Antibody-drug conjugates containing anti-B7-H3 antibodies

Abstract

The present disclosure relates to antibody-drug conjugates (ADCs) in which one or more active agents are conjugated to an anti-B7-H3 antibody via a linker. The linker may comprise a unit that covalently links the active agent to the antibody. The present disclosure further relates to monoclonal antibodies and antigen-binding fragments, variants, multimeric embodiments, or bispecifics thereof that specifically bind to B7-H3, as well as methods of making and using these anti-B7-H3 antibodies and antigen-binding fragments thereof in various therapeutic, diagnostic, and prophylactic applications.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 044,764, filed June 26, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) combine the binding specificity of antibodies with the potency of chemotherapeutic drugs. ADC technology allows drugs to be precisely delivered to targeted cancer cells and released under specific conditions while minimizing collateral damage to healthy cells, thereby increasing the efficacy of therapeutic antibodies and reducing the risk of adverse reactions.

[0003] B7-H3 (CD276) is a novel member of the B7 family, sharing approximately 30% sequence identity with the B7 family. Initially introduced as a costimulatory molecule for T cells, B7-H3 has been shown to be a co-inhibitory checkpoint ligand capable of regulating helper T cells, cytotoxic T cells, and natural killer cells in human immune responses. B7-H3 protein expression is highly restricted in normal tissues, but is induced on the cell surface of antigen-presenting cells and is widespread in a variety of solid tumors, including primary and metastatic cancers. B7-H3 expression has also been detected in multiple cancer cell types, including cancer stem cells and tumor vasculature. B7-H3 overexpression appears to be significantly correlated with disease severity and poor clinical outcomes in tumors.

[0004] Thus, there is a need for improved antibody-drug conjugates that target B7-H3. Summary of the Invention

[0005] In some aspects, the present disclosure relates to antibody-drug conjugates (ADCs). In some embodiments, the present disclosure relates to antibody-drug conjugates comprising an antibody, a linker, and an active agent (e.g., a drug). The antibody-drug conjugate may include, for example, a self-immolative group for use in releasing the active agent from the antibody and linker.

[0006] The present disclosure provides monoclonal antibodies and antigen-binding fragments, or any fragment, variant, multimeric embodiment, or bispecific thereof, that bind to B7-H3. These antibodies and antigen-binding fragments, or any fragment, variant, multimeric embodiment, or bispecific thereof, are collectively referred to herein as anti-B7-H3 monoclonal antibodies, or anti-B7-H3 mAbs, or antigen-binding fragments, or any fragment, variant, multimeric embodiment, or bispecific thereof. Preferably, the monoclonal antibodies and antigen-binding fragments, or any fragment, variant, multimeric embodiment, or bispecific thereof, are specific for at least human B7-H3. In some embodiments, monoclonal antibodies and antigen-binding fragments, or any fragment, variant, multimeric embodiment, or bispecific thereof, that recognize human B7-H3 are also cross-reactive with at least one other non-human B7-H3 protein, for example, by way of non-limiting example, non-human primate B7-H3, e.g., cynomolgus monkey B7-H3, and / or rodent B7-H3.

[0007] In some aspects, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody, at least one branched linker covalently coupled to the antibody, and at least one or two active agents covalently coupled to the branched linker. The branched linker may comprise a branching unit, wherein at least one drug is coupled to the branching unit via a secondary linker, and the branching unit is coupled to the antibody by a primary linker. The primary linker and / or secondary linker may comprise at least one polyethylene glycol unit.

[0008] In some aspects, the present disclosure provides an antibody conjugate represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof: TIFF0007798811000001.tif13160In formula, the Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof, comprising variable heavy chain complementarity determining region 1 (CDRH1), variable heavy chain complementarity determining region 2 (CDRH2), variable heavy chain complementarity determining region 3 (CDRH3), variable light chain complementarity determining region 1 (CDRL1), variable light chain complementarity determining region 2 (CDRL2), and variable light chain complementarity determining region 3 (CDRL3); CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, or 43; CDRH2 comprises the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, or 44; CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, or 45; CDRL1 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, or 46; CDRL2 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, or 47; CDRL3 comprises the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, or 48; each G is independently a chemical moiety comprising an active agent and a linker, the linker connecting the Ab to the active agent; The present invention relates to an antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein n is an integer of 1 to 20. [Brief explanation of the drawings]

[0009] [Figure 1] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in JIMT-1. [Figure 2]Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in JIMT-1. [Figure 3] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-N87. [Figure 4] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in HCT-116. [Figure 5] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in HCT-116. [Figure 6] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in NCI-H23. [Figure 7] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in NCI-H460. [Figure 8] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-H23. [Figure 9] Shown is the IC50 generated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-H460. [Figure 10]1 shows the effect of T-20-AB2.1 and T-21-AB2.1 on tumor volume in JIMT-1 xenografts. [Figure 11] 1 shows the effect of T-20-AB2.1 and T-21-AB2.1 on body weight in JIMT-1 xenografts. [Figure 12] 1 shows the effect of T-Int-102-D1-5 AB2.1 and T-Int-112-AB2.1 on tumor volume in HCT-116 xenografts. [Figure 13] 1 shows the effect of T-Int-102-D1-5 AB2.1 and T-Int-112-AB2.1 on body weight in HCT-116 xenografts. [Figure 14] 1 shows the effect of T-Int-112-AB2.1 on tumor volume in NCI-H23 xenografts. [Figure 15] 1 shows the effect of T-Int-112-AB2.1 on body weight in NCI-H23 xenografts. [Figure 16] 1 shows the effect of T-Int-112-AB2.1 on tumor volume in NCI-H460 xenografts. [Figure 17] 1 shows the effect of T-Int-112-AB2.1 on body weight in NCI-H460 xenografts. DETAILED DESCRIPTION OF THE INVENTION

[0010] The basic structure of an antibody-drug conjugate is as follows: antibody-linker-low molecular weight drug or toxin. The linker ideally allows the drug to exert its effect on the target cancer cells after, for example, separating from the antibody after reaching the target cells. The linker also plays a functional role by connecting the antibody and the drug.

[0011] B7-H3 (CD276) is a member of the B7 family and shows high sequence homology (up to approximately 30%) with this family. B7-H3 expression is highly restricted in normal tissues but widespread in various solid tumors, including breast, lung, pancreatic, prostate, kidney, and colon cancers, as well as melanoma and glioblastoma. B7-H3 has been observed in tumor epithelium and tumor-associated vasculature and stroma. Furthermore, overexpression of B7-H3 correlates with poor outcomes in many cancer diseases. High B7-H3 expression, common in NSCLC (approximately 85%), is associated with metastasis and advanced stage. Higher incidence and expression levels of B7-H3 have been observed in cancers resistant to anti-PD-1 therapy. Therefore, targeting B7-H3 is relevant for relapsed or refractory NSCLC. A series of anti-B7-H3 ADCs were prepared and tested. The key components of ADCs are an OHPAS linker and a benzodiazepine equipped with an OHPAS-compatible functional group. Given their proven plasma stability, ADCs efficiently release toxins in target tumor cells, suggesting the potential for an expanded therapeutic window. ADCs demonstrate excellent efficacy while minimizing in vivo weight changes, providing a new option for NSCLC patients refractory to anti-PD-1 therapy.

[0012] A series of tightly binding anti-B7-H3 mAbs and their thiomab counterparts were generated (Kd approximately 1.7-5.4×10 -11 M). Utilizing the newly discovered OHPAS linker and OHPAS-compatible benzodiazepine payload, a series of anti-B7-H3 ADCs were prepared and tested. Exemplary OHPAS linkers are further described herein and are also disclosed, for example, in International Application Publication No. 2019 / 008441, which is incorporated herein by reference in its entirety. Exemplary OHPAS-compatible benzodiazepine payloads are further described herein and are also disclosed, for example, in U.S. Patent Application Publication No. 2019 / 0367488, which is incorporated herein by reference in its entirety. The ADCs were highly potent in vitro against B7-H3-positive tumor cell lines. The ADCs were efficacious when tested in a mouse xenograft model of NSCLC.

[0013] The ADCs disclosed herein can target certain tumors that express B7-H3 (e.g., breast cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, and colon cancer, as well as melanoma and glioblastoma) (Cancer Cell. 2017 Apr 10; 31(4): 501-515. e8). Overexpression of B7-H3 correlates well with disease severity and poor outcome. B7-H3 is frequently and strongly expressed across a wide range of tumors. Targeting B7-H3 for cancer therapy is beneficial due to its expression on cancer stem cell populations as well as tumor vasculature and stroma (Journal of Clinical Oncology 35, no. 15_suppl). Both tumor cells and tumor vasculature are B7-H3 (CD276) positive (Cancer Cell. 2017 Apr 10; 31(4): 501-515. e8). The disclosed B7-H3 antibodies have improved internalization capabilities as confirmed by Fab-Assay. Thus, the improved antibody-drug conjugates disclosed herein that target B7-H3 are expected to be useful in methods for treating or alleviating symptoms associated with cancer.

[0014] Examples of B7-H3 antibodies and their uses are listed in Table 1. [Table 1] TIFF0007798811000003.tif119161

[0015] B7-H3 expression contributes to tumor invasion and metastasis. Different patterns of B7-H3 fucosylation or expression of different isoforms in cancer cells indicate opposing costimulatory and co-inhibitory functions (Immunological Reviews 2017;276:52-65). B7-H3 is highly expressed in tumor tissues (Figures 26A-26B). B7-H3 expression is significantly associated with poor outcomes in patients with RCC, lung cancer, prostate cancer, colorectal cancer, gallbladder cancer, esophageal squamous cell carcinoma, cervical cancer, osteosarcoma, breast cancer, head and neck cancer, pancreatic cancer, and ovarian cancer (Clin Cancer Res 2008;14:5150-7, J.Cell.Mol.Med.Vol 21,No 9,2017 pp.2199-2210, OncoTargets and Therapy 2014:7 1465-1472, Cell Research volume 27,pages1034-1045(2017), Am J Transl Res 2015;7(12):2646-2660, Clin Cancer Res,2012,18(14):3834-3845).

[0016] B7-H3 is not expressed in many blood cell lines (Tissue Antigens 2005:66:83-92). 44.8% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) cases show B7-H3 expression, and 65% of mantle cell lymphoma (MCL) cases show B7-H3 expression. B7-H3 expression is absent in B cells, T cells, and monocytes (CMI 2005 2(4)307-311). B7-H3 is inducibly expressed in macrophages, DCs, and tumors. B7-H3 is constitutively expressed in monocyte-derived dendritic cells (Mo-DCs). B7-H3 is weakly expressed in monocyte-derived DCs (Clin Cancer Res 18(14);3834-45,2012).

[0017] The present disclosure also provides monovalent and / or bispecific antibodies comprising at least a first arm specific for B7-H3. Preferably, the monovalent and / or bispecific antibodies are specific for at least human B7-H3. In some embodiments, the monovalent and / or bispecific antibodies that recognize human B7-H3 are also cross-reactive with at least one other non-human B7-H3 protein, for example, by way of non-limiting example, non-human primate B7-H3, e.g., cynomolgus monkey B7-H3, and / or rodent B7-H3. The present disclosure also provides antibodies that bind to the same epitope as the anti-B7-H3 monovalent and / or bispecific antibodies disclosed herein.

[0018] Exemplary anti-B7-H3 monoclonal antibodies and antigen-binding fragments thereof of the present disclosure include, for example, the antibodies listed in Tables 19-24.

[0019] In some embodiments, exemplary anti-B7-H3 monoclonal antibodies and antigen-binding fragments thereof of the present disclosure comprise a combination of heavy chain complementarity determining regions (CDRs) selected from the CDR sequences set forth in Table 19 and light chain CDRs selected from the CDR sequences set forth in Table 19. In some embodiments, exemplary anti-B7-H3 monoclonal antibodies and antigen-binding fragments thereof of the present disclosure comprise a combination of heavy chain and light chain domain variable sequences set forth in Tables 20-24. In some embodiments, exemplary anti-B7-H3 monoclonal antibodies of the present disclosure comprise a combination of heavy chain and light chain domain variable sequences set forth in Tables 21-24.

[0020] Antibody-drug conjugates In certain aspects, the antibody-drug conjugates disclosed herein are represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof: TIFF0007798811000004.tif13160In formula, the Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof, comprising variable heavy chain complementarity determining region 1 (CDRH1), variable heavy chain complementarity determining region 2 (CDRH2), variable heavy chain complementarity determining region 3 (CDRH3), variable light chain complementarity determining region 1 (CDRL1), variable light chain complementarity determining region 2 (CDRL2), and variable light chain complementarity determining region 3 (CDRL3); CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, or 43; CDRH2 comprises the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, or 44; CDRH3 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, or 45; CDRL1 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, or 46; CDRL2 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, or 47; CDRL3 comprises the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, or 48; each G is independently a chemical moiety comprising one or more active agents and a linker, the linker covalently linking the Ab to the active agent; n is an integer from 1 to 20.

[0021] In some embodiments, the Ab is a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, a F(ab')2 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, or a bispecific antibody. The Ab can be a rabbit, murine, chimeric, humanized, or fully human monoclonal antibody. In some embodiments, the Ab is an IgG isotype, e.g., an IgG1 isotype.

[0022] In some embodiments, the Ab comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81 in combination with a variable light chain comprising the amino acid sequence of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 83, 85, 87, 89, 91, 93, 95, or 97.

[0023] In some embodiments, Ab is (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 50; (b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52; (c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 54; (d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 56; (e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 58; (f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60; (g) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 62; and (h) a combination of a variable heavy chain sequence and a variable light chain sequence selected from: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 63; and a variable light chain comprising the amino acid sequence of SEQ ID NO: 64.

[0024] In some embodiments, the B7-H3 is human B7-H3.

[0025] In some embodiments, the cleavable group can be cleaved within the target cell. In some embodiments, the cleavable group can release one or more active agents. In some embodiments, the antibody conjugate comprises an Ab, at least one branched linker covalently coupled to the Ab, and at least two active agents covalently coupled to the branched linkers. In some embodiments, at least two branched linkers are coupled to the Ab, and each branched linker is coupled to at least two active agents. In some embodiments, three branched linkers are coupled to the Ab. In other embodiments, four branched linkers are coupled to the Ab. In still other embodiments, exactly one branched linker is coupled to the Ab. In still other embodiments, each branched linker is coupled to exactly two active agents. In some embodiments, the conjugate comprises at least two different active agents. In some embodiments, at least one branched linker is coupled to two different active agents.

[0026] In some embodiments, each active agent is coupled to the branched linker by a cleavable (e.g., hydrolyzable) bond. In some embodiments, each branched linker comprises a branching unit, and each active agent is linked to the branching unit via a secondary linker, and the branching unit is coupled to the anti-B7-H3 antibody by a primary linker. In some embodiments, the branching unit is a nitrogen atom, e.g., an amine or amide nitrogen atom. In some embodiments, the branching unit is an amide and the primary linker comprises an amide carbonyl. In some embodiments, the branching unit is an amide and the secondary linker comprises an amide carbonyl. In some preferred embodiments, the branching unit is a lysine unit.

[0027] Linkers and Conjugation Partners In some preferred embodiments, each G is independently a group having the structure of formula (II): [ka] each Q is independently an active agent linked to L' through a heteroatom, preferably O or N; Z' is a linking group; L' is a spacer moiety attached to SO2 through a heteroatom selected from O, S, and N, preferably O or N, and is selected such that cleavage of the bond between L' and SO2 promotes cleavage of the bond between L' and Q, releasing the active agent; X is -O-, -C(R b )2-, or -N(R c )-, preferably -O-, Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O- or -(CR b 2) y S- and when y is 1, the N, O, or S atom is positioned to be bonded to TG; X and Y' are located on adjacent atoms of Ar; When TG is activated, it reacts with SO2 to form (Q) q -(L') w Replaces X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- or 6-membered ring containing an intervening atom of Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl; 2 R's bform a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the atom to which they are bonded, However, when w is 0, q is 1.

[0028] Each active agent can be any suitable active agent, as described in more detail below. While many conventional conjugation methods require the presence of functional groups such as amine or hydroxyl groups to form stable linkages, the present disclosure provides strategies for forming connections using functional groups previously unavailable for this purpose, such as phenols and tertiary amines. These functional groups form stable bonds in the conjugates disclosed herein while still allowing for release under certain conditions that activate the trigger group.

[0029] Many suitable trigger groups are known in the art, and exemplary trigger groups and the conditions for activating them are discussed below, for example, for moieties such as those described below for Y. Some trigger groups contain an N, O, or S atom but in a non-nucleophilic form. For example, an NO group is a trigger group that is reduced under reducing conditions to an NH or NHOH group capable of reacting with SO, and an acetate group is a trigger group that is hydrolyzed under hydrolysis conditions to a hydroxyl group capable of reacting with SO. Other trigger groups do not contain an N, O, or S atom but are converted to a nucleophilic N, O, or S atom upon activation. For example, a boronate group is a trigger group that is converted under oxidizing conditions (e.g., peroxide) to a hydroxyl group capable of reacting with SO. Preferably, the trigger group is selected so that the conditions for activating it do so selectively without cleaving or degrading other moieties of the conjugate, such as the targeting moiety. Once the nucleophilic N, O, or S atom is generated, it attacks the SO moiety intramolecularly to form a ring, resulting in the formation of the moiety (Q). q -(L') w Ejecting -H, where H is attached to the heteroatom of Q or L' that was previously attached to the SO2 moiety.

[0030] In embodiments where w is 0, q is 1 and Q is directly bonded to SO through a heteroatom. Thus, activation of the trigger group generates a nucleophilic heteroatom that attacks the SO moiety intramolecularly to form a ring and eject the active agent QH, where H is bonded to the heteroatom previously connected to SO.

[0031] In embodiments where w is 1, L' may be selected to allow for the attachment of multiple occurrences of Q, which may be the same or different. Thus, each instance of Q is indirectly attached to SO via a spacer moiety. In such embodiments, activation of the trigger group generates a nucleophilic heteroatom that attacks the SO moiety intramolecularly to form a ring, resulting in the attachment of moiety (Q). q -L'-H (where H is bonded to the heteroatom in L' that was previously attached to SO). In such embodiments, the released heteroatom triggers an intramolecular reaction that releases the active agent Q (e.g., when Q has a tertiary amine attached to L' as a quaternary ammonium) or QH. For example, the heteroatom may undergo an intramolecular cyclization reaction with the ester moiety formed by the hydroxyl of QH to form a ring and release the active agent QH. Alternatively, the heteroatom may undergo intramolecular tautomerization that releases the active agent Q or QH.

[0032] Ar can be any suitable ring, including bicyclic or other polycyclic rings, so that the moieties undergoing intramolecular cyclization are held in close proximity to facilitate reaction after activation of the triggering group. The planar nature of aromatic and heteroaromatic rings is preferred because the rigid geometry of the substituents on such rings ensures the desired placement of the reactive moieties, although other types of rings, such as cycloalkenyl or heterocycloalkenyl, can impose similar geometries. Five- or six-membered rings, and / or the number or identity of heteroatoms in the ring, and / or other substituents on the ring (e.g., electron-donating or electron-withdrawing substituents), can be selected to adjust the cyclization rate based on the resulting bond angle of the ring. Similarly, the more flexible conformation of cycloalkyl and heterocyclyl rings can be useful when it is desired to slow the rate of intramolecular cyclization.

[0033] Z' can be any suitable linking group connecting Ar to one or more Ab groups. Typically, the linking group should be sufficiently hydrophilic to promote water solubility and inhibit aggregation of the conjugate, for example, by including moieties such as polyethylene glycol moieties, peptide sequences, charged moieties (e.g., carboxylates, amines, nitrogen-containing rings, etc.), thereby balancing the hydrophobic properties of any alkyl chains that may be included. Because it is often advantageous to prepare conjugates in a modular manner, Z' can contain a linking unit that is a functional group resulting from the conjugation of one reactive moiety to another. Representative linking units are discussed in more detail below (e.g., in connection with the variable Z), and common linking groups include amides, triazoles, oximes, carbamates, etc. Representative Z' groups include the L groups discussed in more detail below. 1'-Z groups. In some embodiments, all of the G groups attached to each Ab are identical, while in other embodiments, each Ab may be attached to two or more different G groups. For example, some G groups may have trigger groups that are activated under a first condition, while other G groups may have trigger groups that are activated under a second condition, such that, for example, one active agent can be selectively released under a first condition, while a second active agent can be selectively released under a second condition.

[0034] In certain embodiments of Formula (II), -Y' is -(CH2) y NR”-, -(CH2) y O- or -(CH2) y When R″ is S— and y is 1, an N, O, or S atom is positioned to be bonded to TG, R″ is hydrogen or C1-C6-alkyl, and y is an integer having a value of 0 or 1. In some such embodiments, TG is a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0035] In some embodiments of Formula (II), (L')w links each Q to -SO2-, and each Q is an active agent linked to one of the L' groups through a heteroatom, preferably O or N, forming an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- linkage involving the heteroatom of Q. In other embodiments, (Q) q -(L') w -teeth, [ka] is selected from: Q is an active agent linked to L' through a heteroatom, preferably O or N; X 4 forms an —O—, —OC(O)—, —OC(O)O—, or —OC(O)NH— linkage that is absent or includes a heteroatom of Q; X 1 is -O- or -NR a - and X 2 is -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH-, X 3 is -OC(=O)-, w' is an integer having a value of 1, 2, 3, 4, or 5; R 9 and R 10 are each independently hydrogen, alkyl, aryl, or heteroaryl, and the alkyl, aryl, and heteroaryl are unsubstituted or have one or more substituents, for example, alkyl, —(CH) u NH2, -(CH2) u NR u1 R u2 , and -(CH2) u SO2R u3 is substituted with a substituent selected from R u1 , R u2 , and R u3 are each independently hydrogen, alkyl, aryl, or heteroaryl; u is an integer having a value of 1 to about 10.

[0036] In some such embodiments, (Q) q -(L') w -teeth, [ka] is selected from.

[0037] In certain embodiments, Z' comprises a reactive group (e.g., a precursor group as discussed in more detail below with respect to Z) that can be used to connect the compound to a trigger agent, to a solid surface (e.g., to form a solid-supported array or sensor particle), or to any other molecule or support of interest.

[0038] In certain embodiments, Z' is a linking group having the structure of formula (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (IIh): [ka] [ka] During the ceremony, * is the attachment point to Ab, ** is the connection point to Ar, R e is alkyl, X" is -O-, -S-, -NH-, or -CH2-; X 4 is -NHC(O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, W b1 and W b2 are each independently -C(O)NH-, -NHC(O)-, [ka] and L 2 is an optionally present spacer moiety, which may be one or more substituents, e.g., C1-C6 alkyl, C5-C 14 It may be further substituted with aryl and C3 to C8 heteroaryl, and alkyl, aryl, and heteroaryl are, for example, C1 to C 10 Alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 , -(CH2) u CO2H, -(CH2) u CO2R u1 , and -(CH2) u SO2R u3 and R u1 , R u2 , and R u3 are each independently hydrogen, C1 to C 15 Alkyl, C6-C 20 Aryl or C3-C 10heteroaryl, and u is an integer having a value of 1 to about 10; R 12 is hydrogen, a C1-C8 alkyl, or an amino acid moiety such as a naural amino acid moiety; a, b, c, d, e, g, h, o, and qq are each independently an integer having a value of 1 to about 10; s' is an integer having a value of 1 to about 10.

[0039] In a preferred embodiment, W b1 and W b2 are each independently [ka] is.

[0040] In other embodiments, Z' is a linking group having the structure of formula (IIa'), (IIb'), (IIc'), (IId'), (IIe'), (IIf'), (IIg'), or (IIh'): [ka] [ka] During the ceremony, * is the attachment point to Ab, ** is the attachment point for Ar.

[0041] In some preferred embodiments, Z' is [ka] [ka] [ka] [ka] is a linking group selected from the formula: R zais H or methyl, R zb is —OH, ═O, or ═NHOH, [ka] a single or double bond, a" represents the bond between Z' and Ar in formula (II), b" represents the bond between Z' and Ab; Z” is [C10] Oriented in either direction, [ka] is selected from.

[0042] In some embodiments, G is: [ka] wherein Q is an active agent; [ka] is a fragment of the linking group Z' that connects Z' to the substituted phenyl group (represented as Ar in formula (II)).

[0043] In certain embodiments, Ab-(G) n is a compound of formula (III), [ka] or a salt thereof, wherein: A is, [ka] and M, N, CR 30 , or C(-LQ), each L is independently selected from a spacer moiety; Each Q is an active agent, J is a B7-H3 antibody as described herein; R 30 and R 31 are each independently selected from an electron-withdrawing group, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 42 and R 43 are each independently -OH, alkoxy, or -NR 44 R 45 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, and heterocyclyl, wherein R 44 and R 45 can be taken together with the nitrogen atom to which they are attached to form a 5- to 8-membered ring optionally fused to an aryl or heteroaryl ring; R 32 , R 44 , and R 45 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; n is 1 to 4.

[0044] In some embodiments, M is N.

[0045] In certain embodiments, M is CR 30 and R 30 is an electron-withdrawing group.

[0046] In some embodiments, A is [ka] is selected from In the formula, R 31 is an electron withdrawing group, preferably L is coupled to C by an electron withdrawing group selected from an amide or an ester.

[0047] In some embodiments, M is C(-LQ), where L is coupled to C by an electron-withdrawing group.

[0048] In some embodiments, R 30 is -CO2NR 33 R 34 or -CO2R 35 and R 33 , R 34 , and R 35 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0049] In some embodiments, each electron withdrawing group is independently —NO 2 , —CN, -haloalkyl, —CO 2 NR 33 R 34 , -CO2R 35 , -C(=O)R 36 , -S(=O)R 37 , -S(=O)2OR 38 , and -NR 39 R 40 R 41 Selected from R 36 、 R 37 , R 38 , R 39 、 R 40 , and R 41 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0050] In certain embodiments, each electron-withdrawing group is independently —CN, —CONR 33 R 34 , and -CO2R 35 is selected from.

[0051] In some embodiments, each electron-withdrawing group is independently selected from -CN, -CONH2, and -CO2Me.

[0052] In certain embodiments, Q is a drug.

[0053] In some embodiments, Q comprises L' and Q', where L' is a linker and Q' is an active agent.

[0054] In certain embodiments, L′ comprises a coupling group, and the coupling group is coupled to L.

[0055] In some embodiments, the coupling group is —C(═O)NR 32 -, -C(=O)O-, -C(=NR 32 )-, -C=NO-, -NR 32 -C(=O)-NR 32 -, -OC(=O)O-, -SS-, -NR 32 It is selected from -S(=O)2O- and -OS(=O)2O-.

[0056] In certain preferred embodiments, the coupling group is Oriented in either direction, [ka] is selected from.

[0057] In some embodiments, L' further comprises a cleavable group, wherein the cleavable group is coupled to Q'.

[0058] In certain embodiments, the cleavable group -Q' moiety is [ka] is selected from: R 49 is hydrogen or -C(=O)R 50 and R 50 is lower alkyl.

[0059] In some embodiments, L' is a C-C alkyl group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)-. 100 Further includes alkylene.

[0060] In certain embodiments, L is a C-C alkyl group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)-. 100 For example, L can be: [ka] wherein a' is the bond to the M-containing aromatic ring and b' is the bond to L'; n is 2 to 20.

[0061] In some embodiments, A is [ka] For example, A is [ka] Alternatively, A may be [ka] In other embodiments, A may be [ka] In some embodiments, A can be: [ka] is.

[0062] In certain embodiments, R 42 is -OH or -NR 44 R 45 is.

[0063] In some embodiments, the disclosure provides a method of making an ADC disclosed herein, comprising reacting an antibody disclosed herein with a compound of Formula (IV) or Formula (V): [ka] In the formula, A' is [ka] and M, N, CR 30 , or C(-LQ), each L is independently selected from a spacer moiety; each Q is independently selected from an active drug or a reactive group; X is selected from -Cl, -Br, and -I; R 30 and R 31 are each independently selected from an electron withdrawing group, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 46 is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 32 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 47 But, O - alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl; n is 1 to 4.

[0064] In some embodiments, M is N.

[0065] In certain embodiments, M is CR 30 and R 30 is an electron-withdrawing group.

[0066] In some embodiments, A' is [ka] is selected from In the formula, R 31 is an electron withdrawing group, preferably L is coupled to C by an electron withdrawing group selected from an amide or an ester.

[0067] In some embodiments, A' is [ka] where R 46 is C 1~3 It is an aryl group substituted with an alkyl.

[0068] In some embodiments, A' is [ka] is.

[0069] In some embodiments, A' is [ka] where X is —C(O)NH2.

[0070] In some embodiments, M is C(-LQ), where L is coupled to C by an electron-withdrawing group.

[0071] In some embodiments, R 30 is -CO2NR 33 R 34 or -CO2R 35 and R 33 , R 34 , and R 35 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0072] In some embodiments, each electron withdrawing group is independently —NO 2 , —CN, -haloalkyl, —CO 2 NR 33 R 34 , -CO2R 35 , -C(=O)R 36 , -S(=O)R 37 , -S(=O)2OR 38 , and -NR 39 R 40 R 41 Selected from R 36 , R 37 , R 38 , R 39 , R 40 , and R 41 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0073] In certain embodiments, each electron-withdrawing group is independently —CN, —CONR 33 R 34 , and -CO2R 35 is selected from.

[0074] In some embodiments, each electron-withdrawing group is independently selected from -CN, -CONH2, and -CO2Me.

[0075] In certain embodiments, Q is an active agent.

[0076] In some embodiments, Q comprises L' and Q', where L' is a linker and Q' is an active agent.

[0077] In certain embodiments, L′ comprises a coupling group, and the coupling group is coupled to L.

[0078] In some embodiments, the coupling group is —C(═O)NR 32 -, -C(=O)O-, -C(=NR 32 )-, -C=NO-, -NR 32 -C(=O)-NR 32-, -OC(=O)O-, -SS-, -NR 32 It is selected from -S(=O)2O- and -OS(=O)2O-.

[0079] In certain embodiments, the coupling group is Oriented in either direction, [ka] is selected from.

[0080] In some embodiments, L' further comprises a cleavable group, wherein the cleavable group is coupled to Q'.

[0081] In certain embodiments, the cleavable group coupled to Q' is [ka] is selected from: R 49 is hydrogen or -C(=O)R 50 and R 50 is lower alkyl.

[0082] In some embodiments, L' is a C-C alkyl group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)-. 100 Further includes alkylene.

[0083] In certain embodiments, L is a C-C alkyl group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)-. 100 For example, L can be: [ka] wherein a is a bond to the M-containing aromatic ring and b is a bond to L'; n is 2 to 20.

[0084] In some embodiments, Q' is a hormone, an oligonucleotide, a toxin, an affinity ligand, a detector probe, or a combination thereof.

[0085] In certain embodiments, Q' is selected from a cytokine, an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an analgesic agent, or a combination thereof.

[0086] In certain embodiments, Q is a reactive group.

[0087] In some embodiments, the reactive group is -N3, -C≡CH, [ka] , —S(O)2Hal, —NH2, —CO2Hal, —OH, —C(O)H, —SH, —N═C═O, and —N═S═C, where Hal is —Cl, —Br, or —I.

[0088] In some embodiments, A is [ka] is.

[0089] In certain embodiments, R 31 -CN, -CO2NR 33 R 34 , or -CO2R 35 is.

[0090] In certain embodiments, A is [ka] is.

[0091] In some embodiments, R 32 is hydrogen or C 1~3 It is alkyl.

[0092] In some embodiments, A is [ka] is.

[0093] In certain embodiments, R 46 is an optionally substituted C 1~3 Alkyl, optionally substituted C-C 12 It is aryl, or optionally substituted heteroaryl.

[0094] In some embodiments, A is [ka] is.

[0095] In certain embodiments, R 47 O - or C 1~3 It is alkyl.

[0096] In certain embodiments, A is [ka] is.

[0097] Active Agent As noted above, in preferred embodiments of the present disclosure, Q is an active agent forming part of the ADC disclosed herein. In some embodiments, the active agent is independently selected from a chemotherapeutic agent and a toxin. In some embodiments, the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an analgesic agent, or a combination thereof.

[0098] Exemplary Drugs for Conjugation The ADCs of the invention provide targeted therapy, which may reduce side effects common with anti-cancer therapies, for example, as one or more active agents are delivered to specific cells.

[0099] For example, active agents include erlotinib (TARCEVA, Genentech / OSI Pharm.); bortezomib (VELCADE, MillenniumPharm.); fulvestrant (FASLODEX, AstraZeneca); Sutent (SU11248, Pfizer); letrozole (FEMARA, Novartis); imatinib mesylate (GLEEVEC, Novartis), PTK787 / ZK 222584 (Novartis); oxaliplatin (Eloxatin, Sanofi); 5-fluorouracil (5-FU); leucovorin; rapamycin (Sirolimus, RAPAMUNE, Wyeth); lapatinib (TYKERB, GSK572016, GlaxoSmithKline); lonafarnib (SCH 66336); sorafenib (BAY43-9006, Bayer Labs.); gefitinib (IRESSA, Astrazeneca); AG1478, AG1571 (SU 5271, Sugen); alkylating agents (e.g., thiotepa, or CYTOXAN® cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, or piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, or uredopa); ethyleneimine, methylmelamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmethyl lamins; acetogenins (e.g., bullatacin or bullatacinone); camptothecins, including the synthetic analog topotecan; bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, or bizelesin); cryptophycins (e.g., cryptophycin 1 or cryptophycin 8); dolastatin; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, or uracil mustard); nitrousureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimnustine); antibiotics (e.g., calicheamicin gamma 1 I and calicheamicin omega 1 a calicheamicin selected from the group consisting of 1, or dynemicin, including dynemicin A as the enediyne antibiotic; bisphosphonates (e.g., clodronate); esperamicin, neocarzinostatin chromophore, or related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, ADRLIMYCIN® doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, or deoxydoxorubicin), epirubicin, esorubicin, marcellomycin, mitomycin (e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin); antimetabolites (e.g., 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, or trimetrexate);Purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, or thiguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, or floxuridine); androgens (e.g., calsterone, dromostanolone propionate), propionate, epithiostanol, mepitiostane, or testolactone); antiadrenal drugs (e.g., aminoglutethimide, mitotane, or trilostane); folic acid supplements (e.g., folinic acid); aceglatone; aldophoramide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (e.g., maytansine or ansamitocin); trichothecenes (e.g., T-2 toxin, veracrine, urin A, roridin A, or anguidine); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, or anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxoids (e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel, American Pharmaceutical Partners, Schaumber, IL), or TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs (e.g., cisplatin or carboplatin); vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids (e.g., retinoic acid); capecitabine; and pharmaceutically acceptable salts thereof, solvates thereof, acids thereof, or derivatives thereof.

[0100] Mitotic inhibitors In some embodiments, the linkers of the present disclosure may be used to conjugate antibodies to one or more antimitotic agents to form ADCs for the treatment of cancer. The term "antimitotic agent," as used herein, refers to a cytotoxic drug and / or therapeutic agent that blocks mitosis, or cell division, a biological process particularly important to cancer cells. Antimitotic agents often disrupt microtubules so that cell division is prevented by affecting microtubule polymerization or microtubule depolymerization. Thus, in certain embodiments, antibodies are conjugated to one or more antimitotic agents that disrupt microtubule formation by inhibiting tubulin polymerization. In certain embodiments, the antimitotic agent used in the ADCs of the present disclosure is Taxol® (paclitaxel), Taxotere® (docetaxel), or Ixempra® (ixabepilone). Examples of antimitotic agents that can be used in the ADCs disclosed herein are provided below. The genus of antimitotic agents includes the auristatins described above.

[0101] Auristatin The antibody may be conjugated to at least one auristatin using the linker of the present disclosure. Auristatins represent a group of dolastatin analogs that have generally been shown to have anti-cancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analog of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anti-cancer drug vincristine (GR Pettit, Prog. Chem. Org. Nat. Prod, 70:1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides with four amino acids, three of which are unique to the dolastatin group of compounds. Exemplary embodiments of the auristatin subclass of antimitotic agents include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivatives), monomethyl auristatin E (MMAE or auristatin E derivatives), monomethyl auristatin F (MMAF or auristatin F derivatives), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).The synthesis and structure of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, and 2005-0009751, International Patent Publication Nos. 04 / 010957, International Patent Publication No. 02 / 088172, and U.S. Patent Nos. 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,48 Nos. 3, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference.

[0102] Dolastatin In certain embodiments, the active agent in the ADCs described herein is a dolastatin. Dolastatins are short peptide compounds isolated from the Indian Ocean sea hare Dolabella auricularia (see Pettit et al., J. Am. Chem. Soc., 1976, 98, 4677). Examples of dolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15 is a seven-subunit depsipeptide derived from Dolabella auricularia and is a potent antimitotic agent structurally related to the five-subunit antitubulin agent dolastatin 10 obtained from the same organism. Thus, in certain embodiments, the ADCs of the present disclosure comprise an antibody, a linker described herein, and at least one dolastatin. The auristatins described above are synthetic derivatives of dolastatin 10.

[0103] Maytansinoids The linkers of the present disclosure may be used to conjugate an antibody to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as from several species of liverworts (Kupchan et al., J. Am. Chem. Soc. 94:1354-1356

[1972] ; Wani et al., J. Chem. Soc. Chem. Commun 390:

[1973] ; Powell et al., J. Nat. Prod. 46:660-666

[1983] ; Sakai et al., J. Nat. Prod. 51:845-850

[1988] ; and Suwanborirux et al., Experientia 46:117-120

[1990] ). Evidence suggests that maytansinoids inhibit mitosis by inhibiting the polymerization of the microtubule protein tubulin, thereby preventing the formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Furthermore, the cytotoxicity of maytansinoids is 1,000-fold greater than that of conventional chemotherapeutic agents, such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020).

[0104] Maytansinoids include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated herein by reference). C-3 esters of maytansinol may be naturally occurring or synthetically derived. Furthermore, both naturally occurring and synthetic C-3 maytansinol esters can be classified as C-3 esters with simple carboxylic acids or C-3 esters with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogs are described, for example, in Kupchan et al., J. Med. Chem., 21, 31-37 (1978).

[0105] Maytansinoids suitable for use in the ADCs of the present disclosure can be isolated from natural sources, synthetically produced, or semisynthetically produced. Furthermore, maytansinoids can be modified in any suitable manner, so long as sufficient cytotoxicity is retained in the ultimate conjugated molecule. The structure of an exemplary maytansinoid, mertansine (DM1), is provided below. [ka]

[0106] Representative examples of maytansinoids include, but are not limited to, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine, also known as mertansine; drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al. (1992) Cancer Res 52:127), DM2, DM3 (N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine), DM4 (4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (a synthetic maytansinoid analog). Other examples of maytansinoids are described in U.S. Patent No. 8,142,784, incorporated herein by reference.

[0107] Ansamitocins are a group of maytansinoid antibiotics isolated from various bacterial sources. These compounds have potent antitumor activity. Representative examples include, but are not limited to, ansamitocin P1, ansamitocin P2, ansamitocin P3, and ansamitocin P4.

[0108] plant alkaloids The linkers of the present disclosure may be used to conjugate an antibody to at least one plant alkaloid, such as a taxane or vinca alkaloid. Plant alkaloids are chemotherapy treatments made from certain types of plants. Vinca alkaloids are made from the periwinkle plant (Catharanthus rosea), while taxanes are made from the bark of the Pacific Yew tree (Taxus). Both vinca alkaloids and taxanes are also known as anti-microtubule agents and are described in more detail below.

[0109] Taxanes The antibody may be conjugated to at least one taxane using the linker of the present disclosure. As used herein, the term "taxane" refers to a type of antineoplastic agent that has a microtubule-acting mechanism and a structure containing a taxane ring structure and a stereospecific side chain required for cytostatic activity. The term "taxane" also includes various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113, the piperazino and other derivatives described in WO 99 / 14209, the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680, the 6-thio derivatives described in WO 98 / 28288, the sulfenamide derivatives described in U.S. Pat. No. 5,821,263, and the taxol derivatives described in U.S. Pat. No. 5,415,869, each of which is incorporated herein by reference. Taxane compounds are disclosed in U.S. Patent Application Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, and 5,822,264. 40,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference. Further examples of taxanes include, but are not limited to, docetaxel (Taxotere®, Sanofi Aventis), paclitaxel (Abraxane® or Taxol®, Abraxis Oncology), and nanoparticulate paclitaxel (ABI-007 / Abraxene®, Abraxis Bioscience).

[0110] In certain embodiments, the linkers of the present disclosure may be used to conjugate an antibody to at least one docetaxel. In certain embodiments, the linkers of the present disclosure may be used to conjugate an antibody to at least one paclitaxel.

[0111] Vinca alkaloids In certain embodiments, the antibody may be conjugated to at least one vinca alkaloid using a linker of the present disclosure. Vinca alkaloids are a type of cell-cycle-specific drug that inhibits the ability of cancer cells to divide by acting on tubulin and preventing the formation of microtubules. Examples of vinca alkaloids that can be used in the ADCs of the present disclosure include, but are not limited to, vindesine sulfate, vincristine, vinblastine, and vinorelbine.

[0112] Antitumor antibiotics The linkers of the present disclosure may also be used to conjugate antibodies to one or more antitumor antibiotics for the treatment of cancer. As used herein, the term "antitumor antibiotic" refers to an anti-neoplastic drug produced by a microorganism that blocks cell growth by interfering with DNA. In many cases, antitumor antibiotics either break DNA strands or slow or stop DNA synthesis. Examples of antitumor antibiotics that can be included in the ADCs disclosed herein include, but are not limited to, actinomycins (e.g., pyrrolo[2,1-c][1,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins, and are described in more detail below.

[0113] Actinomycin The antibody may be conjugated to at least one actinomycin using a linker of the present disclosure. Actinomycins are a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces. Representative example actinomycins include, but are not limited to, actinomycin D (Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV, and actinomycin C1], Lundbeck, Inc.), anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin B, SG2285, sibanomycin, sibiromycin, and tomaymycin. In certain embodiments, D is a pyrrolobenzodiazepine (PBD). Examples of PBDs include, but are not limited to, anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sibanomycin, sibiromycin, and tomaymycin. Thus, in certain embodiments, D is an actinomycin, e.g., actinomycin D, or a PBD, or a pyrrolobenzodiazepine (PBD) dimer.

[0114] The structure of a PBD can be found, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917 and 2013 / 0028919, and WO2011 / 130598A1, each of which is incorporated by reference in its entirety. The general structure of a PBD is provided below. [ka]

[0115] PBDs vary in the number, type, and position of substituents on both the aromatic A-ring and the pyrrolo C-ring, as well as the degree of saturation of the C-ring. In the B-ring, the N10-C11 position, which is generally the electrophilic center responsible for DNA alkylation, contains an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)). All known natural products have the (S) configuration at the chiral C11α position, giving them a right-handed twist when viewed from the C-ring toward the A-ring. Further examples of PBDs that can be conjugated to antibodies via the linkers disclosed herein can be found, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917A1 and 2013 / 0028919A1, U.S. Patent No. 7,741,319B2, and WO2011 / 130598A1 and WO2006 / 111759A1, each of which is incorporated by reference in its entirety.

[0116] Anthracyclines The linkers of the present disclosure may be used to conjugate an antibody to at least one anthracycline. Anthracyclines are a subclass of antitumor antibiotics isolated from the bacteria of the Streptomyces genus. Representative examples include, but are not limited to, daunorubicin (Cerubicin, Bedford Laboratories), doxorubicin (Adriamycin, Bedford Laboratories, hydroxydaunorubicin, also known as doxorubicin hydrochloride, and Rubex), epirubicin (Ellence, Pfizer), and idarubicin (Idamycin, Pfizer Inc.). Thus, in certain embodiments, D is an anthracycline, e.g., doxorubicin.

[0117] Kalicare sewing machine The linkers of the present disclosure may be used to conjugate an antibody to at least one calicheamicin. Calicheamicins are a family of enediyne antibiotics derived from the soil organism Micromonospora echinospora. Calicheamicins bind to the minor groove of DNA, induce double-stranded DNA breaks, and cause cell death 100-fold greater than other chemotherapeutic agents (Damle et al. (2003) Curr Opin Pharmacol 3:386). Preparation of calicheamicins that can be used as drug conjugates in the present disclosure has been previously described; see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296. Structural analogs of calicheamicin that can be used include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG, and θ1I (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296). Thus, in certain embodiments, D is calicheamicin.

[0118] Duocarmycin The antibody may be conjugated to at least one duocarmycin using a linker of the present disclosure. Duocarmycins are a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces. (See Nagamura and Saito (1998) Chemistry of Heterocyclic Compounds, Vol. 34, No. 12.) Duocarmycins bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position (Boger (1993) Pure and Appl Chem 65(6):1123, and Boger and Johnson (1995) PNAS USA 92:3642). Synthetic analogs of duocarmycin include, but are not limited to, adozelesin, bizeresin, and carzelesin. Thus, in certain embodiments, D is a duocarmycin.

[0119] Other antitumor antibiotics In addition to those mentioned above, additional antitumor antibiotics that may be used in the ADCs of the present disclosure include bleomycin (Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).

[0120] immunomodulators In some embodiments, an antibody may be conjugated to at least one immunomodulatory agent using a linker of the present disclosure. As used herein, the term "immunomodulatory agent" refers to an agent capable of stimulating or modifying an immune response. In certain embodiments, an immunomodulatory agent is an immunostimulatory agent that enhances a subject's immune response. In some embodiments, an immunomodulatory agent is an immunosuppressant that prevents or reduces a subject's immune response. An immunomodulatory agent may modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells), as well as any further differentiated cells thereof. Representative examples include, but are not limited to, bacillus Calmette-Guérin (BCG) and levamisole (ergamisole). Other examples of immunomodulatory agents that may be used in the ADCs of the present disclosure include, but are not limited to, cancer vaccines, cytokines, and immunomodulatory gene therapies.

[0121] Cancer vaccines The linkers of the present disclosure may be used to conjugate antibodies to cancer vaccines. As used herein, the term "cancer vaccine" refers to a composition (e.g., tumor antigens and cytokines) that induces a tumor-specific immune response. The response is elicited from a subject's own immune system by administering the cancer vaccine, or, in the case of the present disclosure, by administering an ADC comprising the antibody and cancer vaccine. In preferred embodiments, the immune response eradicates tumor cells (e.g., primary or metastatic tumor cells) in the body. The use of cancer vaccines generally involves the administration of a specific antigen or group of antigens present, for example, on the surface of specific cancer cells or on the surface of a specific infectious agent shown to promote cancer formation. In some embodiments, the use of cancer vaccines is for prophylactic purposes, while in other embodiments, the use is for therapeutic purposes. Non-limiting examples of cancer vaccines that may be used in the ADCs disclosed herein include recombinant bivalent human papillomavirus (HPV) types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon). Thus, in certain embodiments, D is a cancer vaccine that is either an immunostimulatory agent or an immunosuppressant.

[0122] cytokines The linker of the present disclosure may be used to conjugate an antibody to at least one cytokine. The term "cytokine" generally refers to a protein released by a cell population that acts on another cell as an intercellular mediator. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856). Numerous animal tumor model studies have demonstrated that cytokines have broad anti-tumor activity, which has translated into several cytokine-based approaches for cancer therapy (Lee and Margolin, supra). Recently, several cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have been identified and entered clinical trials for patients with advanced cancer (Lee and Margolin, supra).

[0123] Examples of cytokines that can be used in the ADCs of the present disclosure include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF; platelet growth factor; trans Examples of cytokines include transforming growth factors (TGFs); insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteogenic factors; interferons, such as interferon α, β, and γ, colony-stimulating factors (CSFs); granulocyte-macrophage colony-stimulating factor (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, and IL-12; tumor necrosis factors; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture and biologically active equivalents of native-sequence cytokines. Thus, in certain embodiments, D is a cytokine.

[0124] Colony-stimulating factors (CSFs) The linkers of the present disclosure may be used to conjugate an antibody to at least one colony-stimulating factor (CSF). Colony-stimulating factors (CSFs) are growth factors that assist bone marrow in making red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), colony-stimulating factors can be introduced to help support white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used after bone marrow transplantation to help the new bone marrow begin producing white blood cells. Representative examples of CSFs that may be used in the ADCs disclosed herein include, but are not limited to, erythropoietin (epoetin), filgrastim (Neopogen (also known as granulocyte colony-stimulating factor (G-CSF)), Amgen, Inc.), sargramostim (Leukine (granulocyte-macrophage colony-stimulating factor and GM-CSF), Genzyme Corporation), promegapoietin, and oprelvekin (recombinant IL-11, Pfizer, Inc.). Thus, in certain embodiments, D is a CSF.

[0125] Gene therapy The linkers of the present disclosure may be used to conjugate an antibody to at least one nucleic acid (directly or indirectly via a carrier) for gene therapy. Gene therapy generally refers to the introduction of genetic material into cells, whereby the genetic material is designed to treat a disease. Related to immunomodulators, gene therapy is used to inhibit the growth of cancer cells or stimulate a subject's natural ability to kill cancer cells. In certain embodiments, an ADC of the present disclosure comprises a nucleic acid encoding a functional therapeutic gene used to replace a mutated or otherwise dysfunctional (e.g., truncated) gene associated with cancer. In other embodiments, an ADC of the present disclosure comprises a nucleic acid that encodes or otherwise provides for the production of a therapeutic protein for treating cancer. The nucleic acid encoding the therapeutic gene may be directly conjugated to the antibody, or alternatively, may be conjugated to the antibody via a carrier. Examples of carriers that can be used to deliver nucleic acids for gene therapy include, but are not limited to, viral vectors or liposomes.

[0126] Alkylating agents The linkers of the present disclosure may be used to conjugate an antibody to at least one alkylating agent. Alkylating agents are a type of antineoplastic compound that attaches an alkyl group to DNA. Examples of alkylating agents that can be used in the ADCs of the present disclosure include, but are not limited to, alkylsulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0127] Alkyl sulfonate The linkers of the present disclosure may be used to conjugate an antibody to at least one alkyl sulfonate. Alkyl sulfonates have the general formula R-SO-OR 1 (Wherein R and R 1is a subclass of alkylating agents having a substituted or unsubstituted alkyl group (which is typically an alkyl or aryl group). A representative example of an alkylsulfonate is busulfan (Myleran®, GlaxoSmithKline; Busulfex IV®, PDL BioPharma, Inc.).

[0128] Nitrogen Mustard The linkers of the present disclosure may be used to conjugate an antibody to at least one nitrogen mustard. Representative examples of this subclass of anti-cancer compounds include, but are not limited to, chlorambucil (Leukeran®, GlaxoSmithKline), cyclophosphamide (Cytoxan®, Bristol-Myers Squibb, Neosar, Pfizer, Inc.), estramustine (estramustine phosphate sodium or Estracyt®), Pfizer, Inc.), ifosfamide (Ifex®, Bristol-Myers Squibb), mechlorethamine (Mustargen®, Lundbeck Inc.), and melphalan (Alkeran® or L-Pam®, or phenylalanine mustard, GlaxoSmithKline).

[0129] Nitrosourea The linkers of the present disclosure may be used to conjugate an antibody to at least one nitrosourea. Nitrosoureas are a subclass of lipid-soluble alkylating agents. Representative examples include, but are not limited to, carmustine (BCNU [also known as BiCNU, N,N-bis(2-chloroethyl)-N-nitrosourea, or 1,3-bis(2-chloroethyl)-1-nitrosourea], Bristol-Myers Squibb), fotemustine (also known as Muphoran®), lomustine (CCNU, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb), nimustine (also known as ACNU), and streptozocin (Zanosar®, Teva Pharmaceuticals).

[0130] Triazine and hydrazine The linkers of the present disclosure may be used to conjugate an antibody to at least one triazine or hydrazine. Triazines and hydrazines are subclasses of nitrogen-containing alkylating agents. In some embodiments, these compounds can spontaneously decompose or be metabolized to generate alkyldiazonium intermediates that facilitate the transfer of alkyl groups to nucleic acids, peptides, and / or polypeptides, thereby causing mutagenic, carcinogenic, or cytotoxic effects. Representative examples include, but are not limited to, dacarbazine (DTIC-Dome, Bayer Healthcare Pharmaceuticals Inc.), procarbazine (Mutalane®, Sigma-Tau Pharmaceuticals, Inc.), and temozolomide (Temodar®, Schering Plough).

[0131] Other alkylating agents The linkers of the present disclosure may be used to conjugate an antibody to at least one ethyleneimine, methylamine derivative, or epoxide. Ethylenimines are a subclass of alkylating agents that typically contain at least one aziridine ring. Epoxides represent a subclass of alkylating agents characterized as cyclic ethers with only three ring atoms.

[0132] Representative examples of ethylenimines include, but are not limited to, thiotepa (Thioplex, Amgen), diaziquone (also known as aziridinylbenzoquone (AZQ)), and mitomycin C. Mitomycin C is a natural product that contains an aziridine ring and appears to induce cytotoxicity through cross-linking DNA (Dorr RT, et al. Cancer Res. 1985;45:3510; Kennedy KA, et al. Cancer Res. 1985;45:3541). Representative examples of methylamine derivatives and their analogs include, but are not limited to, altretamine (Hexalen, MGI Pharma, Inc.), also known as hexamethylamine and hexastat. Representative examples of epoxides of this class of anticancer compounds include, but are not limited to, dianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) is chemically related to aziridines and generally facilitates alkyl group transfer via a similar mechanism as described above. Dibromodulcitol is a prodrug for epoxides because it is hydrolyzed to dianhydrogalactitol (Sellei C, et al. Cancer Chemother Rep. 1969;53:377).

[0133] Antiangiogenic agents In some embodiments, the antibody may be conjugated to at least one anti-angiogenic agent using a linker of the present disclosure. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in various ways. In some embodiments, these agents interfere with the ability of growth factors to reach their targets. For example, vascular endothelial growth factor (VEGF) is one of the primary proteins involved in initiating angiogenesis by binding to specific receptors on the cell surface. Therefore, certain anti-angiogenic agents that prevent the interaction of VEGF with its cognate receptor prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, when specific receptors on the cell surface are triggered, a cascade of other chemical signals is initiated to promote blood vessel growth. Thus, for example, certain enzymes, such as several tyrosine kinases, known to promote intracellular signaling cascades that contribute to cell proliferation are targets for cancer therapy. In other embodiments, these agents interfere with intercellular signaling cascades. However, in other embodiments, these agents neutralize specific targets by activating and promoting cell growth or by directly interfering with the growth of vascular cells. Angiogenesis inhibitory properties have been discovered in over 300 substances, with numerous direct and indirect inhibitory effects.

[0134] Representative examples of antiangiogenic agents that may be used in the ADCs of the present disclosure include, but are not limited to, angiostatin, ABX EGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774, erlotinib (Tarceva), angiostatin, arrestin, endostatin, BAY12-9566 and fluorouracil or doxorubicin-containing drugs, canstatin, carboxyamidotriozole and paclitaxel-containing drugs, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, interleukin-12, interleukin-2, NM-3, HuMV833, PTK787, RhuMa b, Angiozyme (Ribozyme), IMC-1C11, Neovastat, Marimstat, Prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, those containing paclitaxel, those containing gemcitabine and cisplatin, and those containing irinotecan and cisplatin, those with radiation, tecogalan, those containing temozolomide and PEG-interferon alpha 2b, tetrathiomolybdate, TNP-470, those containing thalidomide, CC-5013 and Taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (Afinitor, Novartis) Pharmaceutical Corporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.)), nilotinib (Tasigna, Novartis Pharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), and phosphoinositide 3-kinase (PI3K) inhibitors.

[0135] Antimetabolites The linkers of the present disclosure may be used to conjugate an antibody to at least one antimetabolite. Antimetabolites are a type of chemotherapy therapeutic agent that closely resembles normal substances within cells. When a cell incorporates an antimetabolite into its metabolism, the result is negative for the cell, for example, the cell cannot divide. Antimetabolites are classified according to the substance with which they interfere. Examples of antimetabolites that can be used in the ADCs of the present disclosure include, but are not limited to, folate antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, and pentostatin), as described in more detail below.

[0136] antifolates The linkers of the present disclosure may be used to conjugate an antibody to at least one antifolate. Antifolates are a subclass of antimetabolites that are structurally similar to folates. Representative examples include, but are not limited to, methotrexate, 4-amino-folic acid (also known as aminopterin and 4-aminopteroic acid), lometrexol (LMTX), pemetrexed (Alimpta, Eli Lilly and Company), and trimetrexate (Neutrexin, Ben Venue Laboratories, Inc.).

[0137] Purine antagonists The antibody may be conjugated to at least one purine antagonist using the linker of the present disclosure. Purine analogs are a subclass of antimetabolites that are structurally similar to the group of compounds known as purines. Representative examples of purine antagonists include, but are not limited to, azathioprine (Azasan, Salix, Imuran, GlaxoSmithKline), cladribine (leustatin [also known as 2-CdA], Janssen Biotech, Inc.), mercaptopurine (Purinesol [also known as 6-mercaptoethanol], GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation), pentostatin (Nipent, also known as 2'-deoxycoformycin (DCF)), and 6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

[0138] Pyrimidine antagonists The antibody may be conjugated to at least one pyrimidine antagonist using the linker of the present disclosure. Pyrimidine antagonists are a subclass of antimetabolites that are structurally similar to the group of compounds known as purines. Representative examples of pyrimidine antagonists include, but are not limited to, azacitidine (Vidaza, Celgene Corporation), capecitabine (Xeloda, Roche Laboratories), cytarabine (also known as cytosine arabinoside and arabinosylcytosine, Bedford Laboratories), decitabine (Dacogen, Eisai Pharmaceuticals), 5-fluorouracil (Adrsil, Teva Pharmaceuticals, Efudex, Valeant Pharmaceuticals, Inc.), 5-fluoro-2'-deoxyuridine 5'-phosphate (FdUMP), 5-fluorouridine triphosphate, and gemcitabine (Gemzar, Eli Lilly and Company).

[0139] Boron-containing agents The linkers of the present disclosure may be used to conjugate an antibody to at least one boron-containing agent. Boron-containing agents include a class of cancer treatment compounds that interfere with cell proliferation. Representative examples of boron-containing agents include, but are not limited to, borophycin and bortezomib (Velcade, Millenium Pharmaceuticals).

[0140] Chemical protectants The linker of the present disclosure can be used to conjugate the antibody with at least one chemoprotective agent.Chemoprotective drugs are a type of compound that helps protect the body from certain toxic effects of chemotherapy.Chemoprotective agents can be administered together with various chemotherapeutic agents to protect healthy cells from the toxic effects of chemotherapeutic agents, while allowing chemotherapeutic agents to be simultaneously administered to cancer cells being treated. Representative chemoprotective agents include, but are not limited to, amifostine (Ethyol, Medimmune, Inc.), used to reduce nephrotoxicity associated with cumulative doses of cisplatin, dexrazoxane (Totect, Apricus Pharma, Zinecard) for the treatment of extravasation caused by administration of anthracyclines (Totect) and for the treatment of cardiac-related complications caused by administration of the antitumor antibiotic doxorubicin (Zinecard), and mesna (Mesnex, Bristol-Myers Squibb), used to prevent hemorrhagic cystitis during chemotherapy treatment with ifocfamide.

[0141] Hormones The linkers of the present disclosure may be used to conjugate an antibody to at least one hormonal agent. Hormonal agents (including synthetic hormones) are compounds that interfere with the production or activity of endogenously produced hormones of the endocrine system. In some embodiments, these compounds interfere with cell growth or produce cytotoxic effects. Non-limiting examples include androgens, estrogens, medroxyprogesterone acetate (Provera, Pfizer, Inc.), and progestins.

[0142] Antihormonal drugs The antibody may be conjugated to at least one antihormonal agent using the linkers of the present disclosure. An "antihormonal" agent is a drug that suppresses the production and / or prevents the function of certain endogenous hormones. In certain embodiments, the antihormonal agent interferes with the activity of hormones selected from androgen, estrogen, progesterone, and gonadotropin-releasing hormone, thereby interfering with the growth of various cancer cells. Representative examples of antihormonal agents include, but are not limited to, aminoglutethimide, anastrozole (Arimidex, AstraZeneca Pharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals), cyproterone acetate (Ciprostat, Bayer PLC), degarelix (Pharmagon, Ferring Pharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide (Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZeneca Pharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals), letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide (Prostap), lupron, medroxyprogesterone acetate (Provera, Pfizer Inc.), megestrol acetate (Megas, Bristol-Myers Squibb Company), tamoxifen (Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl, Ferring).

[0143] Corticosteroids The antibody may be conjugated to at least one corticosteroid using the linker of the present disclosure. Corticosteroids may be used in the ADCs of the present disclosure to reduce inflammation. Examples of corticosteroids include, but are not limited to, glucocorticoids such as prednisone (Deltasone, Pharmacia & Upjohn Company, a division of Pfizer, Inc.).

[0144] Photoactive Therapeutic Agents The antibody may be conjugated to at least one photoactive therapeutic agent using the linker of the present disclosure. Photoactive therapeutic agents include compounds that can be configured to kill treated cells upon exposure to electromagnetic radiation of a specific wavelength. Therapeutically relevant compounds absorb electromagnetic radiation at wavelengths that penetrate tissue. In preferred embodiments, the compounds are administered in a non-toxic form that can produce photochemical effects that are toxic to cells or tissues upon sufficient activation. In other preferred embodiments, these compounds are retained by cancerous tissue and easily removed from normal tissue. Non-limiting examples include various chromogens and dyes.

[0145] Oligonucleotides The antibody may be conjugated to at least one oligonucleotide using the linker of the present disclosure. Oligonucleotides are made of short nucleic acid strands that function by interfering with the processing of genetic information. In some embodiments, oligonucleotides for use in ADCs are unmodified single- and / or double-stranded DNA or RNA molecules, while in other embodiments, these therapeutic oligonucleotides are chemically modified single- and / or double-stranded DNA or RNA molecules. In certain embodiments, the oligonucleotides used in ADCs are relatively short (19-25 nucleotides) and hybridize to unique nucleic acid sequences in the total pool of nucleic acid targets present in cells. Some important oligonucleotide technologies include antisense oligonucleotides (including RNA interference (RNAi)), aptamers, CpG oligonucleotides, and ribozymes.

[0146] antisense oligonucleotides The antibody may be conjugated to at least one antisense oligonucleotide using a linker of the present disclosure. The antisense oligonucleotide is designed to bind to RNA through Watson-Crick hybridization. In some embodiments, the antisense oligonucleotide is complementary to nucleotides encoding a region, domain, portion, or segment of the conjugated antibody. In some embodiments, the antisense oligonucleotide comprises about 5 to about 100 nucleotides, about 10 to about 50 nucleotides, about 12 to about 35 nucleotides, and about 18 to about 25 nucleotides.

[0147] When an oligonucleotide binds to a target RNA, there are several mechanisms that can be utilized to inhibit the function of the RNA (Crooke ST. (1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterized antisense mechanism results in cleavage of the targeted RNA by endogenous cellular nucleases, such as RNase H or nucleases associated with the RNA interference machinery. However, oligonucleotides that inhibit target gene expression through non-catalytic mechanisms, such as modulation of splicing or translation arrest, can also be potent and selective regulators of gene function.

[0148] Another RNase-dependent antisense mechanism that has recently attracted much attention is RNAi (Fire et al. (1998). Nature, 391, 806-811; Zamore PD. (2002). Science, 296, 1265-1269). RNA interference (RNAi) is a post-transcriptional process in which double-stranded RNA inhibits gene expression in a sequence-specific manner. In some embodiments, the RNAi effect is achieved by introducing relatively long double-stranded RNA (dsRNA), while in preferred embodiments, the RNAi effect is achieved by introducing shorter double-stranded RNA, such as small interfering RNA (siRNA) and / or microRNA (miRNA). In yet another embodiment, RNAi can also be achieved by introducing a plasmid that produces dsRNA complementary to a target gene. In each of the foregoing embodiments, the double-stranded RNA is designed to interfere with gene expression of a specific target sequence in a cell. Generally, this mechanism involves the conversion of dsRNA into short RNA, which directs ribonuclease to homologous mRNA target (reviewed in Ruvkun, Science 2294:797(2001)), and then degrades corresponding endogenous mRNA, thereby resulting in the regulation of gene expression.In particular, dsRNA has been reported to have antiproliferative properties, which may lead to its potential therapeutic use (Aubel et al., Proc.Natl.Acad.Sci.,USA 88:906(1991)). For example, synthetic dsRNA has been shown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA, 62: 357-361 (1969)), and it is active in treating leukemia in mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130: 126-128 (1969)), and inhibits chemically induced tumor formation in mouse skin (Gelboin et al., Science 167: 205-207 (1970)).Therefore, in a preferred embodiment, the present disclosure provides the use of antisense oligonucleotides in ADC for the treatment of breast cancer.In other embodiments, the present disclosure provides compositions and methods for initiating antisense oligonucleotide therapy, wherein the dsRNA interferes with target cell expression of EGFR at the mRNA level. As used above, dsRNA refers to naturally occurring RNA, partially purified RNA, recombinantly produced RNA, synthetic RNA, and modified RNA that differs from naturally occurring RNA by including non-standard nucleotides, non-nucleotide materials, nucleotide analogs (e.g., locked nucleic acids (LNA)), deoxyribonucleotides, and any combination thereof. The RNA of the present disclosure need only be sufficiently similar to natural RNA to have the ability to mediate antisense oligonucleotide-based modulation as described herein.

[0149] Aptamers The linker of the present disclosure may be used to conjugate an antibody to at least one aptamer. Aptamers are nucleic acid molecules selected from a random pool based on their ability to bind to other molecules. Like antibodies, aptamers can bind to target molecules with exceptional affinity and specificity. In many embodiments, aptamers assume a complex, sequence-dependent, three-dimensional shape that allows them to interact with target proteins, resulting in a tightly bound complex similar to antibody-antigen interactions, thereby interfering with the function of the protein. The particular ability of aptamers to bind tightly and specifically to their target proteins highlights their potential as targeted molecular therapeutics.

[0150] CpG oligonucleotides The linker of the present disclosure may be used to conjugate an antibody to at least one CpG oligonucleotide. Bacterial and viral DNA are known to be potent activators of both innate and specific immunity in humans. These immunological characteristics are associated with the unmethylated CpG dinucleotide motifs found in bacterial DNA. Due to the fact that these motifs are rare in humans, the human immune system has evolved the ability to recognize these motifs as early signs of infection and subsequently initiate an immune response. Therefore, oligonucleotides containing this CpG motif can be used to initiate anti-tumor immune responses.

[0151] Ribozymes The linkers of the present disclosure may be used to conjugate an antibody to at least one ribozyme. Ribozymes are catalytic RNA molecules ranging from about 40 to 155 nucleotides in length. The ability of ribozymes to recognize and cleave specific RNA molecules makes them potential therapeutic candidates. Representative examples include angiozymes.

[0152] Radionuclides (radioisotopes) The antibody may be conjugated to at least one radionuclide agent using the linker of the present disclosure.Radionuclide agents include agents characterized by an unstable nucleus that can undergo radioactive decay.The success of radionuclide therapy depends on the sufficient concentration and long-term retention of the radionuclide by cancer cells.Other factors to consider include the half-life of the radionuclide, the energy of the emitted particles, and the maximum range that the emitted particles can move. In a preferred embodiment, the therapeutic agent is a radionuclide selected from the group consisting of In, Lu, Bi, Bi, At, Cu, Cu, Cu, Y, I, I, P, P, Sc, Ag, Ga, Pr, Sm, Tb, Dy, Ho, Re, Re, Re, Pb, Ra, Ac, Fe, Se, As, Sr, Mo, Rh, Pd, Pr, Pm, Er, Ir, Au, Au, and Pb. Radionuclides that substantially decay into Auger-emitting particles are also preferred. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, and Ir-192. Decay energies of useful beta particle-emitting radionuclides are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm-255. Decay energies of useful alpha particle-emitting radionuclides are preferably 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV.Additional potential radioisotopes of use include C, N, , Br, Au, Ru, Ru, Ru, Ru, Hg, Hg, Te, Te, Tm, Tm, Tm, Pt, Pd, Rh, Pr, Pr, Tb, Ho, Au, Co, Co, Cr, Fe, Se, Tl, Ac, Br, Yb, and the like.

[0153] Radiosensitizers The antibody may be conjugated to at least one radiosensitizer using the linker of the present disclosure. The term "radiosensitizer," as used herein, is defined as a molecule, preferably a low-molecular-weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of radiosensitive cells to electromagnetic radiation and / or to promote the treatment of diseases treatable with electromagnetic radiation. A radiosensitizer is an agent that makes cancer cells more sensitive to radiation therapy while typically leaving normal cells much less affected. Thus, a radiosensitizer can be used in combination with a radiolabeled antibody or ADC. The addition of a radiosensitizer can improve efficacy compared to treatment with a radiolabeled antibody or antibody fragment alone. Radiosensitizers are described in D.M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxanes, and cisplatin.

[0154] Radiosensitizers can be activated by the electromagnetic radiation of X-rays. Representative examples of X-ray-activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU1069, SR4233, E09, RB6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives thereof. Alternatively, radiosensitizers can be activated using photodynamic therapy (PDT). Representative examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and therapeutically effective analogs and derivatives thereof.

[0155] Topoisomerase inhibitors The antibody may be conjugated to at least one topoisomerase inhibitor using the linker of the present disclosure. Topoisomerase inhibitors are chemotherapeutic agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control changes in DNA structure by catalyzing, then breaking and reconnecting, the phosphodiester backbone of DNA strands during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecin and its derivatives irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, and teniposide.

[0156] tyrosine kinase inhibitors The linker of the present disclosure may be used to conjugate an antibody to at least one tyrosine kinase inhibitor. Tyrosine kinases are intracellular enzymes that function to attach phosphate groups to the amino acid tyrosine. Blocking the function of protein tyrosine kinases can inhibit tumor growth. Examples of tyrosine kinases that can be used on the ADC of the present disclosure include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxinib, sunitinib, and vandetanib.

[0157] Other drugs Examples of other agents that can be used in the ADCs of the disclosure include, but are not limited to, abrin (e.g., abrin A chain), alpha toxin, Aleurites fordii protein, amatoxin, crotin, curcin, dianthin protein, diphtheria toxin (e.g., diphtheria A chain and non-binding active fragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogellin, modeccin A chain, momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (e.g., exotoxin A chain (from Pseudomonas aeruginosa)), restrictocin, ricin A chain, ribonuclease (Rnase), sapaonaria officinalis inhibitors, saporin, alpha-sarcin, Staphylococcal enterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g., PS-341 [bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck & Company, Inc.)), belinostat, entinostat, mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (e.g., geldanamycin and its many analogs), adrenocortical suppressants, and trichothecenes. (See, e.g., WO 93 / 21232.) Other agents also include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Rizodren, Bristol-Myers Squibb), and tretinoin (Renova, Valeant Pharmaceuticals Inc.).

[0158] It should be noted that the foregoing groups of drug moieties that may be used in the ADCs of the present disclosure are not exclusive, in that particular examples of drugs may be found in more than one category; for example, ansamitocins are both antimitotic and antitumor antibiotics.

[0159] All stereoisomers of the drug moieties described above are contemplated in the compounds of this disclosure, i.e., any combination of R and S configurations at the chiral carbon of D.

[0160] A "detectable moiety" or "marker" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, radioactive, or chemical means. For example, useful labels include: 32 P, 35 Detectable moieties include S, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin-streptavidin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with sequences complementary to the label. Detectable moieties often produce a measurable signal, such as a radioactive, color, or fluorescent signal, which can be used to quantify the amount of detectable moiety bound in a sample. Quantification of the signal can be achieved, for example, by scintillation counting, density gauges, flow cell analysis, ELISA, or direct analysis of the cyclic peptide or subsequently digested peptides by mass spectrometry (one or more peptides can be assayed). Those skilled in the art are familiar with the techniques and detection means for labeling compounds of interest. These techniques and methods are conventional and well known in the art.

[0161] A detector probe refers to (i) a material capable of providing a detectable signal, (ii) a material capable of interacting with a first probe or a second probe to change the detectable signal provided by the first probe or the second probe, e.g., fluorescence resonance energy transfer (FRET), (iii) a material capable of stabilizing interaction with an antigen or a ligand or increasing binding affinity, (iv) a material capable of influencing electromobility or cell invasiveness through physical parameters such as charge or hydrophobicity, or (v) a material capable of adjusting ligand affinity, antigen-antibody binding, or ion complex formation.

[0162] In some embodiments, each active agent is independently: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophos Pholamide (triethiylenethiophosphoramide), trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, Spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, fenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, diazepam Nemycin, dynemycin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antormycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin,Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin migrin, 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 propionate propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophoramide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainin e), maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamt, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, vindesine, dacarbazine, mannomustine,mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin engineered nanoparticle formulations of paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid of any of the foregoing, (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic factor, interferon , interferon-α, interferon-β, interferon-γ, colony stimulating factors ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or any combination of the foregoing; (c) diphtheria toxin, botulinum toxin, tetanus toxin, shiga toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, shiguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, cryptophycin, camptothecin, camptothecin analogs and metabolites, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, azonafide, aplidine, toxoids, or combinations of any of the foregoing; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulant, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) radioactive label; 32 P, 35 S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the above; (f) immunomodulatory compounds, anti-cancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or any combination of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine, (j) aromatase inhibitors, (k) protein kinase inhibitors, (l) lipid kinase inhibitors, (m) antisense oligonucleotides; (n) ribozyme, (o) vaccines, and (p) an anti-angiogenic agent.

[0163] In some preferred embodiments, G is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] wherein: [ka] is the fragment of the linking group Z' that connects Z' to Ar, in this case the substituted Ph group.

[0164] Conjugation Strategies Compounds of formula I can be prepared in a one-step or two-step conjugation procedure.

[0165] One-step conjugation In some embodiments, the present disclosure relates to a method for preparing a compound of formula I, which involves one-step conjugation between an antibody and a linker. The compounds of formula (II) and (III) above are suitable for one-step conjugation with an antibody.

[0166] For example, a precursor containing a methylphenylsulfone moiety (MPS) can undergo conjugation according to the sequence of steps shown in Scheme 1. Step A involves the in situ elimination of the p-methylphenylsulfonyl group to form a reactive intermediate. In Step B, this intermediate undergoes conjugation with a thiol residue of an antibody. [ka]

[0167] The resulting ADC can be further stabilized by treatment with hydroxylamine or a reducing agent, as shown in Scheme 2. [ka]

[0168] In some embodiments, the MPS-containing precursor comprises a moiety that generates an activated Michael acceptor upon elimination of a sulfinic acid. Examples of such precursors are shown in Scheme 3. [ka]

[0169] In some embodiments, the precursor comprises a moiety that acts as an activated Michael acceptor in the conjugation reaction. An example of a conjugation reaction with an activated Michael acceptor is shown in Scheme 4. [ka]

[0170] In some embodiments, precursors for one-step conjugation contain maleimides. An example of conjugation of a thiol-containing antibody with a maleimide-containing precursor is shown in Scheme 5. A precursor containing a maleimidomethylcyclohexane-1-carboxylate (Mal-mcc) linker is shown in Part A, and a precursor with a maleimide moiety directly attached to a PEG spacer is shown in Part B. [ka]

[0171] Two-step conjugation In some embodiments, the present disclosure relates to a two-step conjugation method for preparing a compound of Formula I. The first step involves conjugating an antibody to a linker, where the linker terminates in a reactive group such as an azide or alkyne. In the second step, the antibody-containing precursor undergoes reaction with a precursor containing an active agent to produce the final ADC.

[0172] In some embodiments, the first step of the two-step procedure involves conjugating the antibody with a precursor containing any of the reactive groups disclosed in the "One-Step Conjugation" section above. Exemplary precursors for the first conjugation step are shown in Scheme 6. [ka]

[0173] In some embodiments, the second step of the conjugation process involves reacting the antibody-containing precursor obtained in the first step with an active agent-containing precursor. The active agent-containing precursor contains a reactive group complementary to the reactive group of the precursor obtained in the first step. For example, the antibody-containing precursor is terminated with an azide and the active agent-containing precursor is terminated with an alkyne, or vice versa. An example of an active agent-containing precursor is shown in Scheme 7. [ka] [ka]

[0174] Anti-B7-H3 antibody Exemplary anti-B7-H3 antibodies include those set forth in Tables 19-24 herein, or any fragment, variant, multimeric embodiment, or bispecific variant thereof. Similarly, an anti-B7-H3 antibody can be an antibody that binds to the same epitope as an antibody listed in Tables 19-24, or any fragment, variant, multimeric embodiment, or bispecific variant thereof. Suitable anti-B7-H3 antibodies of the present disclosure include fully human monoclonal antibodies, as well as humanized monoclonal and chimeric antibodies, or any fragment, variant, multimeric embodiment, or bispecific variant thereof. These antibodies exhibit specificity for human B7-H3 and have been shown to modulate, e.g., block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological function or activity of B7-H3.

[0175] An antibody is considered to fully modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one functional activity of B7-H3 if the level of B7-H3 functional activity in the presence of the antibody is reduced by at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, compared to the level of B7-H3 functional activity in the absence of binding with an antibody described herein. An antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one functional activity of B7-H3 if the level of B7-H3 functional activity in the presence of the antibody is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of B7-H3 functional activity in the absence of binding with an antibody described herein.

[0176] Each of the anti-B7-H3 monoclonal antibodies, or any fragment, variant, multimeric embodiment, or bispecific variant thereof described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL) as shown in the amino acid and corresponding nucleic acid sequences listed in Tables 20-24.

[0177] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terminology used in connection with and techniques of chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0178] The methods and techniques of the present disclosure are generally carried out according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, e.g., "Principles of Neural Science," McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics," Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", W.H. Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", W.H. Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0179] Chemical terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified in "The McGraw-Hill Dictionary of Chemical Terms," ​​Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0180] All of the above, and any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including specific definitions herein, will control.

[0181] The term "agent" is used herein to refer to a chemical compound (e.g., an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (e.g., a nucleic acid, an antibody (including a portion thereof), and a humanized, chimeric, and human antibody, and a monoclonal antibody, a protein or a portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as a bacterial, plant, fungal, or animal (e.g., mammalian) cell or tissue. Agents include, for example, agents with known structures and agents with unknown structures. The ability of such agents to inhibit AR or promote AR degradation may make them suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0182] The terms "patient," "subject," or "individual" are used interchangeably and refer to either a human or non-human animal. These terms include mammals such as humans, primates, livestock animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0183] "Treating" a condition or patient refers to taking measures to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stable (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, palliation or palliative suppression of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0184] The term "preventing," when 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 art-recognized and well understood in the art and includes administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects not receiving the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancer growths in a population of patients receiving prophylactic treatment compared to a population of untreated subjects, and / or delaying the appearance of detectable cancer growths in a treated population relative to a population of untreated subjects, e.g., by a statistically and / or clinically significant amount.

[0185] "Administering" or "administering" a substance, compound, or agent to a subject can be accomplished 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, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through skin channels). A compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, e.g., patches and pumps, or formulations that provide extended, delayed, or controlled release of the compound or agent. Administering can be performed, for example, once, multiple times, and / or over one or more extended periods.

[0186] The appropriate method of administering a substance, compound, or agent to a subject will also depend, for example, on the age and / or physical condition of the subject, as well as the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is orally administered to the subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is a sustained-release or timed-release formulation or is administered using a device for such sustained-release or timed-release.

[0187] As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents in which a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., two agents are effective in a patient simultaneously, which may involve a synergistic effect of the two agents). For example, different therapeutic compounds may be administered either simultaneously or sequentially, in either the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effects of the different therapeutic agents.

[0188] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is the amount of the drug or agent that has the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administration of a single dose, but may 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 required for a subject will depend, for example, on the subject's size, health, and age, as well as the nature and extent of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation.

[0189] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples when the event or circumstance occurs and examples when it does not occur. For example, "optionally substituted alkyl" refers to alkyls that may be substituted and also to alkyls that are not substituted.

[0190] It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by one skilled in the art to result in chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and the methods described below. When a substituent is itself substituted with more than one group, it is understood that these multiple groups can be on the same carbon or on different carbons as long as a stable structure results.

[0191] As used herein, the term "optionally substituted" refers to the replacement of 1 to 6 hydrogen atoms in a given structure with a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH-O-alkyl, -OP(O)(O-alkyl), or -CH-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of 1 to 4 hydrogen atoms in a given structure with a substituent as described above. More preferably, 1 to 3 hydrogen substituents are replaced by a substituent, as described above. It is understood that a substituent may be further substituted.

[0192] As used herein, the term "alkyl" includes, but is not limited to, C1 to C 10 Straight chain alkyl group or C1-C 10 It refers to saturated aliphatic groups, including branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. The "alkyl" group may be optionally substituted.

[0193] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0194] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0195] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0196] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0197] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0198] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C for straight chain). 1~30 , C for branched chain 3~30 ), more preferably having 20 or fewer carbon atoms.

[0199] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0200] "C x~y " or "C x ~C yThe term " when used in combination with a chemical moiety, e.g., acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. CO alkyl indicates that the group is a hydrogen in a terminal position, while if internal, it is a bond. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0201] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0202] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0203] The term "amide" as used herein refers to the group [ka] In the formula, R 9 and R 10 each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0204] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, e.g., [ka] or refers to a part that can be represented by R 9 , R 10 and R 10 Each ' independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0205] The term "aminoalkyl," as used herein, refers to an alkyl group substituted with an amino group.

[0206] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0207] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is 5- to 7-membered, more preferably 6-membered. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of the rings is aromatic, and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0208] The term "carbamate" is art-recognized and refers to a group [ka] In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0209] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0210] The term "carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring in a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring in a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Valence permitting, any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any one or more positions that can have a hydrogen atom.

[0211] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0212] The term "carbonate" is art-recognized and refers to an -OCO2- group.

[0213] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0214] The term "ester" as used herein refers to an ester of -C(O)OR 9 R refers to the group 9 represents a hydrocarbyl group.

[0215] The term "ether," as used herein, refers to a hydrocarbyl group linked to another hydrocarbyl group through an oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0216] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo, and iodo.

[0217] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0218] The terms "heteroaryl" and "hetaaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, where the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaaryl" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, at least one of the rings is heteroaromatic, and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0219] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0220] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0221] The terms "heterocyclyl," "heterocycle," and "heterocyclic ring system" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, containing at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocycle" also include polycyclic ring systems having two or more cyclic rings, in which two or more carbons are common to two adjacent rings and at least one of the rings is a heterocycle, and the other cyclic rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0222] The term "hydrocarbyl," as used herein, refers to a group bonded through a carbon atom that does not have an =O or =S substituent, typically having at least one carbon-hydrogen bond and a primarily carbon backbone, but optionally containing heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon) are not considered hydrocarbyl. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0223] The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with a hydroxy group.

[0224] The term "lower," when used in combination with a chemical moiety, e.g., acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer atoms, preferably 6 or fewer atoms, in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, an acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituent defined herein is a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether appearing alone or in combination with other substituents, such as descriptions such as hydroxyalkyl and aralkyl (in which, e.g., the elements in the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0225] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings (e.g., the rings are "fused rings"). Each ring of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10, preferably 5 to 7, atoms in the ring.

[0226] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0227] The term "sulfonamide" is art-recognized and refers to a compound having the general formula [ka] wherein R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0228] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0229] The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0230] The term "sulfone" is art-recognized and refers to a -S(O)2- group.

[0231] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more backbone carbons. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution, subject to the permissible valencies of the substituted atom and substituent, results in a stable compound that does not undergo spontaneous transformation, e.g., by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad respect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatoms. Substituents can include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0232] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0233] The term "thioester" as used herein refers to a group selected from the group consisting of -C(O)SR 9 or -SC(O)R 9 Pointing to the base, In the formula, R 9 represents a hydrocarbyl.

[0234] The term "thioether," as used herein, is the equivalent of an ether, where the oxygen is replaced with a sulfur.

[0235] The term "urea" is art-recognized and has the general formula [ka] wherein R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0236] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0237] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable or compatible with the treatment of a patient.

[0238] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to any non-toxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include monocarboxylic, dicarboxylic, and tricarboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, and sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either monobasic or dibasic acid salts can be formed, and such salts may exist in either hydrated, solvated, or substantially anhydrous form. In general, acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than their free base forms. The selection of an appropriate salt will be known to one skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0239] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any of its intermediates. Exemplary inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxides. Exemplary organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, picoline, or ammonia. The selection of appropriate salts will be known to those skilled in the art.

[0240] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may exist in the R or S configuration, and the R and S designations are used according to the rules set forth in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, including enantiomeric and diastereoisomeric forms, of the compounds, salts, prodrugs, or mixtures thereof, including all possible mixtures of stereoisomers. See, for example, WO01 / 062726.

[0241] In certain embodiments, the compounds of the present disclosure may be racemic. In certain embodiments, the compounds of the present disclosure may be enriched in one enantiomer. For example, the compounds of the present disclosure may have an ee of about 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, 95%, 96% ee, 97% ee, 98% ee, 99% ee or more.

[0242] As is commonly understood in the art, a single bond drawn without stereochemistry does not indicate the stereochemistry of the compound. The compound of Formula I provides an example of a compound where the stereochemistry is not indicated.

[0243] In certain embodiments, compositions or compounds of the present disclosure can be enriched to provide predominantly one enantiomer of a compound. An enantiomerically enriched composition or compound can contain, for example, at least 60 mole percent of one enantiomer, or more preferably at least 75, 90, 95, or even 99 mole percent of one enantiomer. In certain embodiments, a compound enriched in one enantiomer is substantially free of the other enantiomer, where substantially free means that the substance in question constitutes less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the other enantiomer (e.g., in a composition or mixture of compounds). For example, if a composition or compound contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it would be said to contain 98 mole percent of the first enantiomer and only 2 mole percent of the second enantiomer.

[0244] Furthermore, certain compounds containing alkenyl groups can exist as Z (Zusammen) or E (Entgegen) isomers. In each case, the present disclosure includes both mixtures and the separate individual isomers.

[0245] Some of the compounds may also exist in tautomeric forms, and such forms, although not explicitly indicated in the formulae set forth herein, are intended to be included within the scope of the present disclosure.

[0246] A "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in the host after administration to form a compound of the present disclosure (e.g., a compound of Formula I). ​​Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to generate the active compound. Examples of prodrugs that use esters or phosphoramidates as the biologically labile or cleavable (protecting) group are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to generate a compound of Formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0247] As used herein, the terms "Log of Solubility," "LogS," or "logS" are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution properties. Low solubility is often associated with poor absorption. The LogS value is the unitless logarithm (base 10) of solubility measured in moles / liter.

[0248] General Methods for Antibody Preparation Various procedures known in the art can be used to produce polyclonal or monoclonal antibodies directed against a given target, such as B7-H3, a tumor-associated antigen, or other target, or against derivatives, fragments, analogs, homologs, or orthologs thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference.)

[0249] Antibodies can be prepared by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fragment of immune serum. Subsequently, or alternatively, the specific antigen, or epitope thereof, that is the target of the desired immunoglobulin may be immobilized on a column, and the immune-specific antibody purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0250] In some embodiments, the antibody of the present disclosure is a monoclonal antibody. Monoclonal antibodies are produced, for example, by using the procedures described in the examples provided herein. Antibodies can also be produced, for example, by immunizing BALB / c mice with a combination of cell transfectants that express high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusion are then screened for reactivity against the selected target.

[0251] Monoclonal antibodies are prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0252] The immunizing agent will typically include the protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0253] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).)

[0254] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity for the target antigen.

[0255] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0256] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0257] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of a murine antibody). The hybridoma cells of the present disclosure serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of the antibodies of the present disclosure, or for the variable domains of one antigen-combining site of the antibodies of the present disclosure to create a chimeric bivalent antibody.

[0258] Monoclonal antibodies of the present disclosure include humanized or human antibodies. These antibodies are suitable for administration to humans without generating an immune response to the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulins. Humanization can be performed, for example, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). (See also U.S. Pat. No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, for example, residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. 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 CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0259] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including the CDRs, arises from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), or EBV hybridoma technology to produce monoclonal antibodies (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies may be utilized and may be produced by using human hybridomas (Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0260] In addition, human antibodies can also be produced using additional technologies, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).) Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368, 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0261] Furthermore, human antibodies can be produced using transgenic non-human animals engineered to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies. (See PCT Publication No. WO 94 / 02602.) Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals providing all desired modifications are then obtained as progeny by breeding intermediate transgenic animals containing less than the full complement of modifications. One example of such a non-human animal is the Xenomouse™ mouse, as disclosed in PCT Publication Nos. WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with an immunogen of interest, e.g., as polyclonal antibody preparations, or alternatively, from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, e.g., single-chain Fv (scFv) molecules.

[0262] One example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being carried out by a targeting vector containing a gene encoding a selectable marker, and producing from the embryonic stem cells a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0263] One method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0264] In a further improvement to this procedure, methods for identifying clinically relevant epitopes on immunogens and correlating methods for selecting antibodies that specifically bind with high affinity to the relevant epitopes are disclosed in U.S. Publication No. 2003 / 009212.

[0265] The antibodies can be expressed by vectors containing DNA segments encoding the single chain antibodies described above.

[0266] These may include vectors, liposomes, naked DNA, adjuvanted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701 having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins described in U.S. Pat. No. 7,186,697, which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0267] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpes virus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci. USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993), Yang, et al., J. Virol. 69:2004 (1995), and adeno-associated viral vectors (Kaplitt, M. Get al., Nat. Genet. 8:148 (1994)).

[0268] Poxvirus vectors deliver genes into the cytoplasm of cells. Avipoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus, adeno-associated virus, and herpes simplex virus (HSV) vectors are preferred for delivering nucleic acids to neural cells. Adenovirus vectors deliver shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), and shorter than HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be achieved by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene delivery methods include naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0269] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. Furthermore, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system, such as the SynchroMed Infusion System. A bulk flow-based method, called convection, has also proven effective in delivering large molecules to large areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other suitable administration routes.

[0270] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens, where one of the binding specificities is for a target such as B7-H3 or any fragment thereof, and the second binding target is any other antigen, advantageously a cell surface protein or receptor or receptor subunit.

[0271] Many methods for generating bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy / light chain pairs, with the two heavy chains having different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0272] The bispecific and / or monovalent antibodies of the present disclosure can be produced using any of a variety of art-recognized techniques, including those disclosed in WO2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces bispecific antibodies structurally identical to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each combining site exhibits a different antigen specificity, with contributions from both the heavy and light chains. The light chain variable regions may be from the lambda or kappa family and are preferably fused to lambda and kappa constant domains, respectively. This is preferred to avoid the creation of non-natural polypeptide junctions. However, it is also possible to obtain the bispecific antibodies of the present disclosure by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgGκλ antibodies or "κλ bodies" and are a new fully human bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies that are indistinguishable from standard monoclonal antibodies and therefore have favorable characteristics compared to previous formats, indistinguishable from standard IgG molecules.

[0273] An essential step in this method is the identification of two antibody Fv regions (each composed of a variable light chain domain and a variable heavy chain domain) with different antigen specificities that share the same heavy chain variable domain. Numerous methods for the production of monoclonal antibodies and their fragments have been described. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference.) A fully human antibody is an antibody molecule in which both the light and heavy chain sequences, including CDR1 and CDR2, originate from human genes. The CDR3 region can be of human origin or engineered by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared by using trioma technology, human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), EBV hybridoma technology to produce human monoclonal antibodies (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0274] Monoclonal antibodies are produced, for example, by immunizing animals with the target antigen or its immunogenic fragment, derivative, or variant. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. Various suitable techniques for producing xenogenic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entireties.

[0275] Alternatively, antibodies can be obtained by screening libraries containing antibody or antigen-binding domain sequences for binding to the target antigen, prepared in bacteriophage, for example, as protein or peptide fusions to bacteriophage coat proteins that are expressed on the surface of assembled phage particles, with the encoding DNA sequences contained within the phage particle (i.e., "phage display libraries").

[0276] Hybridomas resulting from the myeloma / B cell fusion are then screened for reactivity against the target antigen. Monoclonal antibodies are prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0277] Although not strictly impossible, the accidental identification of different antibodies that have the same heavy chain variable domain but are directed against different antigens is highly unlikely.In fact, in most cases, the heavy chain contributes the most to the antigen-binding surface and is also the most variable in sequence.In particular, CDR3 on the heavy chain is the most diverse CDR in terms of sequence, length, and structure.Therefore, two antibodies specific for different antigens almost always have different heavy chain variable domains.

[0278] The method disclosed in the U.S. Patent No. 9,926,382 application overcomes this limitation and greatly facilitates the isolation of antibodies with the same heavy chain variable domain by using an antibody library in which the heavy chain variable domain is the same for all library members, and therefore diversity is limited to the light chain variable domain. Such libraries are described, for example, in U.S. Patent No. 8,921,281 and Application No. WO 2011 / 084255, each of which is incorporated herein by reference in its entirety. However, because the light chain variable domain is expressed in conjunction with the heavy chain variable domain, both domains can contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and diversity of either lambda or kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach allows the identification of two antibodies that share a common heavy chain, but one possesses a lambda light chain variable domain and the other a kappa light chain variable domain, which can be used as building blocks for the generation of bispecific antibodies in the full immunoglobulin format of the present disclosure. Bispecific antibodies of the present disclosure may be of different isotypes, and their Fc portions may be modified to alter their binding characteristics to different Fc receptors, thus altering the effector functions and pharmacokinetic properties of the antibody. Numerous methods for modifying Fc portions have been described and are applicable to the antibodies of the present disclosure. (See, e.g., Strohl, WR Curr Opin Biotechnol 2009(6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199, filed January 9, 2009.) The methods of the present disclosure can also be used to produce bispecific antibodies and antibody mixtures in the F(ab')2 format, which lack the Fc portion.

[0279] Coexpression of a common heavy chain and two different light chains within a single cell allows for the assembly of the bispecific antibody of the present disclosure. If all polypeptides are expressed at the same level and assemble equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, different light chains may be expressed at different levels and / or may not assemble with the same efficiency. Therefore, means for adjusting the relative expression of different polypeptides are used to compensate for their inherent expression characteristics or different propensities to assemble with a common heavy chain. This adjustment can be achieved through promoter strength, the use of internal ribosome entry sites (IRES) characterized by different efficiencies, or other types of regulatory elements that can act at the transcriptional or translational level and affect mRNA stability. Different promoters with different strengths include CMV (immediate-early cytomegalovirus promoter), EF1-1α (human elongation factor 1 α subunit promoter), Ubc (human ubiquitin C promoter), and SV40 (simian virus 40 promoter). Different IRESs have also been described from mammalian and viral sources (see, for example, Hellen CU and Sarnow P. Genes Dev 2001 15:1593-612). These IRESs can vary greatly in their length and ribosome recruitment efficiency. Furthermore, activity can be further tuned by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97:1536-1541). Regulation of expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus alter their relative expression. The examples provided herein demonstrate that controlling the relative expression of different chains is important for maximizing the assembly and overall yield of bispecific antibodies.

[0280] Coexpression of a heavy chain and two light chains generates a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by using affinity chromatography media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to the antibodies of the present disclosure. This contrasts sharply with specialized purification methods that must be developed and optimized for each bispecific antibody derived from a quadroma or other cell line expressing an antibody mixture. Indeed, if the biochemical characteristics of different antibodies in a mixture are similar, their separation using standard chromatographic techniques, such as ion exchange chromatography, can be difficult or even impossible.

[0281] Other suitable purification methods include those disclosed in US2013 / 0317200, the contents of which are incorporated herein by reference in their entirety.

[0282] In another embodiment for producing bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Fusions preferably are with immunoglobulin heavy-chain constant domains, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0283] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of the antibody constant domains. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A "cavity" of identical, or commensurate size, to the large side chain(s) is created at the interface of a second antibody molecule by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0284] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The resulting bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0285] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise a light chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain.L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H and V L The domain is a complementary V L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0286] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0287] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates from the protein antigen of the present disclosure. Alternatively, the anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or an Fc receptor for IgG (FcγR) (e.g., FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)), to focus cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds a protein antigen described herein and further binds tissue factor (TF).

[0288] Heteroconjugate antibodies are also within the scope of the present disclosure. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (see WO 91 / 00360, WO 92 / 200373, EP 03089). It is contemplated that antibodies can be prepared in vitro using synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0289] It may be desirable to modify the antibodies of the present disclosure with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer and / or other diseases and disorders associated with aberrant B7-H3 expression and / or activity. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, one can engineer an antibody which has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0290] Conjugated Antibodies The present disclosure also relates to conjugated antibodies, including antibodies or antigen-binding fragments thereof conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) (also referred to herein as an immunoconjugate), or a radioactive isotope (i.e., a radioconjugate).

[0291] In some embodiments, the toxin is a microtubule inhibitor or a derivative thereof. In some embodiments, the toxin is a dolastatin or a derivative thereof. In some embodiments, the toxin is auristatin E, auristatin F, AFP, MMAF, MMAE, MMAD, DMAF, or DMAE. In some embodiments, the toxin is a maytansinoid or a maytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid damaging toxin. In some embodiments, the toxin is a duocarmycin or a derivative thereof. In some embodiments, the toxin is a calicheamicin or a derivative thereof. In some embodiments, the drug is a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the drug is exatecan or a derivative thereof.

[0292] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the purification of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131In, 90 Y, and 186 Re is an example.

[0293] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. (See WO94 / 11026).

[0294] Those skilled in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies of this disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989), incorporated herein by reference in its entirety).

[0295] Coupling may be achieved by any chemical reaction that will link the two molecules, so long as the antibody and other moiety retain their respective activities. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. However, covalent bonding is preferred. Covalent bonding can be achieved either by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but rather is illustrative of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0296] Suitable linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies by oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 24510) conjugated to EDC.

[0297] The above-mentioned linkers contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically bulky disulfide bond, which can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0298] The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibodies can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0299] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide-interchange reaction.

[0300] Use of anti-B7-H3 antibodies It is understood that administration of therapeutic entities according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved migration, delivery, tolerability, etc. Many suitable formulations can be found in the following formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles (e.g., lipids (cationic or anionic) containing Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable in treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration.See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance," Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals," Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts," J Pharm Sci. 89(8):967-78 (2000); Powell et al. "Compendium of excipients for parenteral formulations," PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein for additional information related to formulations, excipients, and carriers well known to pharmaceutical chemists.

[0301] Therapeutic formulations of the present disclosure, including conjugates of the present disclosure, are used to treat or alleviate symptoms associated with cancer, including, but not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral cavity and pharyngeal cancer, endometrial cancer, and / or melanoma. The present disclosure also provides methods of treating or alleviating symptoms associated with cancer. Treatment regimens can include, for example, identifying a subject, e.g., a human patient, suffering from (or at risk of developing) cancer, using standard methods.

[0302] Therapeutic formulations of the present disclosure comprising a conjugate of the present disclosure that recognizes B7-H3 and optionally a second target can be used to treat or alleviate symptoms associated with autoimmune and / or inflammatory diseases, such as B-cell mediated autoimmune and / or inflammatory diseases, including, by way of non-limiting example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and / or lupus nephritis.

[0303] Efficacy of treatment can be determined in association with any suitable method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the conjugate confers clinical benefit.

[0304] Conjugates directed against targets such as B7-H3, tumor-associated antigens, or other antigens may be used in methods related to the localization and / or quantification of these targets, for example, for use in measuring the levels of these targets in appropriate physiological samples, for use in diagnostic methods, for use in protein imaging, etc. Conjugates specific for these targets, or any of their derivatives, fragments, analogs, or homologs, containing, for example, antibody-derived antigen-binding domains, can be utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0305] The conjugates of the present disclosure can be used to isolate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. The conjugates of the present disclosure can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H is one example.

[0306] The conjugates of the present disclosure can be used as therapeutic agents. Such agents will generally be used to treat or prevent diseases or conditions associated with the abnormal expression or activation of a given target in a subject. A conjugate preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally produce an effect resulting from binding to its target. Administration of the conjugate can neutralize, inhibit, or interfere with the signaling function of the target. Administration of the conjugate can neutralize, inhibit, or interfere with the binding of the target to its endogenous ligand to which it naturally binds.

[0307] A therapeutically effective amount of a conjugate of the present disclosure generally relates to the amount necessary to achieve a therapeutic goal. As discussed above, this may be due to binding interactions between the antibody and its target antigen that, in certain cases, interfere with target function and / or the effectiveness of an active agent conjugated to the antibody. The amount that needs to be administered will further depend on the binding affinity of the antibody for its specific antigen and / or the potency of the active agent, as well as the rate at which the administered antibody is depleted from the free volume of the subject to which it is administered. A typical range for therapeutically effective dosing of a conjugate of the present disclosure may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice daily to once weekly.

[0308] The conjugates of the present disclosure can be administered in the form of pharmaceutical compositions for the treatment of various diseases and disorders. Principles and considerations involved in the preparation of such compositions, as well as guidance regarding the selection of ingredients, are provided, for example, in Remington: The Science and Practice of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995, Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide and Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0309] The formulation may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may include an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0310] The active ingredient can also be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in microemulsions, in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively.

[0311] Formulations to be used for in vivo administration are preferably sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0312] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid enable release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods.

[0313] Conjugates according to the present disclosure can be used as agents for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the conjugate contains a detectable label. The antibody can be polyclonal, or more preferably, monoclonal. Intact antibodies, or fragments thereof (e.g., F ab , scFv, or F (ab)2The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, the use of the term "biological sample" includes blood and blood fractions or components, including serum, plasma, or lymph. That is, the detection methods of the present disclosure can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995, "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Further, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte conjugate. For example, an antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0314] Pharmaceutical Compositions The antibody-drug conjugates may be used to transfer the active agent to target cells in a subject to treat the subject using any suitable method of preparing a composition. In some aspects, the present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising the antibody-drug conjugates described herein.

[0315] The compositions and methods of the present disclosure may be used to treat individuals 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 as a pharmaceutical composition, e.g., comprising a compound of the present disclosure 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 intended for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion across epithelial barriers), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosage unit form, such as a powder, solution, lyophilized product for reconstitution, injection, or the like.

[0316] Pharmaceutically acceptable carriers can contain physiologically acceptable agents that act, for example, to stabilize compounds, increase the solubility, or enhance absorption of compounds, such as the compounds of the present disclosure. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be a liposome or other polymer matrix, which can incorporate, for example, the compounds of the present disclosure. For example, liposomes, containing phospholipids or other lipids, are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0317] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, 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.

[0318] Pharmaceutical compositions (preparations) can be administered to a subject by any of several routes. For example, the compound can simply be dissolved or suspended in sterile water. Details of suitable administration routes and compositions thereof can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0319] The formulations may be conveniently presented in unit dosage form and may be prepared by any suitable method in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0320] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0321] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (including intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, intracapsular, intrathecal, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds together with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0322] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) 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 dispersions, and by the use of surfactants.

[0323] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0324] In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility.In this case, the absorption rate of the drug depends on the dissolution rate, which in turn may depend on the crystal size and crystalline form.Alternatively, the delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0325] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. 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.

[0326] For use in the methods of the present disclosure, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0327] The introduction method may also be provided by a rechargeable or biodegradable device. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained-release polymeric devices have been developed and tested in vivo in recent years. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0328] Actual dosage levels of the active ingredient in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0329] The selected dosage level will depend upon a variety of factors, including factors well known in the medical arts, such as the activity of the particular compound or combination of compounds, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, etc.

[0330] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start by administering a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of a compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and other types of therapeutic agents administered together with the compound of the present disclosure, if necessary. Multiple administrations of the drug can deliver a larger total dose. Many methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0331] Generally, a suitable daily dose of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0332] The patient receiving this treatment may be any animal in need, including primates, particularly humans, other mammals such as horses, cows, pigs, sheep, cats and dogs, poultry and pets in general.

[0333] In certain embodiments, the compounds of the present disclosure can be administered alone or in combination with other types of therapeutic agents.

[0334] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0335] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0336] The composition can be prepared in an injectable form, either as a liquid solution or suspension. Solid forms suitable for injection can also be prepared, for example, as emulsions or using antibody-drug conjugates encapsulated in liposomes. The antibody-drug conjugates can be combined with a pharmaceutically acceptable carrier, including any carrier that does not induce the production of antibodies harmful to the recipient. Suitable carriers typically include large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0337] The composition may also contain diluents, such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present therein. The composition may be administered parenterally by injection, which may be either subcutaneous or intramuscular. In some embodiments, the composition may be administered into a tumor. The composition may be inserted (e.g., injected) into a tumor. Additional formulations are suitable for other forms of administration, such as suppository administration or oral administration. Oral compositions may be administered as solutions, suspensions, tablets, pills, capsules, or sustained-release formulations.

[0338] The composition may be administered in a manner compatible with the dosage and formulation. The composition preferably contains a therapeutically effective amount of the antibody-drug conjugate. The dosage may vary depending on the subject being treated, the subject's health and physical condition, the degree of protection desired, and other relevant factors. The precise amount of the active ingredient (e.g., antibody-drug conjugate) may depend on the judgment of the physician. For example, a therapeutically effective amount of the antibody-drug conjugate, or a composition containing it, may be administered to a patient suffering from cancer or a tumor to treat the cancer or tumor.

[0339] An antibody-drug conjugate according to the present disclosure, or a composition containing the same, may be administered in the form of its pharmaceutically acceptable salt. In some embodiments, an antibody-drug conjugate according to the present disclosure, or a composition containing the same, may be administered together with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable additive. The effective amount and type of pharmaceutically acceptable salt, excipient, and additive may be determined using standard methods (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th Edition, 1990).

[0340] In some embodiments, the present disclosure relates to a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising an antibody-drug conjugate described herein. In preferred embodiments, the subject is a mammal. For example, the subject may be selected from rodents, lagomorphs, cats, dogs, pigs, sheep, cattle, horses, and primates. In certain preferred embodiments, the subject is a human.

[0341] The conjugates (also referred to herein as "active compounds") of the present disclosure, as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the conjugate and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0342] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, and subcutaneous administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting osmolality such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0343] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol or sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0344] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the above-listed ingredients as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the above-listed ones.For the preparation of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, and obtains the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0345] In certain embodiments, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antigens against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to suitable methods, for example, as described in U.S. Pat. No. 4,522,811.

[0346] It is particularly advantageous to formulate oral or parenteral compositions into dosage unit form for ease of administration and dosage uniformity.Dosage unit form as used herein refers to a physically separate unit suitable as a unit dosage for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the technical field of preparing such active compound for individual treatment.

[0347] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0348] In some aspects, the present disclosure provides pharmaceutical compositions comprising an antibody drug conjugate described herein, and optionally further comprising a therapeutically effective amount of a chemotherapeutic agent.

[0349] In some aspects, the present disclosure provides a method of treating cancer comprising administering an antibody-drug conjugate of the present disclosure, or a pharmaceutical composition thereof. In some such embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, or melanoma.

[0350] In some aspects, the present disclosure provides a method of treating an autoimmune disease or an inflammatory disease, the method comprising administering an antibody drug conjugate of the present disclosure, or a pharmaceutical composition thereof. In some embodiments, the autoimmune disease or inflammatory disease is selected from a B cell-mediated autoimmune disease or inflammatory disease, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis.

[0351] The following examples will explain the configuration of the present disclosure in detail, but the following examples are merely intended to aid in understanding the present disclosure. The scope of the present disclosure is not limited thereto. Furthermore, unless otherwise specifically stated, the reagents, solvents, and starting materials described herein are readily available from commercial suppliers.

[0352] Example The following table shows the abbreviations used throughout the following examples. [Table 2] TIFF0007798811000074.tif238161TIFF0007798811000075.tif18159 [Example]

[0353] Example 1. Synthesis of MPS derivatives Example 1.1. Preparation of MPS-D1 [ka] Preparation of compound MPS-D1a To a solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 mL), piperidine hydrochloride (6.66 g, 54.82 mmol), paraformaldehyde (4.95 g, 164.5 mmol), and concentrated HCl (0.6 mL) were added at room temperature under a N atmosphere. The mixture was stirred at 100 °C for 16 hours, cooled to room temperature, and acetone (90 mL) was added dropwise. The mixture was stirred at 0 °C for 1 hour. The solid was filtered and washed with diethyl ether (30 mL × 2) to obtain compound MPS-D1a (6.11 g, 38%).

[0354] 1 H NMR(400Hz,DMSO-d6)δ 8.08(s,4H),5.73(s,1H),3.65(t,J=7.2Hz,2H),3.35(t,J=7.2Hz,2H),3.31(m,6H),1.74(s,4H).

[0355] Preparation of compound MPS-D1b To a solution of MPS-D1a (6.11 g, 20.52 mmol) in EtOH (40 mL) and MeOH (26 mL) was added 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 mL, 3.08 mmol) at room temperature. The mixture was stirred at 100 °C for 16 h, cooled to 0 °C, and stirred for an additional 1 h. The solid was filtered and washed with ether (30 mL × 2) to give compound MPS-D1b (5.56 g, 90%).

[0356] 1 H NMR(400Hz, CDCl3)δ 8.04-7.99(m,4H),7.27(d,J=8.4Hz,2H),7.15(d,J=7.6Hz,2H),3.39-3.36(m,2H),3.25-3.21(m,2H),2.27(s,3H).

[0357] Preparation of compound MPS-D1 To a solution of MPS-D1b (5.56 g, 18.51 mmol) in MeOH (90 mL) and distilled water (90 mL) was added oxone (25.03 g, 40.72 mmol) under a N atmosphere at 0 °C. After stirring at room temperature for 14 h, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL × 3). The organic layer was washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound MPS-D1 (5.29 g, 86%).

[0358] 1 H NMR(400Hz,CDCl3)δ 8.04-7.99(m,4H),7.81(d,J=8.4Hz,2H),7.46(d,J=8.4Hz,2H),3.63(t,J=7.2Hz,2H),3.41(t,J=7.2Hz,2H),2.44(s,3H). ESI-MS m / z:333(M + ).

[0359] Example 1.2. Preparation of BCN-PNP [ka] (1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (800 mg, 5.3 mmol) was dissolved in DCM (125 mL) at room temperature under a N2 atmosphere. Pyridine (1.22 mL, 15.9 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.74 mmol) were added to it. After the mixture was stirred at the same temperature for 4 hours, the reaction was quenched by adding saturated NH4Cl solution (100 mL) and extracted with EA (100 mL × 4). The organic layer was dried over Na2SO4, filtered, and concentrated under high vacuum. The residue was purified by column chromatography (Hex:EA = 10:1) to give compound BCN-PNP (1.34 g, 84%) as a white solid.

[0360] 1H NMR(600MHz,CDCl3)δ 8.29(d,J=9Hz,2H),7.39(d,J=9Hz,2H),4.41(d,J=8.4Hz,2H),2.36-2.24(m,6H),1.62-1.55(m,2H),1.53-1.49(m,1H),1.07(t,J=10.2Hz,2H).

[0361] Example 1.3. Preparation of MPS-D1-1 [ka] To a solution of compound MPS-D1 (500 mg, 1.50 mmol) in DMF (8 mL) was added propargylamine (106 μL, 1.65 mmol) at room temperature under a N atmosphere. The reaction mixture was cooled to 0 °C, and PyBop (1.17 g, 2.26 mmol) and DIPEA (524 μL, 3.01 mmol) were added. The mixture was stirred at room temperature for 2 hours and diluted with EA (30 mL × 2) and distilled water (20 mL). The organic layer was extracted, washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D1-1 (510 mg, 92%).

[0362] 1 H NMR(400Hz,CDCl3)δ 9.11(t,J=5.2Hz,1H),7.98-7.89(m,4H),7.79(d,J=8.0Hz,2H),7.43(d,J=8.4Hz,2H),4 .05-4.03(m,2H),3.60(t,J=7.6Hz,2H),3.39(t,J=7.2Hz,2H),3.12(s,1H),2.38(s,3H).

[0363] Example 1.4. Preparation of L-2 and L-2a [ka] Compound L-2 was synthesized by a synthetic route similar to that described in Journal of Polymer Science, Part A: Polymer Chemistry, 2012, 50(19), 3986-3995, which is incorporated herein by reference.

[0364] Preparation of Compound L-2-1 30% yield

[0365] 1 H NMR(400Hz, CDCl3)δ 7.80(d,J=8.4Hz,2H),7.34(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.74-3.58(m,14H),2.45(s,3H).

[0366] Preparation of Compound L-2-2 Yield 68%

[0367] 1 H NMR(400Hz, CDCl3)δ 3.74-3.61(m,14H),3.40(t,J=4.8Hz,2H),2.45(t,J=6.0Hz,2H).

[0368] Preparation of Compound L-2-3 Yield 63%

[0369] 1 H NMR(400Hz, CDCl3)δ 4.21(d,J=2.4Hz,2H),3.72-3.67(m,14H),3.39(t,J=5.2Hz,2H),2.43(t,J=2.4Hz,1H).

[0370] Preparation of compound L-2 Yield 76%

[0371] 1 H NMR(400Hz, CDCl3)δ 4.20(d,J=2.4Hz,2H),3.71-3.61(m,12H),3.51(t,J=4.8Hz,2H),2.87(t,J=5.6Hz,2H),2.43(t,J=2.4Hz,1H).

[0372] Preparation of compound L-2a At 0 °C under a N atmosphere, a solution of compound L-2-2 (3.0 g, 13.7 mmol) in acetone (100 mL) was treated with Jones reagent (20 mL) and stirred for 4 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was extracted with DCM (50 mL × 2) and distilled water (15 mL). The organic layer was washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-2a (2.8 g, 88%).

[0373] 1H NMR (400Hz, CDCl3) δ 4.22-4.14 (m, 2H), 3.80-3.64 (m, 10H), 3.42 (t, J = 4.4Hz, 2H).

[0374] Example 1.5. Preparation of L-3 [ka] Preparation of Compound L-3-1 To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was added KI (294 mg, 1.77 mmol) and AgO (4.92 g, 19.48 mmol) under a N atmosphere. The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was filtered through Celite® and washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-3-1 (5.98 g, 73%).

[0375] 1H NMR(400Hz, CDCl3)δ 7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.71-3.58(m,22H),2.88(br,1H),2.45(s,3H).

[0376] Preparation of Compound L-3-2 To a solution of compound L-3-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was added NaN (1.34 g, 20.55 mmol) under a N atmosphere. The mixture was stirred at 110 °C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-3-2 (4.1 g, 97%).

[0377] 1 H NMR (400Hz, CDCl3) δ 3.72-3.60 (m, 22H), 3.39 (t, J=4.8Hz, 2H), 2.78 (br, 1H).

[0378] Preparation of Compound L-3-3 5% Pd / C (1.04 g, 0.49 mmol) was added to a stirred solution of L-3-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) at room temperature. Hydrogen gas was bubbled through the reaction mixture for 4 hours. The mixture was filtered through Celite® to remove Pd / C and concentrated under reduced pressure. The residue was dissolved in DCM (25 mL), and then BOC0 (852.1 mg, 3.9 mmol) was added to it. The mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to produce compound L-3-3 (330 mg, 28%).

[0379] 1 H NMR(400Hz,CDCl3)δ 5.19(brs,1H),3.73(t,J=4.8Hz,2H),3.67(s,12H),3.63-3.60(m,6H),3.54(t,J=5.2Hz,2H),3.34-3.27(m,1H),1.44(s,9H). ESI-MS m / z: 382(M + +1).

[0380] Preparation of compound L-3-4 Compound L-3-2 (1.9 g, 6.18 mmol) was dissolved in DCM (20 mL) under a N atmosphere. Triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added thereto, and the mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-3-4 (2.58 g, 91%).

[0381] 1 H NMR(400Hz,CDCl3)δ 7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.70-3.61(m,16H),3.56(s,1H),3.39(t,J=4.8Hz,2H),2.45(s,3H).

[0382] ESI-MS m / z: 462(M + +1).

[0383] Preparation of compound L-3-5 A homogeneous solution of L-2 (1.1 g, 3.4 mmol) in anhydrous THF (30 mL) was treated with NaH (60% dispersion in mineral oil, 135 mg, 3.4 mmol) under a N atmosphere and cooled to 0 °C. After the mixture was stirred at 0 °C for 20 minutes, L-3-4 (1.56 g, 3.4 mmol) was added to it. The reaction was allowed to warm to room temperature and stirred overnight. The reaction was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-3-5 (1.91 g, 93%).

[0384] ESI-MS m / z: 610(M + +1).

[0385] Preparation of compound L-3 To a solution of compound L-3-5 (906.7 mg, 1.49 mmol) in EA (4 mL) and ether (4 mL) at 0 °C, 5% HCl solution (8 mL) and triphenylphosphine (390 mg, 1.49 mmol) were slowly added under a N atmosphere. The mixture was stirred at 0 °C overnight. The mixture was diluted with DCM (10 mL). The aqueous layer was extracted with DCM (10 mL × 3). The aqueous phase was concentrated under high vacuum to give compound L-3 (495 mg, 54%).

[0386] ESI-MS m / z: 584(M + +1).

[0387] Example 1.6. Preparation of L-4 [ka] Preparation of Compound L-4-1 To a solution of KOtBu (943 mg, 8.41 mmol) in dry THF (50 mL) at −20° C. under a N atmosphere, tetraethylene glycol (4.35 mL, 25.22 mmol) was added, followed by propargyl bromide (1.0 g, 8.41 mL). The reaction was allowed to warm to room temperature and stirred for 17 h. The reaction was quenched by adding MeOH (1 mL) and HO (50 mL) while cooling in an ice bath and extracted with EA (100 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-1 (1.46 g, 75%).

[0388] 1 H NMR (400MHz, CDCl3) δ 4.26-4.20(m, 2H), 3.78-3.60(m, 16H), 2.42-2.40(m, 1H).

[0389] Preparation of Compound L-4-2 To a solution of CBr (1.43 g, 4.31 mmol) in dry DCM (20 mL) cooled in an ice bath, triphenylphosphine (1.13 g, 4.31 mmol) was added, followed by L-4-1 (500 mg, 2.15 mmol). The mixture was warmed to room temperature and stirred for 18 hours. The reaction was diluted with water (50 mL) and extracted with DCM (100 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-2 (410 mg, 65%).

[0390] 1 H NMR(400Hz, CDCl3)δ 4.21(s,2H),3.82(t,J=6.4Hz,2H),3.74-3.64(m,12H),3.45(t,J=6.4Hz,2H),2.45-2.42(m,1H).

[0391] Preparation of compound L-4 To a solution of compound L-4-2 (300 mg, 1.02 mmol) in DMF (10 mL), N,N-dimethylethylenediamine (555 μL, 5.08 mmol) was added at room temperature under a N atmosphere. The mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4 (218 mg, 71%).

[0392] ESI-MS m / z: 303(M +1 ).

[0393] Preparation of Example 1.7.L-5 [ka] Preparation of Compound L-5-1 A homogeneous solution of methyl 2,4-dibromobutyrate (10 g, 38.47 mmol) in dry THF (100 mL) was added dropwise to a mixture of thioacetic acid (2.75 mL, 38.47 mmol, 1.0 equiv.) and DIPEA (8.5 mL, 48.9 mmol, 1.3 equiv.) in dry THF (50 mL) at room temperature under a N atmosphere over 1.5 h. After stirring at −20° C. under a N atmosphere for 4 h, the mixture was concentrated, diluted with water (100 mL), and extracted with EA (200 mL × 3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 12:1) to give compound L-5-1 (9.67 g, 98%) as a white solid.

[0394] 1 H NMR(600MHz, CDCl3)δ 4.38(t,J=7.6Hz,1H),3.46-3.39(m,2H),2.56-2.47(m,1H),2.36(s,3H),2.32-2.23(m,1H).

[0395] Preparation of compound L-5-2 L-5-1 (9.67 g, 37.90 mmol) in AcOH (80 mL) was added to 35% hydrogen peroxide (40 mL) at room temperature under a N atmosphere. The mixture was stirred overnight, then concentrated, diluted with water (20 mL), neutralized with NaHCO, and washed with EA / Hex (1 / 1, 30 mL × 2). The aqueous layer was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 8:1:0.01 to 5:1:0.01) to give compound L-5-2 (7.0 g, 71%) as a white solid.

[0396] 1 H NMR(600MHz,D2O)δ 4.11(dd,J=5.4,4.8Hz,1H),3.82(s,3H),3.65-3.62(m,1H),3.52-3.47(m,1H),2.62-2.48(m,2H).

[0397] Preparation of compound L-5-3 To a solution of L-5-2 (7.0 g, 26.81 mmol) in DMF (20 mL), NaN3 (4.5 g, 69.71 mmol, 2.6 equiv.) was added under a N2 atmosphere, and the mixture was stirred at room temperature overnight. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 7:1:0.01 to 5:1:0.01) to obtain compound L-5-3 (5.4 g, 90%) as a white solid.

[0398] 1 H NMR(600MHz,D2O)δ 3.82(dd,J=4.2,6.0Hz,1H),3.63(s,3H),3.36-3.26(m,2H),2.29-2.02(m,2H).

[0399] Preparation of compound L-5-4 To a 50 mL round-bottom flask were added L-5-3 (500 mg, 2.24 mmol), 10 mL of MeOH, 5% Pd / C (715 mg, 0.34 mmol, 0.15 equiv.), and BocO (538 mg, 2.46 mmol, 1.1 equiv.). After evaporating the air, the mixture was stirred under H2 at room temperature for 15 h. The catalyst was filtered through Celite®, and the Celite® was washed with MeOH (20 mL × 2). The solvent was removed by rotary evaporation, and the residue was purified by column chromatography (DCM:MeOH:AcOH = 7:1:0.01 to 5:1:0.01) to give compound L-5-4 (450.2 mg, 68%) as a white solid.

[0400] 1 H NMR (600MHz, DMSO-d6) δ 6.79 (s, 1H), 4.13 (brs, 1H), 3.55 (s, 3H), 2.88-2.80 (m, 2H), 1.96-1.88 (m, 2H), 1.3 6 (s, 9H).

[0401] Preparation of compound L-5-5 A homogeneous solution of L-5-4 (100 mg, 0.34 mmol) in THF / water (4 mL / 8 mL) was treated with LiOH (21.2 mg, 0.50 mmol, 1.5 equiv.) at room temperature under N2 and stirred for 8 h. The reaction mixture was neutralized with 2 N HCl solution and concentrated under reduced pressure. Compound L-5-5 was used directly in the next step without further purification.

[0402] ESI-MS m / z: 284(M + +1).

[0403] Preparation of compound L-5-6 A homogeneous solution of L-5-5 (0.34 mmol), N-hydroxysuccinimide (77.4 mg, 0.67 mmol, 2.0 equiv.), and EDCI-HCl (260.7 mg, 1.36 mmol, 4.0 equiv.) in DMF (2 mL) was stirred overnight at room temperature under a N atmosphere. The mixture was treated with L-3 (210.8 mg, 0.34 mmol, 1.0 equiv.) and DIPEA (177.6 μL, 1.02 mmol, 3.0 equiv.) and stirred overnight. The reaction was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 12:1:0.01 to 5:1:0.01) to give compound L-5-6 (159.1 mg, 55%) as a yellow oil.

[0404] ESI-MS m / z: 850(M + +1).

[0405] Preparation of compound L-5 A homogeneous solution of L-5-6 (100 mg, 0.12 mmol) in 1,4-dioxane (2 mL) was treated with c-HCl (500 μL) at room temperature under a N atmosphere and stirred for 30 min. The reaction mixture was concentrated under reduced pressure to give compound L-5 (92 mg, 99%) as a yellow oil.

[0406] ESI-MS m / z: 749(M + +1).

[0407] Example 1.8. Preparation of L-6 [ka] Preparation of Compound L-6-1 A homogeneous solution of Boc-L-serine methyl ester (5.0 g, 22.8 mmol) in DCM (30 mL) was treated with pyridine (8 mL) and p-toluenesulfonyl chloride (5.22 g, 27.4 mmol, 1.2 equiv.) at room temperature under a N atmosphere and stirred overnight. The reaction was quenched by adding water (50 mL) and extracted with EA (100 mL × 3). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 9:1 to 2:1) to give compound L-6-1 (7.0 g, 82%) as a white solid.

[0408] 1 H NMR(600MHz,CDCl3)δ 7.76(d,J=8.4Hz,2H),7.35(d,J=7.8Hz,2H),5.29(S,1H),4.53-4.47(m,1H),4. 39(dd,J=2.4,7.8Hz,1H),4.29(d,J=7.2,2.4Hz,1H),3.69(s,3H),2.45(s,3H).

[0409] Preparation of compound L-6-2 A suspension of CsCO (1.05 g, 3.21 mmol, 0.6 equiv) in DMF (12 mL) was treated with thioacetic acid (498 μL, 6.96 mmol, 1.3 equiv) and L-6-1 (2.0 g, 5.36 mmol) in DMF (8 mL) at room temperature under a N atmosphere and stirred overnight. The mixture was quenched by adding water (50 mL) and extracted with EA (100 mL × 3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 5:1) to give compound L-6-2 (1.4 g, 95%) as a white solid.

[0410] 1 H NMR (600MHz, CDCl3) δ 5.24 (s, 1H), 4.53-4.49 (m, 1H), 3.75 (s, 3H), 2.45 (s, 3H), 4.41-4.31 (m, 2H).

[0411] Preparation of compound L-6-3 L-6-2 (1.2 g, 4.33 mmol) in AcOH (10 mL) was added to 35% hydrogen peroxide (4 mL) at room temperature under a N atmosphere. The mixture was stirred for 7 h and then concentrated under reduced pressure. The residue was diluted with water (5 mL) and basified to pH 9 using a saturated aqueous solution of NaHCO at 0 °C. BocO (1.4 g, 6.49 mmol, 1.5 equiv.) was added, and the resulting mixture was stirred overnight. The mixture was neutralized with 2 N HCl solution at 0 °C and concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 8:1:0.01 to 5:1:0.01) to give compound L-6-3 (521.5 mg, 42%) as a white solid.

[0412] 1 H NMR(400MHz,DMSO-d6)δ 6.96(d,J=7.2Hz,1H),4.20(q,J=6.8,4.8Hz,1H),3.58(s,3H),2.84(dd,J=14,6.4Hz,1H),2.76(dd,J=9.2,4.4Hz,1H),1.37(s,9H).

[0413] Preparation of compound L-6-4 A homogeneous solution of L-6-3 (71 mg, 0.25 mmol) in THF / HO (2.0 mL / 4.0 mL) was treated with LiOH (17.3 mg, 0.41, 1.5 equiv.) under N atmosphere at room temperature and stirred for 3 h. The mixture was neutralized with 2 N HCl at 0 °C and concentrated under reduced pressure to give compound L-6-4 (67 mg, 99%) as a white solid.

[0414] 1 H NMR (400MHz, DMSO-d6) δ 6.40 (d, J = 7.2 Hz, 1H), 3.96 (q, J = 6.4, 5.6 Hz, 1H), 2.88-2.78 (n, 2H), 1.36 (s, 9H).

[0415] Preparation of compound L-6-5 L-6-4 (35 mg, 0.13 mmol), N-hydroxysuccinimide (22.4 mg, 0.19 mmol, 1.5 equiv.), and EDCI-HCl (50 mg, 0.26 mmol, 2.0 equiv.) were dissolved in DMF (2 mL) at room temperature under a N atmosphere. After stirring the mixture overnight, compound L-6-5 was used directly in the next step without further purification.

[0416] ESI-MS m / z: 367(M + +1).

[0417] Preparation of compound L-6-6 To a stirred solution of L-6-5 (0.13 mmol) in DMF (2 mL) at room temperature under a N atmosphere, L-2 (0.19 mmol, 1.5 equiv.) and EDCI-HCl (50 mg, 0.26 mmol, 2.0 equiv.) were added. The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (DCM:MeOH:AcOH = 12:1:0.01 to 5:1:0.01) to give compound L-6-6 (34.8 mg, 64%) as a yellow oil.

[0418] ESI-MS m / z: 483(M + +1).

[0419] Preparation of compound L-6 c-HCl (300 μL) was added to a stirred solution of L-6-6 (29.6 mg, 0.061 mmol) in 1,4-dioxane (1.2 mL) at room temperature under a N atmosphere, and the mixture was stirred for 30 min. The mixture was concentrated under reduced pressure to give compound L-6 (25.4 mg, 99%) as a yellow oil.

[0420] ESI-MS m / z: 382(M + +1).

[0421] Example 1.9. Preparation of MPS-D1-10 [ka] Preparation of Compound L-1-1 A clear solution of 11-azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS 134179-38-7, 5.0 g, 22.9 mmol) in 1,4-dioxane (100 mL) and HO (25 mL) was treated with NaHCO (3.8 g, 45.8 mmol, 2.0 equiv.) and BOCO (6.0 g, 27.5 mmol, 1.2 equiv.) under a N atmosphere at room temperature, then stirred for 6 h. The reaction was quenched with water (50 mL) and extracted with DCM (100 mL × 3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (1% to 3% MeOH in DCM) to give compound L-1-1 (7.2 g, 99%) as a colorless oil.

[0422] 1 H NMR (400MHz, CDCl3) δ 5.03 (brs, 1H), 3.72-3.60 (m, 10H), 3.98-3.52 (m, 1H), 3.43-3.36 (m, 1H), 3.35-3.24 (m, 1H), 1.26 (s, 9H).

[0423] ESI-MS m / z: 319(M + +1).

[0424] Preparation of Compound L-1 A clear solution of L-1-1 (7.2 g, 22.6 mmol) in THF (30 mL), ether (15 mL), and HO (15 mL) was treated with triphenylphosphine (6.5 g, 24.9 mmol, 1.1 equiv.) at room temperature under a N atmosphere and then stirred overnight. The reaction mixture was diluted with water (10 mL) and extracted with DCM (60 mL × 3). The aqueous layer was concentrated under reduced pressure to give compound L-1-1 (6.3 g, 95%) as a colorless oil.

[0425] ESI-MS m / z: 293(M + +1)

[0426] Compound MPS-D1-10a was synthesized in a manner similar to the preparation method of compound MPS-D1-1 in Example 2.

[0427] Preparation of compound MPS-D1-10a Yield 71%, pale yellow oil.

[0428] 1 H NMR(400MHz,CDCl3)δ 7.99-7.93(m,4H),7.83(d,J=8.0Hz,2H),7.39(d,J=8.0Hz,2H),7.30(brs,1H),5.0 1(brs,1H),3.74-3.46(m,26H),3.34-3.26(m,2H),2.46(s,3H),1.43(s,9H);ESI-MS m / z:695(M + +1).

[0429] Compound MPS-D1-10b was synthesized in a manner similar to the preparation of compound L-6 in Example 1.8.

[0430] Preparation of compound MPS-D1-10b 99% yield, pale yellow oil.

[0431] 1 H NMR(400MHz,DMSO-D6)δ 8.74(t,J=8.0Hz,1H),7.98(dd,J=12,8.4Hz,2H),7.82(d,J=8.4Hz,2H),7.46 (d,J=8.0Hz,2H),3.68-3.36(m,24H),3.01-2.94(m,2H),2.22(s,3H);ESI-MS m / z:595(M + +1).

[0432] Preparation of Compound MPS-D1-10 A homogeneous solution of MPS-D1-10b (63 mg, 0.10 mmol) and BCN-PNP (31.5 mg, 0.10 mmol, 1.0 equiv.) in anhydrous DMF (2.0 mL) was treated with DIPEA (52 uL, 0.3 mmol, 3 equiv.) and HBTU (57 mg, 0.15 mmol, 1.5 equiv.) under a N atmosphere at room temperature and stirred for 2 h. The reaction was quenched with HO (20 mL) and extracted with EA (30 mL × 3). The combined organic layer was washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC to give compound MPS-D1-10 (57 mg, 74%). ESI-MS m / z: 771 (M + +1).

[0433] Preparation of Example 1.10.L-11 [ka] Preparation of Compound L-11-1 To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL), KI (294 mg, 1.77 mmol), AgO (4.92 g, 19.48 mmol), and p-TsCl (3.7 g, 19.48 mmol) were added under a N atmosphere. The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was filtered through Celite®, and the Celite® plug was washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-11-1 (5.98 g, 73%).

[0434] 1 H NMR(400Hz, CDCl3)δ 7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.71-3.58(m,22H),2.88(br,1H),2.45(s,3H).

[0435] Preparation of Compound L-11-2 To a solution of compound L-11-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was added NaN (1.34 g, 20.55 mmol) under a N atmosphere. The mixture was stirred at 110 °C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-11-2 (4.1 g, 97%).

[0436] 1 H NMR (400Hz, CDCl3) δ 3.72-3.60 (m, 22H), 3.39 (t, J=4.8Hz, 2H), 2.78 (br, 1H).

[0437] Preparation of Compound L-11-2a Compound L-11-2 (1.9 g, 6.18 mmol) was dissolved in DCM (20 mL) under a N atmosphere, and triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added thereto. The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-11-2a (2.58 g, 91%).

[0438] 1 H NMR(400Hz,CDCl3)δ 7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.70-3.61(m,16H),3.56(s,1H),3.39(t,J=4.8Hz,2H),2.45(s,3H).

[0439] ESI-MS m / z: 462(M + +1).

[0440] Preparation of compound L-11-3 To a solution of compound L-11-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) was added 5% Pd / C (1.04 g, 0.49 mmol) under an H atmosphere. The mixture was stirred at room temperature for 4 hours. The mixture was filtered through Celite® to remove Pd / C and concentrated under reduced pressure. The residue was dissolved in DCM (25 mL). BOCO (852.1 mg, 3.9 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to produce compound L-11-3 (330 mg, 28%).

[0441] 1 H NMR(400Hz,CDCl3)δ 5.19(brs,1H),3.73(t,J=4.8Hz,2H),3.67(s,12H),3.63-3.60(m,6H),3.54(t,J=5.2Hz,2H),3.34-3.27(m,1H),1.44(s,9H).

[0442] ESI-MS m / z: 382(M + +1).

[0443] Preparation of compound L-11-4 A homogeneous solution of compound L-11-3 (450 mg, 1.18 mmol) in anhydrous THF (10 mL) was treated with NaH (60% dispersion in mineral oil, 47.2 mg, 1.18 mmol) under a N atmosphere at 0 °C. After the mixture was stirred at 0 °C for 20 minutes, L-11-2a (544.5 mg, 1.18 mmol) was added to it. The reaction was allowed to warm to room temperature and stirred overnight. The reaction was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-11-4 (582.9 mg, 74%).

[0444] Preparation of compound L-11 To a solution of compound L-11-4 (582.9 mg, 0.87 mmol) in DCM (3 mL) was added 4 M HCl (1 mL in 1,4-dioxane) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to give compound L-11 (527.6 mg, quantitative yield).

[0445] ESI-MS m / z: 571(M + +1).

[0446] Table 2 below lists compounds that were synthesized via a synthetic route similar to that described in Example 2. [Table 3] TIFF0007798811000087.tif223159TIFF0007798811000088.tif96159

[0447] Example 2. Synthesis of maleimide derivatives and POS derivatives Example 2.1. Preparation of Mal-1 [ka] Compound L-4 was synthesized by a synthetic route similar to that described in Journal of Medicinal Chemistry, 52(19), 5816-5825; 2009, which is incorporated herein by reference.

[0448] Preparation of compound Mal-1a Yield 55%

[0449] 1 H NMR(400Hz, CDCl3)δ 4.21(d,J=2.0Hz,2H),3.72-3.60(m,24H),2.79(brs,1H),2.43(t,J=2.4Hz,1H).

[0450] Preparation of compound Mal-1 ESI-MS m / z: 400(M + )

[0451] Example 2.2. Preparation of Mal-2 [ka] A homogeneous solution of N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-carboxylate (85.5 mg, 0.26 mmol) and L-2 (75.3 mg, 0.28 mmol) in dry DCM was treated with DIPEA (44.5 μL, 0.26 mmol, 1 equiv.) at room temperature under a N atmosphere and stirred for 45 min until the temperature reached room temperature. The reaction mixture was diluted with DCM (32 mL), washed with 1 N HCl (30 mL), brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated under high vacuum. The residue was purified by preparative HPLC to give the title compound L-5 (70.8 mg, 61%, mixture 9 mg) as a white gum.

[0452] ESI-MS m / z: 451(M +1 )

[0453] Example 2.3. Preparation of Mal-3 [ka] Preparation of compound Mal-3-1 To a solution of BOCO (9.6 g, 44.0 mmol) in THF (50 mL) was added 2,2'-diamino-N-methyldiethylamine (10.3 g, 88.0 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2 h. The mixture was quenched with HO (100 mL) and DCM (150 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mal-3-1 (3.3 g, 35%).

[0454] 1 H NMR(400Hz,CDCl3)δ 5.04(brs,1H),3.26-3.16(m,2H),2.78(t,J=6.0Hz,2H),2.47(t,J=6.0Hz,2H),2.43(t,J=6.0Hz,2H),2.22(s,3H),1.45(s,9H).

[0455] Preparation of compound Mal-3-2 To a solution of Mal-3-1 (500 mg, 2.3 mmol) in AcOH (3.0 mL) was added maleic anhydride (248 mg, 2.53 mmol) at room temperature under a N atmosphere. The mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure, and the residue was dissolved in acetic anhydride (5.0 mL) at room temperature. NaOAc (95.7 mg, 1.17 mmol) was added to the reaction mixture, which was stirred at 75 °C for 5 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mal-3-2 (415 mg, 60%).

[0456] 1 H NMR(400Hz,CDCl3)δ 6.70(s,2H),3.63(t,J=6.4Hz,2H),3.18-3.10(m,2H),2.57(t,J=6.4Hz,2H),2.48(t,J=6.0Hz,2H),2.24(s,3H),1.44(s,9H). ESI-MS m / z: 298(M + ).

[0457] Preparation of compound Mal-3-3 To a solution of compound Mal-3-2 (370 mg, 1.24 mmol) in DCM (4.0 mL) was added TFA (3.0 mL) at 0° C. The reaction mixture was warmed to room temperature and stirred for 2.5 hours. The mixture was concentrated under reduced pressure and used directly in the next step without further purification (387 mg, quantitative).

[0458] ESI-MS m / z: 198(M + ).

[0459] Preparation of compound Mal-3 To a solution of compound Mal-3-3 (50 mg, 0.16 mmol) and BCN-PNP (50.6 mg, 0.16 mmol) in DMF (3.0 mL) was added DIPEA (57 μL, 0.32 mmol) at room temperature under a N atmosphere. The mixture was stirred for 2.5 h, and EA (50 mL × 2) and HO (30 mL) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mal-3 (13.2 mg, 22%).

[0460] 1 H NMR(400Hz,CDCl3)δ 6.70(s,2H),5.12(brs,1H),4.14(d,J=8.0Hz,2H),3.63(t,J=6.0Hz,2H),3.24-3.18(m,2H),2.58 (t,J=6.4Hz,2H),2.50(t,J=6.0Hz,2H),2.30-2.20(m,9H),1.28-1.22(m,3H),0.98-0.94(m,1H).

[0461] ESI-MS m / z: 374(M + ).

[0462] Example 2.4. Preparation of POS-1 [ka] Preparation of compound POS-1-1 To a solution of ethyl 4-hydrobenzoate (20 g, 120.35 mmol) in EtOH (60 mL) was added NHNH·H0 (88 mL, 1805.4 mmol) under a N atmosphere. The mixture was stirred at reflux overnight. The mixture was cooled to room temperature and concentrated under reduced pressure, followed by trituration with EtOH to give compound POS-1-1 (17.54 g, 96%).

[0463] 1 H NMR (400Hz, DMSO-d6) δ 9.50 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 4.37 (s, 2H). ESI-MS m / z:431(M+ +1).

[0464] Preparation of compound POS-1-2 To a solution of compound POS-1-1 (17.54 g, 115.28 mmol) in EtOH (200 mL) and DMF (100 mL) was added CS (45 mL, 749.32 mmol) and KOH (6.5 g, 115.28 mmol) under a N atmosphere. After stirring at 85 °C for 18 h, the reaction mixture was adjusted to pH 4 by adding 1 M HCl solution and diluted with distilled water (500 mL) and EA (500 mL). The organic layer was washed with HO (500 mL) and brine (500 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was subjected to ether / Hex trituration to give compound POS-1-2 (20.7 g, 93%).

[0465] 1 H NMR (400Hz, DMSO-d6) δ 10.44 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H). ESI-MS m / z:195(M + +1).

[0466] Preparation of Compound POS-1-3 To a solution of compound POS-1-2 (5 g, 25.75 mmol) in THF (100 mL) was added EtN (4.3 mL, 30.9 mmol) and MeI (1.76 mL, 28.33 mmol) dropwise at 0° C. After stirring at 0° C. for 10 minutes, the mixture was warmed to room temperature and stirred for 2 hours. The mixture was diluted with HO (150 mL) and extracted with EA (100 mL×2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was subjected to ether trituration to give compound POS-1-3 (5.15 g, 96%).

[0467] 1 H NMR (400Hz, DMSO-d6) δ 7.80 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 2.74 (s, 3H). ESI-MS m / z:209(M + +1).

[0468] Preparation of compounds POS-1-4 To a solution of compound POS-1-3 (3.2 g, 15.37 mmol) in EtOH (150 mL) was added 70% m-CPBA (11.4 g, 46.11 mmol) at 0 °C under a N atmosphere. After stirring at room temperature for 5 hours, 70% m-CPBA (11.4 g, 46.11 mmol) was further added. The mixture was then stirred overnight at room temperature, quenched with HO (500 mL), saturated NaHCO (300 mL), and extracted with EA (500 mL × 2). The organic layer was washed with brine (300 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was subjected to trituration with Hex / EA = 1:1 (100 mL) to give compound POS-1-4 (3.2 g, 89%).

[0469] 1 H NMR (400Hz, DMSO-d6) δ 7.95 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 3.69 (4s, 3H). ESI-MS m / z:241(M + +1).

[0470] Preparation of compound POS-1 To a solution of POS-1-4 (310 mg, 1.29 mmol) and L-8-1 (660 mg, 2.84 mmol) in THF (8 mL) and DMF (0.8 mL) was added PPh (667 mg, 2.58 mmol). The mixture was cooled to 0 °C, DEAD (1.17 mL, 2.58 mmol) was added thereto, and the mixture was stirred at 0 °C for 3 hours. The mixture was diluted with water (15 mL) and extracted with EA (15 mL × 2). The obtained organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound POS-1 (205 mg, 30%).

[0471] ESI-MS m / z: 455(M + +1).

[0472] Example 2.5. Preparation of Int-3 [ka] Preparation of compound Int-3-a A solution of Int-TG (18.5 g, 45.0 mmol), 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol), and molecular sieves (10.0 g) in ACN (150 mL) was treated with AgO (38.0 g, 0.164 mol) under a N atmosphere at room temperature and stirred for 3 h. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-3-a (16.0 g, 86%).

[0473] 1 H NMR (400MHz, CDCl3) δ 9.93 (s, 1H), 7.86 (d, J = 6.8Hz, 2H). 7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H),5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[0474] Preparation of compound Int-3-b A solution of Int-3-a (540 mg, 1.19 mmol) in anhydrous THF (15 mL) was treated with NaBH (113 mg, 2.98 mmol) under N atmosphere at 0 °C and stirred at 0 °C for 10 min. After stirring at room temperature for 4 h, the reaction was diluted with HO and EA. The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EA:HEX = 1:1) to give compound Int-3-b (430 mg, 79%).

[0475] 1 H NMR (400MHz, CDCl3) δ 7.30 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H). 5.51-5.54(m,2H),5.11(dd,J=10.8Hz,1H),5.03(d,J=8.0Hz,1H),4.65(d,J= 5.62H)4.25-4.04(m,3H),2.19(s,3H),2.07(s,3H),2.06(s,3H),2.01(s,3H).

[0476] Preparation of compound Int-3 A solution of Int-3-b (1.0 g, 2.2 mmol) in dry DMF (6.0 mL) was treated with bis(pentafluorophenyl carbonate) (1.3 g, 3.3 mmol) at room temperature under a N atmosphere and stirred for 3 h. The reaction mixture was extracted with EA (20 mL × 2) and HO (30 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The reaction mixture was purified by column chromatography to give Int-3 (1.4 g, 98%). 1 H NMR(400MHz,CDCl3): δ 7.384(d,J=8.8Hz,2H),7.039(d,J=8.4Hz,2H),5.529-5.465(m,2H),5.280(s,2H),5.141-5 .068(m,2H),4.262-4.070(m,4H),2.195(s,3H),2.078(s,3H),2.073(s,3H),2.025(s,3H).

[0477] Example 2.6. Preparation of Int-4 [ka] Compound Int-4 was synthesized by a method similar to that described in Example 2.5. Yield: 72%.

[0478] 1 H NMR (400MHz, CDCl3) δ 9.93 (s, 1H), 7.86 (d, J = 6.8Hz, 2H). 7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H),5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[0479] Example 2.7. Preparation of Int-5 [ka] Preparation of compound Int-5-1 To a solution of 4-hydroxybenzoic acid (5.0 g, 36.2 mmol) in methanol (150 mL) was added thionyl chloride (26.3 mL, 362 mmol) under a N atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 4 hours. The reaction was quenched with aqueous NaHCO and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-5-1 (4.87 g, 89%).

[0480] 1 H NMR(400Hz,CDCl3)δ 7.87(d,J=8.8Hz,2H),6.82(d,J=9.2Hz,2H),3.85(s,3H) ESI-MS m / z: 153(M + +1).

[0481] Preparation of compound Int-5-2 To a solution of compound Int-5-1 (1.0 g, 6.57 mmol) in DCM (22.0 mL) was added DIPEA (2.3 mL, 13.4 mmol) and MOM-Cl (0.55 mL, 7.23 mmol) under a N atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 6 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-5-2 (1.14 g, 88%).

[0482] 1 H NMR(400Hz,CDCl3)δ 8.01-7.97(m,2H),7.07-7.04(m,2H),5.23(s,2H),3.89(s,3H),3.48(s,3H)

[0483] Preparation of compound Int-5 To a solution of compound Int-5-2 (1.14 g, 5.81 mmol) in methanol / HO / 1,4-dioxane (16.0 mL / 8.0 mL / 16.0 mL) was added lithium hydroxide monohydrate (975 mg, 23.2 mmol) under a N atmosphere at 0° C. The reaction mixture was stirred at room temperature for 5 hours. The reaction was quenched with 2 N HCl and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. Compound Int-5 was used in the next step without further purification. (995 mg, 94%)

[0484] 1 H NMR(400Hz,MeOH-D4)δ 7.96(d,J=8.8Hz,2H),7.08(d,J=8.8Hz,2H),5.25(s,2H),3.55(s,3H)

[0485] Example 3. Synthesis of OHPAS-linker derivatives Example 3.1. Preparation of Int-TG [ka] β-D-Galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in 33% HBr in AcOH (20 mL) at 0 °C under a N atmosphere. The mixture was allowed to warm to room temperature. After stirring at room temperature for 4 h, the mixture was concentrated under reduced pressure, and then EA (1000 mL) and saturated sodium bicarbonate (1000 mL) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG (5.2 g, 99%).

[0486] Example 3.1.2 Preparation of Int-TG2 [ka] Compound Int-TG2 was synthesized by a method similar to that described in Example 3.1.1. Yield 80%

[0487] 1 H NMR(400MHz,CDCl3)δ 6.654(d,J=4.0Hz,1H),5.627(t,J=10.0Hz,1H),5.252(dd,J=10.4Hz,9.6Hz,1H),4.865(dd,J=10 .0Hz,4.0Hz,1H),4.593(d,J=10.4Hz,1H),3.777(s,3H),2.113(s,3H),2.071(s,3H),2.065(s,3H)

[0488] Example 3.1.3 Preparation of Int-TG3 [ka] Preparation of compound Int-TG3-1 To a solution of beta-D-galactose pentaacetate (1 g, 2.56 mmol) in THF (10 mL) was added 3-(dimethylamino)1-propylamine (1.61 mL, 12.8 mmol) at room temperature under a N atmosphere. After stirring at the same temperature for 3 h, the reaction mixture was extracted with EA (250 mL × 3) and HO (200 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. Compound Int-TG3-1 (891 mg, 100%) was obtained and used without further purification.

[0489] ESI-MS m / z: 371(M + +Na).

[0490] Preparation of compound Int-TG3 To a solution of Int-TG3-1 (891 mg, 2.56 mmol) in DCM (10 mL) was added trichloroacetonitrile (2.57 mL, 25.6 mmol) and DBU (0.3 mL, 2.05 mmol) at 0 °C under a N atmosphere. After stirring at room temperature for 30 minutes, the reaction mixture was extracted with DCM (250 mL × 3) and HO (200 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG3 (880 mg, 70%).

[0491] 1H NMR(400MHz,CDCl3)δ 8.70(s,1H),6.61(d,J=3.6Hz,1H),5.57(dd,J=2.8,0.8Hz,1H),5.55-5.35(m,2H),4.44 (t,J=7.6Hz,1H),4.19-4.06(m,2H),2.17(s,3H),2.04(s,3H),2.03(s,3H),2.02(s,3H).

[0492] ESI-MS m / z: 515(M + +Na).

[0493] Example 3.1.3 Preparation of Int-TG4 [ka] Preparation of compound Int-TG4-1 To a solution of 4-hydroxybenzoic acid (5.0 g, 36.2 mmol) in methanol (150 mL) was added thionyl chloride (26.3 mL, 362 mmol) under a N atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 4 hours. The reaction was quenched with aqueous NaHCO and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG4-1 (4.87 g, 89%).

[0494] 1 H NMR(400Hz,CDCl3)δ 7.87(d,J=8.8Hz,2H),6.82(d,J=9.2Hz,2H),3.85(s,3H)

[0495] EI-MS m / z: 153(M + +1).

[0496] Preparation of compound Int-TG4-2 To a solution of compound Int-TG4-1 (1.0 g, 6.57 mmol) in DCM (22.0 mL) was added DIPEA (2.3 mL, 13.4 mmol) and MOM-Cl (0.55 mL, 7.23 mmol) at 0 °C under a N atmosphere. The reaction mixture was stirred at room temperature for 6 hours. The reaction was quenched with water and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG4-2 (1.14 g, 88%).

[0497] 1 H NMR(400Hz,CDCl3)δ 8.01-7.97(m,2H),7.07-7.04(m,2H),5.23(s,2H),3.89(s,3H),3.48(s,3H)

[0498] Preparation of compound Int-TG4 To a solution of compound Int-TG4-2 (1.14 g, 5.81 mmol) in methanol / HO / 1,4-dioxane (16.0 mL / 8.0 mL / 16.0 mL) was added lithium hydroxide monohydrate (975 mg, 23.2 mmol) under a N atmosphere at 0° C. The reaction mixture was stirred at room temperature for 5 hours. The reaction was quenched with 2N HCl and extracted with EtOAc. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. Compound Int-TG4 was used in the next step without further purification. (995 mg, 94%)

[0499] 1 H NMR(400Hz,MeOH-D4)δ 7.96(d,J=8.8Hz,2H),7.08(d,J=8.8Hz,2H),5.25(s,2H),3.55(s,3H)

[0500] Example 3.2. Preparation of OHPAS-D1, OHPAS-D1a, and OHPAS-D2 [ka] Preparation of compound OHPAS-D1a-1 To a solution of L-1-1 (2 g, 6.282 mmol) in DMF (25 mL) was added sodium hydride (301 mg, 12.56 mmol, 60%) under N2 atmosphere at 0 °C. After 10 min, iodomethane (3.9 mL, 62.82 mmol) was added under N2 atmosphere at the same temperature. The reaction was stirred at room temperature under N2 atmosphere for 3 h. After the reaction was completed, the reaction mixture was quenched with 2 N HCl (10 mL) and extracted with EA (500 mL × 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. Compound OHPAS-D1a-1 (yellow oil) was used directly in the next step without further purification.

[0501] 1 H NMR (400MHz, CDCl3) δ 3.70-3.62 (m, 12H), 3.4 (t, J=5.2Hz, 4H), 2.91 (s, 3H), 1.46 (s, 9H). ESI-MS m / z:333(M + 1)

[0502] Preparation of compound OHPAS-D1a-2 To a solution of compound OHPAS-D1a-1 (3.3 g, 6.282 mmol) in DCM (70 mL) was added 4N HCl in dioxane (25 mL) at 0° C. under a N atmosphere. The reaction was stirred at 0° C. under a N atmosphere for 1 hour. After the reaction was complete, the reaction mixture was concentrated under reduced pressure. Compound OHPAS-D1a-2 was used directly in the next step without further purification.

[0503] 1 H NMR(400MHz, CDCl3)δ 3.92(t,J=4.8Hz,2H),3.73-3.69(m,10H),3.45(t,J=5.2Hz,2H),3.22-3.16(m,2H),2.77(t,J=5.6Hz,3H),2.35(brs,1H). ESI-MS m / z:233(M + 1)

[0504] Preparation of compound OHPAS-D1-1 To a solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL), benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO (2.53 g, 43.06 mmol) were added at room temperature under a N atmosphere. The mixture was stirred at room temperature overnight under a N atmosphere. The reaction mixture was extracted with EA (200 mL × 2) and distilled water (100 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound OHPAS-D1-1 (2.56 g, 39%).

[0505] 1 H NMR(400Hz,CDCl3)δ 11.41(s,1H),9.95(s,1H),8.34(d,J=2.0Hz,1H),8.23(dd,J=6.4Hz,2.4Hz,1H),7.46-7.35(m,5H),7.04(d,J=9.2Hz,1H),5.37(s,2H).

[0506] Preparation of Compound OHPAS-D1-2 To a solution of compound Int-TG-1 (1.0 g, 3.90 mmol) and compound Int-TG (1.6 g, 3.90 mmol) in anhydrous ACN (30 mL), molecular sieves (8 g) and AgO (3.62 g, 15.61 mmol) were added at room temperature under a N atmosphere. The mixture was stirred at room temperature for 1 hour and then filtered through Celite®. The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D1-2 (2.1 g, 92%).

[0507] 1H NMR(400Hz,CDCl3)δ 10.34(s,1H),8.55(d,J=2.0Hz,1H),8.26(dd,J=6.8,2.0Hz,1H),7.45-7.35(m,5H),7.17(d,J=8.8Hz,1H),5.63-5.60(m,1H),5.50 (d,J=3.6Hz,1H),5.37(s,2H),5.23(d,J=8.0Hz,1H),5.16(dd,J=7.2,3.6Hz,1H)4.24-4.10(m,4H),2.20(s,3H),2.10-2.03(m,9H).

[0508] Preparation of compounds OHPAS-D1-3 To a solution of compound OHPAS-D1-2 (2.1 g, 3.58 mmol) in DCM (30 mL) was added m-CPBA (2.65 g, 10.74 mmol) under a N atmosphere at 0 °C. After stirring at 0 °C for 7 h, the mixture was quenched by adding saturated sodium bicarbonate (40 mL × 2). The mixture was separated, and the organic layer was washed with brine, dried over Na SO , filtered, and concentrated under reduced pressure. The residue was dissolved in DCM (5 mL), and hydrazine hydrate (261 μL, 5.37 mmol) was added under a N atmosphere at 0 °C. After stirring at 0 °C for 1 h, EA (30 mL × 2) and 1 M aqueous HCl (10 mL) were added. The resulting organic layer was dried over anhydrous Na SO , filtered, and concentrated under reduced pressure to give compound OHPAS-D1-3 (1.1 g, 55%).

[0509] ESI-MS m / z: 574(M + +Na)

[0510] Preparation of compounds OHPAS-D1-4 To a solution of compound OHPAS-D1-3 (280 mg, 0.49 mmol) in DCM (5 mL), TBDMS-OTf (224 μL, 0.97 mmol) and EtN (207 μL, 1.46 mmol) were added under a N atmosphere at 0° C. The mixture was stirred at room temperature for 1.5 hours and then quenched by adding citric acid (20 ml). The organic layer was washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D1-4 (246.3 mg, 68%).

[0511] 1 H NMR(400Hz,CDCl3)δ 7.67(d,J=8.4Hz,1H),7.57(s,1H),7.44-7.34(m,5H),7.02(d,J=8.4Hz ,1H),5.49-5.44(m,2H),5.30(s,2H),5.19(d,J=7.6Hz,1H),5.10(dd,J =6.8,3.2Hz,1H)4.20-4.11(m,2H),4.05(t,J=6.8Hz,2H),2.19(s,3H), 2.04(s,3H),2.01(d,J=6.0Hz,6H),1.02(s,9H),0.20(d,J=15.6Hz,6H).

[0512] Preparation of Compound OHPAS-D1-5 To a solution of compound OHPAS-D1-4 (283.2 mg, 0.41 mmol) in EA (5 mL), Pd / C (5%, 87.5 mg, 0.04 mmol) was added at room temperature under H. The mixture was stirred for 1 hour, filtered through Celite®, and then concentrated under reduced pressure. Compound OHPAS-D1-5 was used directly in the next step without further purification (246 mg, quantitative).

[0513] 1H NMR(400Hz,CDCl3)δ 7.67(d,J=8.8Hz,1H),7.57(s,1H),7.05(d,J=8.4Hz,1H),5.49-5.45(m,2H),5.22(d,J=7.6Hz,1H),5.12(dd,J=7.2 ,3.6Hz,1H)4.20-4.06(m,4H),2.19(s,3H),2.05(s,3H),2.02(d,J=7.6Hz,6H),1.01(s,9H),0.21(d,J=15.2Hz,6H).

[0514] Preparation of compound OHPAS-D1 To a solution of compound OHPAS-D1-5 (243.2 mg, 0.41 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS134179-38-7, 89.5 mg, 0.41 mmol) in DMF (5 mL), PyBOP (275 mg, 0.53 mmol) and DIPEA (176 μL, 1.02 mmol) were added at room temperature under a N atmosphere. The mixture was stirred at room temperature for 2 hours under a N atmosphere. The reaction mixture was extracted with EA (30 mL × 2) and distilled water (10 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D1 (272.8 mg, 84%).

[0515] 1 H NMR(400Hz,CDCl3)δ 7.34(s,1H),7.31(d,J=9.2Hz,1H),7.02(d,J=8.0Hz,1H),6.73(s,1H),5 .48-5.44(m,2H),5.19(d,J=7.6Hz,1H),5.10(dd,J=6.4,3.6Hz,1H),4.20 -4.10(m,2H),4.06(t,J=6.4Hz,2H),3.66(s,14H),3.38(t,J=4.4Hz,2H) ,2.19(s,3H),2.02(t,J=8.4Hz,9H),1.00(s,9H),0.20(d,J=14.4Hz,6H).

[0516] ESI-MS m / z: 799(M + +1).

[0517] Compounds OHPAS-D1a and OHPAS-D2 were synthesized in a manner similar to the preparation method of compound OHPAS-D1.

[0518] Preparation of compound OHPAS-D1a Yield: 83%.

[0519] 1 H NMR(400MHz,CDCl3)δ 7.00-6.96(m,2H),6.90(s,1H),5.48-5.43(m,2H),5.16(d,J=8.0Hz,1H), 5.10(dd,J=3.2,10.4Hz,1H),4.20-4.11(m,2H),4.05(t,J=7.2Hz,1H),3. 76-3.49(m,14H),3.46-3.39(m,2H),3.10-3.04(m,3H),2.19(s,3H),2.04 (s,3H),2.03(s,3H),2.01(s,3H),0.99(s,9H),0.21(s,3H),0.17(s,3H). ESI-MS m / z: 813(M + 1)

[0520] Preparation of compound OHPAS-D2 Yield: 81%, ESI-MS m / z: 1152(M +1 ).

[0521] Example 3.3. Preparation of OHPAS-D3, OHPAS-D3a, and OHPAS-D4 [ka] Preparation of compound OHPAS-D3-1 To a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 mL) was added EtN (2.28 mL, 16.38 mmol) at room temperature under a N atmosphere. SOF gas was introduced via a balloon, and the mixture was stirred at room temperature for 2 hours. The mixture was then washed with DCM (30 mL × 3) and brine (30 mL), and the organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D3-1 (790 mg, 63%).

[0522] 1 H NMR (400Hz, CDCl3) δ 10.06 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H).

[0523] Preparation of compound OHPAS-D3-2 To a solution of compound OHPAS-D1 (100 mg, 0.13 mmol) and compound OHPAS-D3-1 (26 mg, 0.13 mmol) in anhydrous ACN (3 mL) was added DBU (4 μL, 25 μmol). The mixture was stirred at room temperature for 1 hour and washed with distilled water (10 mL) and EA (10 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D3-2 (103 mg, 94%).

[0524] ESI-MS m / z: 869(M + ).

[0525] Preparation of compound OHPAS-D3-3 To a solution of compound OHPAS-D3-2 (103 mg, 0.12 mmol) in THF (8 mL) was added NaBH (9 mg, 0.24 mmol) under a N atmosphere at 0 °C. After stirring at room temperature for 2 h, distilled water (10 mL) and EA (10 mL × 2) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound OHPAS-D3-3 (101 mg, 98%).

[0526] ESI-MS m / z: 871(M + ).

[0527] Preparation of compound OHPAS-D3 To a solution of compound OHPAS-D3-3 (320.5 mg, 0.0.37 mmol) in DCM (3 ml), 1 M PBr3 (165 μl, 0.19 mmol) in DCM was added at 0 °C under a N2 atmosphere. After stirring for 2 hours, the mixture was quenched by adding saturated sodium bicarbonate (8 mL × 2). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to produce compound OHPAS-D3 (202.6 mg, 59%).

[0528] ESI-MS m / z: 934 (M+).

[0529] Preparation of compound OHPAS-D4 To a solution of compound OHPAS-D3-3 (47 mg, 54 μmol) in DMF (2 mL), bis(4-nitrophenyl)carbonate (25 mg, 81 μmol) and DIPEA (14 μL, 81 μmol) were added at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature overnight. Distilled water (10 mL) and EA (10 mL × 2) were then added, and the organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D3-4 (53 mg, 94%).

[0530] ESI-MS m / z: 1036(M + ).

[0531] Compounds OHPAS-D3a and OHPAS-D4a were prepared by the same synthetic route as that for preparing compounds OHPAS-D3 or OHPAS-D4.

[0532] Preparation of compound OHPAS-D3a-1 Yield 80%; 1H NMR(400MHz, CDCl3)δ 10.04(s,1H),8.00(d,J=8.8Hz,2H),7.57(d,J=8.4Hz,2H),7.44-7.27(m,3H),5.57-5.51(m,1H),5.47(d,J=3.2Hz,1H),5.14-5.10(m, 2H),4.27-4.09(m,3H),3.76-3.53(m,14H),3.42-3.36(m,2H),3.12-3.04(m,3H),2.19(s,3H),2.07(s,3H),2.06(s,3H),2.02(s,3H). ESI-MS m / z:883(M +1 )

[0533] Modulation of compound OHPAS-D3a-2 Yield 81%; 1 H NMR(400MHz, CDCl3)δ 7.47-7.42(m,2H),7.40-7.31(m,3H),7.24-7.21(m,2H),5.54-5.45( m,2H),5.11-5.07(m,2H),4.74-4.70(m,2H),4.25-4.21(m,1H),4.17 -4.12(m,1H),4.06(t,J=7.2Hz,1H),3.74-3.44(m,12H),3.37(t,J=4.8Hz,2H),3.07-3.04(s,3H),2.20(s,3H),2.06(s,6H),2.02(s,3H). ESI-MS m / z:885(M +1 ).

[0534] Modulation of compound OHPAS-D3a 90% yield; 1H NMR(400MHz,CDCl3)δ 7.48-7.41(m,2H),7.35(d,J=8.4Hz,2H),7.29-7.21(m,2H),5.59-5.55(m,1H),5.47(d,J=3.2Hz,1H),5.13-5.09(m,2H),4.26-4.22(m,1) H),4.18-4.08(m,2H),3.80-3.48(m,12H),3.37(t,J=5.2Hz,2H),3.12-3.06(s,3H),2.19(s,3H),2.07(s,3H),2.06(s,3H),2.02(s,3H). ESI-MS m / z:948(M +1 )

[0535] Preparation of compound OHPAS-D4a Yield 94%; ESI-MS m / z:1036(M + 1)

[0536] Example 3.4. Preparation of OHPAS-D5 [ka] Preparation of compound OHPAS-D5-1 To a solution of compound OHPAS-D3-1 (5 g, 24.49 mmol) in MeOH (40 mL) and THF (245 mL) was added NaBH (1.85 g, 48.98 mmol) at −78° C. under a N atmosphere. After stirring at 0° C. for 1 h, the reaction mixture was quenched by adding 2 N HCl (5 mL) and extracted with HO (250 mL) and EA (250 mL × 3). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D5-1 (5.01 g, 99%).

[0537] 1 H NMR(400MHz, CDCl3)δ 7.50-7.46(m,2H),7.34-7.31(m,2H),4.75(d,J=5.6Hz,2H),1.90(t,J=5.6Hz,1H).

[0538] Preparation of compound OHPAS-D5-2 To a solution of compound OHPAS-D5-1 (2 g, 9.7 mmol) in ether (32 mL), 1.0 M PBr3 (3.88 mL, 3.88 mmol) in DCM was added under a N2 atmosphere at 0 °C. After stirring for 2 hours, ether (100 mL) and NaHCO3 (100 mL × 3) were added and extraction was performed. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound OHPAS-D5-2 (2.35 g, 90%).

[0539] 1 H NMR (400MHz, CDCl3) δ 7.52-7.49(m,2H),7.34-7.31(m,2H),4.49(s,2H).

[0540] Preparation of compound OHPAS-D5-3 To a solution of 4-hydroxyisophathalaldehyde (112 mg, 0.746 mmol, CAS number: 3328-70-9) and sodium hydride (45 mg, 1.12 mmol, 60%) in DMF (5 mL) was added a solution of OHPAS-D5-2 (280 mg, 0.97 mmol) in DMF (2 mL) at 0 °C under a N atmosphere. After stirring at room temperature under a N atmosphere for 4 h, the reaction mixture was quenched by adding HO (10 mL) and extracted with HO (100 mL) and EA (100 mL × 2). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D5-3 (180 mg, 71%) as a white solid.

[0541] 1 H NMR(400MHz,CDCl3)δ 10.54(s,1H),9.98(s,1H),8.38(d,J=2.4Hz,1H),8.14(dd,J=2.0,8.8Hz,1H),7 .60(d,J=9.2Hz,2H),7.43(d,J=8.8Hz,2H),7.19(d,J=8.8Hz,1H),5.33(s,2H).

[0542] Preparation of compound OHPAS-D5 To a solution of compound OHPAS-D5-3 (1 g, 2.96 mmol) in THF (8 mL) was added sodium borohydride (391 mg, 10.35 mmol) in MeOH (1.5 mL) and THF (1 mL) at −78° C. under a N atmosphere. The reaction mixture was stirred at 0° C. for 1 hour under a N atmosphere. After the reaction was completed, the mixture was quenched with 2 N HCl (2 mL) and extracted with HO (100 mL) and EA (100 mL×3). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D5 (850 mg, 85%) as a white solid.

[0543] 1 H NMR(400MHz,CDCl3)δ 7.55(d,J=8.8Hz,2H),7.39-7.37(m,3H),7.30-7.28(m,1H),6.89(d,J=8.4Hz,1H),5.16(s,2H),4.76(d,J=6.0Hz,2H),4.65(d,J=5.6Hz,2H).

[0544] Example 3.5. Preparation of OHPAS-D6 [ka] Preparation of compound OHPAS-D6-1 To a solution of 2,6-dimethoxy-4-hydroxybenzaldehyde (0.5 g, 2.74 mmol) in DCM (8 mL) was added EtN (3.8 mL, 27.4 mmol) at room temperature under a N atmosphere. SOF gas was introduced via a balloon, and the mixture was stirred at room temperature for 2 hours. The mixture was then washed with DCM (30 mL × 3) and brine (30 mL), and the organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D6-1 (728 mg, 99%).

[0545] 99% yield

[0546] ESI-MS m / z:265(M+). 1H-NMR (400MHz, CDCl3) δ10.41 (s, 1H), 6.54 (s, 2H), 3.91 (s, 6H).

[0547] Preparation of compound OHPAS-D6-2 To a solution of compound OHPAS-D6-1 (101 mg, 0.38 mmol) and compound OHPAS-D1 (254 mg, 0.32 mmol) in acetonitrile (6 mL) was added BEMP (19 μl, 0.064 mmol) at room temperature. After 2 hours, the reaction mixture was diluted with aqueous citric acid (8 mL) and extracted with EtOAc (2 × 8 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to produce compound OHPAS-D6-2 (295 mg, 99%). ESI-MS m / z: 929 (M + ).

[0548] Compound OHPAS-D6 was synthesized by a synthetic route similar to that described in Example 3.3.

[0549] Preparation of compound OHPAS-D6-3 Yield 96%; ESI-MS m / z:931(M+).

[0550] Preparation of compound OHPAS-D6 Yield 75%; ESI-MS m / z:750(M+).

[0551] Example 3.6. Preparation of OHPAS-D7 [ka] Preparation of compound OHPAS-D7-1 To a solution of compound OHPAS-D1-3 (3 g, 5.22 mmol) in EA (240 mL), Pd / C (300 mg, 10 wt%) was added at 0 °C, and the mixture was stirred at room temperature for 3 hours while injecting H gas. After the reaction was completed, the mixture was filtered through Celite® and then concentrated under reduced pressure. Compound OHPAS-D7-1 was used directly in the next reaction without further purification (2.84 g, 100%, beige foam).

[0552] ET-MS m / z: 507.2 (M +1 +Na)

[0553] OHPAS-D7-2 was prepared by the same method as that for preparing compound OHPAS-D1 in Example 3.2.

[0554] Preparation of compound OHPAS-D7-2 Yield 84%, white solid; 1 H NMR(400MHz,CDCl3)δ 7.38-7.34(m,2H),7.00(d,J=8.0Hz,1H),6.82(d,J=5.2Hz,1H),6.10(brs,1H),5.49-5.45(m,2H),5.14(dd,J=3.6,10.4Hz,1H),4.9 9(d,J=7.6Hz,1H),4.27-4.08(m,3H),3.74-3.63(m,14H),3.37(t,J=5.2Hz,2H),2.20(s,3H),2.12(s,3H),2.08(s,3H),2.03(s,3H). ET-MS m / z:685.3(M +1 ).

[0555] OHPAS-D7-3 was prepared by a similar method to prepare compound OHPAS-D3-2 in Example 3.3.

[0556] Preparation of compound OHPAS-D7-3 Yield 81%, white solid; 1H NMR(400MHz,CDCl3)δ 7.80(d,J=2.0Hz,1H),7.76(dd,J=2.4,8.8Hz,1H),7.50(d,J=8.4Hz,2H)7.43-7.40(m,2H),7.37(d,J=2.0Hz,1 H),7.29-7.25(m,2H),7.08(d,J=4.8Hz,1H),6.90(d,J=8.4Hz,1H),5.60-5.56(m,1H),5.47(d,J=3.2Hz,1H),5 .17-5.10(m,4H),4.74(d,J=6.4Hz,2H),4.64(d,J=6.0Hz,2H),4.26-4.08(m,3H),3.71-3.58(m,14H),3.34(t, J=4.8Hz,2H), 2.41(t,J=6.4Hz,1H),2.18(s,3H),2.08(s,3H),2.07(s,3H),2.01(s,3H),1.77(t,J=6.0Hz,1H). ET-MS m / z:1007.2(M +1 ).

[0557] Preparation of compound OHPAS-D7-4 To a solution of compound OHPAS-D7-3 (150 mg, 0.15 mmol) in CHCl (3 mL) was added methanesulfonyl chloride (150 mg, 0.15 mmol) under N atmosphere at 0 °C. The reaction mixture was stirred at room temperature under N atmosphere for 24 hours. After the reaction was completed, the mixture was quenched with H0 (50 mL) and extracted with CHCl (50 mL × 3). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to produce compound OHPAS-D7-4 (214 mg, 100%) as a beige foam, which was used directly in the next step without further purification.

[0558] Preparation of compound OHPAS-D7-5 To a solution of compound OHPAS-D7-4 (214 mg, 0.15 mmol) in ACN (3 mL) was added potassium thioacetate (43 mg, 0.37 mmol) at room temperature under N atmosphere. After stirring at room temperature for 3 hours under N atmosphere, the mixture was quenched with HO (50 mL) and extracted with EA (50 mL × 3). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound OHPAS-D7 (147 mg, 88%) as a pale yellow foam.

[0559] 1 H NMR(400MHz,CDCl3)δ 7.87(d,J=2.0Hz,1H),7.78(dd,J=2.0,8.4Hz,1H),7.51(d,J=8.4Hz,2H),7.43-7.41(m,2H),7.31-7.27( m,2H),7.15(dd,J=2.0,8.0Hz,1H),7.07-7.06(m,1H),6.79(d,J=8.4Hz,1H),5.61-5.56(m,1H),5.47(d, J=3.2Hz,1H),5.17(d,J=8.0Hz,1H),5.14-5.10(m,3H),4.26-4.09(m,5H),4.05(s,2H),3.66-3.59(m,14 H),3.34(t,J=5.6Hz,2H),2.34(s,3H),2.32(s,3H),2.18(s,3H),2.08(s,3H),2.07(s,3H),2.01(s,3H). ET-MS m / z:1123.2(M +1 ).

[0560] Preparation of compound OHPAS-D7-6 To a solution of compound OHPAS-D7-5 (100 mg, 0.089 mmol) in ACN (2 mL), N-chlorosuccinimide (90 mg, 0.676 mmol) and 2 N HCl (356 μL, 0.712 mmol) were added under N atmosphere at 0 °C. After stirring at 0 °C for 1 h under N atmosphere, dimethyl sulfide (19.6 μL, 0.267 mmol) was added at room temperature. The reaction mixture was further stirred at the same temperature for 5 min. Extraction was performed with HO (20 mL) and EA (20 mL × 3), and the resulting organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to yield compound OHPAS-D7 (140 mg, 100%) as a white foam, which was used directly in the next step without further purification.

[0561] ET-MS m / z: 1173.9 (M +1 ).

[0562] Preparation of compound OHPAS-D7 To a solution of compound OHPAS-D7-6 (140 mg, 0.089 mmol) in ACN (2 mL), potassium bifluoride (41.7 mg, 0.534 mmol) in HO (0.2 mL) was added at room temperature under a N atmosphere. After stirring at room temperature for 2 hours, the mixture was purified by preparative HPLC to give compound OHPAS-D7 (42 mg, 41%) as a white foam.

[0563] 1H NMR(400MHz,CDCl3)δ 7.86(d,J=2.0Hz,1H),7.78(dd,J=2.0,8.4Hz,1H),7.53-7.43(m,6H),7.29(d,J=8.8Hz,1H),7 .13-7.11(m,1H),7.05(d,J=9.2Hz,1H),5.61-5.56(m,1H),5.48(d,J=2.4Hz,1H),5.20(s,2H) ,5.17(d,J=8.0Hz,1H),5.12(dd,J=3.2,10.4Hz,1H),4.78(d,J=3.6Hz,2H),4.26-4.09(m,3H) ,3.70-3.60(m,14H),3.5(t,J=5.2Hz,2H),2.18(s,3H),2.08(s,3H),2.07(s,3H),2.01(s,3H). ET-MS m / z:1139.1(M +1 ).

[0564] Example 3.7. Preparation of OHPAS-D9 and OHPAS-D10 [ka] Preparation of compound OHPAS-D9-1 A homogeneous solution of compound OHPAS-D1-5 (1.0 g, 0.26 mmol) and L-1 (586 mg, 2.0 mmol, 1.2 equiv.) in DMF (10 mL) was treated with PyBOP (1.13 g, 2.17 mmol, 1.3 equiv.) and DIPEA (873 μL, 5.01 mmol, 3.0 equiv.) at room temperature under a N atmosphere and stirred for 4 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 1:1 to 1:3) to give compound OHPAS-D9-1 (1.05 g, 72%) as a white foamy solid.

[0565] ESI-MS m / z: 874(M + +1).

[0566] Preparation of compound OHPAS-D9-2 A homogeneous solution of compound OHPAS-D9-1 (500 mg, 0.57 mmol) and compound OHPAS-D3-1 (140 mg, 0.69 mmol, 1.2 equiv.) in anhydrous ACN (10 mL) was treated with BEMP (66.3 μL, 0.23 mmol, 0.4 equiv.) at room temperature under a N atmosphere and stirred for 4 hours. The reaction was quenched with water (20 mL) and extracted with EA (30 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (4% MeOH in DCM) to give compound OHPAS-D9-2 (495 mg, 85%) as a white foamy solid.

[0567] ESI-MS m / z: 869(M + +1).

[0568] Preparation of compound OHPAS-D9-3 A solution of compound OHPAS-D9-2 (495 mg, 0.52 mmol) in anhydrous THF (5.0 mL) was treated with NaBH (39.7 mg, 1.05 mmol, 2.0 equiv.) under a N atmosphere at 0 °C and stirred for 2 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (2% to 3% MeOH in DCM) to give compound OHPAS-D9-3 (418 mg, 91%) as a white foamy solid.

[0569] ESI-MS m / z: 945(M + +1).

[0570] Preparation of compound OHPAS-D9-4 A solution of compound OHPAS-D9-3 (214.2 mg, 0.23 mmol) in anhydrous THF (5.0 mL) was treated with methanesulfonyl chloride (24.6 μL, 0.32 mmol, 1.4 equiv.) and TEA (79.2 μL, 0.57 mmol, 1.5 equiv.) under a N atmosphere at 0 °C and stirred at room temperature overnight. The reaction was quenched with water (10 mL) and extracted with DCM (20 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (100% DCM to 5% MeOH in DCM) to give compound OHPAS-D9-4 (164 mg, 70%) as a white foamy solid.

[0571] ESI-MS m / z: 1024(M + +1).

[0572] Preparation of compound OHPAS-D9 A solution of compound OHPAS-D9-4 (164 mg, 0.16 mmol) in anhydrous THF (10 mL) was treated with LiBr (69.6 mg, 0.80 mmol, 5.0 equiv.) under a N atmosphere at room temperature and stirred for 3 hours. The reaction was diluted with water (10 mL) and extracted with DCM (20 mL × 2). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (3% to 5% MeOH in DCM) to give compound OHPAS-D9 (161 mg, 99%) as a white foamy solid.

[0573] ESI-MS m / z: 1008(M + +1).

[0574] Compound OHPAS-D10 was synthesized in a manner similar to the preparation method of compound OHPAS-D9.

[0575] Preparation of compound OHPAS-D10-1 Yield 72%, colorless oil

[0576] ESI-MS m / z: 1226(M + +1).

[0577] Preparation of compound OHPAS-D10-2 Yield 82%, colorless oil

[0578] ESI-MS m / z: 1296(M + +1).

[0579] Preparation of compound OHPAS-D10-3 Yield 75%, colorless oil

[0580] ESI-MS m / z: 1298(M + +1).

[0581] Preparation of compound OHPAS-D10-4 Yield 82%, colorless oil

[0582] ESI-MS m / z: 1376(M + +1).

[0583] Preparation of compound OHPAS-D10 Yield 82%, colorless oil

[0584] ESI-MS m / z: 1361(M + +1).

[0585] Example 3.8. Preparation of OHPAS-D11 [ka] Compound OHPAS-D11 was synthesized in a manner similar to the preparation method of compound OHPAS-D3-1 in Example 3.3.

[0586] Preparation of compound OHPAS-D11 Yield 81%, white foamy solid

[0587] 1H NMR(400Hz,CDCl3)δ 7.88(s,1H),7.68(d,J=8.8Hz,1H),7.30(d,J=8.8Hz,1H),7.05(brs,1H),5.62-5.56(m,1H),5.48(d,J=2.8Hz 1H),5.17(d,J=8.0Hz,1H),5.12(dd,J=7.2,3.2Hz,1H),4.26-4.08(m,3H),3.72-3 .60(m,14H),3.36(t,J=4.8Hz,2H),2.20(s,3H),2.08(s,6H),2.02(s,3H);ESI-MS m / z:767(M + +1).

[0588] Example 3.9. Preparation of OHPAS-D12 and OHPAS-D13 [ka] Compound OHPAS-D12 was synthesized by a method similar to that described in Example 3.2. Compound OHPAS-D12-1 Yield 65%

[0589] 1 H NMR(400MHz,CDCl3)δ 10.32(s,1H),8.54(d,J=2.4Hz,1H),8.28(dd,J=8.8Hz,1H),7.45-7.35(m,5H),7.16( d,J=8.8Hz,1H),5.39-5.34(m,6H),4.28-4.26(m,1H),3.72(s,3H),2.11-2.06(m,9H).

[0590] Compound OHPAS-D12-2 Yield 63%

[0591] 1H NMR(400MHz, CDCl3)δ 7.66(d,J=2Hz,1H),7.60(dd,J=8.4Hz,1H),7.43-7.31(m,5H),7.00(d,J=8.4Hz,1H),6.13(s,1H),5.41-5 .28(m,5H),5.12(d,J=7.2Hz,1H),4.23(d,J=9.2Hz,1H),3.76(s,3H),2.09(s,3H),2.06(d,J=3.6Hz,6H).

[0592] Compound OHPAS-D12-3 Yield 70%

[0593] 1 H NMR(400MHz, CDCl3)δ 7.60(dd,J=2.0,2.0Hz,1H),7.43(d,J=0.8Hz,1H),7.48-7.32(m,5H),7.01(d,J=8. 4Hz,1H),5.40-5.26(m,6H),4.18(d,J=9.2Hz,1H),3.72(s,3H),2.09-2.04(m,9H). 0.99(s,9H),0.18(d,J=12.8Hz,1H).

[0594] Compound OHPAS-D12-4 Yield quantitative

[0595] ESI-MS m / z: 607 (M + +Na)

[0596] Compound OHPAS-D13-1 Yield 96%

[0597] 1 H NMR(400Hz,DMSO-d6)δ 9.73(brs,1H),7.44(d,J=2.0Hz,1H),7.37(dd,J=2.4,6.4Hz,1H),7.08(d,J=8.4Hz,1H),5.61(d,J=7. 6Hz,2H),5.45(t,J=9.6Hz,1H),5.15-5.02(m,2H),4.67(d,J=10Hz,1H)3.63(s,3H),2.04-1.98(m,9H).

[0598] ESI-MS m / z: 785(M + +1)

[0599] Compound OHPAS-D13-2 Yield 78%

[0600] ESI-MS m / z: 685(M + +1)

[0601] Compound OHPAS-D12 Yield: 85%.

[0602] ESI-MS m / z: 785(M + +1)

[0603] Compound OHPAS-D12a Yield 70%

[0604] ESI-MS m / z: 559(M + +1)

[0605] Example 4. Synthesis of drug derivatives Example 4.1.1 Preparation of Q-1 and Q-2 [ka] Q-1-1 and Q-2-1 were prepared from β-amanitin and α-amanitin by the same method as that for preparing compound OHPAS-D3-1 in Example 3.3.

[0606] Preparation of Compound Q-1-1 Yield 89%; ESI-MS m / z:1002(M+1).

[0607] Preparation of Compound Q-2-1 Yield 88%; ESI-MS m / z:1003(M+1).

[0608] Q-1-2 and Q-2-2 were prepared by the same method as that for preparing compound OHPAS-D3-2 in Example 3.3.

[0609] Preparation of Compound Q-1-2 Yield 62%; ESI-MS m / z:1666(M +1 ).

[0610] Preparation of Compound Q-2-2 Yield 41%; ESI-MS m / z:1667(M +1 ).

[0611] Preparation of Compound Q-1 To a solution of compound Q-1-2 (50 mg, 0.30 μmol) in MeOH (4 mL) was added KCO (21 mg, 1.5 μmol) under a N atmosphere at 0° C. After stirring for 0.5 h, the resulting residue was diluted with DMSO (0.5 mL) and purified by preparative HPLC to give compound Q-1 (10.5 mg, 19%) as a pale yellow solid.

[0612] ESI-MS m / z: 1498(M +1 ).

[0613] Preparation of Compound Q-2 61% yield in two steps; ESI-MS m / z: 1499 (M +1 ).

[0614] Preparation of Example 4.1.2.Q-1a Compound Q-1a was synthesized by a synthetic route similar to that described in Example 4.1.1. [ka] Preparation of Compound Q-1a Yield 83%; ESI-MS m / z:756(M / 2 +1 ).

[0615] Preparation of Example 4.2.Q-3 [ka] Preparation of Compound Q-3-1 To a solution of β-amanitin (40 mg, 43.5 μmol) in DMF (3 mL), N,N-dimethylethylenediamine (10 μL, 47.83 μmol), TBTU (46 mg, 0.11 mmol), and TEA (18 μL, 0.13 mmol) were added at room temperature. After stirring overnight at 40°C, the mixture was separated and purified by preparative HPLC to give compound Q-3-1 (28 mg, 65%).

[0616] ESI-MS m / z: 991 (M+1)

[0617] Preparation of Compound Q-3-2 To a solution of compound Q-3-1 (20 mg, 20.2 μmol) and OHPAS-D3 (30 mg, 24.2 μmol) in DMF (2 mL) was added DIPEA (11 μL, 60.6 mmol) dropwise under a N atmosphere. After stirring overnight at room temperature, the mixture was separated and purified by preparative HPLC to give compound Q-3-2 (34 mg, 65% yield), ESI-MS m / z: 992 (M / z). +1 ) was obtained.

[0618] Compound Q-3 was synthesized by a synthetic route similar to that described in Example 4.1.1.

[0619] Preparation of Compound Q-3 Yield 71%; ESI-MS m / z:838(M / 2 +1 )

[0620] Example 4.3. Preparation of Q-4 and Q-4a [ka] Preparation of Compound Q-4-1 To a solution of compound OHPAS-D4 (65 mg, 0.063 mmol) and MMAF-OMe (52 mg, 0.069 mmol) in DMF (1 mL), HOBt (2 mg, 0.013 mmol), DIPEA (12 μL, 0.069 mmol), and pyridine (330 μL) were added at room temperature under a N atmosphere. After stirring overnight, the mixture was adjusted to a pH of 2-3 with 1 N HCl and extracted with EA (8 mL × 2). The organic layer was washed with distilled water (8 mL) and brine (12 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound Q-4-1 (73 mg, 71%).

[0621] ESI-MS m / z: 1644(M +1 ).

[0622] Compound Q-4 was synthesized by a synthetic route similar to that described in Example 4.2.

[0623] Preparation of Compound Q-4 Yield 69%; ESI-MS m / z:1462(M +1 ).

[0624] Compound Q-4a was synthesized by a similar synthetic route as described above.

[0625] Preparation of Compound Q-4-1a Yield 99%; ESI-MS m / z:828(M / 2 +1 ).

[0626] Preparation of Compound Q-4a Yield 46%; ESI-MS m / z:738(M / 2 +1 ).

[0627] Preparation of Example 4.4.Q-5 [ka] Q-5-1 and Q-5-2 were synthesized by a synthetic route similar to that described in Example 3.5.

[0628] Preparation of Compound Q-5-1 Yield: 98%

[0629] 1H NMR(400MHz,CDCl3)δ 8.37(brs,1H)8.02(d,J=8.8Hz,1H),7.75(d,J=8.4Hz,1H),7.61(t,J=7.2,1H),7.51(t,J=8.0Hz,1H),4.32( brs,1H),4.18(t,J=8.8,1H),4.05(m,1H),3.93(dd,J=11.2,2.8Hz,1H),3.52(t,J=10.8Hz,1H),1.61(s,9H). ESI-MS m / z:438.2(M +1 +Na).

[0630] Preparation of Compound Q-5-2 Yield 79%

[0631] 1 H NMR (400 MHz, CDCl3) δ 8.09(brs,1H)7.77(m,3H),7.57(t,J=7.2Hz,1H),7.46(t,J=7.6Hz,1H),7.32( m,1H),6.78(m,1H),5.56(m,1H),5.46(d,J=2.8Hz,1H),5.22(d,J=7.6Hz,1H), 5.12(dd,J=10.4,3.2Hz,1H),4.30(brs,1H),4.25-4.02(m,5H),3.93(m,1H),3 .60(m,15H),3.31(m,2H),2.17(s,3H),2.04(s,3H),1.95(s,6H),1.56(s,9H). ESI-MS m / z: 1080.6(M +1 ).

[0632] Preparation of Compound Q-5 Compound Q-5-2 (50 mg, 0.046 mmol) was dissolved in 4 N HCl in 1,4-dioxane (1 mL) under N2 atmosphere at 0 °C. After stirring at room temperature for 4 h, the mixture was diluted with DCM (5 mL) and concentrated. Compound Q-5 was used directly in the next step without further purification (47 mg, 99%).

[0633] ESI-MS m / z: 980.5(M +1 ).

[0634] Preparation of Example 4.5.Q-6 [ka] Preparation of Compound Q-6a Compound Q-6a was synthesized by a synthetic route similar to that described in the literature [see Mol.Pharmaceutics 2015, 12, 1813-1835].

[0635] Preparation of Compound Q-6-1 Compound Q-6-1 was synthesized by a synthetic route similar to that described in the literature [see Angew. Chem. Int. Ed. 2010, 49, 7336-7339 and WO2015 / 110935A1].

[0636] Preparation of Compound Q-6-2 To a solution of compound Q-6a (80 mg, 0.239 mmol) and compound Q-6-1 (118 mg, 0.239 mmol) in DCM (10 mL) was added molecular sieves and BF OEt (14.8 μL, 0.12 mmol) under a N atmosphere at 0 °C. After stirring for 2 h, the mixture was filtered through Celite®, washed with DCM (50 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Q-6-2 (105 mg, 66%) as a white foam.

[0637] 1H NMR(400MHz,CDCl3)δ 8.12(d,J=8.0H,1H),7.89(brs,1H),7.63(d,J=8.0Hz,1H),7.50(m,1H),7.35(m,1H),5.70(m,1H),5.51(s,1H),5.33(m,1H),5.20(m, 1H),4.23(m,3H),4.11(m,2H),3.93(m,2H),3.42(t,J=10.8Hz,1H),2.18(s,3H),2.08(s,3H),2.04(s,3H),2.00(s,3H),1.55(s,9H). ESI-MS m / z:564.4(M +1 ).

[0638] Preparation of Compound Q-6-3 Compound Q-6-2 (100 mg, 0.15 mmol) was dissolved in DCM (2 mL), and then 4N HCl in 1,4-dioxane (1 mL) was added at 0 °C under an atmosphere of N. After stirring for 4 h, the reaction was concentrated under reduced pressure. The reaction mixture was stirred at room temperature under N for 4 h. Compound Q-6-2 (90 mg, 99%) was used directly in the next step without further purification.

[0639] ESI-MS m / z: 564.2 (M +1 ).

[0640] Preparation of Compound Q-6-4 To a solution of compound Q-6-3 (90 mg, 0.149 mmol) in THF (5 mL), glutaric anhydride (18.8 μL, 0.164 mmol), EtN (52 μL, 0.373 mmol), and 4-DMAP (2 mg, 0.015 mmol) were added at room temperature under a N atmosphere. The reaction mixture was stirred at room temperature for 2 hours and purified by preparative HPLC to give compound Q-6-4 (30 mg, 30%) as a white solid.

[0641] Preparation of Compound Q-6-5 To a solution of compound Q-6-4 (30 mg, 0.043 mmol) and compound Q-5 (51 mg, 0.05 mmol) in DMF (3 mL), EDC·HCl (27.2 mg, 0.142 mmol) was added under a N atmosphere at 0° C. After stirring for 11 h, the mixture was purified by preparative HPLC to give compound Q-6-5 (20 mg, 28%) as a light brown solid.

[0642] ESI-MS m / z: 821.7(M +1 / 2).

[0643] Preparation of Compound Q-6 To a solution of compound Q-6-5 (10 mg, 0.006 mmol) in MeOH (1.5 mL) was added 25% NaOMe in MeOH (11 μL, 0.048 mmol) under N atmosphere at 0 °C. The reaction mixture was stirred at room temperature under N atmosphere for 1 hour and adjusted to pH 7 by adding 5% TFA in ACN. The mixture was purified by preparative HPLC to give compound Q-6 (5 mg, 63%) as a pale yellow solid.

[0644] ESI-MS m / z: 1305.3 (M +1 ).

[0645] Preparation of Example 4.6.Q-7 [ka] Preparation of Compound Q-7a To a solution of PNU-159682 (52 mg, 0.081 mmol) in MeOH (5 mL) / distilled water (3 mL) was added NaIO (18 mg, 0.081 mmol) at room temperature. After stirring for 2 hours, the mixture was concentrated under reduced pressure to yield crude compound Q-7a (51 mg, 99%). ESI-MS m / z: 628 (M +1 ).

[0646] Preparation of Compound Q-7b To a solution of compound Q-7a (51 mg, 0.081 mmol) in dry DCM (5 mL) were added 2-(dimethylamino)ethylamine (6.1 μL, 0.089 mmol), TEA (34 μL, 0.243 mmol), and TBTU (52 mg, 0.162 mmol) at room temperature. After stirring for 1 h, the mixture was diluted with DCM (2 × 8 mL). The organic layer was washed with HO (8 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to yield compound Q-7b (38 mg, 67%).

[0647] ESI-MS m / z: 698(M +1 ).

[0648] Q-7 was prepared by a similar method to prepare compound Q-3-2 in Example 4.2.

[0649] Preparation of Compound Q-7-1 Yield 38%; ESI-MS m / z:1551(M +1 ).

[0650] Preparation of Compound Q-7 Yield 54%; ESI-MS m / z:1383(M +1 ).

[0651] Preparation of Example 4.7.Q-8 [ka] Compound Q-8 was synthesized by a synthetic route similar to that described in Example 4.6.

[0652] Preparation of Compound Q-8-1 Yield 42%; ESI-MS m / z:837(M / 2 +1 ).

[0653] Preparation of Compound Q-8 Yield 81%; ESI-MS m / z:746(M / 2 +1 ).

[0654] Example 4.8. Preparation of Benzodiazepine Monomer Derivatives Example 4.8.1 Preparation of Pyrrolo-benzodiazepine Monomer (hereinafter "PBD-Monomer") [ka] The PBD monomer was obtained by carrying out the reaction in a similar manner as described in EP2007 / 1813614.

[0655] Example 4.8.2 Preparation of Indolino-benzodiazepine Monomers (hereinafter "IBD-Monomers") [ka] The IBD monomer was obtained by carrying out the reaction in a similar synthetic manner as described in WO2010 / 091150.

[0656] Example 4.8.3 Preparation of MCBI-monomer [ka] The IBD monomer was obtained by carrying out the reaction in a similar synthetic manner to that described in US Pat. No. 5,985,908.

[0657] Example 4.8.4 Preparation of Tetrahydroisoquinolino-benzodiazepine Monomer (hereinafter "TBD Monomer") [ka] Preparation of Compound M-1-1 To a solution of (s)-(-)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5.0 g, 28.22 mmol) in MeOH (140 mL), SOCl (2.30 mL, 31.04 mmol) was added dropwise under a N atmosphere until the temperature reached 0 °C. After stirring at 40 °C for 21 h, the mixture was concentrated under reduced pressure. Diethyl ether (50 mL) was added to obtain a precipitate, which was filtered with diethyl ether to obtain compound M-1-1 (6.42 g, 99% yield).

[0658] 1H NMR(400MHz,DMSO-d6)δ 10.02(s,2H),7.27(s,4H),4.60-4.56(m,1H),4.39-4.29(m,2H),3.82(s,3H),3.19-3.12(m,2H);ESI-MS m / z:192(M + +1).

[0659] Preparation of Compound M-1-2 To a solution of compound Int-1 (9.07 g, 28.22 mmol) in anhydrous THF (50 ml), compound M-1-1 (6.42 g, 28.22 mmol) and TEA (7.9 mL, 56.43 mmol) in THF (100 mL) were added at 0° C. After stirring at room temperature for 2 h, the reaction mixture was diluted with distilled water (500 mL) and extracted with EA (800 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound M-1-2 (12.01 g, 90%). ESI-MS m / z: 477 (M + +1).

[0660] Preparation of Compound M-1-3 To a solution of compound M-1-2 (4 g, 8.39 mmol) in anhydrous DCM (18 mL) and toluene (52 mL) was added DIBAL (16.8 mL, 16.79 mmol, 1.0 M in toluene) dropwise at −78° C. under a N atmosphere. After stirring at −78° C. for 4 h, the reaction was quenched with MeOH (0.4 mL) and 2 N HCl (25 mL) at −78° C. The mixture was diluted with water (100 mL) and extracted with EA (500 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound M-1-3 (3.07 g, 82%). ESI-MS m / z: 447 (M + +1).

[0661] Preparation of Compound M-1-4 To a solution of compound M-1-3 (3 g, 6.72 mmol) in THF (130 mL) and distilled water (86 mL), NaSO·2H0 (11.3 g, 53.76 mmol) was added at room temperature. After stirring for 5 h, the reaction mixture was concentrated under reduced pressure four times using toluene as a cosolvent to remove water. The resulting yellow solid was then dissolved in anhydrous MeOH (220 mL), and acetyl chloride (4.8 mL, 67.19 mmol) was added to it. After stirring for 15 min, the reaction mixture was adjusted to pH 7 by adding saturated NaHCO solution, diluted with distilled water (100 mL), and extracted with EA (250 mL × 2). The organic layer was dried over anh...

Claims

1. 1. An antibody conjugate represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof: During the ceremony, the Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof, comprising variable heavy chain complementarity determining region 1 (CDRH1), variable heavy chain complementarity determining region 2 (CDRH2), variable heavy chain complementarity determining region 3 (CDRH3), variable light chain complementarity determining region 1 (CDRL1), variable light chain complementarity determining region 2 (CDRL2), and variable light chain complementarity determining region 3 (CDRL3); CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; CDRH2 comprises the amino acid sequence of SEQ ID NO:2; CDRH3 comprises the amino acid sequence of SEQ ID NO:3; CDRL1 comprises the amino acid sequence of SEQ ID NO:4; CDRL2 comprises the amino acid sequence of SEQ ID NO:5; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 6; or CDRH1 comprises the amino acid sequence of SEQ ID NO:7; CDRH2 comprises the amino acid sequence of SEQ ID NO:8; CDRH3 comprises the amino acid sequence of SEQ ID NO:9; CDRL1 comprises the amino acid sequence of SEQ ID NO: 10; CDRL2 comprises the amino acid sequence of SEQ ID NO: 11; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 12; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 13; CDRH2 comprises the amino acid sequence of SEQ ID NO: 14; CDRH3 comprises the amino acid sequence of SEQ ID NO: 15; CDRL1 comprises the amino acid sequence of SEQ ID NO: 16; CDRL2 comprises the amino acid sequence of SEQ ID NO: 17; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 18; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; CDRL2 comprises the amino acid sequence of SEQ ID NO:23; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 24; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 25; CDRH2 comprises the amino acid sequence of SEQ ID NO: 26; CDRH3 comprises the amino acid sequence of SEQ ID NO:27; CDRL1 comprises the amino acid sequence of SEQ ID NO:28; CDRL2 comprises the amino acid sequence of SEQ ID NO:29; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 30; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 31; CDRH2 comprises the amino acid sequence of SEQ ID NO: 32; CDRH3 comprises the amino acid sequence of SEQ ID NO: 33; CDRL1 comprises the amino acid sequence of SEQ ID NO: 34; CDRL2 comprises the amino acid sequence of SEQ ID NO: 35; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 36; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 37; CDRH2 comprises the amino acid sequence of SEQ ID NO: 38; CDRH3 comprises the amino acid sequence of SEQ ID NO: 39; CDRL1 comprises the amino acid sequence of SEQ ID NO: 40; CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 42; or CDRH1 comprises the amino acid sequence of SEQ ID NO: 43; CDRH2 comprises the amino acid sequence of SEQ ID NO: 44; CDRH3 comprises the amino acid sequence of SEQ ID NO: 45; CDRL1 comprises the amino acid sequence of SEQ ID NO: 46; CDRL2 comprises the amino acid sequence of SEQ ID NO:47; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 48; AND, IN ADDITION: each G is independently a chemical moiety comprising one or more active agents and a linker, said linker connecting Ab to said active agents; An antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein n is an integer from 1 to 20.

2. Ab is, (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 50; (b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52; (c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 54; (d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 56; (e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 58; (f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60; (g) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 62; and (h) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 64 2. The antibody conjugate of claim 1, further comprising a combination of variable heavy and light chain sequences selected from:

3. 2. The antibody conjugate of claim 1, wherein the anti-B7-H3 antibody is AB1, AB2, AB3, AB4, AB5, AB6, AB7, AB8, AB1.1, AB2.1, AB3.1, AB4.1, AB5.1, AB6.1, AB7.1, or AB8.

1.

4. The antibody conjugate of any one of claims 1 to 3, wherein the B7-H3 is human B7-H3.

5. Abs can be monoclonal antibodies, domain antibodies (dAbs), single chain antibodies (scAbs), Fab fragments, F(ab') ２ 2. The antibody conjugate of claim 1, which is a fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, or a multimeric antibody.

6. 6. The antibody conjugate of any one of claims 1 to 3 and 5, wherein the Ab is a rabbit, murine, chimeric, humanized, or fully human monoclonal antibody.

7. 6. The antibody conjugate of claim 1, wherein the Ab is of the IgG isotype.

8. 6. The antibody conjugate of claim 1, wherein the Ab is of the IgG1 isotype.

9. 6. The antibody conjugate of claim 1, wherein the linkage between Ab and the active agent is cleavable.

10. G is represented by Formula II: 【Chemistry 1】 During the ceremony, each Q' is independently an active agent linked to L' by a heteroatom; Z' is a linking group; L' is a heteroatom selected from O, S, and N, and is connected to SO ２ and L′ and SO ２ cleavage of the bond between L′ and Q′ promotes cleavage of the bond between L′ and Q′, releasing the active agent; X is —O—, —C(R ｂ ) ２ -, or -N(R ｃ ) - and Ar represents aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR ｂ ２ ) ｙ N (R ａ ) -, -(CR ｂ ２ ) ｙ O- or -(CR ｂ ２ ) ｙ S- and when y is 1, the N, O, or S atom is positioned to be bonded to TG; X and Y' are located on adjacent atoms of Ar; When TG is activated, SO ２ It reacts with (Q') ｑ -(L') ｗ Replace with X-SO ２ and a trigger group that generates an N, O, or S atom that can form a 5- or 6-membered ring containing the intervening atom of Ar; q is an integer having a value from 1 to 20; w, x, and y are each independently an integer having a value of 0 or 1; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently hydrogen or lower alkyl; Two R's ｂ form a 3- to 5-membered ring together with the atoms to which they are attached, 2. The antibody conjugate of claim 1, wherein when w is 0, q is 1.

11. Ab-(G) ｎ is a compound of formula (III) 【Chemistry 2】 or a salt thereof, wherein: A is, 【Transformation 3】 and M, N, CR ３０ or C(-L-Q), each L is independently selected from a spacer moiety; each Q is an active agent; J is the anti-B7-H3 antibody or antigen-binding fragment thereof; R ３０ and R ３１ are each independently selected from an electron withdrawing group, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R ４２ and R ４３ each independently represents —OH, alkoxy, or —NR ４４ R ４５ , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, and heterocyclyl, wherein R ４４ and R ４５ can be combined with the nitrogen atom to which they are attached to form a 5- to 8-membered ring, R ３２ , R ４４ , and R ４５ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; The antibody conjugate of claim 1, wherein n is 1 to 4.

12. Z' is selected from: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 During the ceremony, R ｚａ is H or methyl, R ｚｂ is —OH, ═O, or ═NHOH; 【Transformation 5】 represents a single bond or a double bond, a" represents the bond between Z' and Ar in formula (II); b" represents the bond between Z' and Ab; m is 1 to 20; Z” is, Oriented in either direction 【Transformation 6】 The antibody conjugate of claim 10, wherein the antibody conjugate is selected from the group consisting of:

13. G comprises a moiety selected from: 【Transformation 7】 During the ceremony, Q is an active agent; 【Transformation 8】 13. The antibody conjugate of claim 10, wherein is a fragment of Z' connecting Z' to Ar.

14. G comprises a moiety selected from: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 【Chemistry 9-4】 【Chemistry 9-5】 【Chemistry 9-6】 【Chemistry 9-7】 【Chemistry 9-8】 【Chemistry 9-9】 【Chemistry 10】 13. The antibody conjugate of claim 10, wherein is a fragment of Z' connecting Z' to Ar.

15. 13. The antibody conjugate of any one of claims 1 to 3, 5 and 10 to 12, wherein the active agent is selected from a chemotherapeutic agent and a toxin.

16. 13. The antibody conjugate of any one of claims 1 to 3, 5 and 10 to 12, wherein the active agent is a chemotherapeutic agent.

17. 13. The antibody conjugate of any one of claims 1 to 3, 5 and 10 to 12, wherein the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an analgesic agent, or a combination thereof.

18. The active agent is (a) erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide amide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, es Tramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antimycin cin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin,Marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate propionate), epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, mitoxantrone, modipanol, nitraerine, penicillin, anthracycline, phenamt, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verrucarin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum,etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid of any of the foregoing; (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic factor, interferon interferon-α, interferon-β, interferon-γ, colony stimulating factors ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate , exatecan, exatecan derivatives, vindesine, SG2285, dolastatin, dolastatin analogs, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, azonafide, aplidine, toxoids, or combinations of any of the foregoing; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulant, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) a radioactive label; ３２ P. ３５ S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the above; (f) an immunomodulatory compound, an anti-cancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, and an antiparasitic agent, or a combination of any of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine, (j) aromatase inhibitors, (k) a protein kinase inhibitor, (l) lipid kinase inhibitors, (m) antisense oligonucleotides; (n) a ribozyme; (o) vaccines, and (p) an anti-angiogenic agent selected from the group consisting of: an antibody conjugate according to any one of claims 1 to 3, 5, and 10 to 12;

19. The active agent is a compound listed in Tables 3-5, auristatin F, PNU, α-amanitin, Q-α-amanitin, β-amanitin, CBI indole, CBI dimer, (CA4-CA4), (CA4-SN38), fenpanstatin, exatecan, dPBD, Q-dPBD, dTBD, Q-dTBD, adTBD, adTBD DMBA, dTBD alkylamine, dThBD, dThBD, Q-dThBD NaSO ３ , dImBD, Q-dFuBD, and ImBD-TBD.

20. A pharmaceutical composition comprising an antibody conjugate according to any one of claims 1 to 3, 5 and 10 to 12.

21. 21. The pharmaceutical composition of claim 20, further comprising a therapeutically effective amount of a chemotherapeutic agent.

22. 21. Use of an antibody conjugate according to any one of claims 1 to 3, 5 and 10 to 12, or a pharmaceutical composition according to claim 20, in the manufacture of a medicament for treating cancer.

23. 23. The use of claim 22, wherein the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, and melanoma.

24. 21. The pharmaceutical composition of claim 20 for use in the treatment of cancer.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, and melanoma.

26. 21. Use of an antibody conjugate according to any one of claims 1 to 3, 5 and 10 to 12, or a pharmaceutical composition according to claim 20, in the manufacture of a medicament for treating an autoimmune or inflammatory disease.

27. 27. The use of claim 26, wherein the autoimmune disease or the inflammatory disease is selected from B-cell mediated autoimmune or inflammatory diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and lupus nephritis.

28. 21. The pharmaceutical composition of claim 20 for use in the treatment of an autoimmune or inflammatory disease.

29. 29. The pharmaceutical composition of claim 28, wherein the autoimmune disease or the inflammatory disease is selected from B-cell mediated autoimmune diseases or inflammatory diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and lupus nephritis.

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