Antibody-drug conjugate containing anti-B7-H3 antibody

Abstract

This provides an improved antibody-drug conjugate that targets B7-H3. [Solution] An antibody conjugate represented by formula I, or a pharmaceutically acceptable salt or solvate thereof, is provided, wherein Ab is an anti-B7-H3 antibody or its antigen-binding fragment, comprising a variable heavy chain complementarity determination region 1 (CDRH1), a variable heavy chain complementarity determination region 2 (CDRH2), a variable heavy chain complementarity determination region 3 (CDRH3), a variable light chain complementarity determination region 1 (CDRL1), a variable light chain complementarity determination region 2 (CDRL2), and a variable light chain complementarity determination region 3 (CDRL3), and each G is independently a chemical moiety comprising one or more active agents and a linker, the linker linking Ab to the active agent, and n being an integer from 1 to 20. TIFF2026053657000304.tif14160

Description

[Technical Field]

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

[0002] Antibody-drug conjugates (ADCs) combine the binding specificity of an antibody with the potency of a chemotherapeutic agent. ADC technology allows for precise delivery of drugs to target cancer cells and release under specific conditions while minimizing collateral damage to healthy cells. Therefore, ADC technology increases the effectiveness of therapeutic antibodies and reduces the risk of adverse reactions.

[0003] B7-H3 (CD276) is a novel member of the B7 family, sharing up to approximately 30% sequence homology. Initially introduced as a T cell co-stimulatory molecule, B7-H3 has proven to be a co-inhibitory checkpoint ligand capable of regulating helper T cells, anotoxic T cells, and natural killer cells in human immunity. B7-H3 protein expression is very limited in normal tissues, but is induced on the cell surface of antigen-presenting cells and is widespread in various solid tumors, including primary and metastatic cancers. B7-H3 expression is also detected in multiple cancer cell types, including cancer stem cells and tumor vascular systems. B7-H3 overexpression appears to be deeply correlated with disease severity and poor clinical outcomes in tumors.

[0004] Therefore, there is a need for improved antibody-drug conjugates that target B7-H3. [Overview of the Initiative]

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

[0006] This disclosure provides monoclonal antibodies and antigen-binding fragments, or any fragments, variants, polymeric forms, or their bispecificity, that bind to B7-H3. These antibodies and antigen-binding fragments, or any fragments, variants, polymeric forms, or their bispecificity, are collectively referred to herein as anti-B7-H3 monoclonal antibodies, anti-B7-H3 mAbs, or antigen-binding fragments, or any fragments, variants, polymeric forms, or their bispecificity. Preferably, the monoclonal antibodies and antigen-binding fragments, or any fragments, variants, polymeric forms, or their bispecificity, are specific to at least human B7-H3. In some embodiments, the monoclonal antibody and antigen-binding fragment that recognize human B7-H3, or any fragment, variant, multimer, or bispecificity thereof, is also cross-reactive to at least one other non-human B7-H3 protein, for example, non-human primate B7-H3, e.g., cynomolgus monkey B7-H3, and / or rodent B7-H3, as an example.

[0007] In some embodiments, the disclosure relates to an antibody-drug conjugate (ADC) 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 include a branched unit, at least one drug being coupled to the branched unit via a secondary linker, and the branched unit being coupled to the antibody by a primary linker. The primary and / or secondary linker may include at least one polyethylene glycol unit.

[0008] In some embodiments, the present disclosure relates to an antibody conjugate represented by formula I, or a pharmaceutically acceptable salt or solvate thereof, TIFF2026053657000001.tif13160 formula, Ab is an anti-B7-H3 antibody or its antigen-binding fragment, comprising variable heavy chain complementarity determination region 1 (CDRH1), variable heavy chain complementarity determination region 2 (CDRH2), variable heavy chain complementarity determination region 3 (CDRH3), variable light chain complementarity determination region 1 (CDRL1), variable light chain complementarity determination region 2 (CDRL2), and variable light chain complementarity determination region 3 (CDRL3). CDRH1 contains the amino acid sequence of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, or 43. CDRH2 contains the amino acid sequence of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, or 44. CDRH3 contains the amino acid sequence of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, or 45. CDRL1 contains the amino acid sequence of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, or 46. CDRL2 contains the amino acid sequence of SEQ ID NOs. 5, 11, 17, 23, 29, 35, 41, or 47. CDRL3 contains the amino acid sequence of SEQ ID NOs. 6, 12, 18, 24, 30, 36, 42, or 48. Each G is independently a chemical part containing an active agent and a linker, the linker linking Ab to the active agent, This relates to antibody conjugates, or pharmaceutically acceptable salts or solvates thereof, where n is an integer between 1 and 20. [Brief explanation of the drawing]

[0009] [Figure 1] The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in JIMT-1 is shown. [Figure 2]The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in JIMT-1 is shown. [Figure 3] The IC50 generated using a sigmoid dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-N87 is shown. [Figure 4] The IC50 generated using a sigmoid dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in HCT-116 is shown. [Figure 5] The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in HCT-116 is shown. [Figure 6] The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in NCI-H23 is shown. [Figure 7] The IC50 generated using a sigmoid dose-response nonlinear regression fit (GraphPad software Inc.) for T-Int-102-D1-5 AB2.1 in NCI-H460 is shown. [Figure 8] The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-H23 is shown. [Figure 9] The IC50 generated using a sigmoid dose-response nonlinear regression curve fit (GraphPad software Inc.) for T-Int-112-AB2.1 in NCI-H460 is shown. [Figure 10]This study demonstrates the effects of T-20-AB2.1 and T-21-AB2.1 on tumor volume in JIMT-1 xenografts. [Figure 11] This study demonstrates the effects of T-20-AB2.1 and T-21-AB2.1 on body weight in JIMT-1 xenografts. [Figure 12] This study demonstrates the effects of T-Int-102-D1-5 AB2.1 and T-Int-112-AB2.1 on tumor volume in HCT-116 xenografts. [Figure 13] This study demonstrates the effects of T-Int-102-D1-5 AB2.1 and T-Int-112-AB2.1 on body weight in HCT-116 xenografts. [Figure 14] This study demonstrates the effect of T-Int-112-AB2.1 on tumor volume in NCI-H23 xenografts. [Figure 15] This demonstrates the effect of T-Int-112-AB2.1 on body weight in NCI-H23 xenografts. [Figure 16] This study demonstrates the effect of T-Int-112-AB2.1 on tumor volume in NCI-H460 xenografts. [Figure 17] This study demonstrates the effect of T-Int-112-AB2.1 on body weight in NCI-H460 xenografts. [Modes for carrying out the invention]

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

[0011] B7-H3 (CD276) is a member of the B7 family and exhibits high sequence homology (up to approximately 30%) with this family. While B7-H3 expression is very limited in normal tissues, it is 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, as well as tumor-associated vascular and stroma. Furthermore, B7-H3 overexpression is correlated with poor outcomes in many cancers. High B7-H3 expression, common in NSCLC (approximately 85%), is associated with metastasis and advanced stage. Even higher incidence and expression levels of B7-H3 have been observed in cancers resistant to anti-PD-1 therapy. Therefore, targeting B7-H3 is appropriate for relapsed or refractory NSCLC. A series of anti-B7-H3 ADCs were prepared and tested. The main components of ADCs are benzodiazepines with an OHPAS linker and an OHPAS-compatible functional group. When plasma stability is demonstrated, ADCs efficiently release toxins in target tumor cells, suggesting the potential for an extended therapeutic window. ADCs demonstrate superior efficacy with minimal in vivo weight changes, offering a new option for patients with refractory NSCLC to anti-PD-1 therapy.

[0012] A series of tightly binding anti-B7-H3 mAbs and their thiomagne forms were generated (Kd approximately 1.7-5.4 × 10⁻⁶). -11 M). A series of anti-B7-H3 ADCs were prepared and tested using a newly discovered OHPAS linker and an OHPAS-compatible benzodiazepine payload. An exemplary OHPAS linker is further described herein and is also disclosed, for example, in International Patent Application Publication 2019 / 008441, which is incorporated herein in its entirety by reference. An exemplary OHPAS-compatible benzodiazepine payload is further described herein and is also disclosed, for example, in U.S. Patent Application Publication 2019 / 0367488, which is incorporated herein in its entirety by reference. The ADCs were highly potent in vitro against B7-H3 positive tumor cell lines. The ADCs were effective when tested in a mouse xenograft model of NSCLC.

[0013] The ADCs disclosed herein can target specific 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 shows a good correlation with disease severity and poor outcomes. B7-H3 is highly and strongly expressed at high frequencies across a wide range of tumors. Targeting B7-H3 for cancer therapy is beneficial due to its expression in cancer stem cell populations, tumor vascular systems, and stroma (Journal of Clinical Oncology 35, no.15_suppl). Both tumor cells and tumor vascular systems are B7-H3 (CD276) positive (Cancer Cell. 2017 Apr 10;31(4):501-515.e8). The disclosed B7-H3 antibody possesses improved internalization capabilities, as confirmed by Fab-Assay. Therefore, the improved antibody-drug conjugates disclosed herein, targeting B7-H3, are expected to be useful in methods of treating or alleviating cancer-related symptoms.

[0014] Examples of B7-H3 antibodies and their uses are listed in Table 1. [Table 1] TIFF2026053657000003.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 exhibit opposing co-stimulatory and co-inhibitory functions (Immunological Reviews 2017;276:52-65). B7-H3 is highly expressed in tumor tissue (Figures 26A-26B). B7-H3 expression has been 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 hematopoietic cell lines (Tissue Antigens 2005:66:83-92). 44.8% of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cases express B7-H3, and 65% of mantle cell lymphoma (MCL) cases express B7-H3. B cells, T cells, and monocytes do not express B7-H3 (CMI 2005 2(4)307-311). B7-H3 is inductively expressed in macrophages, DCsm, 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] This disclosure also provides monovalent and / or bispecific antibodies comprising at least a first arm specific to B7-H3. Preferably, the monovalent and / or bispecific antibodies are specific to at least human B7-H3. In some embodiments, the monovalent and / or bispecific antibodies that recognize human B7-H3 are also cross-reactive to at least one other non-human B7-H3 protein, e.g., non-limiting examples, non-human primate B7-H3, e.g., cynomolgus monkey B7-H3, and / or rodent B7-H3. This disclosure also provides antibodies that bind to the same epitopes as the anti-B7-H3 monovalent and / or anti-B7-H3 bispecific antibodies disclosed herein.

[0018] Exemplary anti-B7-H3 monoclonal antibodies and their antigen-binding fragments described herein include, for example, the antibodies listed in Tables 19-24.

[0019] In some embodiments, the exemplary anti-B7-H3 monoclonal antibodies and their antigen-binding fragments of this disclosure include a combination of a heavy chain complementarity-determining region (CDR) selected from the CDR sequences shown in Table 19 and a light chain CDR selected from the CDR sequences shown in Table 19. In some embodiments, the exemplary anti-B7-H3 monoclonal antibodies and their antigen-binding fragments of this disclosure include a combination of variable sequences of the heavy chain domain and light chain domain shown in Tables 20-24. In some embodiments, the exemplary anti-B7-H3 monoclonal antibodies of this disclosure include a combination of variable sequences of the heavy chain domain and light chain domain shown in Tables 21-24.

[0020] Antibody-drug conjugates In certain embodiments, the antibody-drug conjugates disclosed herein are represented by Formula I or a pharmaceutically acceptable salt or solvate thereof. TIFF2026053657000004.tif13160 formula, Ab is an anti-B7-H3 antibody or its antigen-binding fragment, comprising variable heavy chain complementarity determination region 1 (CDRH1), variable heavy chain complementarity determination region 2 (CDRH2), variable heavy chain complementarity determination region 3 (CDRH3), variable light chain complementarity determination region 1 (CDRL1), variable light chain complementarity determination region 2 (CDRL2), and variable light chain complementarity determination region 3 (CDRL3). CDRH1 contains the amino acid sequence of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, or 43. CDRH2 contains the amino acid sequence of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, or 44. CDRH3 contains the amino acid sequence of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, or 45. CDRL1 contains the amino acid sequence of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, or 46. CDRL2 contains the amino acid sequence of SEQ ID NOs. 5, 11, 17, 23, 29, 35, 41, or 47. CDRL3 contains the amino acid sequence of SEQ ID NOs. 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 Ab to the active agent. n is an integer between 1 and 20.

[0021] In some embodiments, Ab is a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, an F(ab')2 fragment, a single-chain variable fragment (scFv), an scFv-Fc fragment, a single-domain heavy-chain antibody, a single-domain light-chain antibody, a variant antibody, a multimeric antibody, or a bispecific antibody. Ab may be a rabbit, mouse, chimeric, humanized, or fully human monoclonal antibody. In some embodiments, Ab is an IgG isotype, for example, an IgG1 isotype.

[0022] In some embodiments, Ab includes a combination of a variable heavy chain containing the amino acid sequence of SEQ ID NOs. 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81, and a variable light chain containing the amino acid sequence of SEQ ID NOs. 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 containing the amino acid sequence of SEQ ID NO: 49 and a variable light chain containing the amino acid sequence of SEQ ID NO: 50, (b) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a variable light chain containing the amino acid sequence of SEQ ID NO: 52, (c) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a variable light chain containing the amino acid sequence of SEQ ID NO: 54. (d) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 55 and a variable light chain containing the amino acid sequence of SEQ ID NO: 56, (e) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 57 and a variable light chain containing the amino acid sequence of SEQ ID NO: 58, (f) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a variable light chain containing the amino acid sequence of SEQ ID NO: 60, (g) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 61, and a variable light chain containing the amino acid sequence of SEQ ID NO: 62, (h) A combination of a variable heavy chain sequence and a variable light chain sequence selected from a variable heavy chain containing the amino acid sequence of SEQ ID NO: 63 and a variable light chain containing the amino acid sequence of SEQ ID NO: 64.

[0024] In some embodiments, 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 includes Ab, at least one branched linker covalently coupled to Ab, and at least two active agents covalently coupled to the branched linker. In some embodiments, at least two branched linkers are coupled to Ab, and each branched linker is coupled to at least two active agents. In some embodiments, three branched linkers are coupled to Ab. In other embodiments, four branched linkers are coupled to Ab. In yet another embodiment, exactly one branched linker is coupled to Ab. In yet another embodiment, each branched linker is coupled to exactly two active agents. In some embodiments, the conjugate includes 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 a branched linker by a cleavable (e.g., hydrolyzable) bond. In some embodiments, each branched linker contains a branched unit, each active agent is linked to the branched unit via a secondary linker, and the branched unit is coupled to an anti-B7-H3 antibody by a primary linker. In some embodiments, the branched unit is a nitrogen atom, e.g., a nitrogen atom of an amine or amide. In some embodiments, the branched unit is an amide, and the primary linker contains the carbonyl group of the amide. In some embodiments, the branched unit is an amide, and the secondary linker contains the carbonyl group of the amide. In some preferred embodiments, the branched unit is a lysine unit.

[0027] Linker and Conjugation Partners In some preferred embodiments, each G is independently a group having the structure of formula (II), [ka] Each Q is an active agent independently linked to L' via a heteroatom, preferably O or N. Z' is a linking group. L' is a spacer moiety bonded to SO2 via a heteroatom selected from O, S, and N, preferably O or N, and the cleavage of the bond between L' and SO2 is selected to facilitate the cleavage of the bond between L' and Q to release 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 arranged to be bonded to TG. X and Y' are arranged on adjacent atoms of Ar. TG is a trigger group that, when activated, reacts with SO2 to replace (Q) q -(L')[[]] w and generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing X-SO 2 and the intervening atoms of Ar. q is an integer having a value from 1 to about 20, preferably from 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 is independently hydrogen or lower alkyl. Each R b is independently hydrogen or lower alkyl, or Two R bHowever, together with the atoms to which they are bonded, they form a 3- to 5-membered ring, preferably a 3- to 4-membered ring. However, if w is 0, then q is 1.

[0028] Each active agent may be any suitable active agent, as described in more detail below. While many conventional conjugation methods require the presence of functional groups such as amines or hydroxyl groups to form stable links, the disclosure herein provides strategies for forming links using functional groups that have not previously been available for this purpose, such as phenols and tertiary amines. These functional groups form stable links in the conjugates disclosed herein, while still allowing release under certain conditions to 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 considered below, for example, in the part described below for Y. Some trigger groups include N, O, or S atoms, but are in a non-nucleophilic form. For example, the NO2 group is a trigger group that is reduced to an NH2 or NHOH group that can react with SO2 under reducing conditions, and the acetate group is a trigger group that is hydrolyzed to a hydroxyl group that can react with SO2 under hydrolysis conditions. Other trigger groups do not contain N, O, or S atoms, but when activated, they are converted to nucleophilic N, O, or S atoms. For example, the boronate group is a trigger group that is converted to a hydroxyl group that can react with SO2 under oxidizing conditions (e.g., peroxide). Preferably, the trigger group is selected such that the conditions for activating it selectively do so without cleaving or decomposing other parts of the conjugate, such as the targeted moiety. Once a nucleophilic N, O, or S atom is generated, that atom attacks the SO2 moiety intramolecularly to form a ring, and moiety (Q) q -(L') w -H (wherein H is bonded to the heteroatom of Q or L' that was previously connected to the SO2 portion) is released.

[0030] In embodiments where w is 0, q is 1, and Q is directly bonded to SO2 via a heteroatom. Thus, when the trigger group is activated, a nucleophilic heteroatom is generated, which attacks the SO2 moiety intramolecularly to form a ring and expel the active agent QH (wherein H is bonded to the heteroatom that was previously connected to SO2).

[0031] In embodiments where w is 1, L' may be selected to allow connection of multiple occurrences of Q, which may be the same or different. Thus, in each case of Q, it is indirectly connected to SO2 via a spacer portion. In such embodiments, activation of the trigger group generates a nucleophilic heteroatom, which attacks the SO2 moiety intramolecularly to form a ring and portion (Q) q -L'-H (wherein H is bonded to a heteroatom in L' that was previously connected to SO2) is released. In such embodiments, the released heteroatom triggers an intramolecular reaction that releases the active agent Q (for example, if Q has a tertiary amine connected 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, such that the moiety undergoing intramolecular cyclization is held in close proximity to facilitate the reaction after activation of the trigger group. Planar properties of aromatic and heteroaromatic rings are preferred. This is because the rigid geometric shape of substituents on such rings ensures the desired arrangement of the reactive moiety, although other types of rings, e.g., cycloalkenyls or heterocycloalkenyls, can be forced to have similar geometric shapes. Five-membered or six-membered rings, and / or the number or identity of heteroatoms in the ring, and / or the other substituent on the ring (e.g., electron-donating or electron-withdrawing substituents), can be selected to regulate the cyclization rate based on the resulting bond angles of the ring. Similarly, the more flexible conformations of cycloalkyl and heterocyclyl rings may be useful when a slower rate of intramolecular cyclization is desired.

[0033] Z' can be any suitable linking group that connects Ar to one or more Ab groups. Typically, the linking group should be hydrophilic enough to promote water solubility and inhibit conjugate aggregation by including a portion such as a polyethylene glycol moiety, a peptide sequence, or a charged moiety (e.g., a carboxylate, amine, nitrogen-containing ring, etc.), thereby balancing the hydrophobic properties of any alkyl chains that may be included. Since it is often advantageous to prepare conjugates in a modular manner, Z' can contain linking units, which are functional groups obtained by conjugating one reactive moiety to another. Typical linking units are discussed in more detail below (for example, in relation to the variable Z), and common linking groups include amides, triazoles, oximes, and carbamates. Typical Z' groups are discussed in more detail below. 1An example is the '-Z group. In some embodiments, all 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 first conditions, while other G groups may have trigger groups that are activated under second conditions, so that, for example, one active agent can be selectively released under first conditions, while a second active agent can be selectively released under second conditions.

[0034] In a particular embodiment of formula (II), -Y' is -(CH2) y NR"-,-(CH2) y O-, or -(CH2) y When S- and y is 1, an N, O, or S atom is arranged to bond to TG, R'' is hydrogen or a 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 via a heteroatom, preferably O or N, forming an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- linkage containing the heteroatom of Q. In other embodiments, (Q) q -(L') w -teeth, [ka] Selected from, in the formula, Q is an active agent linked to L' via a heteroatom, preferably O or N. X 4 It is either absent or forms -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- links containing a heteroatom of Q. X 1 -O- or -NR a -and, X 2 These are -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH-, X 3 This is -OC(=O)-, w' is an integer with a value of 1, 2, 3, 4, or 5. R 9 and R 10 Each is independently hydrogen, alkyl, aryl, or heteroaryl, and alkyl, aryl, and heteroaryl are either unsubstituted or have one or more substituents, e.g., alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 , and -(CH2) u SO2R u3 Substituted with a substituent selected from, R u1 , R u2 , and R u3 Each of these is independently hydrogen, alkyl, aryl, or heteroaryl. u is an integer with values ​​ranging from 1 to approximately 10.

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

[0037] In certain embodiments, Z' comprises a reactive group (e.g., a precursor group as will be discussed in more detail below with respect to Z) which can be used to encapsulate the compound as a trigger, to a solid surface (e.g., to form a solid-supported array or sensor particles), or to encapsulate 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 a connection point to Ab, ** is a connection point to Ar, R e It is an 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 These are, independently, -C(O)NH-, -NHC(O)-, [ka] And, L 2 This is an optional spacer portion, which may contain one or more substituents, such as C1-C6 alkyl groups, C5-C6 alkyl groups, etc. 14 It may be further substituted with aryl and C3-C8 heteroaryl groups, where alkyl, aryl, and heteroaryl groups are, for example, C1-C 10 Alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 ,-(CH2) u CO2H, -(CH2) u CO2R u1 , and -(CH2) u SO2R u3 R may be further substituted with one or more substituents selected from the group consisting of u1 , R u2 , and R u3 Each of them is independently hydrogen, C1~C 15 Alkyl, C6~C 20 Aryl, or C3~C 10is heteroaryl, and u is an integer having a value of 1 to about 10, R 12 is hydrogen, C1-C8 alkyl, or an amino acid moiety such as a natural 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 [Chemical formula] is as follows.

[0040] In other embodiments, Z’ is a linking group having a structure of formula (IIa’), (IIb’), (IIc’), (IId’), (IIe’), (IIf’), (IIg’), or (IIh’), [Chemical formula] [Chemical formula] wherein * is the connection point to Ab, ** is the connection point to Ar.

[0041] In some preferred embodiments, Z’ is [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] a linking group selected from, wherein R zais H or methyl, R zb is -OH, =O, or =NHOH,

Chemical formula

Chemical formula

[0042] In some embodiments, G is as follows:

Chemical formula

Chemical formula

[0043] In certain embodiments, Ab-(G)<00000\74>is a compound of formula (III),

Chemical formula

Chemical formula

[0044] In some embodiments, M is N.

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

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

[0047] In some embodiments, M is C(−L−Q), 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, carbocyclic, heterocyclic, and haloalkyl.

[0049] In some embodiments, each electron withdrawing group is independently −NO2, −CN, −haloalkyl, −CO2NR 33 R 34 −CO2R 35 −C(=O)R 36 −S(=O)R 37 −S(=O)2OR 38 and −NR 39 R 40 R 41 and R 36 、 R 37 R 38 R 39 、 R 40 and R 41 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic, heterocyclic, and haloalkyl.

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

[0051] In some embodiments, each electron withdrawing group is independently −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' includes a coupling group, which 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 The result is selected from S(=O)2O- and -OS(=O)2O-.

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

[0057] In some embodiments, L' further includes a cleavable group which is coupled to Q'.

[0058] In certain embodiments, the -Q' portion, which is a cleavable group, [ka] Selected from, in the formula, R 49 is hydrogen or -C(=O)R 50 And, R 50 It is a lower alkyl group.

[0059] In some embodiments, L' is a C6-C group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-. 100 It also contains alkylene.

[0060] In certain embodiments, L is a C6-C6 molecule containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-. 100 It contains alkylene. For example, L is [ka] Including, in the formula, a' is a bond to the M-containing aromatic ring, and b' is a bond to L'. n is between 2 and 20.

[0061] In some embodiments, A is [ka] For example, A is [ka] Alternatively, A is [ka] It may also be: In other embodiments, A is [ka] It is possible. In some embodiments, A is [ka] That is the case.

[0062] In a particular embodiment, R 42 -OH or -NR 44 R 45 That is the case.

[0063] In some embodiments, the present disclosure is a method for producing the ADCs disclosed herein, comprising reacting the antibodies disclosed herein with a compound of formula (IV) or formula (V), [ka] In the formula, A' is [ka] And, M is N, CR 30 , or C(-LQ), Each L is independently selected from the spacer portion. Each Q is independently selected from an active agent or a reactive group. X is selected from -Cl, -Br, and -I. R 30 and R 31 However, each is independently selected from electron-withdrawing groups, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl groups. R 46 However, selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl, R 32 However, it is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl. R 47 However, O - These are alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykyl, and heterocyclyl compounds. This concerns methods where n is between 1 and 4.

[0064] In some embodiments, M is N.

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

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

[0067] In some embodiments, A' is [ka] And in the formula, R 46 C 1~3 It is an alkyl-substituted aryl group.

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

[0069] In some embodiments, A' is [ka] And in the formula, 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 Each of these is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl.

[0072] In some embodiments, each electron-withdrawing group is independently -NO2, -CN, -haloalkyl, -CO2NR 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 Each of these is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl.

[0073] In certain embodiments, each electron-withdrawing group is independently -CN, -CONR 33 R 34 , and -CO2R 35 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' includes a coupling group, which 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 The result is selected from S(=O)2O- and -OS(=O)2O-.

[0079] In a particular embodiment, the coupling group is Oriented in either direction, [ka] Selected from.

[0080] In some embodiments, L' further includes a cleavable group which is coupled to Q'.

[0081] In certain embodiments, the cleavable group coupled to Q' is [ka] Selected from, in the formula, R 49 is hydrogen or -C(=O)R 50 And, R 50 It is a lower alkyl group.

[0082] In some embodiments, L' is a C6-C group containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-. 100 It also contains alkylene.

[0083] In certain embodiments, L is a C6-C6 molecule containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-. 100 It contains alkylene. For example, L is [ka] Including, in the formula, a is a bond to the M-containing aromatic ring, and b is a bond to L'. n is between 2 and 20.

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

[0085] In certain embodiments, Q' is selected from cytokines, immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, analgesics, or combinations thereof.

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

[0087] In some embodiments, the reactive group is -N3, -C≡CH, [ka] The following are selected from -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] That is the case.

[0089] In a particular embodiment, R 31 -CN, -CO2NR 33 R 34 , or -CO2R 35 That is the case.

[0090] In a particular embodiment, A is [ka] That is the case.

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

[0092] In some embodiments, A is [ka] That is the case.

[0093] In a particular embodiment, R 46 This is C, which is optionally substituted. 1~3 Alkyl, optionally substituted C6-C 12 It is an aryl or a heteroaryl that has been optionally substituted.

[0094] In some embodiments, A is [ka] That is the case.

[0095] In a particular embodiment, R 47 O - or C 1~3 It is alkyl.

[0096] In a particular embodiment, A is [ka] That is the case.

[0097] Active drug As described above, in preferred embodiments of this disclosure, Q is an active agent that forms part of the ADC disclosed herein. In some embodiments, the active agent is independently selected from chemotherapeutic agents and toxins. In some embodiments, the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an analgesic, or a combination thereof.

[0098] Exemplary drugs for conjugation The ADC of the present invention provides a targeted therapy that can reduce, for example, the side effects commonly seen in anticancer therapies, as one or more active agents are delivered to specific cells.

[0099] For example, active drugs 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); ronafarnib (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 pigosulfan); Aziridines (e.g., benzodopa, carboquan, meturedopa, or uredopa); Ethyleneimine, methylmelamine, altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylol Lamin; acetogenin (e.g., bratacin or bratacinone); camptothecin including its synthetic analogue topotecan; bryostatin; callistatin; CC-1065 (including its synthetic analogues adzeresin, karzeresin, or bizeresin); cryptophycin (e.g., cryptophycin 1 or cryptophycin 8); dorastatin; duocalmycin (including its synthetic analogues KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards (e.g., chlorambucil, chlornafadin, cholophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trophosphamide, or uracil mustard); nitrusureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimnustine); antibiotics (e.g., Kalichemycin gamma 1 I and Kalichemycin omega I) 1. Dynemycin, including dynemycin A as an engine antibiotic selected from 1; bisphosphonates (e.g., clodronate); esperamicin, neocardinostatin chromophore, or related pigment protein engine antibiotic chromophore, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, carninomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRLIMYCIN®, doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin) (e.g., 2-pyrrolinodoxorubicin, liposomal doxorubicin, or deoxydoxorubicin), epirubicin, esorubicin, marcelomycin, mitomycin (e.g., mitomycin C, mycophenolic acid, nogaramycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptomygin, streptozocin, tubercidine, ubenimex, dinostatin, or zorubicin); antimetabolites (e.g., 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, or trimethrexate);Purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, or tiguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, or phloxuridine); androgens (e.g., carsterone, dromostanolone propionate) (propionate), epithiostanol, mepitiostane, or testolactone; anti-adrenal drugs (e.g., aminoglutethimide, mitotane, or trilostane); folic acid supplements (e.g., folic acid); acegraton; aldoforamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diaziquan; elfornithine; eriptinium acetate; epotilone; etogluside; gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids (e.g., mytansin or anthamitosin); trichothecenes (e.g., T-2 toxin, veracrine) Urin A, Loridine A, or Anguidin); Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2"-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, verracurin A, Loridine A, or Anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa;Taxoids (e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremofoll-free, albumin-modified nanoparticle formulation of paclitaxel, American Pharmaceutical Partners, Schaumber, I11, 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 may be selected from the group consisting of pharmaceutically acceptable salts thereof, solvates thereof, acids thereof, or derivatives thereof.

[0100] Mitotic inhibitors In some embodiments, the linker of this disclosure may be used to conjugate an antibody to one or more mitotic inhibitors to form an ADC for the treatment of cancer. The term “mitotic inhibitor,” as used herein, refers to a cytotoxic agent and / or therapeutic agent that blocks mitosis or cell division, which is a particularly important biological process for cancer cells. Mitotic inhibitors often disrupt microtubules so as to prevent cell division by affecting microtubule polymerization or depolymerization. Thus, in certain embodiments, the antibody is conjugated to one or more mitotic inhibitors that disrupt microtubule formation by inhibiting tubulin polymerization. In certain embodiments, the mitotic inhibitors used in the ADCs of this disclosure are Taxol® (paclitaxel), Taxotere® (docetaxel), or Ixempra® (ixabepyrone). Examples of mitotic inhibitors that may be used in the ADCs disclosed herein are provided below. The genus of mitotic inhibitors includes the auristatins described above.

[0101] Auristatin The linker of this disclosure may be used to conjugate an antibody to at least one auristatin. Auristatins represent a group of drastatin analogs that have generally been shown to have anticancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (U.S. Patent No. 5,635,483) is a synthetic analog of the marine natural product drastatin 10, which inhibits tubulin polymerization by binding to the same tubulin site as the anticancer drug vincristine (GRPettit, Prog. Chem. Org. Nat. Prod, 70:1-79 (1997)). Drastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the compounds of the drastatin group. Exemplary embodiments of the auristatin subclass of mitotic inhibitors 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-benzoylvalerate-AE ester (AEVB).The synthesis and structure of auristatin derivatives are described in U.S. Patent Publications 2003-0083263, 2005-0238649, and 2005-0009751, International Patent Publication 04 / 010957, International Patent Publication 02 / 088172, and U.S. Patents 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, and 5,635,48. These are described in 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] Dorastatin In certain embodiments, the active agent in the ADC described herein is dorastatin. Dorastatin is a short peptide compound isolated from the Indian Ocean sea hare Dolabella auricularia (see Pett et al., J. Am. Chem. Soc., 1976, 98, 4677). Examples of dorastatin include dorastatin 10 and dorastatin 15. Dorastatin 15 is a seven-subunit depsipeptide derived from Dolabella auricularia and is a potent antimitotic agent structurally related to the five-subunit antitubulin agent dorastatin 10 obtained from the same organism. Thus, in certain embodiments, the ADC of this disclosure comprises an antibody, a linker described herein, and at least one dorastatin. The above-mentioned auristatin is a synthetic derivative of dorastatin 10.

[0103] Mytansinoids The linker of this disclosure may be used to conjugate an antibody to at least one mytansinoid to form an ADC. Mytansinoids are potent antitumor agents originally isolated from members of the Celastraceae, Rhamnaceae, and Euphorbiaceae families of higher plants, as well as from several species of bryophytes (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 mytansinoids inhibit mitosis by inhibiting the polymerization of the microtubule protein tubulin, thereby preventing microtubule formation (see, e.g., U.S. Patent No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Mytansinoids 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 mytansinoids is 1,000 times greater than that of conventional chemotherapeutic agents, such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Patent No. 5,208,020).

[0104] Mytansinoids include mytansine, mytansinol, the C-3 ester of mytansinol, and other mytansinol analogs and derivatives (see, for example, U.S. Patents 5,208,020 and 6,441,163, each of which are incorporated herein by reference). The C-3 ester of mytansinol may be naturally occurring or synthetically derived. Furthermore, both naturally occurring and synthetic C-3 mytansinol esters can be classified as C-3 esters having a simple carboxylic acid or C-3 esters having a derivative of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic mytansinoid analogs are described, for example, in Kupchan et al., J. Med. Chem., 21, 31-37 (1978).

[0105] Mytansinoids suitable for use in ADCs of this disclosure may be isolated from natural sources, synthesized, or semi-synthetically synthesized. Furthermore, mytansinoids may be modified in any preferred manner, as long as sufficient cytotoxicity is maintained in the ultimate conjugate molecule. The structure of meltansin (DM1), an exemplary mytansinoid, is provided below. [ka]

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

[0107] Anthamitocins are a group of mytansinoid antibiotics isolated from various bacterial sources. These compounds possess potent antitumor activity. Representative examples, though not limited to them, include anthamitocin P1, anthamitocin P2, anthamitocin P3, and anthamitocin P4.

[0108] Plant alkaloids The linker of this disclosure may be used to conjugate an antibody to at least one plant alkaloid, such as a taxane or vinca alkaloid. Plant alkaloids are chemotherapeutic agents derived from specific types of plants. Vinca alkaloids are derived from the periwinkle plant (catharanthus rosea), while taxanes are derived from the bark of the Pacific yew tree (taxus). Both vinca alkaloids and taxanes are also known as antimicrotubule agents and are described in more detail below.

[0109] Taxane The linker of this disclosure may be used to conjugate the antibody to at least one taxane. As used herein, the term "taxane" refers to a type of antineoplastic agent having a microtubule-mediated mechanism and a structure comprising a taxane ring structure and stereospecific side chains necessary for cell proliferation inhibitory activity. The term "taxane" also includes a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Examples of taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. 99 / 18113, piperazino and other derivatives described in WO99 / 14209, taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680, 6-thio derivatives described in WO98 / 28288, sulfenamide derivatives described in U.S. Patent No. 5,821,263, and taxol derivatives described in U.S. Patent No. 5,415,869, each of which is incorporated herein by reference. Taxane compounds are listed in U.S. Patent Applications No. 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 5,8 These are also previously described in Nos. 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 nanoparticle paclitaxel (ABI-007 / Abraxene®, Abraxis Bioscience).

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

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

[0112] Antitumor antibiotics The linkers of this disclosure may be used to conjugate antibodies to one or more antitumor antibiotics for the treatment of cancer. As used herein, the term “antitemiotic antibiotic” means an antineoplastic drug made from microorganisms that blocks cell growth by interfering with DNA. Often, antitumor antibiotics either disrupt DNA strands or slow down or stop DNA synthesis. Examples of antitumor antibiotics that may be included in the ADCs disclosed herein include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracyclines, calichemycin, and duocalmycin, which are described in more detail below.

[0113] Actinomycin The linker of this disclosure may be used to conjugate an antibody to at least one actinomycin. Actinomycin is a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces. Representative examples of actinomycin include, but are not limited to, actinomycin D (cosmegen [also known as actinomycin, dactinomycin, actinomycin IV, actinomycin C1], Lundbeck, Inc.), anthramycin, thikamycin A, DC-81, mazethramycin, neotramycin A, neotramycin B, polotoramycin, protracalcin B, SG2285, cibanomycin, cibilomycin, and tomaimycin. In certain embodiments, D is pyrrolobenzodiazepine (PBD). Examples of PBDs include, but are not limited to, anthramycin, thikamycin A, DC-81, mazetramycin, neotramycin A, neotramycin B, porotoramycin, protracalcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), cibanomycin, cibilomycin, and tomaimycin. Therefore, in certain embodiments, D is actinomycin, for example actinomycin D, or PBD, or pyrrolobenzodiazepine (PBD) dimers.

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

[0115] PBDs differ in the number, type, and position of substituents in 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 a (S) configuration at the chiral C11α position, giving them a right-handed twist when viewed from the C ring towards 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 Publications 2013 / 0028917A1 and 2013 / 0028919A1, U.S. Patent No. 7,741,319B2, and WO2011 / 130598A1 and WO2006 / 111759A1, each of which is incorporated herein by reference in whole.

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

[0117] KariCare Sewing Machine The linker of this disclosure may be used to conjugate an antibody to at least one calichemycin. Calichemycins are a family of enediine antibiotics derived from the soil organism Micromonospora echinospora. Calichemycins bind to small grooves in DNA, inducing double-strand DNA breaks and causing cell death 100 times greater than other chemotherapeutic agents (Damle et al. (2003) Curr Opin Pharmacol 3:386). Preparations of calichemycins that can be used as drug conjugates in this disclosure have already been described; see U.S. Patents 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 analogues of usable calicaceamycin include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG, and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. Patents 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). Therefore, in certain embodiments, D is calicaceamycin.

[0118] Duocalmycin The linker of this disclosure may be used to conjugate an antibody to at least one duocalmycin. Duocalmycin is 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.) Duocalmycin binds to the small groove in DNA and alkylates the nucleic acid base 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 duocalmycin include, but are not limited to, adzeresin, bizeresin, and karzeresin. Therefore, in certain embodiments, D is duocalmycin.

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

[0120] Immunomodulators In some embodiments, the linker of this disclosure may be used to conjugate an antibody to at least one immunomodulator. As used herein, the term “immunomodulator” refers to a drug that can stimulate or modify an immune response. In certain embodiments, an immunomodulator is an immunostimulant that enhances the immune response of a target. In some embodiments, an immunomodulator is an immunosuppressant that prevents or reduces the immune response of a target. Immunomodulators can modulate bone marrow cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphocytes (T cells, B cells, and natural killer (NK) cells), as well as any further differentiated cells thereof. Representative examples, but not limited to, include Bacillus calmette-Guérin (BCG) and levamisole (ergamisole). Other examples of immunomodulators that may be used in the ADCs of this disclosure, but not limited to, include cancer vaccines, cytokines, and immunomodulatory gene therapies.

[0121] Cancer vaccine Antibodies may be conjugated into cancer vaccines using the linkers of this disclosure. 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 induced from the subject’s own immune system by administering the cancer vaccine, or, in this disclosure, by administering an ADC comprising antibodies and the cancer vaccine. In preferred embodiments, the immune response eradicates tumor cells in the body (e.g., primary or metastatic tumor cells). The use of cancer vaccines generally involves the administration of a specific antigen or group of antigens that are present, for example, on the surface of certain cancer cells or on the surface of certain infectious agents that have been shown to promote carcinogenesis. In some embodiments, the use of cancer vaccines is for preventive 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) vaccines for types 16 and 18 (Cervarix, GlaxoSmithKline), recombinant tetravalent human papillomavirus (HPV) vaccines for types 6, 11, 16, and 18 (Gardasil, Merck & Company), and cyplucel-T (Provenge, Dendreon). Thus, in certain embodiments, D is a cancer vaccine that is either an immunostimulant or an immunosuppressant.

[0122] Cytokine The linker of this disclosure may be used to conjugate an antibody to at least one cytokine. The term "cytokine" generally refers to a protein released by a population of cells that acts on other cells as an intercellular mediator. Cytokines directly stimulate immune effector cells and stromal cells at tumor sites 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 antitumor activity, which has translated into several cytokine-based approaches for cancer therapy (Lee and Margolin, above). In recent years, several cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have been recognized and are in clinical trials for patients with advanced cancer (Lee and Margolin, above).

[0123] Examples of cytokines that may be used in the ADCs of this disclosure include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, e.g., 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; integrin; thrombopoietin (TPO); nerve growth factor, e.g., NGF; platelet growth factor; trans Examples include forming growth factor (TGF); insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone induction factor; interferons, e.g., interferon α, β, and γ; colony-stimulating factor (CSF); granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), e.g., IL-1, IL-a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, and biologically active equivalents of naturally occurring cytokines. Therefore, in certain embodiments, D is a cytokine.

[0124] Colony-stimulating factor (CSF) The linker of this 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 producing red blood cells. Since some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), introducing colony-stimulating factors can 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 initiate white blood cell production. 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.), salgramostim (Leukin (granulocyte-macrophage colony-stimulating factor and GM-CSF), Genzyme Corporation), promegapoietin, and oprelbequin (recombinant IL-11, Pfizer, Inc.). Therefore, in certain embodiments, D is a CSF.

[0125] gene therapy The linkers of this 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, thereby designed to treat a disease. It relates to immunomodulators, and gene therapy is used to inhibit the growth of cancer cells or to stimulate the natural ability of a target to kill cancer cells. In certain embodiments, the ADCs of this disclosure include nucleic acids encoding a functional therapeutic gene used to replace a mutated gene or other dysfunctional (e.g., cleaved) gene associated with cancer. In other embodiments, the ADCs of this disclosure include nucleic acids encoding or otherwise providing the production of a therapeutic protein for treating cancer. The nucleic acid encoding the therapeutic gene may be directly conjugated to an antibody, or alternatively, conjugated to an antibody via a carrier. Examples of carriers that may be used to deliver nucleic acids for gene therapy include, but are not limited to, viral vectors or liposomes.

[0126] Alkylating agent The linker of this disclosure may be used to conjugate the antibody to at least one alkylating agent. The alkylating agent is a type of antineoplastic compound that binds an alkyl group to DNA. Examples of alkylating agents that may be used in the ADC of this disclosure include, but are not limited to, alkyl sulfonates, ethylenimime, methylamine derivatives, epoxides, nitrogen mustard, nitrosourea, triazines, and hydrazines.

[0127] Alkyl sulfonate The linker of this disclosure may be used to conjugate the antibody to at least one alkyl sulfonate. The alkyl sulfonate has the general formula R-SO2-OR 1 (wherein R and R 1This is a subclass of alkylating agents that typically have an alkyl or aryl group. A typical example of alkyl sulfonates is busulfan (Myleran®, GlaxoSmithKline, Busulfex IV®, PDL BioPharma, Inc.).

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

[0129] Nitrosourea The linker of this disclosure may be used to conjugate the antibody to at least one nitrosourea. Nitrosourea is a subclass of lipid-soluble alkylating agents. Representative examples, but not limited to, include carmustine (BCNU [BiCNU, also known as 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-chloro-ethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb), nimustine (also known as ACNU), and streptozocin (Zanosar®, Teva Pharmaceuticals).

[0130] Triazines and hydrazines The linkers of this disclosure may be used to conjugate antibodies to at least one triazine or hydrazine. Triazines and hydrazines are subclasses of nitrogen-containing alkylating agents. In some embodiments, these compounds may be spontaneously degraded or metabolized to produce 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, but not limited to, include dacarbazine (DTIC-Dome, Bayer Healthcare Pharmaceuticals Inc.), procarbazine (Mutalane®, Sigma-Tau Pharmaceuticals, Inc.), and temozolomide (Temodar®, Schering Plough).

[0131] Other alkylating agents The linker of this disclosure may be used to conjugate the antibody to at least one ethyleneimine, methylamine derivative, or epoxide. Ethyleneimines are typically a subclass of alkylating agents containing at least one aziridine ring. Epoxides represent a subclass of alkylating agents characterized as cyclic ethers having only three ring atoms.

[0132] Representative examples of ethyleneimines include, but are not limited to, thiotepa (Thioplex, Amgen), diaziquane (also known as aziridinylbenzoquane (AZQ)), and mitomycin C. Mitomycin C is a natural product containing an aziridine ring that appears to induce cytotoxicity via cross-linked 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 analogs include, but are not limited to, altoretamine (Hexalen, MGI Pharma, Inc.), also known as hexamethylamine and hexastat. Representative examples of epoxides of this type of anticancer compound include, but are not limited to, dianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodalucitol) is chemically related to aziridine and generally promotes alkyl group transitions via a similar mechanism as described above. Dibromodulucitol is a prodrug for epoxides because it is hydrolyzed to dianhydrogalactitol (Sellei C, et al. Cancer Chemother Rep. 1969;53:377).

[0133] Anti-angiogenic agents In some embodiments, the linker of this disclosure may be used to conjugate an antibody to at least one anti-angiogenic agent. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in a variety of 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, a specific anti-angiogenic agent that prevents VEGF from interacting with its cognitive receptor prevents VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, when a specific receptor on the cell surface is triggered, a cascade of other chemical signals is initiated to promote blood vessel growth. Therefore, for example, certain enzymes known to promote intracellular signaling cascades that contribute to cell proliferation, such as some tyrosine kinases, are targets for cancer treatment. In other embodiments, these agents interfere with intercellular signaling cascades. However, in other embodiments, these agents inactivate specific targets by activating and promoting cell growth or by directly interfering with the growth of vascular cells. Angiogenesis inhibitory properties have been found in more than 300 substances with numerous direct and indirect inhibitory effects.

[0134] Representative examples of anti-angiogenic agents that may be used in the ADCs of this disclosure include, but are not limited to, angiostatins and 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 those containing fluorouracil or doxorubicin, Canstatin, carboxyamidotriozole and those containing paclitaxel, EMD121974, S-24, Vitaxin, Dimethylxanthenon acetate, IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMa b, Angiozymes (Ribozymes), IMC-1C11, Neovastat, Marimstat, Prinomast, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, Paclitaxel, Gemcitabine and Cisplatin, Irinotecan and Cisplatin, Radioactive substances, Tecogalan, Temozolomide and PEG Interferon α2b, Tetrathiomolybdate, TNP-470, Thalidomide, CC-5013 and Taxotere, Tamstatin, 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.)Examples include nilotinib (Tasigna, Novartis Pharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), and phosphoinositide 3-kinase (PI3K).

[0135] antimetabolites An antibody may be conjugated to at least one antimetabolite using the linker of this disclosure. Antimetabolites are a type of chemotherapeutic agent that is very similar to normal substances in cells. When a cell incorporates an antimetabolite into its cellular metabolism, the result is negative for that cell, for example, the cell is unable to divide. Antimetabolites are classified according to the substances they interfere with. Examples of antimetabolites that may be used in the ADCs of this disclosure, but are not limited to, folate antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, foxlyn, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nerarabine, and pentostatin), as described in more detail below.

[0136] Antifolic acid The linker of this disclosure may be used to conjugate an antibody to at least one antifolate agent. The antifolate agent is a subclass of antimetabolites that are structurally similar to folate. Representative examples, but not limited to, include methotrexate, 4-aminofolate (also known as aminopterin and 4-aminopteroinic acid), lometrexol (LMTX), pemetrexed (Alimpta, Eli Lilly and Company), and trimethrexate (Neutrexin, Ben Venue Laboratories, Inc.).

[0137] Prince Antagonist The linker of this disclosure may be used to conjugate an antibody to at least one purine antagonist. 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 (purinethol [also known as 6-mercaptoethanol], GlaxoSmithKline), fludarabine (Fluda, Genzyme Corporation), pentostatin (nipent, also known as 2'-deoxycoformycin (DCF)), and 6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

[0138] Pyrimidine antagonist The linker of this disclosure may be used to conjugate an antibody to at least one pyrimidine antagonist. 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 (Adolsyl, Teva Pharmaceuticals, Efdex, Valeant Pharmaceuticals, Inc.), 5-fluoro-2'-deoxyuridine 5'-phosphate (FdUMP), 5-fluorouridine triphophosphate, and gemcitabine (Gemzar, Eli Lilly and Company).

[0139] Boron-containing agent The linker of this disclosure may be used to conjugate an antibody to at least one boron-containing agent. The boron-containing agent includes a type of cancer therapeutic compound that interferes with cell proliferation. Representative examples of boron-containing agents include, but are not limited to, boroficin and bortezomib (Velcade, Millenium Pharmaceuticals).

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

[0141] Hormone drugs The linker of this disclosure may be used to conjugate an antibody to at least one hormonal agent. The hormonal agent (including synthetic hormones) is a compound that interferes with the production or activity of endogenously produced hormones in 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] Anti-hormone drugs The linker of this disclosure may be used to conjugate an antibody to at least one antihormone agent. The “antihormone” agent is an agent that suppresses the production of a particular endogenous hormone and / or prevents its function. In certain embodiments, the antihormone agent interferes with the activity of a hormone selected from androgens, estrogens, progesterone, and gonadotropin-releasing hormone, thereby interfering with the growth of various cancer cells. Representative examples of antihormone drugs, though not limited to these, include aminoglutethimide, anastrozole (Arimidex, AstraZeneca Pharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals), cyproterone acetate (Cyprostat, 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, and medroxyprogesterone acetate (Provera, Pfizer Inc.). Examples include megestrol acetate (Megas, Bristol-Myers Squibb Company), tamoxifen (Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl, Ferring).

[0143] corticosteroids The linker of this disclosure may be used to conjugate the antibody to at least one corticosteroid. The ADC of this disclosure may be used with a corticosteroid to reduce inflammation. Examples of corticosteroids, but not limited to, include glucocorticoids, such as prednisone (deltazone, a division of Pharmacia & Upjohn Company, Pfizer, Inc.).

[0144] Photoactivating agents The linker of this disclosure may be used to conjugate an antibody to at least one photoactive agent. Examples of photoactive agents include compounds that can be positioned to kill therapeutic cells upon exposure to electromagnetic radiation of a specific wavelength. The therapeutic compound absorbs electromagnetic radiation at wavelengths that penetrate tissue. In a preferred embodiment, the compound is administered in a non-toxic form that, upon sufficient activation, can produce a photochemical effect toxic to cells or tissues. In another preferred embodiment, these compounds are retained by cancerous tissue and readily removed from normal tissue. Examples of various chromogens and dyes are not limited to these.

[0145] Oligonucleotides The linkers of this disclosure may be used to conjugate antibodies to at least one oligonucleotide. Oligonucleotides are made from short nucleic acid strands that function by processing and interfering with genetic information. In some embodiments, the oligonucleotides for use in ADCs are unmodified single-stranded and / or double-stranded DNA or RNA molecules, while in other embodiments, these therapeutic oligonucleotides are chemically modified single-stranded 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 specific nucleic acid sequences in the entire 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 An antibody may be conjugated to at least one antisense oligonucleotide using the linker of this disclosure. The antisense oligonucleotide is designed to bind to RNA via Watson-Crick hybridization. In some embodiments, the antisense oligonucleotide is complementary to the nucleotide 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 oligonucleotides bind to target RNA, several mechanisms can be utilized to inhibit RNA function (Crooke ST. (1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterized antisense mechanism involves cleavage of the targeted RNA by endogenous cellular nucleases such as RNase H or nucleases associated with RNA interference mechanisms. However, oligonucleotides that inhibit target gene expression through non-catalytic mechanisms such as splicing regulation 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, this 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 generates a dsRNA complementary to the target gene. In each of the embodiments described above, the double-stranded RNA is designed to interfere with the gene expression of a specific target sequence within the cell. Generally, this mechanism involves the conversion of dsRNA to a shorter RNA that directs ribonucleases to homologous mRNA targets (summarized in Ruvkun, Science 2294:797 (2001)), followed by the degradation of the corresponding endogenous mRNA, thereby regulating gene expression. In particular, dsRNA has been reported to possess antiproliferative properties, which makes its therapeutic applications possible (Aubel et al., Proc. Natl. Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNAs have been shown to inhibit tumor growth in mice (Levy et al. Proc.Nat.Acad.Sci.USA, 62:357-361 (1969)), and are active in the treatment of leukemia mice (Zeleznick et al., Proc.Soc.Exp.Biol.Med. 130:126-128 (1969)), inhibiting chemically induced tumorigenesis in mouse skin (Gelboin et al., Science 167:205-207 (1970)). Therefore, in preferred embodiments, this disclosure provides the use of antisense oligonucleotides in ADCs for the treatment of breast cancer.In other embodiments, the Disclosure provides compositions and methods for initiating antisense oligonucleotide therapy, wherein dsRNA interferes with the target cell expression of EGFR at the mRNA level. When used above, dsRNA refers to naturally occurring RNA, partially purified RNA, recombinant 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 (LNAs)), deoxyribonucleotides, and any combination thereof. The RNAs of the Disclosure only need to be sufficiently similar to naturally occurring RNA to have the ability to mediate the antisense oligonucleotide-based regulation described herein.

[0149] Aptamer An antibody may be conjugated to at least one aptamer using the linker of this disclosure. The aptamer is a nucleic acid molecule selected from a random pool based on its ability to bind to other molecules. Like antibodies, aptamers can bind to target molecules with outstanding affinity and specificity. In many embodiments, the aptamer is envisioned as a complex, sequence-dependent, three-dimensional shape that enables interaction with the target protein, resulting in a tightly bound complex similar to an antibody-antigen interaction, thereby interfering with the function of the aforementioned protein. The particular ability of aptamers to bind tightly and specifically to their target proteins highlights their potential as targeted molecular therapies.

[0150] CpG oligonucleotides The linker of this disclosure may be used to conjugate an antibody to at least one CpG oligonucleotide. Bacterial DNA and viral DNA are known to be potent activators of both innate and specific immunity in humans. These immunological features are associated with 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 an early sign of infection and to initiate a subsequent immune response. Therefore, oligonucleotides containing this CpG motif can be used to initiate an antitumor immune response.

[0151] Ribozyme The linker described herein may be used to conjugate an antibody to at least one ribozyme. Ribozymes are catalytic RNA molecules ranging in length from approximately 40 to 155 nucleotides. The ability of ribozymes to recognize and cleave specific RNA molecules makes them potential therapeutic candidates. Typical examples include angiozymes.

[0152] Radioactive radionuclides (radioisotopes) The linker of this disclosure may be used to conjugate an antibody to at least one radionuclide agent. The radionuclide agent includes a drug characterized by an unstable nucleus that can undergo radioactive decay. The basis for the success of radionuclide therapy depends on 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 over which the emitted particles can travel. In preferred embodiments, the therapeutic agent is a radionuclide selected from the group consisting of 111In, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 64Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, and 211Pb. Radionuclides that substantially decay with Auger-emitting particles are also preferred. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111-1, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192. The decay energies of useful β-particle emitting nuclides 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. The decay energies of useful α-particle emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioactive isotopes that can be used include 11C, 13N, 150, 75Br, 198Au, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, and 169Yb.

[0153] Radiosensitizer The linker of this disclosure may be used to conjugate an antibody to at least one radiosensitizer. 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 dose to increase the sensitivity of cells sensitized to electromagnetic radiation and / or to facilitate the treatment of diseases treatable with electromagnetic radiation. Radiosensitizers are agents that make cancer cells more sensitive to radiotherapy, while typically having a much lower effect on normal cells. Therefore, radiosensitizers may be used in combination with radiolabeled antibodies or ADCs. The addition of radiosensitizers can improve efficacy compared to treatment with radiolabeled antibodies or antibody fragments alone. Radiosensitizers are described in DMGoldberg (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 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 their therapeutically effective analogs and derivatives. Alternatively, radiosensitizers can be activated using photodynamic therapy (PDT). Representative examples of photoelectric radiation sensitizers include, but are not limited to, hematoporphyrin derivatives, photofrin(r), benzoporphyrin derivatives, NPe6, tin ethithioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and their therapeutically effective analogs and derivatives.

[0155] Topoisomerase inhibitors The linker of this disclosure may be used to conjugate an antibody to at least one topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent designed to interfere with the action of topoisomerase enzymes (topoisomerases I and II), which regulate changes in DNA structure by catalyzing, then cleaving and rejoining 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, such as irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrin, daunorubicin, doxotrubicin, epipodophyllotoxin, ellipticin, epirubicin, etoposide, razoxane, and teniposide.

[0156] Tyrosine kinase inhibitors The linker of this disclosure may be used to conjugate an antibody to at least one tyrosine kinase inhibitor. Tyrosine kinases are intracellular enzymes that function to bind a phosphate group to the amino acid tyrosine. Tumor growth may be inhibited by blocking the ability of protein tyrosine kinases to function. Examples of tyrosine kinases that may be used on the ADCs of this disclosure include, but are not limited to, axitinib, bosutinib, sediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, restaurtinib, nilotinib, semaxinib, sunitinib, and vandetanib.

[0157] Other drugs Other drugs that may be used in the ADCs of this 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 unbound active fragments of diphtheria toxin), deoxyribonuclease (DNase), geronin, mitogellin, modesin A chain, momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana protein (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (e.g., exotoxin A chain (derived from Pseudomonas aeruginosa)), restrictosin, lysine A chain, ribonuclease (RNase), and sapaonaria. Examples include officinalis inhibitors, saporins, 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, mosetinostat, and panobinostat), COX-2 inhibitors, urea substitutions, heat shock protein inhibitors (e.g., geldanamycin and many analogues), corticosteroids, and trichothecenes. (See, for example, WO93 / 21232.) Other drugs 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 aforementioned group of drug portions that may be used in the ADCs of this disclosure are not exclusive in that specific examples of drugs may be found in more than one category, for example, anthamitocin is both a mitotic inhibitor and an antitumor antibiotic.

[0159] All stereoisomers of the drug portion described above are intended for any combination of R and S conformations at the chiral carbon of the compound disclosed herein.

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

[0161] A detection probe refers to (i) a material capable of providing a detectable signal, (ii) a material capable of interacting with a first or second probe to alter the detectable signal provided by the first or second probe, such as fluorescence resonance energy transfer (FRET), (iii) a material capable of stabilizing interactions with an antigen or ligand or increasing binding affinity, (iv) a material capable of influencing electrical mobility or cellular invasiveness through physical parameters such as charge or hydrophobicity, or (v) a material capable of regulating ligand affinity, antigen-antibody binding, or ionic complex formation.

[0162] In some embodiments, each active agent is used independently. (a) Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787 / ZK 222584, Oxaliplatin, 5-Fluorouracil, Leucovorin, Rapamycin, Lapatinib, Ronafarnib, Sorafenib, Gefitinib, AG1478, AG1571, Thiotepa, Cyclophosphamide, Busulfan, Improsulfan, Piposulfan, Benzodopa, Carboquan, Meturedopa, Uredopa, Ethyleneimine, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylentione Foramide (triethiylenethiophosphoramide), trimethylolomelamine, buratacin, buratacinone, camptothecin, topotecan, bryostatin, calistatin, CC-1065, adzeresin, karzeresin, bizeresin, cryptophycin 1, cryptophycin 8, dorastatin, duocalmycin, KW-2189, CB1-TM1, eleuterobin, pancratistatin, sarcodictyin, Spongistatin, chlorambucil, chlornafadin, cholophosphamide, estramustine, ifosfamide, mechloretamine, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, Kalicaremycin, Kalicaremycin gamma 1, Kalicaremycin omega 1, Dy Nemycin, Dynemycin A, Clodronate, Esperamicin, Neocardinostatin Chromophore, Acrasinomycin, Actinomycin, Antremycin, Azaserin, Bleomycin, Kakutinomycin, Carabicin, Carninomycin, Cardinophilin, 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, nogaramycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomygrin migrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimethrexate, fludarabine, 6-mercaptopurine, thiamiprine, tiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone propionate (propionate), epithiostanol, mepitiostane, testactone, aminoglutethimide, mitotane, trilostane, folinic acid, acegraton, aldoforamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, defofamine, demecorsin, diazicone, elfornithine, eriptinium acetate, etogluside, gallium nitrate, hydroxyurea, lentinan, lonidainin e) Mytansine, Anthamitosine, Mitoguazone, Mitoxantrone, Mopidanmol, Nitraerine, Pentostatin, Fenamet, Pirarubicin, Rosoxantrone, 2-Ethylhydrazide, Procarbazine, Polysaccharide-K, Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadiquan, 2,2',2"-Trichlorotriethylamine, T-2 Toxin, Verracurin A, Loridine A, and Anguidin, Urethane, Vindesine, Dacarbazine, Mannomustine,Mitobronitol, Mitractol, Pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-modified 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, topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or any pharmaceutically acceptable salt, solvate, or acid of any of the above. (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 inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone induction factor, interferon Interferon-α, interferon-β, interferon-γ, colony-stimulating factor ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factors, LIF, kit ligand, or any combination of the above. (c) Diphtheria toxin, botulinum toxin, tetanus toxin, shiga toxin, cholera toxin, amanitin, amanitin derivative, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivative, tetrodotoxin, brevetoxin, sxtiotoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, drastatin, drastatin analog, cryptophycin, camptothecin, camptothecin analog and metabolite, lysocine, lysocine derivative, CC-1065, CC-1065 analog or derivative, duocarmycin, enediyne antibiotic, esperamicin, epothilone, azonaphthide, aplidine, toxoid, or any combination of the above, (d) An affinity ligand, wherein the affinity ligand is a substrate, inhibitor, stimulant, neurotransmitter, radioisotope, or any combination of the above, (e) A radioactive label, 32 P, 35 S, fluorescent dye, high electron density reagent, enzyme, biotin, streptavidin, digoxigenin, hapten, immunogenic protein, nucleic acid molecule having a sequence complementary to the target, or any combination of the above, (f) An immunomodulatory compound, anticancer agent, antiviral agent, antibacterial agent, antifungal agent, and antiparasitic agent, or any combination of the above, (g) Tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole, (i) Flutamide, nilutamide, bicalutamide, leuprorelin, goserelin, or troxacitabine, (j) An aromatase inhibitor, (k) A protein kinase inhibitor, (l) A lipid kinase inhibitor, (m) Antisense oligonucleotide, (n) Ribozyme, (o) vaccines, and (p) Select from anti-angiogenic agents.

[0163] In some preferred embodiments, G is as follows: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The formula includes a portion selected from, [ka] This is a fragment of a linking group Z' that connects Z' to Ar, in this case to a substituted Ph group.

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

[0165] One-step conjugation In some embodiments, this disclosure relates to a method for preparing compounds of formula I, involving a one-step conjugate between an antibody and a linker. The compounds of formulas (II) and (III) described above are suitable for one-step conjugate with an antibody.

[0166] For example, a precursor containing a methylphenylsulfone moiety (MPS) can be conjugated according to a series of steps shown in Scheme 1. Step A involves the in-situ elimination of the p-methylphenylsulfonyl group, forming a reactive intermediate. In Step B, this intermediate is conjugated with a thiol residue of the 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 includes a moiety that generates an activated Michael receptor upon sulfinic acid elimination. An example of such a precursor is shown in Scheme 3. [ka]

[0169] In some embodiments, the precursor includes a moiety that acts as an activated Michael receptor in the conjugate reaction. An example of a conjugate reaction mediated by an activated Michael receptor is shown in Scheme 4. [ka]

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

[0171] Two-step conjugation In some embodiments, the present disclosure relates to a method for preparing a compound of formula I, involving a two-step conjugation. The first step involves the conjugation of an antibody with a linker, the linker having a reactive group such as an azide or alkyne at its terminus. In the second step, the antibody-containing precursor is reacted with a precursor containing an active agent to produce the final ADC.

[0172] In some embodiments, the first step of a two-step procedure involves conjugation of an antibody with a precursor containing one of the reactive groups disclosed in the "One-Step Conjugation" section above. An exemplary precursor for the first conjugation step is shown in Scheme 6. [ka]

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

[0174] Anti-B7-H3 antibody Exemplary anti-B7-H3 antibodies include those shown in Tables 19-24 herein, or any fragments, variants, multimers, or bispecificities thereof. Similarly, anti-B7-H3 antibodies may be antibodies that bind to the same epitopes as the antibodies listed in Tables 19-24, or any fragments, variants, multimers, or bispecific variants thereof. Preferred anti-B7-H3 antibodies of this disclosure include fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies, or any fragments, variants, multimers, or bispecificities thereof. These antibodies exhibit specificity to human B7-H3 and have been shown to modulate, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological function or activity of B7-H3.

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

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

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

[0178] The methods and techniques described herein are generally performed in accordance with the prior art methods known in the art, unless otherwise indicated, and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, “Principles of Neural Science”, McGraw-Hill Medical, New York, NY (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, WH Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, WH Freeman & Co., NY (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

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

[0180] All of the foregoing references made in this application, as well as any other publications, patents, and published patent applications, are incorporated herein by reference. In the event of any conflict, this Specified Provision shall prevail, including the specific definitions herein.

[0181] The term “agent” is used herein to refer to chemical compounds (e.g., organic or inorganic compounds, mixtures of chemical compounds), biological macromolecules (e.g., nucleic acids, antibodies (including some thereof), and humanized antibodies, chimeric antibodies and human antibodies, and monoclonal antibodies, proteins or parts thereof, e.g., peptides, lipids, carbohydrates), or extracts made from biological materials such as bacterial, plant, fungal, or animal (especially mammalian) cells or tissues. Examples of agents include agents with known structures and agents with unknown structures. The ability of such agents to inhibit AR or to promote AR degradation may make them suitable as “therapeutic agents” in the methods and compositions of this disclosure.

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

[0183] "Treating" a condition or patient means taking measures to obtain a beneficial or desirable outcome, including clinical outcomes. As used herein and as is well understood in the art, "treatment" is an approach to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, whether detectable or undetectable; reduction of disease severity; stable (i.e., non-exacerbating) state of disease; prevention of disease spread; delay or stabilization of disease progression; alleviation or temporary suppression of disease conditions; and remission (whether partial or total). "Treatment" may also mean extending survival compared to the expected survival time without treatment.

[0184] The term “prevent” is recognized in the art and, when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or any other medical condition, is well understood in the art and includes the administration of a composition that reduces the frequency of symptoms of a medical condition in a subject or delays the onset of the condition compared to a subject that has not received the composition. Therefore, cancer prevention could include, for example, reducing the number of detectable cancer growths in a patient population receiving prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancer growths in a treated population compared to an untreated control population to, for example, a statistically and / or clinically significant amount.

[0185] The “administration” of a substance, compound, or drug to a subject, or the “administration of” them to a subject, can be carried out using one of the various methods known to those skilled in the art. For example, a compound or drug may be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intrathecally, intracerebrally, and transdermally (by absorption, e.g., via skin ducts). The compound or drug may also be appropriately introduced by rechargeable or biodegradable polymer devices or other devices, e.g., patches and pumps, or formulations, that provide prolonged, delayed, or controlled release of the compound or drug. Administration can also be carried out, for example, once, multiple times, and / or over a prolonged period of time.

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

[0187] As used herein, the term “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (for example, the two agents may be effective simultaneously in the patient and may include a synergistic effect between the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially, in the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effect of different therapeutic agents.

[0188] The "therapeutic effective dose" of a drug or medication is the amount of the drug or medication that, when administered to a subject, is intended to produce the desired therapeutic effect. Complete therapeutic effect may not necessarily occur with a single dose, but may only occur after a series of doses. Therefore, the therapeutic effective dose may be administered in one or more doses. The exact effective dose required for a subject will depend, for example, on the subject's size, health, and age, as well as the nature and severity of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective dose for a given situation through routine experimentation.

[0189] As used herein, the terms “optional” or “optionally” mean that the event or situation described thereafter may or may not occur, and that the description includes examples of cases in which the event or situation occurs and examples in which it does not occur. For example, “optionally substituted alkyl” means that the alkyl may be substituted, or that the alkyl is not substituted.

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

[0191] As used herein, the term “optionally substituted” means replacing 1 to 6 hydrogen atoms in a given structure with specific substituents, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” means replacing 1 to 4 hydrogen atoms in a given structure with the substituents described above. More preferably, as described above, 1 to 3 hydrogen substituents are replaced by substituents. It should be understood that substituents may be further substituted.

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

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

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

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

[0196] The term "alkoxy" refers to an alkyl group in which oxygen is bonded to an alkyl group. Representative alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[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 linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, linear or branched alkyl groups have 30 or fewer carbon atoms in their skeleton (for example, C in the case of a linear group). 1~30 In the case of a branched chain, C 3~30 ), more preferably having 20 or fewer carbon atoms.

[0199] Furthermore, as used herein, in the examples and throughout the claims, the term “alkyl” is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter referring to alkyl moieties having substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0200] "C x~y " or "C x ~C yThe term "alkyl" means, when used in combination with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, that the group contains x to y carbon atoms in the chain. C0 alkyl indicates that the group has hydrogen atoms at the terminal position, and internally it is a bond. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in their chain.

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

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

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

[0204] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines and their salts, for example, [ka] Does it refer to the part that can be represented by, R 9 , R 10 and R 10 ' represents either a hydrogen atom or a hydrocarbyl group, or R 9 and R 10These atoms, together with the N atoms to which they are bonded, complete a heterocycle having 4 to 8 atoms in the ring structure.

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

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

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

[0208] The term "carbamate" is recognized in the art and is based on [ka] It refers to, and in the formula, R 9 and R 10 However, it independently represents hydrogen or a hydrocarbyl group.

[0209] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0210] The term "carbocyclic ring" includes monocyclic rings with 5 to 7 members and bicyclic rings with 8 to 12 members. Each ring in a bicyclic carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. Carbocyclic rings include bicyclic molecules in which one, two, or three or more atoms are shared between two rings. The term "condensed carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring in a condensed carbocyclic ring may be selected from saturated, unsaturated, and aromatic rings. In exemplary embodiments, an aromatic ring, e.g., phenyl, may be condensed with a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of a carbocyclic ring, as long as the valence allows. Examples of “carbocyclic” rings include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octa-3-ene, naphthalene, and adamantane. Examples of condensed carbocyclic rings include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hepta-3-ene. The “carbocyclic” rings can be substituted at any one or more positions where hydrogen atoms may be present.

[0211] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0212] The term "carbonate" is recognized in the relevant technical field and refers to the -OCO2- group.

[0213] As used herein, the term "carboxyl" refers to the group represented by the formula -CO2H.

[0214] The term "ester" as used herein means -C(O)OR 9 It refers to the base, R 9 This represents a hydrocarbyl group.

[0215] As used herein, the term "ether" refers to a hydrocarbyl group linked to another hydrocarbyl group via oxygen. Therefore, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers may be symmetric or asymmetric. Examples of ethers, but not limited to, include heterocyclic-O-heterocyclic and aryl-O-heterocyclic groups. Ethers may also contain "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0216] As used herein, the terms "halo" and "halogen" mean halogens, including chloro, fluoro, bromo, and iodine.

[0217] As used herein, the terms "hetaralkyl" and "hetaryl" refer to alkyl groups substituted with a hetaryl group.

[0218] The terms "heteroaryl" and "hetaaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, in which the ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaaryl" 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 heteroaromatic, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0219] As used herein, the term “heteroatom” means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0220] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclic group.

[0221] The terms "heterocyclyl," "heterocycle," and "heterocyclic system" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, in which the ring structure contains 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, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like.

[0222] As used herein, the term "hydrocarbyl" refers to a group that is bonded via a carbon atom that does not have an =O or =S substituent, and typically has at least one carbon-hydrogen bond and mainly a carbon skeleton, but may optionally include heteroatoms. Therefore, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is bonded via oxygen rather than carbon) are not considered hydrocarbyl. Examples of hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0223] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

[0224] The term “lower” means, when used in combination with a chemical moiety, e.g., acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, a group having 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, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are, each, a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether alone or in combination with other substituents, such as hydroxyalkyl and aralkyl (in which case, e.g., elements in the aryl group are not counted when counting carbon atoms in the alkyl substituent).

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

[0226] The term "sulfate" is recognized in the art and refers to the -OSO3H group or its pharmaceutically acceptable salts.

[0227] The term "sulfonamide" is recognized in the art and has a general formula. [ka] This refers to the group represented by, in the formula, R 9 and R 10 However, it independently represents hydrogen or hydrocarbyl.

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

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

[0230] The term "sulfone" is recognized in the relevant technical field and refers to the -S(O)2- group.

[0231] The term “substituted” refers to a portion having a substituent that replaces one or more hydrogens on one or more carbons of the skeleton. “Substituted” or “substituted with ~” should be understood to imply the implicit condition that such substitution results in a stable compound that does not undergo spontaneous transformation, for example, by rearrangement, cyclization, elimination, etc., depending on the acceptable valences of the substituted atom and the substituent. As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. In a broad view, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. For a given organic compound, there may be one or more, the same, or different acceptable substituents. For the purposes of the present invention, heteroatoms such as nitrogen may have any acceptable substituent of the organic compound described herein that satisfies the valences of the hydrogen substituent and / or the heteroatom. The substituents may include any substituents described herein, for example, halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that, where appropriate, the substituted moieties on the hydrocarbon chain themselves may be substituted.

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

[0233] The term "thioester" as used herein refers to -C(O)SR 9 or -SC(O)R 9 It refers to the base, In the formula, R 9 However, this represents hydrocarbyl.

[0234] As used herein, the term "thioether" refers to an ether equivalent in which oxygen is replaced by sulfur.

[0235] The term "urea" is recognized in the relevant technical field, and the general formula [ka] It may also be expressed as, in the formula, R 9 and R 10 However, it independently represents hydrogen or hydrocarbyl.

[0236] As used herein, the term “modulate” includes inhibition or suppression of function or activity (such as cell proliferation), as well as enhancement of function or activity.

[0237] "Pharmacologically acceptable salt" or "salt" is used herein to mean an acid addition salt or basic addition salt that is suitable for or appropriate for the treatment of a patient.

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

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

[0240] Many of the compounds useful for the methods and compositions of this disclosure have at least one stereocenter in their structure. This stereocenter may be in the R or S conformation, and the notations R and S are used in accordance with the rules set out in Pure Appl. Chem. (1976), 45, 11-30. This disclosure intends all stereoisomer forms, including enantiomers and diastereoisomers, of 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 Disclosure may be racemic. In certain embodiments, the compounds of the Disclosure may be rich in one enantiomer. For example, the compounds of the Disclosure may have 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 greater ee.

[0242] As is generally 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 whose stereochemistry is not shown.

[0243] In certain embodiments, the compositions or compounds of the present disclosure may be concentrated to provide primarily one enantiomer of the compound. Enantiomerically concentrated compositions or compounds may, for example, contain at least 60 mol percent of one enantiomer, or more preferably at least 75, 90, 95, or even 99 mol percent of one enantiomer. In certain embodiments, a compound rich in one enantiomer may be substantially free of the other enantiomer, by which case 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%, compared to the amount of the other enantiomer (e.g., in the composition or mixture compound). 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 mol percent of the first enantiomer and only 2 mol% of the second enantiomer.

[0244] Furthermore, certain compounds containing alkenyl groups may exist as Z (tuzamen) or E (entgegen) isomers. In each case, this disclosure includes both mixtures and distinct individual isomers.

[0245] Some of the compounds may also exist in tautomeristic forms. Such forms are not expressly shown in the formulas described herein, but are intended to be included within the scope of this disclosure.

[0246] A “prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that, after administration, is metabolized in the host, for example, by hydrolysis or oxidation, to form a compound of the Disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds having a biologically unstable or cleavable (protecting) group on the functional moiety of an active compound. Examples of prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramides as biologically unstable or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, which are incorporated herein by reference. The prodrugs of the Disclosure are metabolized to produce a compound of formula I. The Disclosure, to its extent, includes prodrugs of the compounds described herein. Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0247] As used herein, the terms “Log,” “LogS,” or “logS” refer to solubility in water, and are used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution characteristics. 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 within the art may be used to produce polyclonal or monoclonal antibodies directed against a given target, such as B7-H3, tumor-associated antigens, or other targets, or against their derivatives, fragments, analog homologs, or orthologues. (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.)

[0249] Antibodies can be prepared by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide IgG fragments of immunoserum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin may be immobilized on a column, and the immunospecific antibody may be purified by immunoaffinity chromatography. The purification of immunoglobulins 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 antibodies of this disclosure are monoclonal antibodies. Monoclonal antibodies are generated, for example, by using the procedures described in the examples provided herein. Antibodies are also generated, for example, by immunizing BALB / c mice with a combination of cell plasma transfer agents that express a given target at high levels on their surface. Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to a selected target.

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

[0252] Immunotherapeutic agents typically include protein antigens, their fragments, or fusion proteins. Generally, peripheral blood lymphocytes are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with immortalized cell lines using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), and these substances prevent the growth of HGPRT-deficient cells.

[0253] Preferred immortalized cell lines are those that efficiently fuse, support stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to media such as HAT medium. More preferred immortalized cell lines include, for example, mouse myeloma lines obtainable from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for monoclonal antibody production. (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] Next, the culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed towards the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

[0255] After the desired hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and grown using 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 Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0256] Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0257] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of this disclosure can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of this disclosure serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not otherwise produce immunoglobulin proteins, to obtain synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567, Morrison, Nature 368,812-13 (1994)), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domains of the antibodies of this disclosure, or in place of the variable domain of one antigen combination site of the antibodies of this disclosure, to produce a chimeric bivalent antibody.

[0258] Examples of monoclonal antibodies in this disclosure include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without inducing an immune response by humans to the administered immunoglobulin. The humanized form of the antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) that mainly consists of human immunoglobulin sequences and contains minimal sequences derived from non-human immunoglobulins. Humanization is carried out, for example, by substituting a rodent CDR or CDR sequence with the corresponding sequence of the human antibody, according to the methods of Winter and collaborators (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. Patent No. 5,225,539.) In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies also contain residues not found, for example, in the recipient antibody, in the transferred CDR, or in the framework sequence. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin, and all or substantially all of the framework region being that of the human immunoglobulin consensus sequence. Humanized antibodies also optimally contain at least a portion (Fc) of the immunoglobulin constant region, typically at least a portion of the immunoglobulin constant region of human immunoglobulin (Jones et al., 1986, Riechmann et al., 1988, and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0259] Fully human antibodies are antibody molecules in which both the light and heavy chain sequences, including the CDR, are derived from human genes. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), or EBV hybridoma technology for producing monoclonal antibodies (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies may be used, and they 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 with Epstein-Barr virus in vitro (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 techniques, 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 produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar in all aspects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and 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 It is described in 65-93 (1995).

[0261] Furthermore, human antibodies can be produced using transgenic non-human animals modified to produce fully human antibodies, rather than endogenous antibodies, in response to antigenic challenge. (See PCT Publication WO94 / 02602). Endogenous genes encoding heavy and light chain immunoglobulins in the non-human host are neutralized, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host genome. Human genes are incorporated, for example, using a yeast artificial chromosome containing the required human DNA segment. Animals providing all the desired modifications are then obtained as offspring by mating intermediate transgenic animals containing less complement than the complete complement of the modifications. An example of such a non-human animal is a mouse called Xenomouse®, as disclosed in PCT Publication WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, directly from animals after immunization with the desired immunogen as a preparation of polyclonal antibodies, or alternatively, from immortalized B cells derived from animals such as hybridomas that produce monoclonal antibodies. Furthermore, antibodies can be obtained directly by recovering and expressing genes encoding immunoglobulins with human variable regions, or by further modifying these genes to obtain antibody analogs, such as single-stranded Fv(scFv) molecules.

[0262] An example of a method for producing a non-human host, exemplified as a mouse, lacking the 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 locus rearrangement and the formation of a transcript of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeted vector containing a gene encoding a selectable marker; and producing a transgenic mouse from embryonic stem cells containing a gene encoding a selectable marker in both somatic and germ cells.

[0263] One method for producing a target antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in a 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 both a heavy chain and a light chain.

[0264] Further improvements to this procedure, including a method for identifying clinically relevant epitopes on an immunogen and a correlation method for selecting antibodies that specifically bind to the relevant epitopes with high affinity, are disclosed in U.S. Publication No. 2003 / 009212.

[0265] The antibody can be expressed using a vector containing a DNA segment encoding the single-strand antibody described above.

[0266] These may include vectors, liposomes, naked DNA, adjuvant-utilized DNA, gene guns, catheters, etc. Examples of 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), a viral vector (e.g., a DNA or RNA viral vector), a fusion protein containing a targeting moiety (e.g., an antibody specific to a target cell) and a nucleic acid binding moiety (e.g., protamine), a plasmid, a phage, etc., as described in U.S. Patent No. 7,186,697. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0267] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. A retroviral vector is Moloney's mouse leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex virus type 1 (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 (see 1990)), and adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993), Davidson, et al., Nat. Genet Examples include 3:219 (1993), Yang, et al., J. Virol. 69:2004 (see 1995), and adeno-associated virus vectors (Kaplitt, MGet al., Nat. Genet. 8:148 (see 1994)).

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

[0269] Vectors can be used to target virtually any desired target cell. For example, stereotactic injection can be used to direct a vector (e.g., adenovirus, HSV) to a desired location. Furthermore, particles can be delivered by intraventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. Methods based on bulk flow, known as convection, have 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 available methods include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injections, as well as oral or other suitable routes of administration.

[0270] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. In this case, one binding specificity is to a target such as B7-H3 or any fragment thereof. The second binding target is any other antigen, and advantageously, it is a cell surface protein, receptor, or receptor subunit.

[0271] Many methods for producing bispecific antibodies are known in the art. Conventionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have 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. Similar procedures are disclosed in WO93 / 08829, published on 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0272] The bispecific antibodies and / or monovalent antibodies of this disclosure can be prepared using any of the various techniques known in the art, including those disclosed in application WO2012 / 023053, filed on 16 August 2011, the details of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces a bispecific antibody that is structurally identical to human immunoglobulin. This type of molecule consists 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 combination site exhibits different antigen specificity to which both the heavy and light chains contribute. The light-chain variable regions may be of the lambda or kappa family and are preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the formation of non-natural polypeptide junctions. However, it is also possible to obtain the bispecific antibodies of this disclosure by fusing a kappa light chain variable domain to a constant lambda domain for first specificity, and by fusing a lambda light chain variable domain to a constant kappa domain for second specificity. The bispecific antibodies described in WO2012 / 023053 are referred to as IgGκλ antibodies or "κλ antibodies" and are a novel fully human bispecific IgG format. This κλ format enables affinity purification of bispecific antibodies that are indistinguishable from standard monoclonal antibodies and therefore have preferred characteristics compared to previous formats, and are 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) that share the same heavy chain variable domain and have different antigen specificities. Numerous methods for generating monoclonal antibodies and their fragments are 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). Fully human antibodies are antibody molecules in which both the light chain and heavy chain sequences, including CDR1 and CDR2, originate from human genes. The CDR3 region may be of human origin or may be designed by synthetic means. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Human monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology for producing 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 used, or they 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 with Epstein-Barr virus in vitro (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 a 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 so that the target antigen is expressed and associates with the surface of the transfected cells. Various preferred techniques for producing heterologous non-human animals are well known in the art. See, for example, U.S. Patents 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

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

[0276] Next, hybridomas arising from myeloma / B cell fusions are screened for their reactivity to target antigens. Monoclonal antibodies are prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes that produce antibodies that will specifically bind to the immunizing agent, or lymphocytes capable of producing such antibodies. Alternatively, lymphocytes can be immunized in vitro.

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

[0278] The method disclosed in U.S. Patent Application No. 9,926,382 overcomes this limitation and greatly facilitates the isolation of antibodies having 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 the 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. WO2011 / 084255, each of which is incorporated herein by reference in its entirety. However, since the light chain variable domain is expressed in conjunction with the heavy chain variable domain, both domains may contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and diversity in either the lambda variable light chain or the kappa variable light chain can be used in parallel for in vitro selection of antibodies against different antigens. This approach enables the identification of two antibodies that have a common heavy chain, but one possessing a lambda light chain variable domain and the other possessing a kappa light chain variable domain, and can be used as building blocks for the generation of bispecific antibodies in the complete immunoglobulin format of the present disclosure. The bispecific antibodies of this disclosure may be of different isotypes, and their Fc moieties can be modified to alter their binding properties to different Fc receptors, and thus to alter the effector function and pharmacokinetic properties of the antibodies. Numerous methods for modifying the Fc moiety are described and applicable to the antibodies of this disclosure. (See, for example, Strohl, WR Curr Opin Biotechnol 2009(6):685-91, U.S. Patent No. 6,528,624, PCT / US2009 / 0191199, filed January 9, 2009.) Furthermore, bispecific antibodies and antibody mixtures of the F(ab')2 form lacking the Fc moiety can be produced using the methods of this disclosure.

[0279] Co-expression of a common heavy chain and two different light chains within a single cell enables the assembly of the bispecific antibodies of this disclosure. If all polypeptides are expressed at the same level and assembled equally well to form immunoglobulin molecules, the ratio of monospecificity (same light chain) to bispecificity (two different light chains) should be 50%. However, different light chains may be expressed at different levels and / or not assembled with the same efficiency. Therefore, means for regulating the relative expression of different polypeptides are used to compensate for their inherent expression characteristics or different tendencies to assemble with the common heavy chain. This regulation can be achieved through promoter strength, the use of internal ribosome entry sites (IRESs) characterized by different efficiencies, or other types of regulatory elements that can act at the transcriptional or translational level and act on mRNA stability. Examples of different promoters of different strengths include CMV (pre-early cytomegalovirus promoter), EF1-1α (human elongation factor 1α subunit promoter), Ubc (human ubiquitin C promoter), and SV40 (Simian virus 40 promoter). Different IRESs have also been described from mammalian and viral origins (see, for example, Hellen CU and Sarnow P. Genes Dev 2001 15:1593-612). These IRESs may differ significantly in their length and ribosome recruitment efficiency. Furthermore, activity can be further tuned by introducing multiple copies of the 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 modify 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] Co-expression of the 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 the constant domains of the kappa or lambda light chain, 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 this disclosure. This stands in sharp contrast to specialized purification methods that must be developed and optimized for each bispecific antibody derived from the quadroma or other cell lines expressing the antibody mixture. Indeed, when the biochemical characteristics of different antibodies in a mixture are similar, their separation using standard chromatography techniques such as ion-exchange chromatography can be difficult or impossible.

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

[0282] In other embodiments for producing bispecific antibodies, an antibody-variable domain (antibody-antigen binding site) having the desired binding specificity can be fused to an immunoglobulin constant domain sequence. The fusion is preferably with an immunoglobulin heavy chain constant domain that includes at least a portion of the hinge, CH2, and CH3 regions. Preferably, at least one of the fusions has a first heavy chain constant region (CH1) containing the sites necessary for light chain binding. The immunoglobulin heavy chain fusions, and optionally DNA encoding the immunoglobulin light chain, are inserted into a separate expression vector and co-transfected into a suitable host organism. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0283] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. A preferred interface includes at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A "cavity" equal in size to or similar to the larger side chain is created at the interface of the second antibody molecule by replacing the larger 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 undesirable end products such as homodimers.

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

[0285] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J.Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' portion of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, and then reoxidized to form an antibody heterodimer. This method can also be used to produce antibody homodimers. The “diabody” technique described by Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) provides an alternative mechanism for producing bispecific antibody fragments. The fragment is linked by a linker that is too short to form pairs between two domains on the same chain, thereby separating the light chain variable domain (VL ) connected to the heavy chain variable domain (V H ) includes. Therefore, V of one fragment H and V L The domain is a complementary V of another fragment. L and V H The domain is forced to pair with another, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J.Immunol. 152:5368 (1994).

[0286] Antibodies with a valency greater than 2 are intended. For example, triplicate antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

[0287] An exemplary bispecific antibody can bind to two different epitopes, at least one of which is derived from the protein antigen of this disclosure. Alternatively, the anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or a trigger molecule on a leukocyte such as an Fc receptor for IgG (FcγR) (e.g., FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16)) to concentrate 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 have an antigen-binding arm and an arm that binds to a cytotoxic agent or radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigen described herein and further binds to tissue factor (TF).

[0288] Heteroconjugate antibodies are also within the scope of this disclosure. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to undesirable cells (see U.S. Patent No. 4,676,980) and for the treatment of HIV infection (see WO91 / 00360, WO92 / 200373, EP03089). Antibodies are intended to be prepared in vitro using synthetic protein chemistry, including those containing crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutylimidate, as disclosed, for example, in U.S. Patent No. 4,676,980.

[0289] For example, it may be desirable to modify the antibodies of this disclosure with respect to effector function to enhance the efficacy of the antibody in the treatment of cancer and / or other diseases and disorders associated with abnormal B7-H3 expression and / or activity. For example, a cysteine ​​residue can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. Homodimerated antibodies thus produced may have improved internalization ability and / or increased complement-mediated cell death and antibody-dependent cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992).) Alternatively, antibodies having a double Fc region, thereby capable of enhanced complement lysis and ADCC ability, can be manipulated. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989).)

[0290] Conjugated antibody This disclosure also relates to conjugated antibodies, including antibodies or antigen-binding fragments thereof that are conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) (also referred to herein as immunoconjugates), or to radioisotopes (i.e., radioconjugates).

[0291] In some embodiments, the toxin is a microtubule inhibitor or a derivative thereof. In some embodiments, the toxin is drastatin 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 mytansinoid or a mytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid damage toxin. In some embodiments, the toxin is duocalmycin or a derivative thereof. In some embodiments, the toxin is calichemycin or a derivative thereof. In some embodiments, the drug is pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the drug is exatecan or a derivative thereof.

[0292] Available enzymatically active toxins and their fragments include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecenes. Various radionuclides are available for the purification of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131In, 90 Y, and 186 Re is one example.

[0293] Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azide compounds (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine 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 parts can be coupled to the antibodies obtained in this disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989), the full text of which is incorporated herein by reference).

[0295] Coupling may be achieved by any chemical reaction that will bind the two molecules together, insofar as the antibody and the other part retain their respective activities. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. However, the preferred binding is covalent bonding. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of an external crosslinking molecule. Many divalent or polyvalent coupling agents are useful for coupling protein molecules, such as the antibodies of this disclosure, to other molecules. For example, typical coupling agents include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to cover all types of coupling agents known in the art, but rather to be an example of more general 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. (For example, see Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies with oligopeptide linkers. Particularly preferred linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G)), and (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co.) Examples include (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem.Co. catalog number 2165-G) and (v) sulfo-NHS (N-hydroxysulfosuccinimide) conjugated to EDC (Pierce Chem.Co. catalog number 24510).

[0297] The linkers described above contain components with different attributes and therefore result 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, SMPT linkers can contain sterically bulky disulfide bonds and 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 carbodimide coupling. When used in combination with sulfo-NHS, carbodimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than carbodimide coupling reactions alone.

[0298] Furthermore, the antibodies disclosed herein may be formulated as immunoliposomes. Liposomes containing these 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. Patents 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent 5,013,556.

[0299] Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to obtain liposomes of the desired diameter. The Fab' fragment of the antibody of this disclosure can be conjugated to liposomes described by Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide exchange reaction.

[0300] Use of anti-B7-H3 antibodies It should be understood that the therapeutic entities described herein are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in the following formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles (e.g., lipids containing Lipofectin® (cationic or anionic)), 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 carbowaxes. Any of the aforementioned mixtures may be suitable in the treatments and therapies according to this disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible, has a suitable route of administration, and is tolerable.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 the citations therein for additional information on formulations, excipients, and carriers that are well known to pharmaceutical chemists.

[0301] The therapeutic formulations of the Disclosure, including the conjugates of the Disclosure, are used to treat or alleviate symptoms associated with cancer, for example, in non-limited examples, 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 and pharyngeal cancer, endometrial cancer, and / or melanoma. The Disclosure also provides methods for treating or alleviating symptoms associated with cancer. A treatment regimen may include, for example, identifying a subject, for example, a human patient who has cancer (or is at risk of developing cancer), using standard methods.

[0302] A therapeutic formulation of the Disclosure, comprising a conjugate of the Disclosure that recognizes B7-H3 and selectively recognizes a second target, can be used to treat or alleviate symptoms associated with B-cell-mediated autoimmune and / or inflammatory diseases, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenström hypergammaglobulinemia, Sjögren's syndrome, multiple sclerosis (MS), and / or lupus nephritis.

[0303] The effectiveness of the treatment can be determined in relation to any preferred method for diagnosing or treating a specific immune-related disorder. Relief of one or more symptoms of an immune-related disorder indicates that the conjugate provides clinical benefit.

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

[0305] Using the conjugates of this disclosure, specific targets can be isolated using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. The conjugates of this disclosure can be used diagnostically, for example, as part of a clinical trial procedure to monitor protein levels in tissues to determine the effectiveness of a given therapeutic regimen. Detection can be facilitated by coupling (i.e., physically linking) an antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetics, 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 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; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H can be mentioned.

[0306] The conjugates of this disclosure are available for use as therapeutic agents. Such agents would 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 having high specificity and high affinity for its target antigen, would be administered to a subject and would generally produce an effect resulting from its binding to the target. Administration of a conjugate can inactivate, inhibit, or interfere with the signaling function of the target. Administration of a conjugate can inactivate, inhibit, or interfere with the binding of the target to an endogenous ligand to which it naturally binds.

[0307] The therapeutically effective dose of the conjugates in this disclosure generally relates to the amount required to achieve the therapeutic objective. As described above, this may, in particular, be the binding interaction between the antibody and its target antigen, which interferes with the function of the target and / or the effect of the active agent conjugated to the antibody. The amount required to be administered further depends on the binding affinity of the antibody to 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 other target to which it is administered. A general range for therapeutically effective dosing of the conjugates in this disclosure may, as a non-limiting example, be about 0.1 mg / kg body weight to about 50 mg / kg body weight. A general dosing frequency may be, for example, in the range of twice a day to once a week.

[0308] The conjugates of this disclosure may 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 on the selection of components, 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 necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are preferably 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, for example, by coacervation technology or by interfacial polymerization, in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively.

[0311] Formulations used for in vivo administration should preferably be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0312] Sustained-release preparations can be prepared. Preferred examples of sustained-release preparations include semipermeable solid hydrophobic polymer matrices containing antibodies in the form of molded 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 (US Patent 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 (an injectable microsphere composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow for molecular release over periods longer than 100 days, while certain hydrogels release proteins over shorter periods.

[0313] The conjugates according to this disclosure can be used as agents for detecting the presence of a given target (or its protein fragment) in a sample. In some embodiments, the conjugate contains a detectable label. The antibody may be polyclonal, or more preferably monoclonal. Intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2The following can be used. The 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 the subject. Therefore, the use of the term “biological sample” includes blood, and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present disclosure can be used to detect analytes such as mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detecting the analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting the analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting the 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, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting analyte proteins include introducing a labeled anti-analyte conjugate into the target. For example, the antibody may be labeled with a radiomarker whose presence and location in the target can be detected by standard imaging techniques.

[0314] Pharmaceutical composition The antibody-drug conjugate may be used to treat the target by transferring the active drug to the target cells of interest using any preferred method for preparing the composition. In some embodiments, this disclosure relates to compositions (e.g., pharmaceutical compositions) comprising the antibody-drug conjugate described herein.

[0315] The compositions and methods of this disclosure may be used to treat individuals in need. In certain embodiments, the individuals are mammals such as humans, or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered as a pharmaceutical composition comprising, for example, the compounds of this disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or oils such as glycol, glycerol, olive oil, or other solvents or vehicles such as injectable organic esters. In preferred embodiments, when such a pharmaceutical composition is for human administration, particularly for invasive administration routes (i.e., routes such as injection or implantation that avoid transport or diffusion across the epithelial barrier), the aqueous solution is pyrogenic or substantially pyrogenic. Excipients may be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical compositions may be in the form of dosing units such as reconstitutes, powders, solutions, or lyophilized products for injection.

[0316] A pharmaceutically acceptable carrier may contain, for example, a physiologically acceptable agent that acts to stabilize, increase the solubility of, or increase the absorption of a compound, such as the compounds of this disclosure. Examples of such physiologically acceptable agents include 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 selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may be a liposome or other polymer matrix, which may incorporate, for example, the compounds of this disclosure. Liposomes, for example, including phospholipids or other lipids, are non-toxic, physiologically acceptable, and metabolizable carriers that can be prepared and administered relatively easily.

[0317] The term "pharmaceutically acceptable" is used herein to mean, within reasonable medical judgment, a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues, free from excessive toxicity, irritation, allergic reactions, or other problems or complications, and commensurate with a reasonable benefit-to-risk ratio.

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

[0319] The formulation 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-dose formulation varies depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single-dose formulation is the amount of the compound that produces the therapeutic effect. Generally, out of 100 percent, this amount is in the range of about 1 percent to about 99 percent, preferably about 5 percent to about 70 percent, and most preferably about 10 percent to about 30 percent of the active ingredient.

[0320] Methods for preparing these formulations or compositions include the step of associating an active compound, such as the compounds of the Disclosure, with a carrier and optionally one or more minor components. Generally, formulations are prepared by homogeneously and closely associating the compounds of the Disclosure with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, forming the product.

[0321] As used herein, the terms “parenteral administration” and “administered parenterally” typically refer to, but are not limited to, modes of administration other than enteral and topical administration by injection, including intravenous, intraocular (including intravitreous), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, intracapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds together with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injection solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes to make the formulation isotonic with the blood of the recipient to whom the formulation is intended, or suspending agents or thickeners.

[0322] Suitable aqueous and non-aqueous carriers usable in the pharmaceutical compositions of this 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. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0323] These compositions may contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin can lead to prolonged absorption of the injectable drug form.

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

[0325] Injectable depot formulations are prepared by forming a microencapsulation matrix of the target compound in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Injectable depot formulations can also be prepared by capturing the drug in liposomes or microemulsions that are compatible with biological tissues.

[0326] For use in the methods of this disclosure, the active compound may be given by itself 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 delivery method may also be provided by a rechargeable or biodegradable device. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein-based biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-biodegradable polymers, can be used to form implants for the sustained release of compounds at specific target sites.

[0328] The actual dosage level of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, while remaining non-toxic to the patient.

[0329] The selected dosage level will depend on a variety of factors, including the activity of the specific compound or combination of compounds used, or its ester, salt, or amide; the route of administration; the time of administration; the rate of excretion of the specific compound used; the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific compound used; and factors well known in the medical field, such as the age, sex, weight, condition, overall health, and prior medical history of the patient being treated.

[0330] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the therapeutically effective dose of the required pharmaceutical composition. For example, a physician or veterinarian can start administration of the pharmaceutical composition or compound at a lower level than necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. "Therapeutic dose" means a concentration of the compound sufficient to induce the desired therapeutic effect. Generally, the effective dose of a compound is understood to vary depending on the subject's weight, sex, age, and medical history. Other factors influencing the effective dose may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and any other type of therapeutic agent administered together with the compound of this 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 13th ed., 1814–1882, incorporated herein by reference).

[0331] Generally, a preferred daily dose of the active compound used in the compositions and methods of this disclosure would be the minimum effective dose of the compound that produces a therapeutic effect. Such an effective dose generally depends on the factors described above.

[0332] Patients receiving this treatment may be primates, especially humans, as well as any animal in need, including other mammals such as horses, cattle, pigs, sheep, cats, and dogs, poultry, and pets in general.

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

[0334] Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0335] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, 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 may be prepared in an injectable form, either as a liquid solution or a suspension. A solid form suitable for injection may also be prepared, for example, as an emulsion or using an antibody-drug conjugate encapsulated in liposomes. The antibody-drug conjugate may be combined with a pharmaceutically acceptable carrier, which may include any carrier that does not induce the production of antibodies harmful to the recipient. Suitable carriers typically include large macromolecules that are metabolized slowly, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and lipid aggregates.

[0337] The composition may also contain diluents, such as water, saline solution, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., may also be present. The composition may be administered parenterally by injection, which may be subcutaneous or intramuscular. In some embodiments, the composition may be administered to a tumor. The composition may be inserted (e.g., injected) into the tumor. Additional formulations are suitable for other forms of administration, such as suppositories 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 suitable for the dosage and formulation. The composition preferably contains a therapeutically effective amount of antibody-drug conjugate. The dosage may vary depending on the subject being treated, the subject's health and physical condition, the desired degree of protection, and other relevant factors. The exact amount of the active ingredient (e.g., antibody-drug conjugate) may depend on the physician's judgment. For example, a therapeutically effective amount of antibody-drug conjugate, or a composition containing it, may be administered to a patient with cancer or a tumor to treat the cancer or tumor.

[0339] The antibody-drug conjugates or compositions containing the same according to this disclosure may be administered in the form of a pharmaceutically acceptable salt thereof. In some embodiments, the antibody-drug conjugates or compositions containing the same according to this disclosure may be administered together with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable additive. The effective amounts and types of pharmaceutically acceptable salts, excipients, and additives 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, this disclosure relates to a method for 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 of this disclosure (also referred to herein as “active compounds”), as well as their derivatives, fragments, analogues, and homologs, can be incorporated into pharmaceutically suitable compositions for administration. Such compositions typically comprise a conjugate and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder, etc., that are suitable for pharmaceutically administered substances. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is intended for use in this composition unless it is incompatible with the active compound. Auxiliary active compounds may also be incorporated into the composition.

[0342] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, and subcutaneous administration. The following components may be used as solutions or suspensions for parenteral, intradermal, or subcutaneous application: Sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and agents for adjusting osmotic pressure such as sodium chloride or dextrose. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0343] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble), or dispersions and sterile powders for the immediate preparation of sterile injections or dispersions. 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 fluid enough to allow for easy injection. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. The prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols (mannitol, sorbitol, etc.), and sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0344] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination thereof of the components listed above, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, from which powders of the active component and any additional desired components are obtained from the solution that has been previously sterile filtered.

[0345] In certain embodiments, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be obvious to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to infected cells with monoclonal antigens against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to preferred methods, for example, as described in U.S. Patent No. 4,522,811.

[0346] It is particularly advantageous to formulate oral or parenteral compositions into dosing unit forms for ease of administration and uniformity of dosage. As used herein, dosing unit forms refer to physically distinct units suitable as unit doses for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dosing unit forms in this disclosure depend on and directly on the inherent characteristics of the active compound, the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such active compounds for the treatment of an individual.

[0347] The pharmaceutical composition may be contained in a container, pack, or dispenser, along with instructions for administration.

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

[0349] In some embodiments, the Disclosure provides a method for treating cancer, comprising administering an antibody-drug conjugate 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, uterine cancer, or melanoma.

[0350] In some embodiments, the present disclosure provides a method for treating an autoimmune or inflammatory disease, comprising administering an antibody-drug conjugate or a pharmaceutically active composition thereof. In some embodiments, the autoimmune or 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), Waldenström hypergammaglobulinemia, Sjögren's syndrome, multiple sclerosis (MS), or lupus nephritis.

[0351] The configuration of this disclosure will be described in detail below through examples, but these examples are intended only to aid in understanding this disclosure. The scope of this 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] TIFF2026053657000074.tif238161TIFF2026053657000075.tif18159 [Examples]

[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 an N2 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 x 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), 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 mL, 3.08 mmol) were added at room temperature. The mixture was stirred at 100 °C for 16 hours, cooled to 0 °C, and stirred for another hour. The solid was filtered and washed with ether (30 mL x 2) to obtain 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), Oxone (25.03 g, 40.72 mmol) was added at 0°C under an N2 atmosphere. After stirring at room temperature for 14 hours, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL x 3). The organic layer was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 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]nona-4-in-9-ylmethanol (800 mg, 5.3 mmol) was dissolved in DCM (125 mL) at room temperature under an N2 atmosphere. Pyridine (1.22 mL, 15.9 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.74 mmol) were added. The mixture was stirred at the same temperature for 4 hours, then 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 pressure. The residue was purified by column chromatography (Hex:EA=10:1) to obtain 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), propargylamine (106 μL, 1.65 mmol) was added at room temperature under a N2 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 x 2) and distilled water (20 mL). The organic layer was extracted, washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 Yield 30%

[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 an N2 atmosphere, compound L-2-2 (3.0 g, 13.7 mmol) was treated with Jones' reagent (20 mL) in acetone solution (100 mL) and stirred for 4 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was extracted with DCM (50 mL x 2) and distilled water (15 mL). The organic layer was washed with brine (50 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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] Preparation of Example 1.5.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), KI (294 mg, 1.77 mmol) and Ag2O (4.92 g, 19.48 mmol) were added under a N2 atmosphere. The mixture was stirred overnight at room temperature. After the reaction was complete, 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 obtain 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), NaN3 (1.34 g, 20.55 mmol) was added under an N2 atmosphere. The mixture was stirred at 110°C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 EtOH (5 mL) solution of L-3-2 (1.0 g, 3.25 mmol) 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 BOC2O (852.1 mg, 3.9 mmol) was added thereto. 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 an N2 atmosphere. Triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 an N2 atmosphere and cooled to 0°C. The mixture was stirred at 0°C for 20 minutes, after which L-3-4 (1.56 g, 3.4 mmol) was added. The reaction mixture was heated to room temperature and stirred overnight. The reaction mixture was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-3-5 (1.91 g, 93%).

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

[0385] Preparation of compound L-3 At 0°C, a solution of compound L-3-5 (906.7 mg, 1.49 mmol) in EA (4 mL) and ether (4 mL) was slowly added with a 5% HCl solution (8 mL) and triphenylphosphine (390 mg, 1.49 mmol) under a N2 atmosphere. The mixture was stirred overnight at 0°C. The mixture was diluted with DCM (10 mL). The aqueous layer was extracted with DCM (10 mL x 3). The aqueous phase was concentrated under high pressure to obtain compound L-3 (495 mg, 54%).

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

[0387] Preparation of Example 1.6.L-4 [ka] Preparation of compound L-4-1 At -20°C under an N2 atmosphere, tetraethylene glycol (4.35 mL, 25.22 mmol), followed by propargyl bromide (1.0 g, 8.41 mL), was added to a 50 mL dry THF solution of KOtBu (943 mg, 8.41 mmol). The reaction mixture was heated to room temperature and stirred for 17 hours. The reaction mixture was quenched by adding MeOH (1 mL) and H2O (50 mL) while cooling in an ice bath, and extracted with EA (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 Triphenylphosphine (1.13 g, 4.31 mmol), followed by L-4-1 (500 mg, 2.15 mmol), was added to a 20 mL solution of dried DCM cooled in an ice bath of CBr4 (1.43 g, 4.31 mmol). The mixture was heated to room temperature and stirred for 18 hours. The reaction product was diluted with water (50 mL) and extracted with DCM (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 N2 atmosphere. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 over 1.5 hours at room temperature under an N2 atmosphere to a mixture of thioacetic acid (2.75 mL, 38.47 mmol, 1.0 equivalent) and DIPEA (8.5 mL, 48.9 mmol, 1.3 equivalent) in dry THF (50 mL). After stirring at -20°C for 4 hours under an N2 atmosphere, the mixture was concentrated, diluted with water (100 mL), and extracted with EA (200 mL × 3). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=12:1) to obtain 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 9.67 g (37.90 mmol) of L-5-1 in 80 mL of AcOH was mixed with 40 mL of 35% hydrogen peroxide under a N2 atmosphere at room temperature. The mixture was stirred overnight, then concentrated, diluted with 20 mL of water, neutralized with NaHCO3, and washed with EA / Hex (1 / 1, 30 mL x 2). The aqueous layer was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 8:1:0.01~5:1:0.01) to obtain 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 equivalents) was added under an N2 atmosphere, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 7:1:0.01~5:1:0.01) 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 In a 50 mL round-bottom flask, L-5-3 (500 mg, 2.24 mmol), 10 mL of MeOH, 5% Pd / C (715 mg, 0.34 mmol, 0.15 equivalents), and Boc2O (538 mg, 2.46 mmol, 1.1 equivalents) were added. After removing the air by aspirator, the mixture was stirred under H2 at room temperature for 15 hours. The catalyst was filtered through Celite®, and the Celite® was washed with MeOH (20 mL x 2). The solvent was removed by rotary evaporator, and the residue was purified by column chromatography (DCM:MeOH:AcOH = 7:1:0.01~5:1:0.01) to obtain 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 Under N2 conditions at room temperature, 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 equivalents) and stirred for 8 hours. The reaction mixture was neutralized with 2N 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 equivalents), and EDCI-HCl (260.7 mg, 1.36 mmol, 4.0 equivalents) in DMF (2 mL) was stirred overnight at room temperature under a N2 atmosphere. The mixture was treated with L-3 (210.8 mg, 0.34 mmol, 1.0 equivalent) and DIPEA (177.6 μL, 1.02 mmol, 3.0 equivalents) and stirred overnight. The reaction product was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 12:1:0.01~5:1:0.01) to obtain compound L-5-6 (159.1 mg, 55%) as a yellow oily substance.

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

[0405] Preparation of compound L-5 At room temperature under an N2 atmosphere, 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) and stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure to obtain compound L-5 (92 mg, 99%) as a yellow oily substance.

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

[0407] Preparation of Example 1.8.L-6 [ka] Preparation of compound L-6-1 At room temperature under an N2 atmosphere, 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 equivalents), and stirred overnight. The reaction mixture was quenched by adding water (50 mL) and extracted with EA (100 mL x 3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 9:1~2:1) to obtain 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 At room temperature under an N2 atmosphere, a suspension of CsCO3 (1.05 g, 3.21 mmol, 0.6 equivalents) in DMF (12 mL) was treated with thioacetic acid (498 μL, 6.96 mmol, 1.3 equivalents) and L-6-1 (2.0 g, 5.36 mmol) in DMF (8 mL), and the mixture was stirred overnight. The mixture was quenched by adding water (50 mL) and extracted with EA (100 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=5:1) to obtain 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 1.2 g (4.33 mmol) of L-6-2 was added to 10 mL of AcOH under a N2 atmosphere at room temperature, and 4 mL of 35% hydrogen peroxide was added. The mixture was stirred for 7 hours and then concentrated under reduced pressure. The residue was diluted with water (5 mL) and basicized to pH 9 using a saturated aqueous solution of NaHCO3 at 0°C. 1.4 g (6.49 mmol, 1.5 equivalents) of Boc2O was added, and the resulting mixture was stirred overnight. The mixture was neutralized with 2N HCl solution at 0°C and concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 8:1:0.01~5:1:0.01) to obtain 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 / H2O (2.0 mL / 4.0 mL) was treated with LiOH (17.3 mg, 0.41 mL, 1.5 equivalents) at room temperature under an N2 atmosphere and stirred for 3 hours. The mixture was neutralized with 2N HCl at 0°C and concentrated under reduced pressure to obtain 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 equivalents), and EDCI-HCl (50 mg, 0.26 mmol, 2.0 equivalents) were dissolved in DMF (2 mL) at room temperature under an N2 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 At room temperature under an N2 atmosphere, L-2 (0.19 mmol, 1.5 equivalents) and EDCI-HCl (50 mg, 0.26 mmol, 2.0 equivalents) were added to a stirred DMF (2 mL) solution of L-6-5 (0.13 mmol). 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 = 12:1:0.01~5:1:0.01) to obtain compound L-6-6 (34.8 mg, 64%) as a yellow oily substance.

[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) under a N2 atmosphere at room temperature, and the mixture was stirred for 30 minutes. The mixture was concentrated under reduced pressure to obtain compound L-6 (25.4 mg, 99%) as a yellow oily substance.

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

[0421] Example 1.9. Preparation of MPS-D1-10 [ka] Preparation of compound L-1-1 Under an N2 atmosphere at room temperature, a clear solution of 11-azido-3,6,9-trioxaundecane-1-amine (Aldrich, CAS 134179-38-7, 5.0 g, 22.9 mmol) in 1,4-dioxane (100 mL) and H2O (25 mL) was treated with NaHCO3 (3.8 g, 45.8 mmol, 2.0 equivalents) and BOC2O (6.0 g, 27.5 mmol, 1.2 equivalents), and then stirred for 6 hours. The reaction mixture was quenched with water (50 mL) and extracted with DCM (100 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (1%-3% MeOH in DCM) to obtain 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 H2O (15 mL) was treated with triphenylphosphine (6.5 g, 24.9 mmol, 1.1 equivalents) at room temperature under an N2 atmosphere, and then stirred overnight. The reaction mixture was diluted with water (10 mL) and extracted with DCM (60 mL x 3). The aqueous layer was concentrated under reduced pressure to obtain 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 the same manner as the preparation method for 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 the same manner as the preparation method for 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 equivalent) in anhydrous DMF (2.0 mL) was treated with DIPEA (52 μL, 0.3 mmol, 3 equivalents) and HBTU (57 mg, 0.15 mmol, 1.5 equivalents) under an N2 atmosphere at room temperature and stirred for 2 hours. The reaction mixture was quenched with H2O (20 mL) and extracted with EA (30 mL x 3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC to obtain 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), Ag2O (4.92 g, 19.48 mmol), and p-TsCl (3.7 g, 19.48 mmol) were added under an N2 atmosphere. The mixture was stirred overnight at room temperature. After the reaction was complete, 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 obtain 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), NaN3 (1.34 g, 20.55 mmol) was added under an N2 atmosphere. The mixture was stirred at 110°C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 an N2 atmosphere. Triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), 5% Pd / C (1.04 g, 0.49 mmol) was added under an H2 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). BOC2O (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 Under an N2 atmosphere at 0°C, 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). The mixture was stirred at 0°C for 20 minutes, after which L-11-2a (544.5 mg, 1.18 mmol) was added. The reaction mixture was heated to room temperature and stirred overnight. The reaction mixture was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), 4 M HCl (1 mL in 1,4-dioxane) was added at 0°C under an N2 atmosphere. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to obtain compound L-11 (527.6 mg, quantitative).

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

[0446] Table 2 below lists the compounds synthesized via the same synthetic route as described in Example 2. [Table 3] TIFF2026053657000087.tif223159TIFF2026053657000088.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 the 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 equivalent) at room temperature under an N2 atmosphere and stirred for 45 minutes until it reached room temperature. The reaction mixture was diluted with DCM (32 mL), washed with 1 N HCl (30 mL) and brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under high pressure. The residue was purified by preparative HPLC to obtain the title compound L-5 (70.8 mg, 61%, 9 mg of mixture) as a white gum-like substance.

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

[0453] Example 2.3. Preparation of Mal-3 [ka] Preparation of compound Mal-3-1 2,2'-diamino-N-methyldiethylamine (10.3 g, 88.0 mmol) was added to a solution of BOC2O (9.6 g, 44.0 mmol) in THF (50 mL) at 0°C under an N2 atmosphere. The mixture was stirred at room temperature for 2 hours. The mixture was quenched with H2O (100 mL) and DCM (150 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 At room temperature, maleic anhydride (248 mg, 2.53 mmol) was added to a solution of Mal-3-1 (500 mg, 2.3 mmol) in AcOH (3.0 mL) under a N2 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, and the mixture was stirred at 75°C for 5 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), TFA (3.0 mL) was added 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), DIPEA (57 μL, 0.32 mmol) was added at room temperature under a N2 atmosphere. The mixture was stirred for 2.5 hours, and EA (50 mL x 2) and H2O (30 mL) were added. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), NH2NH2·H2O (88 mL, 1805.4 mmol) was added under a N2 atmosphere. The mixture was stirred overnight under reflux. The mixture was cooled to room temperature, concentrated under reduced pressure, and subsequently ground with EtOH to obtain 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), CS2 (45 mL, 749.32 mmol) and KOH (6.5 g, 115.28 mmol) were added under an N2 atmosphere. After stirring at 85°C for 18 hours, the reaction mixture was adjusted to pH 4 by adding 1 M HCl solution and diluted with distilled water (500 mL) and EA (500 mL²). The organic layer was washed with H2O (500 mL) and brine (500 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to ether / Hex grinding to obtain 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), Et3N (4.3 mL, 30.9 mmol) and MeI (1.76 mL, 28.33 mmol) were added 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 H2O (150 mL) and extracted with EA (100 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to ether grinding to obtain 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 compound POS-1-4 To a solution of compound POS-1-3 (3.2 g, 15.37 mmol) in EtOH (150 mL), 70% m-CPBA (11.4 g, 46.11 mmol) was added at 0°C under an N2 atmosphere. After stirring at room temperature for 5 hours, another 70% m-CPBA (11.4 g, 46.11 mmol) was added. The mixture was then stirred overnight at room temperature, quenched with H2O (500 mL) and saturated NaHCO3 (300 mL), and extracted with EA (500 mL x 2). The organic layer was washed with brine (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was ground to a hex / EA ratio of 1:1 (100 mL) to obtain 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 solutions 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), PPh3 (667 mg, 2.58 mmol) was added. The mixture was cooled to 0°C, DEAD (1.17 mL, 2.58 mmol) was added, 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 x 2). The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 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 Under an N2 atmosphere at room temperature, a solution of Int-TG (18.5 g, 45.0 mmol), 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol), and molecular sieve (10.0 g) in ACN (150 mL) was treated with Ag2O (38.0 g, 0.164 mol) and stirred for 3 hours. The reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 Under an N2 atmosphere at 0°C, Int-3-a (540 mg, 1.19 mmol) in anhydrous THF (15 mL) was treated with NaBH4 (113 mg, 2.98 mmol) and stirred at 0°C for 10 minutes. After stirring at room temperature for 4 hours, the reaction product was diluted with H2O and EA. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EA:HEX = 1:1) to obtain 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) under an N2 atmosphere at room temperature and stirred for 3 hours. The reaction mixture was extracted with EA (20 mL x 2) and H2O (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The reaction mixture was purified by column chromatography to obtain 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 the same method as 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 methanol (150 mL) solution of 4-hydroxybenzoic acid (5.0 g, 36.2 mmol), thionyl chloride (26.3 mL, 362 mmol) was added under a N2 atmosphere at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The reaction product was quenched with aqueous NaHCO3 solution and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), DIPEA (2.3 mL, 13.4 mmol) and MOM-Cl (0.55 mL, 7.23 mmol) were added at 0°C under a N2 atmosphere. The reaction mixture was stirred at room temperature for 6 hours. The reaction product was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 methanol / H2O / 1,4-dioxane (16.0 mL / 8.0 mL / 16.0 mL) solution of compound Int-5-2 (1.14 g, 5.81 mmol), lithium hydroxide monohydrate (975 mg, 23.2 mmol) was added under an N2 atmosphere at 0°C. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was quenched with 2N HCl and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, 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 an N2 atmosphere. The mixture was warmed to room temperature. After stirring at room temperature for 4 hours, 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 Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG (5.2 g, 99%).

[0486] Example 3.1.2 Preparation of Int-TG2 [ka] Compound Int-TG2 was synthesized by the same method as 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), 3-(dimethylamino)1-propylamine (1.61 mL, 12.8 mmol) was added at room temperature under an N2 atmosphere. After stirring at the same temperature for 3 hours, the reaction product was extracted with EA (250 ml x 3) and H2O (200 ml). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Compound Int-TG3-1 (891 mg, 100%) was produced 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), trichloroacetonitrile (2.57 mL, 25.6 mmol) and DBU (0.3 mL, 2.05 mmol) were added at 0°C under an N2 atmosphere. After stirring at room temperature for 30 minutes, the reaction product was extracted with DCM (250 ml x 3) and H2O (200 ml). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 methanol (150 mL) solution of 4-hydroxybenzoic acid (5.0 g, 36.2 mmol), thionyl chloride (26.3 mL, 362 mmol) was added under a N2 atmosphere at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The reaction product was quenched with aqueous NaHCO3 solution and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), DIPEA (2.3 mL, 13.4 mmol) and MOM-Cl (0.55 mL, 7.23 mmol) were added at 0°C under a N2 atmosphere. The reaction mixture was stirred at room temperature for 6 hours. The reaction product was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 methanol / H2O / 1,4-dioxane (16.0 mL / 8.0 mL / 16.0 mL) solution of compound Int-TG4-2 (1.14 g, 5.81 mmol), lithium hydroxide monohydrate (975 mg, 23.2 mmol) was added under an N2 atmosphere at 0°C. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was quenched with 2N HCl and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium 2SO4, 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 Sodium hydride (301 mg, 12.56 mmol, 60%) was added to a solution of L-1-1 (2 g, 6.282 mmol) in DMF (25 mL) under an N2 atmosphere at 0°C. After 10 minutes, iodomethane (3.9 mL, 62.82 mmol) was added under an N2 atmosphere at the same temperature. The reaction mixture was stirred under an N2 atmosphere at room temperature for 3 hours. After the reaction was complete, the reaction mixture was quenched with 2N HCl (10 mL) and extracted with EA (500 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. Compound OHPAS-D1a-1 (yellow oily substance) 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), 4N HCl in dioxane (25 mL) was added under a N2 atmosphere at 0°C. The reaction mixture was stirred under a N2 atmosphere at 0°C 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 NaHCO3 (2.53 g, 43.06 mmol) were added at room temperature under a N2 atmosphere. The mixture was stirred overnight at room temperature under a N2 atmosphere. The reaction product was extracted with EA (200 mL x 2) and distilled water (100 mL). The resulting organic layer was dried over anhydrous Na2SO4, 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 sieve (8 g) and Ag2O (3.62 g, 15.61 mmol) were added at room temperature under a N2 atmosphere. The mixture was stirred at room temperature for 1 hour and then filtered using Celite®. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 compound OHPAS-D1-3 To a solution of compound OHPAS-D1-2 (2.1 g, 3.58 mmol) in DCM (30 mL), m-CPBA (2.65 g, 10.74 mmol) was added under an N2 atmosphere at 0°C. After stirring at 0°C for 7 hours, the mixture was quenched by adding saturated sodium bicarbonate (40 mL x 2). The mixture was separated, the organic layer was washed with brine, dried over Na2SO4, 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 an N2 atmosphere at 0°C. After stirring at 0°C for 1 hour, EA (30 mL x 2) and 1 M aqueous HCl (10 mL) were added. The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound OHPAS-D1-3 (1.1 g, 55%).

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

[0510] Preparation of compound 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 Et3N (207 μL, 1.46 mmol) were added at 0°C under an N2 atmosphere. 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 Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 5 mL 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 under H2 at room temperature. 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-trioxaundecane-1-amine (Aldrich, CAS 134179-38-7, 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 N2 atmosphere. The mixture was stirred at room temperature under a N2 atmosphere for 2 hours. The reaction product was extracted with EA (30 mL x 2) and distilled water (10 mL). The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 using the same method as the preparation 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), Et3N (2.28 mL, 16.38 mmol) was added at room temperature under a N2 atmosphere. SO2F2 gas was introduced by balloon, and the mixture was stirred at room temperature for 2 hours. The mixture was then washed with DCM (30 mL x 3) and brine (30 mL), the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), DBU (4 μL, 25 μmol) was added. The mixture was stirred at room temperature for 1 hour and washed with distilled water (10 mL) and EA (10 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), NaBH4 (9 mg, 0.24 mmol) was added under an N2 atmosphere at 0°C. After stirring at room temperature for 2 hours, distilled water (10 mL) and EA (10 mL x 2) were added. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 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, 0.037 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 x 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 obtain 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 overnight at room temperature. Then, distilled water (10 mL) and EA (10 mL x 2) were added, the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound OHPAS-D3-4 (53 mg, 94%).

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

[0531] Compounds OHPAS-D3a and OHPAS-D4a were prepared using a similar synthetic route to that used to prepare 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), NaBH4 (1.85 g, 48.98 mmol) was added at -78 °C under an N2 atmosphere. After stirring at 0 °C for 1 hour, the reaction mixture was quenched by adding 2N HCl (5 mL), and extracted with H2O (250 mL) and EA (250 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound OHPAS-D5-1 (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 at 0°C under an N2 atmosphere. After stirring for 2 hours, ether (100 mL) and NaHCO3 (100 mL x 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), a solution of OHPAS-D5-2 (280 mg, 0.97 mmol) in DMF (2 mL) was added under an N2 atmosphere at 0°C. After stirring at room temperature under an N2 atmosphere for 4 hours, the reaction mixture was quenched by adding H2O (10 mL) and extracted with H2O (100 mL) and EA (100 mL x 2). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound OHPAS-D5-3 (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), MeOH (1.5 mL) and sodium borohydride (391 mg, 10.35 mmol) in THF (1 mL) were added under an N2 atmosphere at -78 °C. The reaction mixture was stirred under an N2 atmosphere at 0 °C for 1 hour. After the reaction was complete, the mixture was quenched with 2N HCl (2 mL) and extracted with H2O (100 mL) and EA (100 mL x 3). 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 (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), Et3N (3.8 mL, 27.4 mmol) was added at room temperature under a N2 atmosphere. SO2F2 gas was introduced by balloon, and the mixture was stirred at room temperature for 2 hours. The mixture was then washed with DCM (30 mL x 3) and brine (30 mL), the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), BEMP (19 μl, 0.064 mmol) was added at room temperature. After 2 hours, the reaction mixture was diluted with aqueous citric acid (8 mL) and extracted with  (2 × 8 mL). The combined organic layer was dried over anhydrous Na₂SO₄, 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 using the same synthetic route as 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 H2 gas. After the reaction was complete, 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 used to prepare 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 the same method used 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 CH2Cl2 (3 mL), methanesulfonyl chloride (150 mg, 0.15 mmol) was added at 0°C under a N2 atmosphere. The reaction mixture was stirred at room temperature under a N2 atmosphere for 24 hours. After the reaction was complete, the mixture was quenched with H2O (50 mL) and extracted with CH2Cl2 (50 mL x 3). The organic layer was dried over Na2SO4, 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 Potassium thioacetate (43 mg, 0.37 mmol) was added to a solution of compound OHPAS-D7-4 (214 mg, 0.15 mmol) in ACN (3 mL) under a N2 atmosphere at room temperature. After stirring for 3 hours under a N2 atmosphere at room temperature, the mixture was quenched with H2O (50 mL) and extracted with EA (50 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 2N HCl (356 μL, 0.712 mmol) were added under an N2 atmosphere at 0°C. After stirring for 1 hour under an N2 atmosphere at 0°C, dimethyl sulfide (19.6 μL, 0.267 mmol) was added at room temperature. The reaction mixture was stirred for another 5 minutes at the same temperature. Extraction was carried out by adding H2O (20 mL) and EA (20 mL x 3). The resulting organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to produce 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 hydrogen fluoride (41.7 mg, 0.534 mmol) in H2O (0.2 mL) was added at room temperature under a N2 atmosphere. After stirring at room temperature for 2 hours, the mixture was purified by preparative HPLC to obtain 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 compounds OHPAS-D1-5 (1.0 g, 0.26 mmol) and L-1 (586 mg, 2.0 mmol, 1.2 equivalents) in DMF (10 mL) was treated with PyBOP (1.13 g, 2.17 mmol, 1.3 equivalents) and DIPEA (873 μL, 5.01 mmol, 3.0 equivalents) at room temperature under an N2 atmosphere, and stirred for 4 hours. The reaction mixture was quenched with water (20 mL) and extracted with EA (30 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 1:1~1:3) to obtain 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 Under an N2 atmosphere at room temperature, homogeneous solutions of compounds OHPAS-D9-1 (500 mg, 0.57 mmol) and OHPAS-D3-1 (140 mg, 0.69 mmol, 1.2 equivalents) in anhydrous ACN (10 mL) were treated with BEMP (66.3 μL, 0.23 mmol, 0.4 equivalents) and stirred for 4 hours. The reaction mixture was quenched with water (20 mL) and extracted with EA (30 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (4% MeOH in DCM) to obtain 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 Under an N2 atmosphere at 0°C, compound OHPAS-D9-2 (495 mg, 0.52 mmol) was treated with NaBH4 (39.7 mg, 1.05 mmol, 2.0 equivalents) in a 5.0 mL solution of anhydrous THF and stirred for 2 hours. The reaction mixture was quenched with water (20 mL) and extracted with EA (30 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (2%-3% MeOH in DCM) to obtain 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 equivalents) and TEA (79.2 μL, 0.57 mmol, 1.5 equivalents) under a N2 atmosphere at 0°C, and stirred overnight at room temperature. The reaction mixture was quenched with water (10 mL) and extracted with DCM (20 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (100% DCM to 5% MeOH in DCM) to obtain 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 Under an N2 atmosphere at room temperature, 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 equivalents) and stirred for 3 hours. The reaction mixture was diluted with water (10 mL) and extracted with DCM (20 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (3%-5% MeOH in DCM) to obtain 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 using the same method as that used to prepare 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 the same manner as the preparation method for 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 the same method as 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 used to prepare 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 used to prepare 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), K2CO3 (21 mg, 1.5 μmol) was added under an N2 atmosphere at 0°C. After stirring for 0.5 hours, the resulting residue was diluted with DMSO (0.5 mL) and purified by preparative HPLC to obtain 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 2 steps; ESI-MS m / z: 1499(M +1 ).

[0614] Example 4.1.2. Preparation of Q-1a Compound Q-1a was synthesized using the same synthetic route as 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 heating and stirring overnight at 40 °C, the mixture was separated and purified by preparative HPLC to obtain 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), DIPEA (11 μL, 60.6 mmol) was added dropwise under an N2 atmosphere. After stirring overnight at room temperature, the mixture was separated and purified by preparative HPLC to obtain compound Q-3-2 (34 mg, yield 65%), ESI-MS m / z: 992 (M / 2 +1 ) was obtained.

[0618] Compound Q-3 was synthesized using the same synthetic route as 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 compounds 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 an N2 atmosphere. After stirring overnight, the mixture was adjusted to a pH of 2-3 with 1N 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 Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain compound Q-4-1 (73 mg, 71%).

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

[0622] Compound Q-4 was synthesized using the same synthetic route as 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 using the same 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 using the same synthesis route as 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 1H NMR (400MHz, 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 1 mL of 4N HCl in 1,4-dioxane at 0°C under an N2 atmosphere. After stirring at room temperature for 4 hours, 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 using the same synthetic route as described in the document [see Mol. Pharmaceutics 2015, 12, 1813-1835].

[0635] Preparation of compound Q-6-1 Compound Q-6-1 was synthesized using the same synthetic route as described in the document [see Angew.Chem.Int.Ed.2010,49,7336-7339 and WO2015 / 110935A1].

[0636] Preparation of compound Q-6-2 To a 10 mL solution of compound Q-6a (80 mg, 0.239 mmol) and compound Q-6-1 (118 mg, 0.239 mmol) in DCM, molecular sieves and BF3·OEt2 (14.8 μL, 0.12 mmol) were added under an N2 atmosphere at 0°C. After stirring for 2 hours, the mixture was filtered through Celite®, washed with DCM (50 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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 (1 mL) in 1,4-dioxane was added at 0°C under a nitrogen atmosphere. After stirring for 4 hours, the reaction product was concentrated under reduced pressure. The reaction mixture was stirred at room temperature under nitrogen for 4 hours. Compound Q-6-2 was used directly in the next step without further purification (90 mg, 99%).

[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), Et3N (52 μL, 0.373 mmol), and 4-DMAP (2 mg, 0.015 mmol) were added at room temperature under a N2 atmosphere. The reaction mixture was stirred at room temperature for 2 hours and purified by preparative HPLC to obtain 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 N2 atmosphere at 0°C. After stirring for 11 hours, the mixture was purified by preparative HPLC to obtain 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), 25% NaOMe (11 μL, 0.048 mmol) in MeOH was added under a N2 atmosphere at 0°C. The reaction mixture was stirred at room temperature under a N2 atmosphere for 1 hour, and the pH was adjusted to 7 by adding a 5% TFA ACN solution. The mixture was purified by preparative HPLC to obtain 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), NaIO4 (18 mg, 0.081 mmol) was added at room temperature. After stirring for 2 hours, the mixture was concentrated under reduced pressure to produce 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), 2-(dimethylamino)ethylamine (6.1 μl, 0.089 mmol), TEA (34 μl, 0.243 mmol), and TBTU (52 mg, 0.162 mmol) were added at room temperature. After stirring for 1 hour, the mixture was diluted with DCM (2 × 8 mL). The organic layer was washed with H2O (8 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Q-7b (38 mg, 67%).

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

[0648] Q-7 was prepared by the same method as that used 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 using the same synthetic route as 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 referred to as "PBD-monomer") [ka] The PBD monomer was obtained by carrying out the reaction using a similar method described in EP2007 / 1813614.

[0655] Example 4.8.2 Preparation of indolino-benzodiazepine monomer (hereinafter referred to as "IBD-monomer") [ka] The IBD monomer was obtained by carrying out a reaction using a similar synthesis method described in WO2010 / 091150.

[0656] Example 4.8.3 Preparation of MCBI monomers [ka] IBD monomers were obtained by carrying out a reaction using a similar synthetic method described in US5985908.

[0657] Example 4.8.4 Preparation of tetrahydroisoquinolino-benzodiazepine monomer (hereinafter referred to as "TBD monomer") [ka] Preparation of compound M-1-1 (s)-(-)-1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid (5.0 g, 28.22 mmol) was dissolved in MeOH (140 mL), to which SOCl2 (2.30 mL, 31.04 mmol) was added dropwise under an N2 atmosphere until the temperature reached 0°C. After stirring at 40°C for 21 hours, the mixture was concentrated under reduced pressure. Diethyl ether (50 mL) was added to obtain a precipitate, which was filtered through 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 Compound Int-1 (9.07 g, 28.22 mmol) was dissolved in anhydrous THF (50 ml) to which compound M-1-1 (6.42 g, 28.22 mmol) and TEA (7.9 mL, 56.43 mmol) from THF (100 mL) were added at 0°C. After stirring at room temperature for 2 hours, the reaction product was diluted with distilled water (500 mL) and extracted with EA (800 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 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), DIBAL (16.8 mL, 16.79 mmol, 1.0 M in toluene) was added dropwise at -78°C under an N2 atmosphere. After stirring at -78°C for 4 ho...

Claims

1. An antibody conjugate represented by formula I, or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is an anti-B7-H3 antibody or its antigen-binding fragment, comprising variable heavy chain complementarity determination region 1 (CDRH1), variable heavy chain complementarity determination region 2 (CDRH2), variable heavy chain complementarity determination region 3 (CDRH3), variable light chain complementarity determination region 1 (CDRL1), variable light chain complementarity determination region 2 (CDRL2), and variable light chain complementarity determination region 3 (CDRL3). CDRH1 contains the amino acid sequence of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, or 43. CDRH2 contains the amino acid sequence of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, or 44. CDRH3 contains the amino acid sequence of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, or 45. CDRL1 contains the amino acid sequence of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, or 46. CDRL2 contains the amino acid sequence of SEQ ID NOs. 5, 11, 17, 23, 29, 35, 41, or 47. CDRL3 contains the amino acid sequence of SEQ ID NOs. 6, 12, 18, 24, 30, 36, 42, or 48. Each G is independently a chemical part comprising one or more active agents and a linker, wherein the linker links Ab to the active agent. An antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein n is an integer between 1 and 20.

2. The antibody conjugate according to claim 1, further comprising a combination of a variable heavy chain containing the amino acid sequence of SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81, and a variable light chain containing the amino acid sequence of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 83, 85, 87, 89, 91, 93, 95, or 97, where Ab further comprises a combination of a variable heavy chain containing the amino acid sequence of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 83, 85, 87, 89, 91, 93, 95, or 97.

3. Ab is, (a) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a variable light chain containing the amino acid sequence of SEQ ID NO: 50 (b) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a variable light chain containing the amino acid sequence of SEQ ID NO: 52 (c) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 53 and variable light chain containing the amino acid sequence of SEQ ID NO: 54 (d) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 55 and variable light chain containing the amino acid sequence of SEQ ID NO: 56 (e) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 57 and a variable light chain containing the amino acid sequence of SEQ ID NO: 58 (f) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 59 and variable light chain containing the amino acid sequence of SEQ ID NO: 60 (g) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 61 and variable light chain containing the amino acid sequence of SEQ ID NO: 62 (h) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 63 and variable light chain containing the amino acid sequence of SEQ ID NO: 64 (i) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 65 and a variable light chain containing the amino acid sequence of SEQ ID NO: 83, (j) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 67 and variable light chain containing the amino acid sequence of SEQ ID NO: 83 (k) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 69 and variable light chain containing the amino acid sequence of SEQ ID NO: 85 (l) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 71 and a variable light chain containing the amino acid sequence of SEQ ID NO: 87, (m) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 73 and variable light chain containing the amino acid sequence of SEQ ID NO: 89, (n) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 75 and a variable light chain containing the amino acid sequence of SEQ ID NO: 91, (o) Variable heavy chain containing the amino acid sequence of SEQ ID NO: 77 and variable light chain containing the amino acid sequence of SEQ ID NO: 93 (p) A variable heavy chain containing the amino acid sequence of SEQ ID NO: 79 and a variable light chain containing the amino acid sequence of SEQ ID NO: 95, (q) The antibody conjugate according to claim 1, further comprising a combination of a variable heavy chain sequence and a variable light chain sequence selected from a variable heavy chain having the amino acid sequence of SEQ ID NO: 81 and a variable light chain having the amino acid sequence of SEQ ID NO:

97.

4. The antibody conjugate according to claim 1, wherein the anti-B7-H3 antibody is AB1, AB2, AB3, AB4, AB5, AB6, AB7, or AB8.

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

6. Ab can be a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, or F (ab'). ２ The antibody conjugate according to 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.

7. The antibody conjugate according to any one of claims 1 to 6, wherein Ab is a rabbit, mouse, chimeric, humanized, or fully human monoclonal antibody.

8. The antibody conjugate according to any one of claims 1 to 7, wherein Ab is an IgG isotype.

9. The antibody conjugate according to any one of claims 1 to 8, wherein Ab is an IgG1 isotype.

10. The antibody conjugate according to any one of claims 1 to 9, wherein the link between Ab and the active agent is cleavable.

11. G is expressed by equation II, 【Chemistry 1】 During the ceremony, Each Q is an active agent independently linked to L' by a heteroatom, preferably O or N. Z' is a linking group, L' is SO via a heteroatom selected from O, S, and N, preferably O or N. ２ This is a spacer portion connected to L' and SO ２ The cleavage of the bond between L' and Q is selected to facilitate the cleavage of the bond between L' and Q, thereby releasing the active agent. X is -O-, -C(R ｂ ) ２ -, or -N(R ｃ ) -, preferably -O-, Ar represents a ring, for example, an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably an 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 arranged to be bonded to TG. X and Y' are arranged on adjacent atoms of Ar, When TG is activated, SO ２ And in response (Q) ｑ - (L') ｗ Replace X-SO ２ and a trigger group that generates N, O, or S atoms capable of forming a 5-6 membered ring containing an intervening Ar atom, q is an integer having a value of 1 to about 20, preferably 1 to about 10. w, x, and y are integers that each independently have a value of either 0 or 1. Each R ａ and R ｃ However, independently, they are hydrogen or lower alkyl, Each R ｂ However, independently, it is hydrogen or a lower alkyl, or Two R ｂ However, together with the atoms to which they are bonded, they form a 3- to 5-membered ring, preferably a 3- to 4-membered ring. The antibody conjugate according to any one of claims 1 to 10, wherein when w is 0, q is 1.

12. Ab-(G) ｎ However, the compound of formula (III) 【Chemistry 2】 Or represented by its salt, in the formula, A, 【Transformation 3】 And, M is N, CR ３０ , or C(-L-Q), Each L is independently selected from the spacer portion. Each Q is an active agent, X is selected from -Cl, -Br, and -I. J is Ab, R ３０ and R ３１ However, each is independently selected from electron-withdrawing groups, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl groups. R ４２ and R ４３ However, each is independent of -OH, alkoxy, and -NR ４４ R ４５ Selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykyl, and heterocyclyl, where R ４４ and R ４５ However, together with the nitrogen atom to which they are bonded, they can optionally form a 5- to 8-membered ring fused with an aryl or heteroaryl ring. R ３２ , R ４４ , and R ４５ However, each is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykryl, heterocyclyl, and haloalkyl. The antibody conjugate according to any one of claims 1 to 11, wherein n is 1 to 4.

13. Z' is selected from the following: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 During the ceremony, R ｚａ However, it is H or methyl, R ｚｂ However, -OH, =O, or =NHOH, 【Transformation 5】 Single bond or double bond, a'' represents the combination between Z' and Ar in equation (II), b'' represents the combination between Z' and Ab, Z" is Oriented in either direction 【Transformation 6】 An antibody conjugate according to claim 11 or 12, selected from the above.

14. G includes the portion selected from the following: 【Transformation 7】 During the ceremony, Q is the active agent, 【Transformation 8】 The antibody conjugate according to any one of claims 1 to 13, wherein Z' is a fragment of Z' that connects to Ar.

15. G includes the portion selected from the following: 【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】 The antibody conjugate according to any one of claims 11 to 14, wherein Z' is a fragment of Z' that connects to Ar.

16. The antibody conjugate according to any one of claims 1 to 15, wherein the active agent is selected from chemotherapeutic agents and toxins.

17. The antibody conjugate according to any one of claims 1 to 16, wherein the active agent is a chemotherapeutic agent.

18. The antibody conjugate according to any one of claims 1 to 17, 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.

19. The aforementioned active agent, (a) Erlotinib, Bortezomib, Fulvestrant, Sutent, Letrozole, Imatinib mesylate, PTK787 / ZK222584, Oxaliplatin, 5-Fluorouracil, Leucovorin, Rapamycin, Lapatinib, Ronafarnib, Sorafenib, Gefitinib, AG1478, AG1571, Thiotepa, Cyclophosphamide, Busulfan, Improsulfan, Piposulfan, Benzodopa, Carboquan, Metsuredopa, Uredopa, Ethyleneimine, Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylentione Foramide (triethylenethiophosphoramide), trimethylolomelamine, bratacin, bratacinone, camptothecin, topotecan, bryostatin, calistatin, CC-1065, adzeresin, karzeresin, bizeresin, cryptophycin 1, cryptophycin 8, dorastatin, duocalmycin, KW-2189, CB1-TM1, eleuterobin, pancratistatin, sarcodicin Spongistatin, chlorambucil, chlornafadin, cholophosphamide, estramustine, ifosfamide, mechloretamine, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, Kaliceamicin, Kaliceamicin gamma 1, Kaliceamicin omega 1, Dy Nemycin, Dynemycin A, Clodronate, Esperamicin, Neocardinostatin Chromophore, Acrasinomycin, Actinomycin, Antremycin, Azaserin, Bleomycin, Kakutinomycin, Carabicin, Carninomycin, Cardinophilin, 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, nogaramycin, olibomycin, peplomycin, potphylomycin, puromycin, queramycin, rhodorubicin, streptomygrin migrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimethrexate, fludarabine, 6-mercaptopurine, thiamiprine, tiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolonone propionate (propionate), epithiostanol, mepitiostane, testactone, aminoglutethimide, mitotane, trilostane, folinic acid, acegraton, aldoforamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, defofamine, demecolsin, diazicone, elfornithine, eriptinium acetate, etogluside, gallium nitrate, hydroxyurea, lentinan, lonidainin e) Mytansine, Anthamitosine, Mitoguazone, Mitoxanthrone, Mopidanmol, Nitraerine, Pentostatin, Fenamet, Pirarubicin, Rosoxanthrone, 2-Ethylhydrazide, Procarbazine, Polysaccharide-K, Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadiquan, 2,2',2"-Trichlorotriethylamine, T-2 Toxin, Verracurin A, Loridine A, and Anguidin, Urethane, Vindesine, Dacarbazine, Mannomustine,Mitobronitol, Mitractol, Pipobroman, Gacytosine, Arabinoside, Cyclophosphamide, Thiotepa, Paclitaxel, Albumin-modified nanoparticle formulations of Paclitaxel, Doxetaxel, Chlorambucil, Gemcitabine, 6-Thiogunine, Mercaptopurine, Cisplatin, Carboplatin, Vinblastine, Platinum, Etoposide, Ifosfamide, Mitoxantrone, Vincristine, Vinorelbine, Novantrone, Teniposide, Edatrexate, Daunomycin, Aminopterin, Xeloda, Ibandronate, CPT-11, Topoisomerase inhibitor RFS2000, Difluoromethylornithine, Retinoic acid, Capecitabine, or any pharmaceutically acceptable salt, solvate, or acid of any of the above. (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 inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone induction factor, interferon Interferon-α, interferon-β, interferon-γ, colony-stimulating factor ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or any combination of the above. (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, lysine, AM toxin, auristatin, tubulisin, geldanamycin, mytansinoids, calichemycin, daunomycin, doxorubicin, methotrexate Exatecan, exatecan derivatives, vindesine, SG2285, drastatin, drastatin analogs, cryptophycin, camptothecin, camptothecin analogs and metabolites, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocalmycin, engine antibiotics, esperamicin, epotilon, azonafide, apridin, toxoids, or any combination of the above. (d) affinity ligands wherein the affinity ligand is a substrate, inhibitor, stimulant, neurotransmitter, radioisotope, or any combination of the above, (e) a radioactive label; ３２ P, ３５ S, fluorescent dyes, high electron density reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to the target, or any combination of the above. (f) Immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or any combination of the above, (g) Tamoxifen, raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, or toremifene (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine (j) Aromatase inhibitors, (k) Protein kinase inhibitors, (l) Lipid kinase inhibitors, (m) Antisense oligonucleotide, (n) Ribozyme, (o) vaccines, and (p) An antibody conjugate according to any one of claims 1 to 16, selected from anti-angiogenic agents.

20. The active agent is one of the compounds 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 An antibody conjugate according to any one of claims 1 to 16, selected from dImBD, Q-dFuBD, and ImBD-TBD.

21. A pharmaceutical composition comprising an antibody conjugate according to any one of claims 1 to 20.

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

23. A method for treating cancer, comprising administering to a subject in need of such treatment an antibody conjugate according to any one of claims 1 to 20, or a pharmaceutical composition according to claim 21 or 22.

24. The method according to claim 23, 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, uterine cancer, or melanoma.

25. A method for treating an autoimmune disease or an inflammatory disease, comprising administering to a subject in need of such treatment an antibody conjugate according to any one of claims 1 to 20, or a pharmaceutical composition according to claim 21 or 22.

26. The method according to claim 25, wherein the autoimmune disease or 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), Waldenström hypergammaglobulinemia, Sjögren's syndrome, multiple sclerosis (MS), or lupus nephritis.

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