, GE healthcare), and the protein fraction was collected and concentrated. Thus, a total of 13.5 mg of ADC2 was obtained. The production process and its structure are shown in Figure 2

[0630] Oxime bond reaction of ADC 3 (by oxime bond formation conjugation)

[0631] The oxime bond formation reaction between the prenylated antibody and the linker-drug was performed by mixing 100 mM sodium acetate buffer (pH 5.2), 10% DMSO, 14.4 mg of the prenylated antibody (total 24 mM) and 10 equivalents of the linker-drug (compound 2 of Example 2) at 30°C under stirring at 500 to 600 rpm for 6 hours. After the reaction, the excess low molecular weight compounds were removed by FPLC (AKTA purifier, GE healthcare), and the protein fraction was collected and concentrated. Thus, a total of 13.5 mg of ADC2 was obtained. The production process and its structure are shown in Figure 3

[0632] To confirm the characteristics of the three ADCs produced above, analysis was performed using an HPLC instrument with a HIC column, using a mobile phase prepared with ammonium sulfate, acetonitrile and potassium phosphate, and the results are shown in Figure 4 Figure 5

[0633] As shown in Figure 4 , ADC1, ADC2 and ADC3 all exhibited different levels of hydrophobicity, confirming that the hydrophobicity was in the order of ADC1>ADC2>ADC3, and confirming that the purity was 97% or more at the same DAR (drug-antibody ratio).

[0634] 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.

[0635] The ADCs produced by the above two steps are summarized in Table 9.

[0636] Table 9. Exemplary ADCs

[0637] ​​​​

[0638] Example 4: Assessment of Cancer Cell Binding of Anti-L1CAM Antibodies

[0639] The cell binding affinity of the AFF4-CaaX antibody produced 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 line (SK-N-F1).

[0640] In particular, the above cancer cell lines were cultured, and the cells were divided into two groups: a control antibody-treated group and an anti-L1CAM antibody-treated group. Subsequently, the cells were incubated in a phosphate buffered saline solution containing 2% fetal bovine serum at 4°C for 1 hour to remove non-specifically bound antibodies. Next, the control antibody and the anti-L1CAM antibody were incubated with each group of cell lines at 4°C for 1 hour to allow the antibodies to bind to the L1CAM protein on the surface of the cancer cells. Subsequently, the antibodies that were not bound 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 was not bound to the antibody using a phosphate buffered saline solution, the fluorescence intensity of the cells was measured using a flow cytometer to calculate the fluorescence intensity of the cell lines in the anti-L1CAM antibody group compared to the fluorescence intensity of the cell lines in the control group, thereby confirming the binding affinity of the antibodies to the cancer cell lines. The results are presented in Tables 10 to 15 below.

[0641] Table 10. Fluorescence intensity of JIMT-1, MDA-MB-231, SK-BR-3, and HCC1395 cells

[0642]

[0643] Table 11. Fluorescence intensity of MCF7, SK-OV-3, OVCAR-3, and SNU-840 cells

[0644]

[0645] Table 12. Fluorescence intensity of SNU-8, PANC-1, PATU-8988s, and H69 cells

[0646]

[0647] Table 13. Fluorescence intensity of PC-9, NCI-H460, Calu-6 and HCT116 cells

[0648]

[0649] Table 14. Fluorescence intensity of SW480, SNU-1197, A2058 and MeWo cells

[0650]

[0651] Table 15. Fluorescence intensity of G361 and SK-N-F1 cells

[0652]

[0653] As shown in Tables 10 to 15, the AFF4-CaaX antibody can strongly bind to all cancer cell lines.

[0654] Example 5: In vitro cytotoxicity evaluation

[0655] The three ADCs produced in Example 3 were measured for their cell proliferation inhibitory activity against cancer cell lines.

[0656] In particular, 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, and commercially available human kidney cells (HK-2, HEK293T), skin cells (HaCaT), and liver cells (Fa2N-4) were used as control normal cell lines.

[0657] In addition, each cancer cell line was seeded in a 96-well culture plate at 500 to 9,000 cells / well and cultured for 24 hours. Subsequently, the cells were treated with ADCs at a concentration range of 0.256 pM to 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 to 18 below.

[0658] Table 16. 50% cytotoxic concentrations of ADCs in MT-1, MDA-MB-231, SK-BR-3, HCC1395, MCF7, OVCAR-3, and SNU-840 cells

[0659]

[0660] Table 17. 50% cytotoxic concentrations of ADCs in SNU-8, PANC-1, PATU-8988s, H69, PC-9, NCI-H460, Calu-6, and HCT116 cells

[0661]

[0662] Table 18. 50% cytotoxic concentrations of ADCs in SW480, SNU-1197, A2058, MeWo, and G-361 cells

[0663]

[0664] As shown in Tables 16 and 17, the results of cytotoxicity analysis of ADC1 and ADC2 indicate that pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC2) exhibited significantly stronger cytotoxicity than auristatin-based antibody-drug conjugate (ADC1) in most of the cancer cell lines except colorectal cancer cell line HCT116 and breast cancer cell lines HCC1395 and MCF7. In addition, as shown in Table 18, the results of cytotoxicity analysis of ADC1, ADC2, and ADC3 indicate that the order of cytotoxicity strength shown in colorectal cancer cell line SW480 and melanoma cell line MeWo was ADC2 > ADC1 > ADC3.

[0665] In addition, the results of cytotoxicity in normal cells are shown in Table 19.

[0666] Table 19. 50% cytotoxic concentrations of ADCs in HK-2, Fa2N-4, and HEK293T cells

[0667]

[0668] As shown in Table 19, ADCs exhibited weak cytotoxicity in normal cell lines. In particular, ADC2 showed weaker cytotoxicity than ADC1 in normal cell lines, while ADC1 and ADC2 showed similar cytotoxicity in Fa2N-4 and HEK293T cells.

[0669] These results indicate that the exemplary ADCs exhibit weak cytotoxicity in normal cells, while showing strong cytotoxicity in cancer cells. In particular, ADC2 exhibits weaker cytotoxicity in normal cells and stronger cytotoxicity in cancer cells than other ADCs.

[0670] Example 6: Assessment of Anti-cancer Efficacy in vivo

[0671] The tumor growth inhibition efficacy of the ADCs produced in Example 3 was analyzed in a tumor xenograft mouse model.

[0672] In particular, a breast cancer cell line (JIMT-1), a colorectal cancer cell line (SW480), a melanoma cell line (MeWo), a colorectal cancer cell line (HT29), a small cell lung cancer cell line (NCI-H146, NCI-H69), an ovarian cancer cell line (SNU-840), a pancreatic cancer cell line (PATU-8988s), and a non-small cell lung cancer cell line (Calu-6) were used for cell culture, and 2,000,000 to 5,000,000 cells were mixed with 100 μl of a PBS solution and 100 μl of Matrigel. Subsequently, these mixtures were subcutaneously implanted into Balb / c nude mice to produce a tumor xenograft mouse model. When the tumor size reached 100 to 300 mm 3 , intravenous injection of the ADCs was performed according to the dose and administration schedule shown in Table 20. The tumor size was measured just before the initial administration (day 1) and periodically measured for a certain period of time (up to 56 days), and the results are shown in Figures 6 to 13

[0673] Table 20. Administration schedule of ADCs

[0674]

[0675] As shown in Figure 6 , the ADCs exhibited significant tumor growth inhibition in the JIMT-1 model of a breast cancer cell line. In particular, the auristatin-based antibody-drug conjugate (ADC1) showed tumor growth inhibition superior to the control group at 5 mg / kg, and the pyrrolobenzodiazepine-based antibody-drug conjugate (ADC2) showed tumor growth inhibition at 1 mg / kg.

[0676] In addition, as shown in Figure 7 , in the SW480 model of a colorectal cancer cell line, both ADC1 and ADC2 exhibited concentration-dependent tumor growth inhibition compared to the control group. Furthermore, ADC2 exhibited significant tumor growth inhibition even at a low dose.

[0677] In addition, as shown in Figure 8 ​​​​​​As shown in Table 2, both ADC1 and ADC2 exhibited concentration-dependent tumor growth inhibition in the MeWo model of melanoma cell line compared to the control group.

[0678] In addition, as shown in Table 3, 4 mg / kg (BIW*2) dose group of ADC1 and 4 mg / kg (QD*1, BIW*2) dose group of ADC3 showed significant tumor growth inhibition in the Calu-6 model of non-small cell lung cancer cell line compared to the control group. Figure 9

[0679] In addition, as shown in Table 4, 4 mg / kg ADC1 and 8 mg / kg ADC3 (QD*1 and BIW*2) groups showed significant tumor growth inhibition in the SNU840 model of ovarian cancer cell line compared to the control group. Figure 10

[0680] In addition, as shown in Table 5, 4 mg / kg ADC1 showed significant tumor growth inhibition in the Patu8988s model of pancreatic cancer cell line compared to the control group. Figure 11

[0681] In addition, as shown in Table 6, 4 mg / kg ADC1 and 16 mg / kg ADC3 groups showed significant tumor growth inhibition in the NCI-H146 model of small cell lung cancer cell line compared to the control group. Figure 12

[0682] In addition, as shown in Table 7, 2 mg / kg ADC1 and 16 mg / kg ADC3 showed significant tumor growth inhibition in the NCI-H69 model of small cell lung cancer cell line compared to the control group. Figure 13

[0683] Example 7: Analysis of pharmacokinetics in mouse model

[0684] To analyze the pharmacokinetics of the ADCs, ADC1 and ADC3 were intravenously administered to nude mice at a single dose of 2 mg / kg and 8 mg / kg, respectively. Blood samples were collected at 0 minutes, 3 minutes, 3 hours, 1 day, 2 days, 3 days, 7 days, 9 days, and 14 days after administration to measure the serum concentrations of the ADCs and antibodies using LC-MS. Five nude mice were used for each time point.

[0685] Figure 14 The results of the pharmacokinetic analysis of ADC1 and ADC3 in the mouse model are shown in Table 8, and ADC1 and ADC3 have excellent half-lives. In particular, there is little difference in the half-life of the ADCs from that of the antibody used as a control.

[0686] Example 8: Single-dose acute toxicity study in rats ​​​​​

[0687] To analyze the acute toxicity of the ADCs, ADC1 (10 mg / kg, 20 mg / kg) and ADC3 (100 mg / kg, 150 mg / kg) were administered intravenously as a single dose to the tail vein of Sprague-Dawley rats (n=5) and the rats were observed for 4 weeks.

[0688] Figure 15 It was shown that the administration of ADC1 or ADC3 in rats at the highest concentration did not result in significant toxic symptoms (measured as body weight change) and there were no abnormal findings in the necropsy compared to the control group.

[0689] Incorporation by Reference

[0690] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually incorporated by reference herein. In case of conflict between the present application, including any definitions and the incorporated publications, the present application, including any definitions, controls.

[0691] Equivalents

[0692] While particular embodiments of the present application have been discussed, the above specification is illustrative and not restrictive. Many variations of the application will become apparent to those of skill in the art upon review of this specification and the claims below. The full scope of the application should be determined by reference to the claims, along with their full scope of equivalents, and the specification.

Claims

1. A conjugate having a structure represented by Formula I: [Formula I] wherein Ab is an anti-human neural cell adhesion molecule LI (LI CAM) antibody or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, each B is independently an active agent; each W is independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'-, or -PO2NR'-, wherein C, S, or P is directly bound to the phenyl ring and NR' is bound to L; each L is independently a linker and comprises: (i) one or more unsaturated bonds; (ii) a heteroarylene (e.g., a heteroarylene in an alkylene or heteroalkylene chain); or (iii) a combination thereof; and B) at least one isoprenyl unit having a structure represented by Formula II: [Formula II] n1 and n2 are each independently an integer selected from 1 to 20; and n3 is an integer from 0 to 3.

2. The conjugate of claim 1, wherein Ab comprises: (i) a variable heavy chain framework region from a heavy chain of a human antibody or a human consensus framework, wherein the variable heavy chain framework region comprises one or more of the following mutations: T34A, Y58F, N61H, and S103G; and (ii) a variable light chain framework region from a light chain of the human antibody or the human consensus framework.

3. The conjugate of claim 1 or 2, wherein Ab comprises a LALA mutation in a heavy chain constant region.

4. The conjugate of claim 3, wherein the LALA mutation comprises L236A and L237A.

5. The conjugate of any one of claims 1-4, wherein Ab is any one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, a Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a bivalent antibody, a bispecific antibody, and a multispecific antibody.

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

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

8. The conjugate of any one of claims 1-6, wherein n1 is 1 or 2. ​ ​ ​ each G is independently a glucuronic acid moiety or R 3 is hydrogen or a carboxyl protecting group; Each R 4 are independently hydrogen or a hydroxy protecting group; R 1 and R 2 each independently is hydrogen, C 1-8 alkyl or C 3-8 cycloalkyl; ​ R' and R" are each independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono-C 1-8 alkylamino or di-C 1-8 alkylamino, C 3-10 heteroaryl or C 6-10 aryl; each Z is independently C 1-8 alkyl, halo, cyano or nitro; ​ A) C 1-50 alkylene or 1- to 50-membered heteroalkylene comprising: ​ ​ (iii) C 1-20 alkyl substituents; and / or ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 9. The conjugate of any one of claims 1-8, wherein n2 is 1, 2, 3, or 4.

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

11. The conjugate of any one of claims 1 to 10, wherein each G is and R 3 and R 4 is each H.

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

13. The conjugate of any one of claims 1-12, wherein each W is independently -C(O)NR'- 14. The conjugate of any one of claims 1-13, wherein R' is H.

15. The conjugate of any one of claims 1-14, wherein R" is H.

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

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

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

19. The conjugate of any one of claims 1-18, wherein L comprises C 1-50 alkylene.

20. The conjugate of any one of claims 1-19, wherein L comprises C 1-50 heteroalkylene.

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

22. The conjugate according to any one of claims 1 to 21, wherein L is replaced by C 1-20 Alkyl substitution.

23. The conjugate of any one of claims 1-22, wherein L comprises an isoprenyl group having a structure represented by Formula Ilia: wherein n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

24. The conjugate of any one of claims 1-23, wherein L comprises a peptide.

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

26. The conjugate of claim 24 or 25, wherein the peptide comprises an amino acid having a side chain with a moiety (e.g., an amine, guanidine, or carboxyl moiety) that carries a charge in a neutral aqueous solution at neutral pH.

27. The conjugate of any one of claims 24-26, wherein the peptide comprises an amino acid selected from the group consisting of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

28. The conjugate of any one of claims 1-27, wherein the antibody comprises a cysteine residue and L is covalently attached to the antibody via a thioether bond through a sulfur atom in the cysteine residue (e.g., through a sulfur atom in the side chain of the cysteine residue).

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

30. The conjugate of claim 29, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyltransferase (GGTase).

31. The conjugate of claim 29 or 30, wherein the amino acid motif comprises a cysteine residue and L is covalently attached to the antibody via a thioether linkage through a sulfur atom in the cysteine residue (e.g., through a sulfur atom in the side chain of the cysteine residue).

32. The conjugate of claim 31, wherein the amino acid motif comprises the following sequence: CYYX wherein: C is cysteine; each Y is an aliphatic amino acid; and X is selected from the group consisting of glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine.

33. The conjugate of claim 32, wherein each Y is independently selected from the group consisting of alanine, isoleucine, leucine, methionine, and valine.

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

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

36. The conjugate of any one of claims 29-35, wherein the amino acid motif comprises the sequence GGGGGGGCVIM.

37. The conjugate of any one of claims 1-36, wherein L comprises an oxime.

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

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

40. The conjugate of any one of claims 1-39, wherein L comprises a structure represented by Formula IVa or IVb: Formula IVa, Formula IVb; each r is independently an integer from 1 to 10, preferably 2; each p is independently an integer from 0 to 12, preferably 2; q is an integer from 1 to 20, preferably 2, 5, or 11; and w is an integer from 1 to 20, preferably 6 to 20.

41. The conjugate of claim 40, wherein V is -0-.

42. The conjugate of claim 40 or 41, wherein X is -0-.

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

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

50. The conjugate of any one of claims 40-49, wherein q is 2 or 5. -(CH2) r (V(CH2) p ) q - 51. The conjugate of any one of claims 40-50, wherein w is 6 to 20. - (CH2CH2X) w - 53. The conjugate of claim 52, wherein L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyethylene glycol units. V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 , preferably -O-; X is -O-, C 1-8 alkylene or -NR 21 - and preferably -O-. R 21 to R 25 each independently H, C 1-6 alkyl, C 1-6 alkyl C 6-20 aryl or C 1-6 alkyl C 3-20 heteroaryl; 54. The conjugate of any one of claims 1-53, wherein L comprises an oxime and at least one polyethylene glycol unit covalently binds the oxime to the active agent. ​ ​ ​ ​ ​ 43. The conjugate of any one of claims 40-42, wherein R 21 is H.

44. The conjugate of any one of claims 40-43, wherein R 22 is H.

45. The conjugate of any one of claims 40-44, wherein R 23 is H.

46. The conjugate of any one of claims 40-45, wherein R 24 is H.

47. The conjugate of any one of claims 40-46, wherein R 25 is H. ​ ​ ​ ​ 52. according to the described conjugate of any one of claim 40 to 51, wherein L comprises at least one by Represents polyethylene glycol units. ​ ​ 55. The conjugate of any one of claims 1-54, wherein L comprises a moiety formed from the reaction of an alkyne with an azide or the reaction of an aldehyde or ketone group with a hydrazine or hydroxylamine.

56. The conjugate of any one of claims 1-55, wherein L comprises a unit having a structure represented by Formula Va, Vb, Vc, Vd, or Ve: L 1 is a single bond or C 1-30 alkylene; and R 11 is H or C 1-10 alkyl.

57. The conjugate of claim 56, wherein L 1 is a single bond.

58. The conjugate of claim 56, wherein L is C 1-30 alkylene.

59. The conjugate of any one of claims 56-58, wherein R 11 is H.

60. The conjugate of any one of claims 56-58, wherein R 11 is C 1-10 alkyl (e.g., methyl).

61. The conjugate of any one of claims 1-60, wherein L comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch of the branching unit to which a first B is coupled; and iiia) a second branch of the branching unit to which a second B is coupled; or iiib) a second branch of the branching unit comprising an alkyl or heteroalkyl group (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer).

62. The conjugate of claim 61, wherein L comprises a second branch of the branching unit to which a second B is coupled via a second cleavage group.

63. The conjugate of claim 61, wherein L comprises a second branch of the branching unit comprising an alkyl or heteroalkyl group (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer).

64. The conjugate of any one of claims 61-63, wherein the branching unit has a structure represented by Formula Via, VIb, VIc, or VId: wherein 65. The conjugate of any one of claims 61-63, wherein the branching unit has a structure represented by Formula Vie:

66. The conjugate of any one of claims 61-63, wherein the branching unit has a structure represented by Formula VIf: G 1 , G 2 , G 3 each independently is a bond, R 30 is H or alkyl; R 40 is H, alkyl or L 5 -CO2R 50 ; R 50 is H or alkyl; and L 2 , L 3 , L 4 and L 5 are each independently a bond or alkylene.

67. The conjugate of any one of claims 1-66, wherein the conjugate comprises 1, 2, 3, or 4 branched linkers. wherein R 30 is H or alkyl.

68. The conjugate of claim 67, wherein each branched linker comprises at least two active agents. wherein R 30 is H or alkyl.

69. The conjugate of claim 67, wherein each branched linker comprises two active agents.

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

71. The conjugate of any one of claims 1-70, wherein the conjugate is capable of being cleaved in a target cell (e.g., the conjugate is cleaved to release one or more active agents).

72. The conjugate of any one of claims 1-71, wherein the conjugate comprises a structure represented by: n11, n22, and n33 are each independently an integer from 0 to 30; and AA represents an amino acid group.

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

74. The conjugate of any one of claims 1-72, wherein each active agent is independently an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof. ​ ​ 75. The conjugate of any one of claims 1-72, wherein the active agent is independently an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, or an anti-parasitic agent.

76. The conjugate of any one of claims 1-72, wherein the active agent is independently selected from: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1,1) ) Cytostatic / cytotoxic agents: cytarabine, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, etoposide, teniposide, topotecan, irinotecan, sobuzoxane, 5-fluorouracil, floxuridine, tegafur, carmofur, doxifuridine, palafoxur, gemcitabine, 6-mercaptopurine, thioguanine, pentostatin, cladribine, 2-chlorodeoxyadenosine, nelarabine, cladribine, clofarabine, fludarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabine, nelarabinemustard), carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, alacinomysin, actinomycin, antrmycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, Marcelo marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, plicamycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like.acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, ro dorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone, enalapril, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamide, mitoguazone, mitomycin, mitosporin, myriaporfin, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK®, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2'-trichlorotriethylamine, trichothecene, troxacitabine, urogenital cancer vaccine, vincristine, vinorelbine and zinostatin stimalamer.acetate), etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, lo soxantrone, 2-ethylhydrazide, procarbazine, polysaccharide k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, canarypine, cactinomycin, carabicin, cararnine, aclarubicin, CC-1065, chloranmbucil, cytochalasin B, dacarbazine, daunorubicin, 6-diazo-5-oxo-l-norleucine, elaiplomycin, elliptinium acetate, epirubicin, esorubicin, grebleomycin, hydroxyitraconazole, izonici acid, mitoxantrone, nitoxantrone, nitrinex, peplomycin, potfiromycin, puromycin, quelamycin, swinomycin, zinostatin, zorubicin, ibronucleic acid, olomucine, temiserte, triptorelin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carzinophilin, chromomycin A3, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine, adriamycin, mycophenolic acid, nogalamycin, oltipraz, peplomycin, potfiromycin, puromycin, quelamycin, swinomycin, zorubicin, ibronucleic acid, olomucine, temiserte, triptorelin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carzinophilin, chromomycin A3, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine,A) anguidine, ethyl carbamate, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, SN-38, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxy camptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan, topoisomerase inhibitor RFS2000, topoisomerase inhibitor S39625, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid thereof; (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-alpha, tumor necrosis factor-beta, mullerian inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factors, interferons, interferon-alpha, interferon-beta, interferon-gamma, colony stimulating factors (CSFs), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins (ILs), IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, polypeptide factors, LIF, kit ligand, or combinations thereof; (c) diphtheria toxin, botulinum toxin, tetanus toxin, decentritoxin, cholera toxin, amanitin, alpha-amanitin, pyrrolobenzo-diazepine, pyrrolobenzo-diazepine derivatives, indolinobenzo-diazepine, pyridinobenzo-diazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, cyanobacterium, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotic, esperamycin, epothilone, toxoid, or combinations thereof; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an activator, a neurotransmitter, a radioisotope, or a combination thereof; (e) a radiolabel, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target site, or a combination thereof; (f) an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, and an anti-parasitic agent, 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, leuprolide, 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) a vaccine; (p) an anti-angiogenic agent; and (q) a topoisomerase I inhibitor.

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

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

79. The conjugate of any one of claims 1 to 77, wherein the conjugate comprises wherein MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F; and the dashed line indicates attachment to the remainder of the conjugate.

80. The conjugate of any one of claims 1-77, wherein the conjugate comprises wherein MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F; and the dashed line indicates attachment to the remainder of the conjugate.

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

82. The conjugate of claim 1, wherein the conjugate is: or a salt thereof, wherein: For use in the treatment of a disease or disorder associated with anti-human neural cell adhesion molecule Ll (LlCAM) antibodies.

83. The conjugate of claim 1, wherein the conjugate is: or a salt thereof, wherein: For use in the treatment of a disease or disorder associated with anti-human neural cell adhesion molecule Ll (LlCAM) antibodies.

84. The conjugate of claim 1, wherein the conjugate is: or a salt thereof, wherein: For use in the treatment of diseases associated with the human neural cell adhesion molecule Ll (LlCAM).

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

61.

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

87. A method of treating or preventing a proliferative, cancerous, or angiogenic disease in an individual, comprising administering to the individual the conjugate of any one of claims 1-85, or a pharmaceutically acceptable salt thereof.

88. The method of claim 87, wherein the method treats a cancer.

89. The method of 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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