Compositions and methods related to anti-CD19 antibody drug conjugates

Abstract

To provide monoclonal antibodies and antigen binding fragments that specifically bind to CD 19, or any fragments, variants, multimeric versions, or bispecific antibodies thereof.SOLUTION: The present disclosure relates to antibody-drug conjugates (ADCs) where one or more active agents are conjugated to an anti-CD19 antibody through a linker. The linker may comprise a unit that covalently links active agents to the antibody.SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] The present invention relates to compositions and methods related to anti-CD19 antibody drug conjugates.

[0002] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 669,183, filed May 9, 2018, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] background Antibody-drug conjugate (ADC) technology is a targeted technology that enables selective apoptosis of cancer cells. ADCs generally work by targeting cancer cells with antibodies, where they release a toxic substance (i.e., a drug) that results in cell death. Because ADC technology enables precise delivery of drugs to targeted cancer cells and their release under specific conditions while minimizing collateral damage to healthy cells, ADC technology enhances the efficacy of therapeutic antibodies and reduces the risk of adverse reactions.

[0004] B cells express a wide variety of cell surface molecules throughout their differentiation and proliferation. Examples include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, and CD86 leukocyte surface markers. These markers are commonly identified as therapeutic targets for the treatment of B cell disorders or diseases, such as B cell malignancies, autoimmune diseases, and transplant rejection. CD19 is a surface protein found on B cells and certain cancer cells derived from B cells, such as many B cell lymphomas. Anti-CD19 monoclonal antibodies are produced in mice. However, mouse-derived antibodies are generally immunogenic in humans, and humanized antibodies may be immunogenic in humans.

[0005] Therefore, there is a need for improved antibody-drug conjugates that target CD19. Summary of the Invention

[0006] Disclosure summary In some aspects, the present disclosure relates to antibody-drug conjugates (ADCs). In some embodiments, the present disclosure relates to antibody-drug conjugates comprising an antibody, a linker, and an active agent (e.g., a drug). The antibody-drug conjugate may include a self-immolative group, for example, for use in releasing the active agent from the antibody and linker.

[0007] The present specification provides monoclonal antibodies and antigen-binding fragments, or any fragments, variants, multimeric forms, or bispecific antibodies thereof, that bind to CD19. These antibodies and antigen-binding fragments, or any fragments, variants, multimeric forms, or bispecific antibodies thereof, are collectively referred to herein as anti-CD19 monoclonal antibodies or anti-CD19 mAbs or antigen-binding fragments, or any fragments, variants, multimeric forms, or bispecific antibodies thereof. Preferably, the monoclonal antibodies and antigen-binding fragments, or any fragments, variants, multimeric forms, or bispecific antibodies thereof, are specific for at least human CD19. In some embodiments, the monoclonal antibodies and antigen-binding fragments, or any fragments, variants, multimeric forms, or bispecific antibodies thereof that recognize human CD19 also cross-react with at least one other non-human CD19 protein, such as, but not limited to, non-human primate CD19, e.g., cynomolgus monkey CD19, and / or rodent CD19.

[0008] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADCs) containing an antibody, at least one branched linker covalently bound to the antibody, and at least one or two active agents covalently bound to the branched linker. The branched linker may include a branching unit, and at least one drug is bound to the branching unit via a secondary linker; the branching unit is bound to the antibody by a primary linker. The primary linker and / or secondary linker may include at least one polyethylene glycol unit.

[0009] In some embodiments, the present specification relates to an antibody conjugate represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof, Ab-(X) y Formula I During the ceremony: The Ab is an anti-CD19 antibody or antigen-binding fragment thereof, or a bispecific antibody comprising a first arm that binds to CD19, the Ab comprising heavy chain variable region complementarity determining region 1 (CDRH1), heavy chain variable region complementarity determining region 2 (CDRH2), heavy chain variable region complementarity determining region 3 (CDRH3), light chain variable region complementarity determining region 1 (CDRL1), light chain variable region complementarity determining region 2 (CDRL2), and light chain variable region complementarity determining region 3 (CDRL3); CDRH1 comprises the amino acid sequence of SEQ ID NO: 23 or 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 24 or 30; CDRH3 comprises the amino acid sequence of SEQ ID NO: 25, 26, 27, 28, or 31; CDRL1 comprises the amino acid sequence of SEQ ID NO: 32, 37, 41, or 44; CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, 38, 42, or 45; CDRL3 comprises the amino acid sequence of SEQ ID NO: 34, 35, 36, 40, 43, or 46; each X is independently a chemical moiety comprising an active agent and a linker, the linker connecting the Ab to the active agent; and y is an integer between 1 and 20. [Brief explanation of the drawings]

[0010] [Figure 1-1] 1A-1F are a series of graphs showing the ability of various anti-CD19 antibodies herein to bind to six different B lymphocyte cell lines (Raji, Ramos, Nalm6, SU-DHL6, SU-DHL4, Mec2), a CD19-silencing cell line (Raji siRNA), and a negative control cell line (Jurkat), as measured by FACS analysis. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-1. [Figure 2] FIG. 2 is a series of graphs showing the ability of various anti-CD19 antibodies herein to bind to cynomolgus monkey CD19 expressed by transfected CHO cells or a negative control cell line (CHO), as measured by FACS analysis. [Figure 3] Figure 3A shows a graph depicting the ability of various anti-CD19 antibodies herein to bind to human T cells and monocytes at concentrations of 30 μg / mL or 3 μg / mL. Figure 3B shows a graph depicting the ability of various anti-CD19 antibodies herein to bind to cynomolgus monkey B cells at concentrations of 30 μg / mL or 3 μg / mL. Figure 3C shows a graph depicting the ability of various anti-CD19 antibodies herein to bind to human T cells and monocytes at concentrations of 30 μg / mL or 3 μg / mL. [Figure 4-1]Figures 4A-4G show tumor volume over time in CB17-SCID mice implanted with Ramos cells and then treated with the 9G8 anti-CD19 ADC, a non-tumor-specific human IgG1 anti-HER2 ADC, or rituximab. Mice treated with the 9G8 anti-CD19 ADC showed regression of tumor growth. Figure 4H shows the average body weight over time in CB17-SCID mice implanted with Ramos cells and then treated with the human IgG1 isotype, the 9G8 anti-CD19 ADC, a non-tumor-specific human IgG1 anti-HER2 ADC, or rituximab. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-1. [Figure 4-4] This is a continuation of Figure 4-1. [Figure 5A] Figure 5A shows the mean tumor volume over time in CB17-SCID mice implanted with Ramos cells and then treated with the 9G8 anti-CD19 ADC, a non-tumor-specific human IgG1 anti-HER2 ADC, or rituximab. Mice treated with the 9G8 anti-CD19 ADC showed regression of tumor growth out to 70 days. [Figure 5B] Figure 5B shows the percent survival of CB17-SCID mice implanted with Ramos cells and treated with 9G8 anti-CD19 ADC (human IgG1 isotype, 9G8 anti-CD19 CaaX antibody), a non-tumor-specific human IgG1 anti-HER2 ADC, or rituximab. Mice treated with 9G8 anti-CD19 ADC showed 100% survival out to 70 days. [Figure 6A] Figures 6A and 6B show the inhibition rates of anti-CD19 ADC or dPBD (SG2057). The inhibition rates of the ADC were comparable to those of the dPBD. [Figure 6B] This is a continuation of Figure 6A.

[0011] Detailed Description of the Invention The basic structure of an antibody-drug conjugate is as follows: antibody-linker-low molecular weight drug or toxin. The linker ideally allows the drug to exert its effect on the target cancer cells after it reaches the target cells, for example, after separation from the antibody (e.g., by enzyme-mediated hydrolysis). The linker also serves a functional role by connecting the antibody and the drug. The efficacy and toxicity of an antibody-drug conjugate depend in part on the linker, and therefore the linker plays an important role in drug safety, as described in U.S. Pat. No. 9,919,057, International Publication No. WO 2017 / 089890, and International Publication No. WO 2017 / 089895, the contents of which are incorporated herein by reference in their entirety.

[0012] Linkers in antibody-drug conjugates can be broadly classified as non-cleavable or cleavable. Most non-cleavable linkers are attached to antibodies using thioethers containing antibody cysteines. The pendant drug generally cannot dissociate from the antibody in vivo. However, in the case of the widely used thiol-maleimide method, the antibody-drug conjugate is unstable, which can cause the drug to dissociate from the conjugate before or after it reaches the target cell.

[0013] Cleavable linkers are, for example, linkers that can be hydrolyzed by lysosomal enzymes. Cleavable linkers can include, for example, disulfide bonds involving cysteines of antibodies. Disulfide linkers allow dissociation via thiol exchange reactions, and rely in part on the uptake of the antibody-drug conjugate into target cells and the exposure of the disulfide to the cytosol, which is a reducing environment. However, because various types of thiols (e.g., albumin and glutathione) are present in the blood, the drug may dissociate from the antibody before reaching the target.

[0014] Recently, a new approach to making antibody-drug conjugates has been described using protein prenylation of the C-terminal amino acid sequence to introduce a modified isoprenoid unit that allows for the attachment of drugs or other active agents to antibodies in a gentle and site-specific manner (see, e.g., U.S. Patent Publication No. 2012 / 0308584, the entire contents of which are incorporated herein by reference). Further improvements are possible, and descriptions of additional cleavable linkers can be found in the following documents: U.S. Patent No. 9,919,057; WO 2017 / 089890; and WO 2017 / 089895, the contents of which are incorporated herein by reference in their entireties.

[0015] The present disclosure provides antibody-drug conjugates of antibodies that bind to CD19. These antibodies, including anti-CD19 monoclonal antibodies or anti-CD19 mAbs and antigen-binding fragments thereof, are described in U.S. Patent Application No. 15 / 804,517, published as US 2018 / 0142018 A1, the contents of which are incorporated herein by reference in their entirety. Preferably, the monoclonal antibodies are specific for at least human CD19. In some embodiments, monoclonal antibodies that recognize human CD19 also exhibit cross-reactivity with at least one other non-human CD19 protein, such as, but not limited to, non-human primate CD19, e.g., cynomolgus monkey CD19, and / or rodent CD19. The present disclosure also includes antibodies that bind to the same epitope as the anti-CD19 monoclonal antibodies described herein.

[0016] The present specification also provides monovalent antibodies specific for CD19 and / or bivalent antibodies comprising at least a first arm specific for CD19. Preferably, the monovalent and / or bivalent antibodies are specific for at least human CD19. In some embodiments, the monovalent and / or bivalent antibodies that recognize human CD19 also exhibit cross-reactivity with at least one other non-human CD19 protein, such as, but not limited to, non-human primate CD19, e.g., cynomolgus monkey CD19, and / or rodent CD19. The present specification also provides antibodies that bind to the same epitope as the anti-CD19 monovalent and / or anti-CD19 bivalent antibodies described herein.

[0017] The bispecific antibodies herein allow two antibody arms to simultaneously bind to two antigens on the cell surface (referred to as "co-engagement"), resulting in an additive or synergistic increase in affinity through an avidity mechanism. Thus, co-engagement confers high selectivity for cells expressing both antigens compared to cells expressing only one antigen. Additionally, the affinities of the two arms of the bispecific antibody for their respective targets can be configured so that binding to target cells is primarily driven by one of the antibody arms. In some embodiments, the bispecific antibody comprises a first arm that binds to CD19 and a second arm that binds to a second target other than CD19. In some embodiments, the bispecific antibody comprises a first arm that binds to CD19 and a second arm that binds to a tumor-associated antigen (TAA). In some embodiments, the bispecific antibody comprises a first arm that binds to CD19 and a second arm that binds to a tumor-associated antigen (TAA), wherein the first arm binds to CD19 with high affinity and the second arm binds to the TAA with low affinity. In some embodiments, the TAA is an antigen expressed on the cell surface of a cancer cell. In some embodiments, the cancer cell is selected from lung cancer cells, bronchial cancer cells, prostate cancer cells, breast cancer cells, colon cancer cells, pancreatic cancer cells, ovarian cancer cells, leukemia cancer cells, lymphoma cancer cells, esophageal cancer cells, liver cancer cells, urinary tract and / or bladder cancer cells, renal cancer cells, oral cancer cells, pharyngeal cancer cells, uterine cancer cells, and / or melanoma cancer cells. In some embodiments, suitable second targets include, by way of non-limiting example, CD47, CD20, CD22, CD40, BAFFR, CD5, CD32b, ICOSL, IL6R, and / or IL21R.

[0018] In some embodiments, the bispecific antibody is a fully human bispecific IgG format, for example, the κλ-body format described in PCT Publication WO 2012 / 023053, the contents of which are incorporated herein by reference in their entirety.

[0019] Specific examples of the anti-CD19 monoclonal antibodies and antigen-binding fragments thereof herein include, for example, the 5F5 antibody, the 7F11 antibody, the 9G8 antibody, the F6 antibody, the 7F1 antibody, and the 10D8 antibody, or antigen-binding fragments thereof.

[0020] Specific examples of anti-CD19 bispecific antibodies herein, in which at least one binding site is specific for CD19, include, for example, the 5F5 antibody, the 7F11 antibody, the 9G8 antibody, the F6 antibody, the 7F1 antibody, and the 10D8 antibody, or antigen-binding fragments thereof.

[0021] In some embodiments, specific examples of anti-CD19 monoclonal antibodies and antigen-binding fragments thereof herein comprise a combination of heavy chain complementarity-determining regions (CDRs) selected from the CDR sequences set forth in Table 1, and light chain CDRs selected from the CDR sequences set forth in Table 2, where the CDRs set forth in Tables 1 and 2 are defined according to the IMGT nomenclature.

[0022] In some embodiments, the anti-CD19 monoclonal, monospecific, monovalent, and / or bispecific antibodies herein comprise a combination of heavy chain complementarity determining regions (CDRs) selected from the CDR sequences set forth in Table 1, and light chain CDRs selected from the CDR sequences set forth in Table 2, where the CDRs set forth in Tables 1 and 2 are defined according to the IMGT nomenclature. [Table 1] [Table 2]

[0023] In certain embodiments, the antibody-drug conjugate described herein is represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof, Ab-(X) y Formula I During the ceremony, The Ab is an anti-CD19 antibody or antigen-binding fragment thereof, or a bispecific antibody comprising a first arm that binds to CD19, the Ab comprising heavy chain variable region complementarity determining region 1 (CDRH1), heavy chain variable region complementarity determining region 2 (CDRH2), heavy chain variable region complementarity determining region 3 (CDRH3), light chain variable region complementarity determining region 1 (CDRL1), light chain variable region complementarity determining region 2 (CDRL2), and light chain variable region complementarity determining region 3 (CDRL3); CDRH1 comprises the amino acid sequence of SEQ ID NO: 23 or 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 24 or 30; CDRH3 comprises the amino acid sequence of SEQ ID NO: 25, 26, 27, 28, or 31; CDRL1 comprises the amino acid sequence of SEQ ID NO: 32, 37, 41, or 44; CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, 38, 42, or 45; CDRL3 comprises the amino acid sequence of SEQ ID NO: 34, 35, 36, 40, 43, or 46; each X is independently a chemical moiety comprising one or more active agents and a linker, the linker connecting the Ab to the active agent(s); and y is an integer between 1 and 20.

[0024] In some embodiments, the Ab is a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a scFv-Fc fragment, a single-domain heavy chain antibody, a single-domain light chain antibody, a variant antibody, a multimeric antibody, or a bispecific antibody. The Ab can be a rabbit, murine, chimeric, humanized, or fully human monoclonal antibody. In some embodiments, the Ab is an IgG isotype, such as an IgG1 isotype.

[0025] In some embodiments, the Ab comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 6, 12, 16, or 20 in combination with a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 8, 10, 14, 18, or 22.

[0026] In some embodiments, the Ab comprises a combination of heavy and light chain variable region sequences selected from the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; and (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.

[0027] In some embodiments, the anti-CD19 antibody is 5F5, 7F11, 9G8, F6, 7F1 or 10D8. In some embodiments, the CD19 is human CD19.

[0028] Preferably, the bond between the Ab and the active agent is cleavable. Generally, the linker is represented by Formula II: [ka] G is a glucuronic acid moiety or [ka] wherein R 3 is hydrogen or a carboxyl protecting group, and R 4 are each independently hydrogen or a hydroxyl protecting group; B is the active agent; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl; or W is —C(O)—, —C(O)NR′—, —C(O)O—, —SONR′—, —P(O)R″NR′—, —SONR′—, or —PONR′—, where C, S, or P is directly attached to the phenyl ring, and R′ and R″ are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 is aryl; Each Z is independently 1-8 alkyl, halogen, cyano, or nitro; n is an integer from 0 to 3; and L is a linker connecting Ab and W.

[0029] In some embodiments, L is C 1-50 In some embodiments, L satisfies at least one of the following: (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the two atoms bridging them, complete a heteroarylene; (iii) L is a heteroalkylene of 1-50 atoms; or (iv) alkylene is one or more C 1-20 It is substituted with alkyl.

[0030] In some embodiments, L comprises at least one isoprenyl derivative unit, represented by Formula III, which is recognized by an isoprenoid transferase: [ka] .

[0031] In some such embodiments, the linker has Formula II: [ka] G is a glucuronic acid moiety or [ka] wherein R 3 is hydrogen or a carboxyl protecting group, and R 4 are each independently hydrogen or a hydroxyl protecting group; B is the active agent; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl; or W is —C(O)—, —C(O)NR′—, —C(O)O—, —SONR′—, —P(O)R″NR′—, —SONR′—, or —PONR′—, where C, S, or P is directly attached to the phenyl ring, and R′ and R″ are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 is aryl; Each Z is independently 1-8 alkyl, halogen, cyano, or nitro; n is an integer from 0 to 3; During the ceremony: A) L is C 1-50 alkylene or heteroalkylene of 1-50 atoms, satisfying at least one of the following: (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the bridging atoms, complete (constitute) a heteroarylene; (iii) L is a heteroalkylene of 1-50 atoms; (iv) alkylene is one or more C 1-20 substituted with alkyl; or B) L comprises at least one isoprenyl derivative unit, represented by formula III, which is recognized by an isoprenoid transferase: [ka] .

[0032] In some embodiments, G is [ka] R 3is hydrogen or a carboxyl protecting group; and R 4 are each independently hydrogen or a hydroxyl protecting group. In some preferred embodiments, each R 1 and R 2 is hydrogen.

[0033] In some embodiments, each Z is independently C 1-8 It is alkyl, halogen, cyano, or nitro.

[0034] In some preferred embodiments, n is 0.

[0035] In some embodiments, W is —C(O)—, —C(O)NR′—, —C(O)O—, —SONR′—, —P(O)R″NR′—, —SONR′—, or —PONR′—, where C, S, or P is directly attached to the phenyl ring, and R′ and R″ are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl, or C 6-20 In some preferred embodiments, W is —C(O)—, —C(O)NR′—, or —C(O)O—. In some further preferred embodiments, W is —C(O)NR′—, where C(O) is attached to the phenyl ring and NR′ is attached to L.

[0036] In some embodiments, G is [ka] W is —C(O)NR′—, where C(O) is bonded to the phenyl ring and NR′ is bonded to L; and R 1 and R 2 represents hydrogen.

[0037] In some embodiments, L is C 1-50 alkylene or heteroalkylene of 1-50 atoms, satisfying at least one of the following: (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the two atoms bridging them, complete a heteroarylene; (iii) L is a heteroalkylene of 1 to 50 atoms; and (iv) alkylene is one or more C 1-20 It is substituted with alkyl.

[0038] In some embodiments, L comprises an oxime and at least one polyethylene glycol unit covalently links the oxime to the active agent.

[0039] In some embodiments, L is a nitrogen-containing heteroalkylene of 1-50 atoms, the linker includes at least two atoms of a hydrophilic amino acid, and the nitrogen forms a peptide bond with the carbonyl of the hydrophilic amino acid.

[0040] In some preferred embodiments, W represents -C(O)NR'-, and the nitrogen of W is a nitrogen atom of a hydrophilic amino acid. In some embodiments, the hydrophilic amino acid is an amino acid containing a side chain with a moiety that retains a charge in aqueous solution at neutral pH. In some embodiments, the hydrophilic amino acid is arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In some preferred embodiments, the hydrophilic amino acid is arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In some preferred embodiments, the hydrophilic amino acid is aspartic acid or glutamic acid. In other preferred embodiments, the hydrophilic amino acid is ornithine or lysine. In yet another preferred embodiment, the hydrophilic amino acid is arginine. In some embodiments, the amino acid covalently links the oxime of the linker to the polyethylene glycol unit of the linker.

[0041] In some embodiments, the linker comprises a peptide, and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid with a side chain having a moiety (e.g., an amine, guanidine, or carboxyl moiety) that carries a charge in aqueous solution at neutral pH. In some embodiments, each amino acid of the peptide is independently selected from alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, ornithine, proline, serine, and threonine. In some embodiments, the peptide comprises at least one aspartic acid or glutamic acid.

[0042] In some preferred embodiments, W represents -C(O)NR'-, and the nitrogen of W is the nitrogen of the N-terminal amino acid in the peptide.

[0043] In some embodiments, the peptide covalently links the oxime of the linker to the polyethylene glycol unit of the linker.

[0044] In some embodiments, the peptide consists of 2 to 20 amino acids.

[0045] In some embodiments, the linker is covalently attached to the Ab by a thioether bond, wherein the thioether bond comprises a sulfur atom of a cysteine ​​of the Ab. In some embodiments, the Ab comprises an amino acid motif recognized by an isoprenoid transferase, preferably at the C-terminus of the Ab; and The thioether bond involves the sulfur atom of the cysteine ​​amino acid motif.

[0046] In some embodiments, the amino acid motif is the sequence CYYX; C represents cysteine; Y represents, independently in each instance, an aliphatic amino acid such as alanine, isoleucine, leucine, methionine, or valine; X represents, independently in each instance, glutamine, glutamic acid, serine, cysteine, methionine, alanine, or leucine; and The thioether bond involves the sulfur atom of the cysteine ​​amino acid motif.

[0047] In some embodiments, the amino acid motif is the sequence CVIM or CVLL.

[0048] In some embodiments, at least one of the seven amino acids preceding the amino acid motif is glycine. In some embodiments, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine and proline. In some embodiments, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine, aspartic acid, arginine, and serine. In some embodiments, all 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids preceding the amino acid motif are glycine. In some preferred embodiments, L comprises the amino acid sequence GGGGGGCVIM, preferably at the C-terminus.

[0049] In some embodiments, L comprises at least one isoprenyl derivative unit represented by Formula III, which is recognized by an isoprenoid transferase: [ka]

[0050] In some embodiments, L is a 3-50 heteroalkylene containing oxime, wherein the oxygen atom of the oxime is on the side bonded to W of L and the carbon atom of the oxime is on the side bonded to Ab of L; or The carbon atom of the oxime is on the side of L that is bonded to W, and the oxygen atom of the oxime is on the side of L that is bonded to Ab.

[0051] In some preferred embodiments, L comprises an oxime and at least one isoprenyl derivative unit covalently links the oxime to Ab. [ka] In some embodiments, L includes: [ka] In some preferred embodiments, L includes: [ka] Includes:

[0052] In some embodiments, L further comprises a linking unit represented by formula VIII or IX: -(CH2) r (V(CH2) p ) q - Formula VIII, -(CH2CH2X) w - Formula IX; V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 -is; X is -O-, C 1-8 Alkylene, or -NR 21 -is; R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl, or C 1-6 Alkyl C 3-20 is heteroaryl; r is an integer between 1 and 10; p is an integer between 0 and 12; q is an integer from 1 to 20; and w is an integer from 1 to 20.

[0053] In some embodiments, q is an integer from 4 to 20. In some embodiments, q is an integer from 2 to 12. In some embodiments, q is an integer from 6 to 20. In some embodiments, q is 2, 5, or 11. In some embodiments, r is 2. In some embodiments, p is 2. In some preferred embodiments, V is -O-. In some embodiments, r is 2; p is 2; q is 2, 5, or 11; and V is -O-. In some preferred embodiments, X is -O-.

[0054] In some embodiments, w is an integer from 6 to 20. In some embodiments, L is [ka] or [ka] In some embodiments, L contains 1 to 12 -OCH2CH2- units. In some embodiments, L contains 3 to 12 -OCH2CH2- units. In some embodiments, L contains 5 to 12 -OCH2CH2- units. In some embodiments, L contains 6 or 12 -OCH2CH2- units. In some preferred embodiments, L contains 3 -OCH2CH2- units.

[0055] In some embodiments, L comprises an oxime and at least one polyethylene glycol unit covalently links the oxime to the active agent. In some embodiments, L comprises a linking unit formed by a 1,3-dipolar cycloaddition reaction, a hetero Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, an addition to a carbon-carbon multiple bond, an oxidation reaction, or a click reaction.

[0056] Click chemistry reactions are carried out under mild conditions, allowing proteins to be easily handled. Click chemistry reactions exhibit significantly high reaction specificity. Therefore, even if a protein has other functional groups (e.g., at side chain residues or the C- or N-terminus), these functional groups are not affected by the click chemistry reaction. For example, a click chemistry reaction between an azide group and an acetylene group on a protein can occur, even though other functional groups on the protein are not affected by the click chemistry reaction. Furthermore, click chemistry reactions can occur specifically regardless of the type of ligand involved. In some cases, ligands can be selected to improve overall reaction efficiency. For example, the azide-acetylene click chemistry reaction can produce triazoles in high yields (References: Rhiannon K. Hia et al, Chem. Rev. 2009, 109, 5620; Morten Meldal and Christian Wenzel Tornoe, Chem Rev., 2008, 108, 2952; Hartmuth C. Kolb et al, Angew. Chemie Int. Ed. Engl., 2001, 40, 2004, all of which are incorporated herein by reference).

[0057] In some embodiments, the linking unit is formed by the reaction between an acetylene and an azide, or between an aldehyde or ketone group and a hydrazine or alkoxyamine.

[0058] In some embodiments, L comprises a linking unit represented by formula IV, V, VI, or VII: [ka] L 1 is a single bond or C 1-30 alkylene; and R 11 is hydrogen or C 1-10 It is alkyl.

[0059] In some embodiments, L 1 is a single bond. In other embodiments, L 1 is C 11 In yet another embodiment, L is alkylene. 1 is C 12 It is alkylene.

[0060] In some embodiments, L is: [ka] Contains; V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 -, but -O- is preferred; R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl, or C 1-6 Alkyl C 3-20 is heteroaryl; r is an integer between 1 and 10; p is an integer between 0 and 10; q is an integer from 1 to 20; and L1 is a single bond.

[0061] In some embodiments, r is 2 or 3. In some embodiments, p is 1 or 2. In some embodiments, q is 1 to 6. In some embodiments, r is 2 or 3; p is 1 or 2; and q is 1 to 6.

[0062] In some embodiments, the linker is: [ka] Including, where Ab represents an anti-CD19 antibody; B represents an active agent; and n is an integer from 1 to 20.

[0063] In other embodiments, the linker is: [ka] Including, where Ab represents an anti-CD19 antibody; B represents an active agent; and n is an integer from 1 to 20.

[0064] In yet another embodiment, the linker is: [ka] Including, where Ab represents an anti-CD19 antibody; B represents an active agent; and n is an integer from 0 to 20.

[0065] In yet another embodiment, the linker is: [ka] Including, where Ab represents an anti-CD19 antibody; B represents an active agent; and n is an integer from 1 to 20.

[0066] In some embodiments, the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

[0067] In some embodiments, L further comprises: [ka] In some embodiments, [ka] is the binding moiety.

[0068] In some embodiments, L comprises one or more branched linkers covalently linked to Ab: i) Each branched linker comprises a branching unit (BR) covalently linked to an Ab by a primary linker (PL); ii) each branched linker contains a first branch (B1), which connects a first active agent to the branched unit, and contains a secondary linker (SL) and a cleavage group (CG); and iii) each branched linker further contains a second branch (B2), wherein a) a second active agent is covalently linked to the branching unit by a secondary linker (SL) and a cleavage group (CG); or b) a polyethylene glycol moiety is covalently attached to the branching unit, and Each cleavable group can be hydrolyzed to release the active agent from the antibody conjugate.

[0069] In some embodiments, at least one branching unit is [ka] wherein L 2 , L 3 , L 4 are each independently a direct bond or -C n H 2n -, and n is an integer of 1 to 30; 1 , G 2 , G 3 are each independently a direct bond (bond), [ka] R 30 is hydrogen or C 1-30 alkyl; and 40 L 5 -COOR 50 , where L 5 is a direct bond (bond) or C 1-10alkylene, and R 50 is hydrogen or C 1-30 It is alkyl.

[0070] In yet another embodiment, the linker is: [ka] wherein: B and B' represent active agents, which may be the same or different; n represents, independently for each instance, an integer from 0 to 30; f represents, independently in each instance, an integer from 0 to 30; and L represents binding to Ab.

[0071] In some embodiments, n is an integer from 1 to 10. In some embodiments, n is an integer from 4 to 20.

[0072] In some embodiments, the cleavable group is capable of cleavage within a target cell. In some embodiments, the cleavable group is capable of releasing one or more active agents. In some embodiments, the antibody conjugate (antibody complex) comprises an Ab; at least one branched linker covalently linked to the Ab; and at least two active agents covalently linked to the branched linkers. In some embodiments, at least two branched linkers are linked to the Ab, and each branched linker is linked to at least two active agents. In some embodiments, three branched linkers are linked to the Ab. In other embodiments, four branched linkers are linked to the Ab. In still other embodiments, exactly one branched linker is attached to the Ab. In still other embodiments, each branched linker is linked to exactly two active agents. In some embodiments, the complex comprises at least two different active agents. In some embodiments, at least one branched linker is linked to two different active agents.

[0073] In some embodiments, each active agent is linked to the branched linker by a cleavable (e.g., hydrolyzable) bond. In some embodiments, each branched linker contains a branching unit, and each active agent is linked to the branching unit via a secondary linker, and the branching unit is linked to the anti-CD19 antibody by a primary linker. In some embodiments, the branching unit is a nitrogen atom, e.g., an amine or amide nitrogen atom. In some embodiments, the branching unit is an amide and the primary linker comprises an amide carbonyl. In some embodiments, the branching unit is an amide and the secondary linker comprises an amide carbonyl. In some preferred embodiments, the branching unit is a lysine unit.

[0074] In some preferred embodiments, B is an active agent. In some embodiments, the active agent is independently selected from a chemotherapeutic agent and a toxin. In some embodiments, the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

[0075] In some embodiments, the active agent is a chemotherapeutic agent or a toxin. (a) Erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, kale Amputatecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, colofsphamide, estramustine, ifosfamide, mechlorethamine, melphalan, nobe tambitine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antimycin (antr mycin), azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin,Esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, Mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defofamine, demecolcine, diaziconazole, eflornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, mayta ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziconazole, 2,2',2''-trichlorotriethylamine, T-2 toxin, velaculin A, Roridin A and Anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-modified nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine,Vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine or a pharmaceutically acceptable salt, solvate or acid of any of the foregoing, (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone (parathyroid hormone), thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor interferon, interferon-alpha, interferon-beta, interferon-gamma, colony stimulating factor ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin ("IL"), IL-1, IL-1alpha, 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-alpha, TNF-beta, polypeptide factor, LIF, kit ligand, or any combination of the foregoing; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, azonafide, aplidine, toxoids, or combinations of any of the foregoing. (d) an affinity ligand, which is a substrate, an inhibitor, a stimulator, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) radioactive label; 32 P, 35 S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, an incomplete antigen, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; (f) immunomodulatory compounds, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents and anti-parasitic agents, or a combination of any of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxacitabine, (j) aromatase inhibitors, (k) protein kinase inhibitors, (l) lipid kinase inhibitors, (m) antisense oligonucleotides; (n) ribozyme, (o) vaccines; and (p) anti-angiogenic agents.

[0076] In some embodiments, the Ab is an anti-CD19 antibody; The active drug is a pyrrolobenzodiazepine dimer; The linker connects the Ab to the N10 or N'10 position of the pyrrolobenzodiazepine dimer; and y is an integer from 1 to 20.

[0077] In some embodiments, the active agent is a pyrrolobenzodiazepine dimer; the pyrrolobenzodiazepine dimer is substituted at the N10 position with X or at the N'10 position with X', where X or X' connects the pyrrolobenzodiazepine dimer to the linker; X and X' each independently represent -C(O)O-*, -S(O)O-*, -C(O)-*, or -C(O)NR X -*, -S(O)2NR X -*, -(P(O)R')NR X -*, -S(O)NR X -*, or -PO2NR X -Selected from *; R X is H, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 3-20 Heteroaryl, or C 5-20 is aryl; R X ' is OH, N3, CN, SH, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 aryl, or amino; and * represents the attachment point between the pyrrolobenzodiazepine dimer and the linker. In some embodiments, X and X′ are each independently —C(O)O—*, —C(O)—*, or —C(O)NR X -* is selected.

[0078] In some embodiments, the pyrrolobenzodiazepine dimer is represented by Formula X or Formula XI: [ka] During the ceremony: The dotted lines indicate double bonds that may be present between C1 and C2 or between C2 and C3; and between C'1 and C'2 or between C'2 or C'3; R X1 and R X1’are independently H, OH, =O, =CH2, CN, R m , OR m , =CH-R m' =C(R m' )2, O-SO2-R m , CO2R m , C.O.R. m , halo, and dihalo; R m' are independent, R m , CO2R m , C.O.R. m , CHO, COH, and halo; R m are each independently, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R X2 , R X2’ , R X3 , R X3’ , R X5 , and R X5’ are independently H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3Sn and halo; R X4 and R X4’ are independently H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3Sn, Halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-12 Aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n’ , -OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n’ , -S(O)NR n R n’ , -OS(O)NR n R n’ , -OS(O)2NR n R n’ , -NR n R n’ , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o’ , -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o’ , -NR n S(O)NR o R o’ , -C(O)R n , -C(O)OR n and -C(O)NR n R n’ Selected from; R X and R X ' are independently H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 Aryl, or mono- or di-C 1-8selected from alkylamino; Y and Y' are independently selected from O, S, and N(H); R X6 is C 3-12 Alkylene, C 3-12 Alkenylene, or C 3-12 is heteroalkylene; R X7 and R X7’ are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C6- 10 Aryl, 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r’ , -OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r’ , -S(O)NR r R r’ , -OS(O)NR r R r’ , -OS(O)2NR r R r’ , -NR r R r’ , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s’ , -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s’ , -NR r S(O)NR s R s , -C(O)Rr , -C(O)OR s or -C(O)NR r R r’ Selected from; R r , R r’ , R s , and R s ' are independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 aryl, and 5- to 7-membered heteroaryl; R X8 and R X8’ are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Heteroalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -S(O)R m , -S(O)2R m , -S(O)NR m R m’ , -S(O)NR m R m’ , -NR m R m’ , -NR m C(O)R m , -NR m C(O)OR n , -NR m C(O)NR n R n' , -NR m S(O)R n , -NR m S(O)2R n , -NR m S(O)NR n R n’ , -NR m S(O)NR n R n’ , -C(O)R m , -C(O)OR m and -C(O)NR m Rm’ Selected from; Z a is OR X12a , N.R. X12a R X12a , or SR X12a Selected from; Z b is OR X13a , N.R. X13a R X13a , or SR X13a Selected from; Z a ' is OR X12a , N.R. X12a R X12a , or SR X12a Selected from; Z b ' is OR X13a ', NR X13a 'R X13a ', or SR X13a ' selected from; R X12a , R X12a’ , R X13a’ , and R X13a’ are independently None, H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -C(O)R X15a , -C(O)OR X15a and -C(O)NR X15a R X15a ' selected from; and R X15a and R X15a ' are each independently 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R in the formula X13a and RX14a may combine with the atom to which they are attached to form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; and R X13a’ and R X14a’ may combine with the atom to which they are attached to form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; and R in the formula n , R n ', R o , R o ', R p , and R p ' are independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 aryl, and 5- to 7-membered heteroaryl.

[0079] In some embodiments, R m are each independently C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 c is selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; In this case, R m is one or more C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 c may be substituted with cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl.

[0080] In some embodiments, R X4 and R X4’ are independently H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m R m' , NO2, Me3Sn, Halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n’ , -OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n’ , -S(O)NR n R n’ , -OS(O)NR n R n’ , -OS(O)2NR n R n’ , -NR n R n’ , -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o’ , -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o’ , -NR n S(O)NR o R o’ , -C(O)R n , -C(O)OR nand -C(O)NR n R n’ is selected from where R X4 or R X4’ But C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 When it is an aryl or a 5- to 7-membered heteroaryl, it may optionally contain one or more C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3- C6 cycloalkyl, 3-7 membered heterocycloalkyl, C5- 10 Aryl, 5- to 7-membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p’ , -OS(O)R p , -OS(O)2R p , -SR p , -S(O)R p , -S(O)2R p , -S(O)NR p R p’ , -S(O)NR p R p’ , -OS(O)NR p R p’ , -OS(O)2NR p R p’ , -NR p R p’ , -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q R q’ , -NR p S(O)R q , -NR p S(O)2R q , -NR p S(O)NRq R q’ , -NR p S(O)NR q R q’ , -C(O)R p , -C(O)OR p or -C(O)NR p R p may be substituted with.

[0081] In some embodiments, R X7 and R X7’ are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r’ , -OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r’ , -S(O)NR r R r’ , -OS(O)NR r R r’ , -OS(O)2NR r R r’ , -NR r R r’ , -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s’ , -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s’ , -NR r S(O)NRs R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r’ is selected from where R X7 or R X7’ C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 When it is an aryl or a 5- to 7-membered heteroaryl, it may optionally contain one or more C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t’ , -OS(O)R t , -OS(O)2R t , -SR t , -S(O)R t , -S(O)2R t , -S(O)NR t R t’ , -S(O)NR t R t’ , -OS(O)NR t R t’ , -OS(O)2NR t R t’ , -NR t R t’ , -NR t C(O)R u , -NR t C(O)OR u , -NR t C(O)NR u R u’ , -NR t S(O)R u , -NR t S(O)2R u , -NR tS(O)NR u R u’ , -NR t S(O)NR u R u’ , -C(O)R t , -C(O)OR t or -C(O)NR t R t’ may be substituted with The above R r , R r’ , R s , R s ', R t , R t ', R u and R u ' are independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 aryl, and 5- to 7-membered heteroaryl.

[0082] In some embodiments, R X1 and R X1’ are independent, R m selected from; and R m is C 1-6 Alkyl, C 2-6 Alkenyl, C 5-7 Aryl, and C 3-6 heteroaryl.

[0083] In some embodiments, R X2 , R X2’ , R X3 , R X3’ , R X5 and R X5’ are independently selected from H or OH.

[0084] In some embodiments, R X4 and R X4’ is independently R m selected from; and R m is C 1-6 In some preferred embodiments, R X4 and R X4’ is independently selected from methoxy, ethoxy, or butoxy. In some embodiments, Y and Y' are O.

[0085] In some embodiments, R X6 is C 3-12 Alkylene, C 3-12 Alkenylene, C 3-12 heteroalkylene, where: R X6 -NH2, -NHR m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R XX , or -[CH2CH2O] n -R XX is replaced by; This R XX are H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 aAlkylthio, C 3-20 Heteroaryl, C 5-20 Aryl, or mono- or di-C 1-8 alkylamino; and n is an integer from 1 to 6.

[0086] In some embodiments, the active agent is a pyrrolobenzodiazepine dimer represented by Formula XII or Formula XIII: [ka] During the ceremony: X a and X a’ are independently a bond or C 1-6 selected from alkylene; ZX’ and Z X Independently, hydrogen, C 1-8 Alkyl, halogen, cyano, nitro, [ka] or -(CH2) m -OCH3; selected from; R 80 , R 90 and R 100 are each independently hydrogen, C 1-8 Alkyl, C 2-6 Alkenyl, and C 1-6 alkoxy; and m is an integer between 0 and 12.

[0087] In some embodiments, Z X’ and Z X are each independently hydrogen, [ka] and -(CH2) m - selected from OCH3; R 80 , R 90 and R 100 are each independently hydrogen, C 1-3 Alkyl, and C 1-3 selected from alkoxy; m is an integer from 1 to 6.

[0088] In some preferred embodiments, the active agent is [ka] TIFF0007771235000033.tif241151TIFF0007771235000034.tif241151TIFF0007771235000035.tif241161.

[0089] X may be linked to Ab via a coupling group, which can be formed by reacting two individual coupling groups. For example, a coupling group can be formed by reaction of an amine or hydroxylamine with an electrophile, forming an amide or N-C bond, etc. In some embodiments, X comprises a coupling group and is linked to Ab via a coupling group (e.g., an amine, amide, hydroxylamine, triazole, alkyne, disulfide, or thioether). A triazole can be formed by reacting an azide with an alkyne. A succinimide can be formed by reaction of a thiol with a maleimide. A disulfide can be formed by reaction of a thiol with a maleimide.

[0090] In some embodiments, at least one X comprises a moiety formed from one of the following structural formulas: While the coupling groups in the structures below are depicted as structurally complete formulas, it is understood that the coupling groups in the structures are linked to Ab by appropriate reactions. For example, the -NH group below is understood to comprise an -N(H)- group when attached to the Ab moiety. Similarly, the azide or ethynyl groups are understood to comprise triazole: [ka] TIFF0007771235000037.tif196170TIFF0007771235000038.tif215167TIFF000 7771235000039.tif215166TIFF0007771235000040.tif224168TIFF00077712350 00041.tif226168TIFF0007771235000042.tif159169TIFF0007771235000043.t if182168TIFF0007771235000044.tif229170TIFF0007771235000045.tif132169

[0091] In another aspect, the present specification provides a pharmaceutical composition containing the antibody-drug conjugate described herein, which may optionally further contain a therapeutically effective amount of a chemotherapeutic agent.

[0092] In yet another aspect, the description provides a method of treating cancer, comprising administering an antibody-drug conjugate of the description, or a pharmaceutical composition thereof. In some such embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, or melanoma.

[0093] In yet another aspect, the description provides a method of treating an autoimmune disease or an inflammatory disease, comprising administering an antibody-drug conjugate of the description, or a pharmaceutical composition thereof. In some embodiments, 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), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis.

[0094] Anti-CD19 antibody Examples of anti-CD19 antibodies include the antibodies designated herein as 5F5, 7F11, 9G8, F6, 7F1, and 10D8, or any fragment, variant, multimeric, or bispecific antibody thereof. Similarly, anti-CD19 antibodies can be antibodies that bind to the same epitope as 5F5, 7F11, 9G8, F6, 7F1, and 10D8, or any fragment, variant, multimeric, or bispecific antibody thereof. These antibodies, or fragments, variants, multimeric, or bispecific antibodies thereof, are each referred to herein as "huCD19" antibodies. HuCD19 antibodies herein include fully human monoclonal antibodies, as well as humanized monoclonal and chimeric antibodies, or any fragment, variant, multimeric, or bispecific antibody thereof. These antibodies exhibit specificity for human CD19 and have been shown, for example, to modulate, e.g., block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological function or activity of CD19.

[0095] Biological functions or activities of CD19 include, by way of non-limiting example, binding to one or more Src family kinases and functioning as a B cell coreceptor with CD21 and / or CD81 when in an activated, phosphorylated state; and / or recruiting PI-3 kinase. An antibody is considered to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one functional activity of CD19 if the level of functional activity of CD19 in the presence of the antibody is reduced by at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, compared to the level of functional activity of CD19 in the absence of binding to an antibody described herein. An antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one functional activity of CD19 if the level of functional activity of CD19 in the presence of the antibody is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of functional activity of CD19 in the absence of binding to an antibody described herein.

[0096] Each huCD19 monoclonal antibody, or fragment, variant, multimeric, or bispecific antibody thereof described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL) as shown in the amino acid sequences and corresponding nucleic acid sequences below. The CDR sequences according to IMGT are embedded within each VH and VL sequence below.

[0097] The 5F5 antibody comprises a heavy chain variable region (VH) (SEQ ID NO:2) encoded by the nucleic acid sequence set forth in SEQ ID NO:1, and a light chain variable region (VL) (SEQ ID NO:4) encoded by the nucleic acid sequence set forth in SEQ ID NO:3: TIFF0007771235000046.tif204160

[0098] The 7F11 antibody comprises a heavy chain variable region (VH) (SEQ ID NO: 6) encoded by the nucleic acid sequence set forth in SEQ ID NO: 5, and a light chain variable region (VL) (SEQ ID NO: 8) encoded by the nucleic acid sequence set forth in SEQ ID NO: 7: TIFF0007771235000047.tif204160

[0099] The 9G8 antibody comprises a heavy chain variable region (VH) (SEQ ID NO: 6) encoded by the nucleic acid sequence set forth in SEQ ID NO: 39, and a light chain variable region (VL) (SEQ ID NO: 10) encoded by the nucleic acid sequence set forth in SEQ ID NO: 9: TIFF0007771235000048.tif183160

[0100] The F6 antibody comprises a heavy chain variable region (VH) (SEQ ID NO: 12) encoded by the nucleic acid sequence set forth in SEQ ID NO: 11, and a light chain variable region (VL) (SEQ ID NO: 14) encoded by the nucleic acid sequence set forth in SEQ ID NO: 13: TIFF0007771235000049.tif183160

[0101] The 7F1 antibody comprises a heavy chain variable region (VH) (SEQ ID NO: 16) encoded by the nucleic acid sequence set forth in SEQ ID NO: 15, and a light chain variable region (VL) (SEQ ID NO: 18) encoded by the nucleic acid sequence set forth in SEQ ID NO: 17: TIFF0007771235000050.tif30159TIFF0007771235000051.tif145160

[0102] The 10D8 antibody comprises a heavy chain variable region (VH) (SEQ ID NO: 20) encoded by the nucleic acid sequence set forth in SEQ ID NO: 19, and a light chain variable region (VL) (SEQ ID NO: 22) encoded by the nucleic acid sequence set forth in SEQ ID NO: 21: TIFF0007771235000052.tif66160TIFF0007771235000053.tif116160

[0103] In some embodiments, the anti-CD19 antibody sequences or antigen-binding fragments thereof described herein are used to generate monovalent antibodies. The monovalent antibodies herein comprise a common heavy chain sequence, one arm that specifically recognizes CD19, and a second arm, referred to herein as a dummy arm. The dummy arm comprises an amino acid sequence that does not bind to or otherwise cross-react with human proteins. In some embodiments, the dummy arm comprises an amino acid sequence that does not bind to or otherwise cross-react with human proteins present in whole blood. In some embodiments, the dummy arm comprises an amino acid sequence that does not bind to or otherwise cross-react with human proteins present in solid tissues. Preferably, the monovalent antibody is specific for at least human CD19. In some embodiments, the monovalent antibody that recognizes human CD19 is also cross-reactive with at least one other non-human CD19 protein, for example, by way of non-limiting example, non-human primate CD19, e.g., cynomolgus monkey CD19, and / or rodent CD19.

[0104] In some embodiments, an anti-CD19 antibody sequence or antigen-binding fragment thereof is used in conjunction with a second antibody sequence or antigen-binding fragment thereof that binds to a target other than CD19 to generate a bispecific antibody referred to herein as an "anti-CD19 bispecific antibody."

[0105] The following antibody sequences are given herein as examples, but it will be understood that these sequences can be used to generate bispecific antibodies using any of a variety of art-recognized techniques. Examples of bispecific formats include fully human bispecific antibodies containing a common heavy chain, a kappa-type light chain, and a lambda-type light chain (PCT Publication WO 2012 / 023053), bispecific IgG based on Fab arm exchange (Gramer et al., 2013 MAbs. 5(6)); CrossMab format (Klein C et al., 2012 MAbs 4(6)); SEED technology (Davis JH et al., 2010 Protein Eng Des Sel. 23(4):195-202), electrostatic steering (Gunasekaran K et al., J Biol Chem. 2010 285(25):19637-46.) or knob-into-hole (Ridgway JB et al., Protein Eng. 1996 Several formats are based on forced heterodimerization approaches, such as α- and β-blockers (Von Kreudenstein TS et al., 2013 MAbs. 5(5):646-54), or other mutations that prevent homodimer formation (Von Kreudenstein TS et al., 2013 MAbs. 5(5):646-54); fragment-based bispecific formats (e.g., BiTEs) like tandem scFvs (Wolf E et al., 2005 Drug Discov. Today 10(18):1237-44); bispecific tetravalent antibodies (Poertner LM et al., 2012 Cancer Immunol Immunother. 61(10):1869-75); dual affinity re-targeting molecules (Moore PA et al., 2011 Blood. 117(17):4542-51), and diabodies (Kontermann RE et al., Nat Biotechnol. 1997 15(7):629-31), but are not limited to these.

[0106] Definition: Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms used in connection with, and techniques relating 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.

[0107] The methods and techniques herein are generally carried out according to conventional methods well known in the art, unless otherwise indicated, as described in 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).

[0108] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, the terminology used in connection with, and techniques relating to, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry, and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification methods are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with, and the laboratory procedures and techniques related to, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0109] As used in accordance with this specification, the following terms shall have the following meanings, unless otherwise indicated:

[0110] As used herein, the term "antibody" refers to immunoglobulin molecules and portions of immunoglobulin (Ig) molecules that are immunologically active, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the antigen of interest but does not react with other polypeptides, or does so with a much lower affinity (Kd > 10 -6 ) means that the antibody binds only to the antibody at the nucleotide sequence 110 of the present invention. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, any fragment, variant, multimeric, or bispecific antibody, including Fab, Fab', and F(ab')2 fragments, scFv, and Fab expression libraries. Antibodies may belong to any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, termed alpha, delta, epsilon, gamma, and mu, respectively. The various classes of immunoglobulins have different and well-known subunit structures and configurations. The term "antibody" does not refer to molecules that do not share homology with immunoglobulin sequences. For example, "antibody" as used herein does not include "repebodies."

[0111] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100 to 110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Antibody molecules obtained from humans generally belong to one of the following classes: IgG, IgM, IgA, IgE, and IgD, and differ from each other depending on the nature of the heavy chain present in the molecule. Specific classes have subclasses, e.g., IgG1, IgG2, etc. Furthermore, in humans, light chains can be kappa or lambda chains.

[0112] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), Fd, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0113] As used herein, "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a specific epitope of an antigen, characterized by a unique binding affinity for it. The term "monoclonal antibody" refers to a homogeneous population of antibodies involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which contain different antibodies typically directed against a variety of different antigenic determinants. The term "monoclonal antibody" includes, but is not limited to, antibody fragments (e.g., Fab, Fab', F(ab')2, Fd, Fv), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen-recognition site, as well as both intact and full-length monoclonal antibodies. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0114] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable regions ("V") of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, termed "hypervariable regions," are sandwiched between adjacent, more conserved sections known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally present between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." The amino acid assignments for each domain are based on the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0115] An antibody "specifically binds" to an epitope or antigenic molecule means that the antibody interacts or binds to the epitope or antigenic molecule more frequently, rapidly, for longer duration, with higher affinity, or a combination of the above, than to other substances, such as unrelated proteins. In certain embodiments, "specific binding" refers to, for example, an antibody that binds to an epitope or antigenic molecule with a K of about 0.1 mM or less, more typically about 1 μM or less. D In certain embodiments, "specifically binds" means that the antibody binds to a protein having a K of about 0.1 μM or less. D and sometimes binds to proteins with a K of about 0.01 μM or less.D "Specific binding" refers to binding to a protein having the sequence identity of the target. Because of the sequence identity between homologous proteins in different species, specific binding can include antibodies that recognize specific proteins in more than one species. It is understood that an antibody or binding moiety that specifically binds to a first target may or may not specifically bind to a second target. As noted above, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Generally, the term "binding" as used herein refers to, but is not necessarily, specific binding.

[0116] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequence. Generally, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, and hamster) having the desired specificity, affinity, and capacity (see, e.g., Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In some instances, Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues in an antibody of the non-human species having the desired specificity, affinity, and / or binding capacity. Humanized antibodies can be further modified by substituting additional residues in the Fv framework regions and / or within the replaced non-human residues to improve and optimize antibody specificity, affinity, and / or binding capacity. Generally, a humanized antibody comprises substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDRs corresponding to a non-human immunoglobulin, while all or substantially all of the framework regions (FRs) are of the consensus sequence of a human immunoglobulin. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, incorporated herein by reference.

[0117] As used herein, the term "human antibody" refers to an antibody encoded by a human nucleotide sequence or an antibody having an amino acid sequence corresponding to an antibody artificially generated using any suitable technique. This definition of a human antibody includes intact, full-length antibodies and / or fragments thereof.

[0118] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species, one of which is preferably human. Generally, the variable regions of both the light and heavy chains are derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) and correspond to the variable regions of antibodies with the desired specificity, affinity, and capacity, while the constant regions are similar to sequences in antibodies derived from another species (usually human), for example, to avoid eliciting an immune response in that species.

[0119] Antibodies, including fragments / derivatives thereof and monoclonal antibodies, can be produced using any suitable technique (e.g., McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628; Marks et al., J. Mol. Biol. 222:581-597 (1991); Marks et al., Bio / Technology 10:779-783 (1992); Waterhouse et al., Nucleic Acids Res. 21:2265-2266 (1993); Morimoto et al., J. Biochemical & Biophysical Methods 24:107-117 (1992); Brennan et al., Science 229:81 (1985); Carter et al., Bio / Technology 10:163-167 (1992); Kohler et al., Nature, 256:495 (1975); Kilpatrick et al., Hybridoma 16(4):381-389 (1997); Wring et al., J. Pharm. Biomed. Anal. 19(5):695-707 (1999); Bynum et al., Hybridoma 18(5):407-411 (1999), Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year Immuno. 7:33 (1993); Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol.226:889-896 (1992); U.S. Pat. Nos. 4,816,567, 5,514,548, 5,545,806, 5,569,825, 5,591,669, 5,545,807; PCT Patent Application Publication No. WO 97 / 17852, each of which is incorporated herein by reference in its entirety.

[0120] In certain preferred embodiments, the antibody does not specifically bind to CD19 or EGFR (epidermal growth factor receptor). In other embodiments, the antibody may be an anti-CD19 or EGFR antibody.

[0121] When an antibody comprises at least one light chain and at least one heavy chain, at least one light chain of the antibody, or at least one heavy chain of the antibody, or both, may contain an amino acid region having an amino acid motif capable of being recognized by an isoprenoid transferase. Because an antibody may comprise four polypeptide chains (e.g., two heavy chains and two light chains), the antibody may comprise four amino acid motifs, each of which can be used to conjugate an active agent to the antibody via a linker. Thus, an antibody-drug conjugate may comprise four linkers, each conjugated to an active agent, for example, each conjugated to the C-terminus of a different antibody chain. Thus, an antibody-drug conjugate may comprise at least one linker and at least one active agent. An antibody-drug conjugate may comprise at least two linkers, and an antibody-drug conjugate may comprise at least two active agents. An antibody-drug conjugate may comprise multiple linkers. An antibody-drug conjugate may comprise multiple active agents. In antibody-drug conjugates that include two or more active agents, the active agents may all be the same, all different, or may be present in any mixture or ratio.

[0122] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants typically consist of surface groupings of chemically active molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide.

[0123] As used herein, the terms "immunological binding" and "immunological binding characteristics" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and the antigen for which that immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd indicating a higher affinity. The immunological binding characteristics of a selected polypeptide can be quantified using any suitable method. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculating the concentrations and the observed rates of on- and off-coupling. (See Nature 361:186-87 (1993)). The ratio Koff / Kon allows for the elimination of all parameters unrelated to affinity and is equal to the dissociation constant Kd. (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) An antibody herein specifically binds to its target when the equilibrium binding constant (Kd) is 1 μM or less, e.g., 100 nM or less, preferably 10 nM or less, and more preferably 1 nM or less, as measured in an appropriate assay, such as a radioligand binding assay or similar assay known to one of skill in the art.

[0124] As used herein, the term "isolated polynucleotide" is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof, but depending on its origin, an "isolated polynucleotide" is (1) not associated, in whole or in part, with a polynucleotide with which it is naturally contained; (2) operably linked to a polynucleotide with which it is not linked in nature; or (3) not found in nature as part of a larger sequence. Polynucleotides herein include nucleic acid molecules encoding heavy chain immunoglobulin molecules and nucleic acid molecules encoding light chain immunoglobulin molecules, as described herein.

[0125] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or a combination thereof, and depending on its origin or source, an "isolated protein" is (1) not associated with naturally occurring proteins, (2) free from other proteins of the same origin, (3) expressed by cells of a different species, or (4) not naturally occurring.

[0126] The term "polypeptide" is used herein as a generic term to refer to naturally occurring proteins, fragments, or analogs that are polypeptide sequences. Naturally occurring protein fragments and analogs are therefore species of polypeptides. Polypeptides herein include the heavy and light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and combinations comprising light chain immunoglobulin molecules with heavy chain immunoglobulin molecules, as well as fragments and analogs thereof.

[0127] As used herein, the phrase "operably linked" means that the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0128] As used herein, the term "control sequence" refers to a polynucleotide sequence necessary to effect the expression and processing of a coding sequence to which it is ligated. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression and processing, but may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. As referred to herein, the term "polynucleotide" refers to a polymer of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of both types of nucleotides. This term includes single- and double-stranded forms of DNA.

[0129] The 20 common amino acids and their abbreviations used herein follow conventional convention. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the 20 common amino acids (e.g., D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptides of the present invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0130] The term "substantial identity" as applied to polypeptides means that two peptide sequences, when optimally aligned, e.g., by the GAT or BESTFIT programs using default gap weights, share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

[0131] Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0132] Conservative amino acid substitution refers to the residues that have similar side chains that can be exchanged.For example, the amino acid group that has aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; the amino acid group that has aliphatic hydroxyl side chains is serine and threonine; the amino acid group that has amide-containing side chains is asparagine and glutamine; the amino acid group that has aromatic side chains is phenylalanine, tyrosine and tryptophan; the amino acid group that has basic side chains is lysine, arginine and histidine; and the amino acid group that has sulfur-containing side chains is cysteine ​​and methionine.Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

[0133] Minor variations in the amino acid sequences of the antibodies or immunoglobulin molecules discussed herein are considered to be encompassed herein, provided that the amino acid sequence changes are at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are made within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods are preferably used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Many methods are known for identifying protein sequences that fold into known three-dimensional structures. Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to reveal structural and functional domains in accordance with the present specification.

[0134] Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins with sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain(s) that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Art-recognized examples of polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed. W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds. Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0135] Chemical terms used herein, unless otherwise defined, are used in accordance with conventional usage in the art as exemplified by "The McGraw-Hill Dictionary of Chemical Terms," ​​Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0136] The term "agent" is used herein to refer to a compound (e.g., an organic or inorganic compound, a mixture of compounds), a biopolymer (e.g., a nucleic acid, an antibody (including portions thereof, as well as humanized, chimeric, and human antibodies, and monoclonal antibodies, etc.), a protein or portion thereof (e.g., a peptide), a lipid, a carbohydrate), or an extract made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (e.g., mammals). Agents include, for example, agents with known structures and agents with unknown structures. The ability of such agents to inhibit AR or promote its degradation may qualify them as "therapeutic agents" in the methods and compositions herein.

[0137] 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, livestock animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0138] "Treating" a disease or patient refers to taking measures to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or disease, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or palliation of disease symptoms, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0139] As used herein, a therapeutic agent that "prevents" a disorder or disease refers to a compound that reduces the occurrence of the disorder or disease in a treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or disease compared to an untreated control sample. The term "preventing," while art-recognized, when used in the context of a disease, such as a local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure, or any other disease, is well understood in the art and includes administration of a composition that reduces the frequency or delays the onset of symptoms of the disease in a subject compared to subjects who do not receive the composition. Thus, cancer prevention includes, for example, reducing, to a statistically and / or clinically significant extent, the number of detectable cancerous growths in a population of patients receiving prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population compared to an untreated control population.

[0140] "Administering" or "administration" of a substance, compound, or agent to a subject can be by any suitable method or route. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intrathecally, intracerebrally, or transdermally (e.g., by absorption through the skin's ducts). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations that provide sustained, sustained, or controlled release of the compound or agent. Administration can also be carried out, for example, in a single dose, multiple doses, and / or over one or more extended periods of time.

[0141] The appropriate method of administering a substance, compound, or agent to a subject will also depend, for example, on the age and / or health of the subject, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a sustained-release or timed-release formulation or is administered using a sustained-release or timed-release device, as described above.

[0142] As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents in which a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., two therapeutic agents are effective in a patient simultaneously, which may include a synergistic effect of the two therapeutic agents). For example, different therapeutic compounds can be administered simultaneously or sequentially, either in the same formulation or in separate formulations. Thus, an individual receiving such treatment can benefit from the combined effect of the different therapeutic agents.

[0143] The term "therapeutically effective amount" refers to a single dose or a composition administered in a multiple dose schedule effective for treating or preventing a disease or disorder. The term "therapeutically effective amount" in reference to cancer or tumor refers to an amount that can reduce the number of cancer cells; reduce the size of cancer cells; inhibit or reduce the invasion of cancer cells into the peripheral system; inhibit or reduce the spread of cancer cells to other systems; inhibit the proliferation of cancer cells; and / or ameliorate at least one symptom associated with cancer. In treating cancer, the effectiveness of a drug can also be assessed by the time to tumor progression (TTP) and / or response rate (RR).

[0144] As used herein, the term "pharmaceutically acceptable salt" includes organic and inorganic salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogensulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis-(2-hydroxy-3-naphthoic acid)) salts. A pharmaceutically acceptable salt may include another molecule (e.g., an acetate ion, a succinate ion, and / or other counter ion).

[0145] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and the description is meant to encompass both cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" refers to cases where the alkyl may be substituted and cases where it is not substituted.

[0146] It will be appreciated that those skilled in the art can select substituents and substitution patterns on the compounds herein to result in chemically stable compounds that can be readily synthesized from readily available starting materials by any suitable method, such as those described below. When a substituent is itself substituted with two or more groups, it will be appreciated that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0147] As used herein, the term "optionally substituted" refers to the replacement of 1 to 6 hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH-O-alkyl, -OP(O)(O-alkyl) or -CH-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of 1 to 4 hydrogen radicals in a given structure with the substituent. More preferably, 1 to 3 hydrogen radicals are replaced with the substituent. Of course, the substituent may be further substituted.

[0148] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0149] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0150] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0151] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, trifluoromethoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0152] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0153] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having substituents replacing hydrogen on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons that are or are not included in one or more double bonds. Furthermore, such substituents include all those contemplated for alkyl groups, as described below, except where stability would be impaired. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0154] An "alkyl" group or "alkane" is a fully saturated, straight-chain or branched non-aromatic hydrocarbon. Typically, a straight-chain or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise specified. Examples of straight-chain or branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups.

[0155] Furthermore, the term "alkyl" (or "lower alkyl"), as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, unless otherwise specified, for example, halogen (e.g., fluoro), hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetic acid, or thioformic acid), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. In a preferred embodiment, a substituent on a substituted alkyl is C 1-6 Alkyl, C 3-6 The substituents on the substituted alkyl are selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In a more preferred embodiment, the substituents on the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted, if appropriate. For example, the substituents on the substituted alkyl may include substituted and unsubstituted amino, azido, imino, amido, phosphoryl (including phosphate and phosphinate), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate), and silyl groups, as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylic acid, and ester), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. The cycloalkyl may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, and the like.

[0156] "C x-yThe term "C" when used in connection with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy means that the group contains from x to y carbons in the chain. For example, "C x-y The term "alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, such as a straight-chain alkyl group or a branched-chain alkyl group, containing from x to y carbons in the chain, including haloalkyl groups. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. CO alkyl refers to hydrogen when the group is in a terminal position and to a bond when the group is in an internal position. "C 2-y alkenyl" and "C 2-y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond respectively.

[0157] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0158] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0159] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter of which refers to an alkynyl moiety having substituents replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may be present on one or more carbons that are or are not included in one or more triple bonds. Furthermore, such substituents include all those contemplated for alkyl groups, as described above, except where stability would be impaired. For example, substitution of alkynyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0160] As used herein, the term "amide" refers to the following group: [ka] refers to, In the formula, R A each independently represents hydrogen or a hydrocarbyl group, or two R A together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0161] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, and salts thereof, and include, for example, the moiety represented by [ka] refers to, In the formula, R A each independently represents hydrogen or a hydrocarbyl group, or two R A together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0162] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0163] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0164] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which all ring atoms are carbon. Preferably, the ring is a 6-membered or 10-membered ring, with a 6-membered ring being more preferred. The term "aryl" also includes polycyclic ring systems having two or more rings, in which two or more carbon atoms of the ring system are common to two adjacent rings, and at least one of the rings is aromatic, and for example, the other ring can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0165] The term "carbamic acid" is art-recognized and refers to the following group: [ka] Although, In the formula, R A each independently represents hydrogen or a hydrocarbyl group such as an alkyl group, or two R A together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0166] As used herein, the terms "carbocycle" and "carbocyclic" refer to saturated or unsaturated rings in which all ring atoms are carbon. The term "carbocycle" includes both aromatic and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings in which all carbon atoms in the ring are saturated and cycloalkene rings that contain at least one double bond. "Carbocycle" includes monocyclic 5- to 7-membered rings and bicyclic 8- to 12-membered rings. Each ring in a bicyclic carbocycle can be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring in a fused carbocycle can be selected from saturated, unsaturated, and aromatic rings. In a typical embodiment, an aromatic ring, such as phenyl, may be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Depending on the valence, any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle. Examples of "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Examples of fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions that can have a hydrogen atom.

[0167] A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. A "cycloalkyl" group includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, unless otherwise specified, and more typically from 3 to about 8 carbon atoms. The second ring of a bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each ring shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0168] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0169] The term "carbonate" is art-recognized and refers to -OCO-R A refers to the group, but R A represents a hydrocarbyl group.

[0170] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0171] As used herein, the term "ester" refers to an ester of -C(O)OR A refers to the group, but R A represents a hydrocarbyl group.

[0172] The term "ether" as used herein refers to a hydrocarbyl group linked to another hydrocarbyl group via an oxygen atom. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether can be a symmetrical or asymmetrical ether. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0173] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo. The term "dihalo," when referring to substitution, refers to two halogens bonded to a single carbon atom.

[0174] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0175] As used herein, the term "heteroalkyl" refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom. In certain embodiments, the two heteroatoms in a heteroalkyl are not adjacent.

[0176] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, which ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, and more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more ring structures in which two or more carbons are shared between two adjacent rings, at least one of which is heteroaromatic, and the other rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0177] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0178] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably 3- to 10-membered, more preferably 3- to 7-membered, which ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more rings, where two or more carbons of the ring system are common to two adjacent rings, at least one of the rings is heterocyclic, and the other rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactones, lactams, and the like.

[0179] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0180] As used herein, the term "hydrocarbyl" refers to a group bonded through a carbon atom that does not have an =O or =S substituent, typically has at least one carbon-hydrogen bond, and a primarily carbon backbone, but may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl in this application, while substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon) are not considered hydrocarbyl. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0181] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0182] The term "lower," when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups having 10 or fewer, preferably 6 or fewer, non-hydrogen atoms in the substituent. "Lower alkyl," for example, refers to alkyl groups containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents described herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether listed alone or in combination with other substituents, such as those described for hydroxyalkyl and aralkyl (e.g., in the example of aralkyl, when counting the carbon atoms in the alkyl substituent, the atoms in the aryl group are not counted).

[0183] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each ring of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains from 3 to 10 atoms, preferably from 5 to 7 atoms, within the ring.

[0184] The term "silyl" refers to a silicon moiety having three hydrocarbyl moieties attached thereto.

[0185] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more backbone carbons. It should be understood that the terms "substituted" or "substituted with" implicitly include the proviso that such substitution is consistent with the permissible valences of the replaced atom and substituent, and that the substitution results in a stable compound that does not undergo spontaneous transformation, e.g., by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In various embodiments, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. As used herein, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituent of organic compounds described herein, which satisfy the valence of the heteroatom. The substituents can include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetic acid, or thioformic acid), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In a preferred embodiment, the substituent on the substituted alkyl is C 1-6 Alkyl, C 3-6In a more preferred embodiment, the substituent on the substituted alkyl is selected from fluoro, carbonyl, cyano, or hydroxyl. As will be appreciated by those skilled in the art, the substituent may itself be substituted, if appropriate. Unless specifically stated as "unsubstituted," reference to a chemical moiety herein is understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0186] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0187] The term "sulfonamide" is art-recognized and refers to a group that may be represented by the general formula: [ka] In the formula, R A each independently represents hydrogen or a hydrocarbyl such as alkyl, or two R A together with the atom(s) in between complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0188] The term "sulfoxide" is art-recognized and refers to an -S(O)-R A This R A represents a hydrocarbyl.

[0189] The term "sulfonic acid" is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0190] The term “sulfone” is art-recognized and refers to the group —S(O)—R A refers to the group, but R A represents a hydrocarbyl.

[0191] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0192] As used herein, the term "thioester" refers to a thioester of -C(O)SR A group or -SC(O)R A This R A represents a hydrocarbyl.

[0193] The term "thioether" as used herein corresponds to an ether where the oxygen has been replaced with a sulfur.

[0194] The term "urea" is art-recognized and may be represented by the general formula: [ka] In the formula, R A each independently represents hydrogen or a hydrocarbyl such as alkyl, or any of the R A The existence of another R A and together with the atom(s) in between complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0195] "Protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces, or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as needed during synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, aryloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), etc. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0196] As used herein, the term "modulate" includes not only inhibiting or suppressing a function or activity (such as cell proliferation), but also enhancing a function or activity.

[0197] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, this term includes compositions, additives, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0198] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable or compatible with the treatment of a patient.

[0199] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to any non-toxic organic or inorganic salt of any base compound. Examples of inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Examples of organic acids that form suitable salts include mono-, di-, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, and sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. While either mono- or di-acid salts can be formed, such salts may exist in hydrated, solvated, or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than their free base forms. The selection of appropriate salts is within the skill of the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, for isolation of experimental compounds or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0200] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acid compound. Examples of inorganic bases which form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Examples of organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline, or ammonia. The selection of appropriate salts is known to those skilled in the art.

[0201] Many of the compounds useful in the methods and compositions herein have at least one asymmetric center in their structure. This asymmetric center can be represented by R or S configuration, and the R and S designations are used according to the rules set forth in Pure Appl.Chem. (1976), 45, 11-30. This specification contemplates all stereoisomers, such as enantiomers and diastereoisomers of compounds, salts, prodrugs, or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 01 / 062726.

[0202] Furthermore, certain compounds containing alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers, and in each case, the specification includes both mixtures and individual isomers.

[0203] Some compounds may exist as tautomers and such forms, although not explicitly indicated in the formulas given herein, are intended to be included within the scope herein.

[0204] A "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in a host after administration to form a biologically active molecule. Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on a functional group of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs that use esters or phosphoramidates as biologically labile or cleavable (protecting) groups are described in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the specifications of which are incorporated herein by reference. The present specification includes within its scope prodrugs of the active agents described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0205] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid filler, diluent, additive, solvent, or encapsulating material, that is useful in formulating a medicinal or therapeutic drug. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) starches, such as corn starch, potato starch, and the like; Cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) additives, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0206] As used herein, the terms "logarithm of solubility," "LogS," or "logS" are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound greatly affects its absorption and distribution characteristics.

[0207] Low solubility often leads to poor absorption. LogS values ​​are the unit-free logarithm (base 10) of the solubility measured in moles / liter.

[0208] As used herein, "substantially pure" means that the species of interest is present as the predominant species (i.e., more abundant than other individual species in the composition on a molar basis); preferably, a substantially purified fraction is a composition in which the species of interest constitutes at least about 50% (on a molar basis) of all macromolecular species present.

[0209] Generally, a substantially pure composition will contain greater than about 80%, more preferably greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. Most preferably, the species of interest is purified to complete homogeneity such that the composition consists essentially of a single macromolecular species (contaminant species cannot be detected in the composition by conventional detection methods).

[0210] Isoprenoid transferase that recognizes antibodies The antibodies described herein may comprise, for example, an amino acid motif recognized by an isoprenoid transferase, preferably at the C-terminus of the antibody, wherein the thioether bond comprises the sulfur atom of a cysteine ​​in the amino acid motif. CXC , XCXC, XXCC, and CYYX, where C represents cysteine, Y represents, independently in each instance, an aliphatic amino acid, and X represents, independently in each instance, glutamine, glutamic acid, serine, cysteine, methionine, alanine, or leucine. In a preferred embodiment, the thioether bond involves the sulfur atom of the cysteine ​​of the amino acid motif.

[0211] In some embodiments, the amino acid motif is the sequence CYYX, where Y represents, independently in each instance, alanine, isoleucine, leucine, methionine, or valine. For example, the amino acid motif can be CVIM or CVLL.

[0212] In a preferred embodiment, at least one of the seven amino acids preceding the amino acid motif is glycine. In a preferred embodiment, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine and proline. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids preceding the amino acid motif are all glycine, preferably 7. In a specific preferred embodiment, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine, aspartic acid, arginine, and serine.

[0213] In some embodiments, the antibody comprises the amino acid sequence GGGGGGCVIM, preferably at the C-terminus.

[0214] In preferred embodiments, the antibody comprises an amino acid motif that can be recognized by an isoprenoid transferase. For example, at least one C-terminus of the antibody may comprise an amino acid motif that can be recognized by an isoprenoid transferase (e.g., before forming the antibody-drug conjugate, e.g., as a substrate, or, e.g., as a product of the isoprenoid transferase after forming the antibody-drug conjugate). The antibody may further comprise a spacer, e.g., an amino acid or stretch of amino acids that connects the antibody peptide chain to the amino acid motif. The spacer may consist of 1 to 20 consecutive amino acids, preferably 7 to 20 amino acids. In some embodiments, glycine and proline are preferred amino acids for the spacer, and can be used in any combination, e.g., a stretch of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycines, or a stretch of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycines. In other embodiments, the amino acid motifs are each independently selected from glycine, aspartic acid, arginine, and serine. The antibody may, for example, contain additions or deletions at the carboxy terminus compared to a form of the antibody that is not included in the ADC.

[0215] Examples of isoprenoid transferases include protein farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase), which can also catalyze the transfer of a farnesyl or geranyl-geranyl group to at least one C-terminal cysteine ​​of a target protein. GGTases can be classified as GGTase I or GGTase II. FTase and GGTase I can recognize the CAAX motif, and GGTase II can recognize the XXCC, XCXC, or CXX motif, where C represents cysteine, A represents an aliphatic amino acid (e.g., isoleucine, valine, methionine, leucine), and each X independently represents, for example, glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine (Nature Rev. Cancer, 5(5):405-12 (2005); Nature Chemical Biology 17:498-506 (2010); Lane KT, Bees LS, J. Lipid Research, 47:681-699 (2006); Kasey PJ, Seabra MC, J. Biological Chemistry, 271(10):5289-5292 (1996), each of which is incorporated herein by reference in its entirety.

[0216] Antibody-drug conjugates according to the present disclosure may comprise an amino acid motif such as CYYX, XXCC, XCXC, or CXX, preferably CYYX, where C represents cysteine, Y represents an aliphatic amino acid such as leucine, isoleucine, valine, and / or methionine, and X represents an amino acid that determines the substrate specificity of an isoprenoid transferase, such as glutamine, glutamate, serine, cysteine, methionine, alanine, and / or leucine.

[0217] Isoprenoid transferases from a variety of sources may be used. For example, isoprenoid transferases may be obtained from humans, animals, plants, bacteria, viruses, or other sources. In some embodiments, naturally occurring isoprenoid transferases are used. In some embodiments, naturally or artificially modified isoprenoid transferases may be used. For example, the isoprenoid transferase may include one or more amino acid substitutions, additions, and / or deletions, and / or the isoprenoid transferase may be modified by the addition of at least one tag, such as a histidine tag, GST, GFP, MBP, CBP, isopep tag, BCCP, Myc tag, calmodulin tag, FLAG tag, HA tag, maltose binding protein tag, Nus tag, glutathione-S-transferase tag, green fluorescent protein tag, thioredoxin tag, S tag, Softag1, Softag3, strep tag, SBP tag, Ty tag, etc.

[0218] Isoprenoid transferases recognize isosubstrates and / or substrates. The term isosubstrate refers to substrate analogs containing chemical modifications. Isoprenoid transferases can alkylate specific amino acid motifs (e.g., CAAX motifs) at the C-terminus of antibodies (see, e.g., Duckworth, BP et al., ChemBioChem, 8:98 (2007); Uyen TT et al., ChemBioChem, 8:408 (2007); Labadie, GR et al., J. Org. Chem., 72(24):9291 (2007); Wollack, JW et al., ChemBioChem, 10:2934 (2009), each of which is incorporated herein by reference). Functionalized antibodies can be generated using isoprenoid transferases and isosubstrates that can alkylate C-terminal cysteines.

[0219] The isosubstrate may be, for example, a compound of the formula: [ka] The cysteine ​​of the C-terminal CAAX motif can be attached to an isosubstrate using an isoprenoid transferase. In some embodiments, a portion of the motif, e.g., AAX, can then be removed, e.g., by a protease, leaving only the isoprenoid-attached cysteine. The cysteine ​​can optionally be methylated at the carboxyl terminus, e.g., by an enzyme (see, e.g., Bell, IM, J. Med. Chem., 47(8):1869 (2004), which is incorporated herein by reference).

[0220] The antibody-drug conjugates of the present invention can be prepared by any suitable method, including molecular biology and cell biology.For example, transient transfection or stable transfection can be used.The gene sequence encoding the specific amino acid motif that can be recognized by isoprenoid transferase can be inserted into a plasmid vector, of which many suitable vectors are known, by standard PCR and / or ligation techniques, so as to express the antibody that has this specific amino acid motif at its C-terminus.In this way, the antibody that has at least one amino acid motif that can be recognized by isoprenoid transferase can be expressed in a suitable host, for example, CHO cells or E. coli.

[0221] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporating a radioactively labeled amino acid or by attaching a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain circumstances, the label or marker may be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art, and any suitable method can be used. Examples of polypeptide labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. As used herein, "pharmaceutical or drug" refers to a compound or composition capable of producing a desired therapeutic effect when properly administered to a patient.

[0222] The active agent can be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof. The active agent can be an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof. In some embodiments, the active agent can be a chemotherapeutic agent and a toxin, as described herein. For example, in some embodiments, the active agent can be amanitin, auristatin, calicheamicin, camptothecin, cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansinoid, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, a toxoid, or a derivative of any of the foregoing. In some embodiments, at least one active agent can be taltubulin. In some embodiments, at least one active agent can be azonafide. In some embodiments, the active agent can be a pyrrolobenzodiazepine dimer, as described herein.

[0223] In some embodiments, the active agent is a chemotherapeutic drug or toxin. The active agent may be erlotinib; bortezomib; fulvestrant; sutent; letrozole; imatinib mesylate; PTK787 / ZK 222584; oxaliplatin; 5-fluorouracil; leucovorin; rapamycin (sirolimus); lapatinib; lonafarnib; sorafenib; gefitinib; AG1478; AG1571; alkylating agents (e.g., thiotepa or cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, or piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, or uredopa); ethylenimines, methyl Melamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins (e.g., bullatacin or bullatacinone); camptothecin; derivatives or metabolites of camptothecin (e.g., SN-38); topotecan; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, or bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin, cryptophycin 1 or cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, e.g., KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, fenesteryl nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimnustine); antibiotics (e.g., enediyne antibiotics, such as calicheamycins selected from calicheamycin gamma 1I and calicheamycin omega 1I, or dynemicins, including dynemicin A); bisphosphonates (e.g., clodronate;esperamicin, neocarzinostatin chromophore, or related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, or deoxydoxorubicin), epirubicin, esorubicin, marcellomycin, mitomycin (e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rhodrubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; antimetabolites (e.g., 5-fluorouracil); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, or trimetrexate); purine analogs (e.g., fludarabine, 6-mercapto purines, thiamiprine, or tiguanine; pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, or floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane) or testolactone); antiadrenal agents (e.g., aminoglutethimide, mitotane, or trilostane); folic acid replacement acid replenishers) (e.g., folinic acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elfornithine; elliptinium acetate; epothilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin;Maytansinoids (e.g., maytansine or ansamitocin); trichothecenes (especially T-2 toxin, veraculin A, roridin A, or anguidine); mitoguazone; mitoxantrone; mopidammol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide K complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol acetaminophen; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids (e.g., paclitaxel), cremophor-free ABRAXANE™, albumin-modified nanoparticle formulations of paclitaxel, doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs (e.g., cisplatin or carboplatin); vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors (RFS 2000); difluoromethylornithine; retinoids (e.g., retinoic acid); capecitabine, and pharmaceutically acceptable salts, solvates, acids, or derivatives thereof.

[0224] Active agents include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators, including tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene; (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, and anastrozole; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; and (v) protein kinase inhibitors. (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in adherent cells, such as PKC-alpha, Raf, H-Ras; (viii) ribozymes, such as VEGF inhibitors, including ribozymes and HER2 expression inhibitors; (ix) vaccines, such as gene therapy vaccines; ALLOVECTIN® vaccine, LEUVECTIN vaccine, VAXID vaccine; PROLEUKIN® rlL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; (x) anti-angiogenic agents, such as bevacizumab; (xi) affinity ligands: affinity ligands are substrates, inhibitors, stimulators, neurotransmitters, radioisotopes, or any combination thereof; (xii) radiolabels: 32 P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or any combination thereof; (xii) an immunomodulatory compound, an anticancer drug, an antiviral drug, an antibacterial drug, an antifungal drug, an antiparasitic drug, or any combination thereof; (xiv) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene; (xv) (xvi) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (xvii) aromatase inhibitors; (xvii) protein kinase inhibitors; (xix) lipid kinase inhibitors; (xx) antisense oligonucleotides; (xxi) ribozymes; (xxii) vaccines; (xxiii) angiogenesis inhibitors; and (xxiv) pharmaceutically acceptable salts, solvates, acids, or derivatives thereof.

[0225] In some embodiments, at least one active agent is taltubulin or azonafide.

[0226] In some embodiments, the active agent is amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansinoid, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, or a derivative of any one of these. In certain embodiments, the active agent is amanitin, MMAE, or MMAF, or a derivative of any one of the foregoing.

[0227] Additionally, cytokines may be used as active agents. Cytokines are small cell-signaling protein molecules secreted by many cells and belong to a class of signaling molecules widely used in intercellular communication. Cytokines include monokines, lymphokines, and traditional polypeptide hormones. Examples of cytokines include growth hormones (e.g., human growth hormone, N-methionyl human growth hormone, or bovine growth hormone); parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones (e.g., follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), or luteinizing hormone (LH)); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α, tumor necrosis factor-β; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins, thrombopoietin (TPO); nerve growth factor (e.g., NGF-β); platelet growth factor; transforming growth factor (TGF) (e.g., TGF-α or TGF-β). interferons (e.g., interferon-α, interferon-β, or interferon-γ); colony-stimulating factors (CSFs) (e.g., macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), or granulocyte-CSF (G-CSF)); interleukins (ILs) (e.g., IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, or IL-12); tumor necrosis factors (TNFs) (e.g., TNF-α or TNF-β); and polypeptide factors (e.g., LIF or kit ligand). Furthermore, the term "cytokine" also includes cytokines from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0228] The term "toxin" refers to a substance that is toxic to living cells or organisms. A toxin can be a small molecule, peptide, or protein that can cause cellular dysfunction or cell death after contact with or absorption by body tissue, for example, through interaction with one or more biological macromolecules, such as enzymes or cellular receptors. Toxins include plant and animal toxins. Examples of animal toxins include, but are not limited to, diphtheria toxin, botulinum toxin, tetanus toxin, shiga toxin, cholera toxin, tetrodotoxin, brevetoxin, and ciguatoxin. Examples of plant toxins include, but are not limited to, ricin and AM-toxin.

[0229] Examples of small molecule toxins include auristatins, tubulysins, geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784), maytansinoids (EP 1391213, ACR 2008, 41, 98-107), calicheamicin (US Patent Publication No. 2009 / 0105461, Cancer Res. 1993, 53, 3336-3342), daunomycin, doxorubicin, methotrexate, vindesine, SG2285 (Cancer Res. 2010, 70(17), pp. 6849-6858), dolastatin, dolastatin analogs, auristatin (U.S. Pat. No. 5,635,483), cryptophycin, camptothecin, camptothecin derivatives or metabolites (e.g., SN-38), rhizoxin derivatives, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotics, esperamicin, epothilones, pyrrolobenzodiazepine (PBD) derivatives, amanitin, amanitin derivatives, α-amanitin, aplidine, azonafide, and toxoids. Toxins can exhibit cytotoxicity and cell growth inhibitory activity through tubulin binding, DNA binding, topoisomerase inhibition, etc.

[0230] A "detectable moiety" or "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, radioactive, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin-streptavidin, digoxigenin, haptens, proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules having a sequence complementary to a target. Detectable moieties often generate a measurable signal, e.g., a radioactive, chromogenic, or fluorescent signal, which is used to quantify the amount of bound detectable moiety in a sample. Quantitation of the signal can be achieved, for example, by scintillation counting, densitometry, flow cytometry, ELISA, or direct analysis of intact or subsequently digested peptides by mass spectrometry (one or more peptides can be evaluated).

[0231] As used herein, the term "probe" refers to a material that (i) exhibits a detectable signal, (ii) interacts with a first probe or a second probe to modify the detectable signal exhibited by the first or second probe, e.g., fluorescence resonance energy transfer (FRET), (iii) stabilizes interactions with or increases binding affinity to an antigen or ligand, (iv) affects electrophoretic mobility or cell penetration activity through physical parameters, e.g., charge, hydrophobicity, etc., or (v) can modulate ligand affinity, antigen-antibody binding, or ionic complex formation.

[0232] The active agent may be an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof.

[0233] The immunomodulatory compound may be selected from aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, cyclosporine, cyclosporine A, danazol, dehydroepiandrosterone, dexamethasone, etanercept, hydrocortisone, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenylate mofetil, prednisone, sirolimus, and tacrolimus.Anticancer drugs include 1-methyl-4-phenylpyridinium ion, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), 5-fluorouracil, 9-aminocamptothecin, actinomycin D, asparaginase, bicalutamide, bis-chloroethylnitrosourea (BCNU), bleomycin, bleomycin A2, bleomycin B2, busulfan, camptothecin, derivatives or metabolites of camptothecin, e.g., SN -38, carboplatin, carmustine, CB1093, chlorambucil, cisplatin, crisnatol, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin, dacarbazine, deferoxamine, demethoxyhypocrelin A, docetaxel, doxifluridine, doxorubicin, EB1089, epirubicin, etoposide, floxuridine, fluoxetine Darabine, flutamide, gemcitabine, goserelin, hydroxyurea, idarubicin, ifosfamide, interferon-α, interferon-γ, irinotecan, KH1060, leuprolide acetate, lomustine, lovastatin, megestrol, melphalan, mercaptopurine, methotrexate, mitomycin, mitomycin C, mitoxantrone, mycophenolic acid, nitrogen mustard, nitrosourea, paclitaxel, peplomycin, photosensitizers It may be selected from Pe4, phthalocyanine, pirarubicin, plicamycin, procarbazine, raloxifene, raltitrexed, revlimid, ribavirin, staurosporine, tamoxifen, teniposide, thalomid, thapsigargin, thioguanine, tiazofurin, topotecan, threosulfan, trimetrexate, tumor necrosis factor, velcade, verapamil, verteporfin, vinblastine, vincristine, vinorelbine and zorubicin.The antiviral agent may be selected from pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin, idoxuridine, vidarabine, trifluridine, acyclovir, famcicyclovir, amantadine, rimantadine, cidofovir, antisense oligonucleotides, immunoglobulins, and interferons. The antibacterial agent may be selected from chloramphenicol, vancomycin, metronidazole, trimethoprim, sulfamethazole, quinupristin, dalfopristin, rifampin, spectinomycin, and nitrofurantoin. Antifungal agents include amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, and ravconazole. The antiparasitic agent may be selected from mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, niclosamide, praziquantel, albendazole, rifampin, amphotericin B, melarsoprol, eflornithine, metronidazole, tinidazole, and miltefosine.

[0234] The antibody may comprise an amino acid motif selected from Ab-HC-(G)zCVIM, Ab-HC-(G)zCVLL, Ab-LC-(G)zCVIM and Ab-LC-(G)zCVLL, Ab-HC-(G)zCVIM / LC-(G)zCVIM, Ab-HC-(G)zCVLL / LC-(G)zCVIM, Ab-HC-(G)zCVIM / LC-(G)zCVLL and Ab-HC-(G)zCVLL / LC-(G)zCVLL. wherein Ab represents an antibody (e.g., one disclosed herein), Ab-HC- represents a heavy chain of the antibody (e.g., a heavy chain disclosed herein), Ab-LC- represents a light chain of the antibody (e.g., a light chain disclosed herein), G represents glycine, C represents cysteine, V represents valine, I represents isoleucine, M represents methionine, L represents leucine, and z is an integer from 0 to 20, preferably 1 to 10.

[0235] General Methods for Producing Antibodies Various methods known in the art can be used to produce polyclonal or monoclonal antibodies against a given target, such as CD19, a tumor-associated antigen, or other target, or against derivatives, fragments, analogs, homologs, or orthologs thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference).

[0236] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which primarily yield the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen or epitope targeted by the desired immunoglobulin can be immobilized on a column, and the immune-specific antibody purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by Wilkinson (The Scientist, Published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0237] In some embodiments, the antibodies of the present disclosure are monoclonal antibodies. Monoclonal antibodies are produced, for example, by using the methods described in the Examples herein. Antibodies can also be produced, for example, by immunizing BALB / c mice with a combination of cell transfectants expressing high levels of a given target on their surface. Hybridomas obtained from myeloma / B cell fusions are then screened for reactivity against the selected target.

[0238] Monoclonal antibodies are produced using the hybridoma method, e.g., as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0239] The immunizing agent typically contains a protein antigen, a fragment thereof, or a fusion protein thereof. Generally, peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevent growth of HGPRT-deficient cells.

[0240] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0241] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0242] After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0243] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0244] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the present disclosure serve as a preferred source of such DNA. Once the DNA is isolated, it can be placed into an expression vector, which can then be transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to achieve the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of the antibodies of the present disclosure, or for the variable domains of one antigen-binding site of an antibody of the present disclosure, to obtain a chimeric bivalent antibody.

[0245] The monoclonal antibodies of the present disclosure include humanized or human antibodies. These antibodies are suitable for administration to humans without eliciting an immune response against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulins. Humanization can be achieved, for example, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter et al. (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 instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, for example, residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Humanized antibodies optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0246] A fully human antibody is an antibody molecule in which the entire sequences of both the light and heavy chains, including the CDRs, are derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be produced using trioma technology; human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4: 72); and EBV hybridoma technology for producing monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus (see Cole et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0247] Human antibodies can also be produced using additional technologies, including phage display libraries (see Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; and Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0248] Human antibodies can also be produced using transgenic non-human animals modified to produce fully human antibodies in response to antibody challenge rather than the animal's endogenous antibodies (see PCT Publication WO 94 / 02602). Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Intermediate transgenic animals containing less than the full complement of modifications are then crossed to obtain progeny animals that provide all of the desired modifications. One example of such a non-human animal is the mouse known as the Xenomouse™, disclosed in PCT Publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with an immunogen of interest, e.g., as polyclonal antibody preparations, or from immortalized B cells derived from animals, such as hybridomas that produce monoclonal antibodies. Genes encoding immunoglobulins containing human variable regions can also be recovered and expressed to obtain antibodies directly, or the genes can be further modified to obtain antibody analogs, such as single-chain Fv (scFv) molecules.

[0249] One example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method comprising deleting J segment genes from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and prevent the formation of transcripts of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeting vector containing a gene encoding a selectable marker, and producing from the embryonic stem cells a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0250] One method for producing a desired antibody, e.g., a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0251] In a further refinement of this approach, methods for identifying clinically relevant epitopes on immunogens and correlating methods for selecting antibodies that specifically bind to the relevant epitopes with high affinity are disclosed in PCT Publication WO 99 / 53049.

[0252] The antibodies can be expressed by vectors containing DNA segments encoding the single chain antibodies described above.

[0253] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701 having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid-binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0254] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F. et al., DNA Cloning: Mammalian Systems, edited by D. Glover, (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet, 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995)), and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994)).

[0255] Poxvirus vectors deliver genes into the cytoplasm. Avipoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus, adeno-associated virus, and herpes simplex virus (HSV) vectors are preferred for delivering nucleic acids into neural cells. The adenovirus vectors deliver expression for a shorter period (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than HSV vectors. The particular vector selected depends on the target cell and the condition being treated. Delivery can be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene delivery methods include naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0256] Vectors can be used to target virtually any desired target cell. For example, stereotactic injection can be used to guide vectors (e.g., adenovirus, HSV) to the desired location. Particles can also be delivered by intracerebroventricular (icv) injection using a miniature pump infusion system such as the SynchroMed Infusion System. A bulk flow-based method called convection has also been shown to be effective in delivering large molecules to widespread areas of the brain and may be useful for delivering vectors to target cells (see Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous injection, parenteral injection, intraperitoneal injection, and subcutaneous injection, as well as oral or other suitable administration routes.

[0257] A bispecific antibody is an antibody that has binding specificities for at least two different antigens. In this case, one of the binding specificities is for a target such as CD19 or any fragment thereof. The second binding target is any other antigen, preferably a cell surface protein or receptor or receptor subunit.

[0258] Numerous methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the coexpression 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 unselected assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out by affinity chromatography steps. Similar methods are disclosed in WO 93 / 08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0259] The bispecific and / or monovalent antibodies of the present disclosure can be produced using any of a variety of art-recognized techniques, including those disclosed in WO 2012 / 023053, filed August 16, 2011, the entire contents of which are incorporated herein by reference. The method described in WO 2012 / 023053 produces bispecific antibodies structurally identical to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, with the first light chain variable region fused to a constant kappa domain and the second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity contributed by both the heavy and light chains. The light chain variable region can be from the lambda or kappa family and is preferably fused to lambda and kappa constant domains, respectively. This is preferred to avoid the creation of non-natural polypeptide junctions. However, it is also possible to obtain the bispecific antibodies of the present disclosure by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgGκλ antibodies or "κλ bodies," and are a new, fully human, bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, with properties indistinguishable from standard monoclonal antibodies, and is therefore preferred over previous formats.

[0260] An essential step in this method is the identification of two antibody Fv regions (each composed of a variable light chain and a variable heavy chain domain) with different antigen specificities that share the same heavy chain variable domain. Numerous methods for the production of monoclonal antibodies and their fragments have been described (see, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). A fully human antibody is an antibody molecule in which both the light and heavy chain sequences, including CDR1 and CDR2, are derived from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be produced 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 (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus (see Cole et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0261] Monoclonal antibodies are produced, for example, by immunizing animals with the target antigen or its immunogenic fragment, derivative, or variant. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen so that the target antigen is expressed and bound to the surface of the transfected cells. Various suitable techniques for producing xenogeneic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (the entireties of which are incorporated herein by reference).

[0262] Alternatively, antibodies are obtained by screening libraries containing antibody or antigen-binding domain sequences for binding to the target antigen, for example, prepared in bacteriophage as protein or peptide fusions to bacteriophage coat proteins with the encoding DNA sequences contained within the phage particle and expressed on the surface of the assembled phage particle (i.e., "phage display libraries").

[0263] Hybridomas resulting from the myeloma / B cell fusion are then screened for reactivity to the target antigen. Monoclonal antibodies are produced using the hybridoma method, such as that described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0264] Although it is not strictly impossible, it is almost impossible to accidentally identify different antibodies that have the same heavy chain variable domain but are directed to different antigens.In fact, in most cases, heavy chain mainly contributes to antigen binding surface, and is also the most variable in sequence.In particular, CDR3 on heavy chain is the most diverse CDR in sequence, length and structure.Therefore, two antibodies that are specific to different antigens almost always have different heavy chain variable domains.

[0265] The method disclosed in application WO 2012 / 023053 overcomes this limitation and significantly facilitates the isolation of antibodies with identical heavy chain variable domains. This method relies on the use of an antibody library in which the heavy chain variable domains are identical for all library members, and therefore diversity is limited to the light chain variable domains. Such libraries are described, for example, in applications WO 2010 / 135558 and WO 2011 / 084255 (each of which is incorporated by reference in its entirety). However, because the light chain variable domain is expressed together with the heavy chain variable domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing identical heavy chain variable domains and diverse lambda or kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach allows for the identification of two antibodies with a common heavy chain, one with a lambda light chain variable domain and the other with a kappa light chain variable domain, which can be used as building blocks for the production of bispecific antibodies in the complete immunoglobulin format of the present disclosure. The bispecific antibodies of the present disclosure can be of different isotypes, and their Fc portions can be modified to alter their binding characteristics to different Fc receptors, thus altering the antibody's effector functions and its pharmacokinetic properties. Numerous methods for modifying Fc portions have been described and are applicable to the antibodies of the present disclosure (see, e.g., Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199, filed January 9, 2009). The methods of the present disclosure can also be used to produce bispecific antibodies and antibody mixtures in the F(ab')2 format, which lack the Fc portion.

[0266] To enable the construction of the bispecific antibodies of the present disclosure, a common heavy chain and two different light chains are coexpressed in a single cell. If all polypeptides were expressed at the same level and assembled equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, different light chains may be expressed at different levels and / or may not assemble with the same efficiency. Therefore, means are used to adjust the relative expression of different polypeptides to compensate for their inherent expression characteristics or different propensities to combine with the common heavy chain. This adjustment can be achieved through promoter strength, the use of internal ribosome entry sites (IRES) with different efficiencies, or other types of regulatory elements that act at the transcriptional or translational level and can affect mRNA stability. Various promoters of varying strength could include CMV (immediate-early cytomegalovirus promoter), EF1-1α (human elongation factor 1 α subunit promoter), Ubc (human ubiquitin C promoter), and SV40 (simian virus 40 promoter). Various IRESs of mammalian and viral origin have also been described (see, e.g., Hellen CU and Sarnow P. Genes Dev 2001 15: 1593-612). These IRESs can vary significantly in their length and ribosome recruitment efficiency. Furthermore, activity can be further tuned by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). Expression regulation 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 alter their relative expression. The examples described herein demonstrate that controlling the relative expression of the different chains is crucial for maximizing the construction and overall yield of bispecific antibodies.

[0267] Coexpression of a heavy chain and two light chains generates a mixture of three distinct antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. To obtain the desired molecule, the latter must be purified from the mixture. The method described herein significantly facilitates this purification procedure by using affinity chromatography media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multistep affinity chromatography purification approach is efficient and generally applicable to the antibodies disclosed herein. This contrasts with specific purification methods that require the development and optimization of each bispecific antibody derived from a quadroma or other antibody mixture-expressing cell line. Indeed, if the biochemical properties of the different antibodies in the mixture are similar, their separation using standard chromatographic techniques, such as ion exchange chromatography, can be difficult or even impossible.

[0268] Other suitable purification methods include those disclosed in US2013 / 0317200, the contents of which are incorporated herein by reference in their entirety.

[0269] In another embodiment of producing bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Fusions preferably are with immunoglobulin heavy-chain constant domains, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on producing bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0270] According to another approach, described in WO 96 / 27011, the boundary (interface) between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred boundary comprises at least a part of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the boundary of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain is created at the boundary of the second antibody molecule. This provides a means of increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0271] Techniques for producing bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0272] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described in Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative means for producing bispecific antibody fragments. The fragments comprise a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain.L ) linked to a heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H and V L The domain is the complementary V of another fragment. L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0273] Antibodies with more than two valencies are envisioned, for example, trispecific antibodies can be made (Tutt et al., J. Immunol. 147:60 (1991)).

[0274] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which is derived from the protein antigen of the present disclosure. Alternatively, an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor (FcγR) for IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), can be combined with an anti-antigenic arm of an immunoglobulin molecule to focus cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to target cytotoxic substances to cells expressing a specific antigen. These antibodies have an antigen-binding arm and an arm that binds a cytotoxic substance or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigen described herein and also binds tissue factor (TF).

[0275] Heteroconjugate antibodies are also within the scope of the present disclosure. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and to treat HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies can be produced in vitro using synthetic protein chemistry methods, including those involving the use of crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0276] It may be desirable to modify the antibodies of the present disclosure with respect to effector function to enhance the antibody's effectiveness in treating cancer and / or other diseases and disorders associated with aberrant CD19 expression and / or activity. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation within this region. Homodimeric antibodies produced in this manner may exhibit improved internalization capability and / or enhanced complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies can be designed with dual Fc regions, thereby exhibiting enhanced complement cytolysis and ADCC functions (see Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0277] Conjugated Antibodies The present disclosure also relates to conjugated antibodies (also referred to herein as immunoconjugates) comprising an antibody or antigen-binding fragment thereof conjugated (bound) to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin) or a radioactive isotope (i.e., in the case of a radioconjugate).

[0278] In some embodiments, the toxin is a microtubule inhibitor or a derivative thereof. In some embodiments, the toxin is a dolastatin or a derivative thereof. In some embodiments, the toxin is auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, or DMAE. In some embodiments, the toxin is a maytansinoid or a maytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid-damaging toxin. In some embodiments, the toxin is a duocarmycin or a derivative thereof. In some embodiments, the toxin is a calicheamicin or a derivative thereof. In some embodiments, the substance is a pyrrolobenzodiazepine or a derivative thereof.

[0279] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitgelin, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides are available for the preparation of radioconjugates. Specific examples include:212 Bi, 131 I, 131 In, 90 Y and 186 Re is included.

[0280] Conjugates of antibodies and cytotoxic substances can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), 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, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionucleotides to antibodies (see WO94 / 11026).

[0281] Those skilled in the art will recognize that a wide variety of possible moieties may be coupled to the antibodies provided in this disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0282] Coupling can be achieved by any chemical reaction that links two molecules, so long as the antibody and the other moiety retain their respective activities. This binding can involve numerous chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, covalent bonding is preferred. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of external crosslinking molecules. Many bivalent or polyvalent linking agents are useful for coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is illustrative of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0283] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. (21558G)); #21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6-[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0284] The linkers contain components with different properties and therefore yield 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 not as soluble as sulfo-NHS esters. Furthermore, the linker SMPT contains a disulfide bond that provides steric hindrance, potentially forming conjugates with improved stability. Because disulfide bonds are cleaved in vitro, they are generally less stable than other bonds, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC), when used in combination with sulfo-NHS, forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0285] The antibodies disclosed herein can be formulated as immunoliposomes. Liposomes containing the antibodies can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0286] Particularly useful liposomes can be prepared by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

[0287] Use of anti-CD19 antibodies It will be understood that administration of therapeutic agents according to the present disclosure will be administered with appropriate carriers, excipients, and other materials incorporated into the formulation to provide improved uptake, delivery, tolerance, and the like. Many suitable formulations can be found in the 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 therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable in treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and acceptable for the route of administration. 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), as well as other formulations, excipients, and other information familiar to pharmaceutical chemists. See also the references cited therein for additional information related to carriers.

[0288] Therapeutic formulations of the present disclosure comprising conjugates of the present disclosure are used to treat or alleviate symptoms associated with cancer, including, but not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral cavity and pharynx cancer, endometrial cancer, and / or melanoma. The present disclosure also provides methods for treating or alleviating symptoms associated with cancer. Treatment regimens can include identifying a subject, e.g., a human patient, suffering from (or at risk of developing) cancer, e.g., using standard methods.

[0289] Therapeutic formulations of the present disclosure comprising a conjugate of the present disclosure that recognizes CD19 and optionally a second target may be used to treat or alleviate symptoms associated with autoimmune and / or inflammatory diseases, such as B-cell mediated autoimmune and / or inflammatory diseases, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and / or lupus nephritis.

[0290] Efficacy of treatment can be determined in association with any suitable method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the conjugate provides clinical benefit.

[0291] Conjugates to targets, such as CD19, tumor-associated antigens, or other antigens, may be used in methods related to the localization and / or quantification of these targets, for example, for use in measuring the levels of these targets in appropriate physiological samples, for use in diagnostic methods, for use in imaging of the protein, etc. Conjugates specific for these targets or any of their derivatives, fragments, analogs, or homologs, containing, for example, antibody-derived antigen-binding domains, may be utilized as pharmacologically active compounds (hereinafter referred to as "therapeutics").

[0292] The conjugates of the present disclosure can be used to isolate specific targets using standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. The conjugates of the present disclosure can be used diagnostically, for example, to monitor protein levels in tissues as part of a clinical testing procedure, to determine the effectiveness of a given therapeutic regimen, etc. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131 I, 35 S or 3 Contains H.

[0293] The conjugates of the present disclosure can be used as therapeutic substances. Such substances are generally used to treat or prevent diseases or conditions associated with the abnormal expression or activation of a given target in a subject. A conjugate preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and generally exerts its effect by binding to the target. Administration of the conjugate can inhibit, suppress, or interfere with the signaling function of the target. Administration of the conjugate can inhibit, suppress, or interfere with the target's binding to the endogenous ligand to which it naturally binds.

[0294] A therapeutically effective amount of a conjugate of the present disclosure generally refers to the amount necessary to achieve a therapeutic goal. As noted above, this may be the effect of the binding interaction between the antibody and its target antigen and / or the active agent bound to the antibody, in some cases interfering with the target's function. The amount required to be administered will further depend on the binding affinity of the antibody for its particular antigen and / or the potency of the active agent, as well as the rate at which the administered antibody is depleted from the free volume of the subject to which it is administered. Typical therapeutically effective dosages of conjugates of the present disclosure may range, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical administration frequencies may range, for example, from twice daily to once weekly.

[0295] The conjugates of the present disclosure can be administered in the form of pharmaceutical compositions for the treatment of various diseases and disorders.The principles and considerations for the preparation of such compositions and guidelines for the selection of ingredients are described, for example, in Remington: The Science and Practice of Pharmacy 19th ed. (Alfonso R. Gennaro et al., ed.) 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.

[0296] The formulations may also contain multiple active compounds, as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-suppressing agent. Such molecules are suitably present in combination in amounts effective for the purpose intended.

[0297] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, prepared by coacervation techniques or by boundary polymerization, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively.

[0298] The formulations to be used for in vivo administration are preferably sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0299] Sustained-release preparations can be manufactured. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for over 100 days, some hydrogels release proteins for shorter periods.

[0300] The conjugates of the present disclosure can be used as agents to detect the presence of a given target (or protein fragment thereof) in a sample. In some embodiments, the conjugate comprises a detectable label. The antibody can be polyclonal or, more preferably, monoclonal. An intact antibody or a fragment thereof (e.g., F ab , scFv or F (ab)2) may 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 a subject. Thus, the use of the term "biological sample" includes blood, blood fractions, or components (including serum, plasma, or lymph). That is, the detection methods of the present disclosure may be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro or in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detection of analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R. Crowther (ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Further, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte conjugate. For example, an antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0301] Pharmaceutical Composition The antibody-drug conjugates can be used to introduce an active agent into target cells of a subject to treat the subject using any suitable method for preparing the composition. In some aspects, the present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising the antibody-drug conjugates described herein.

[0302] The compositions and methods of the present disclosure can be used to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When the composition or compound is administered to an animal, such as a human, it is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for human administration, particularly for invasive administration routes (i.e., routes that avoid transport or diffusion through epithelial barriers, such as injection or implantation), the aqueous solution is pyrogen-free or substantially pyrogen-free. The excipient can be selected, for example, to provide delayed release of the substance or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in unit dosage form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized products for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0303] Pharmaceutically acceptable carriers may include physiologically acceptable substances that act to stabilize, increase the solubility, or enhance the absorption of compounds such as the compounds of the present disclosure. Such physiologically acceptable substances include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable substance, depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix, which may incorporate, for example, a compound of the present disclosure. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0304] The term "pharmaceutically acceptable" is used herein to mean those compounds, substances, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio and without excessive toxicity, irritation, allergic response, or other problem or complication.

[0305] Pharmaceutical compositions (formulations) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., liquids such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., patches applied to the skin); and topically (e.g., creams, ointments, or sprays applied to the skin). The compounds can also be formulated for inhalation. In certain embodiments, the compounds can 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. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0306] The formulations can be conveniently provided in unit dosage form and can be prepared by any suitable method in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0307] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present disclosure, with the carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0308] Formulations of the present disclosure suitable for oral administration can be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), lyophilisates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (with an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present disclosure as an active ingredient. The composition or compound can also be administered as a bolus, electuary, or paste.

[0309] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; and (4) disintegrants. Examples of suitable additives include, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, for example, paraffin; (6) absorption accelerators, for example, quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, for example, kaolin and bentonite clay; (9) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, for example, modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft- and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0310] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be made using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0311] Tablets and other solid dosage forms of pharmaceutical compositions, such as sugar-coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be prepared with or scored, if desired, with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical arts. They can also be used to effect sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to produce the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by adding a sterilizing agent or some other sterile injectable medium in the form of a sterile solid composition that can be dissolved in sterile water immediately before use. These compositions can also optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0312] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized products for reconstitution (reconstitution), microemulsions, solutions, suspensions (suspensions), syrups and elixirs.In addition to active ingredients, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0313] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0314] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0315] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0316] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0317] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0318] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present disclosure to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to promote the flux of the compound across the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0319] As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration contain one or more active compounds together with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0320] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, and in the case of dispersions, by maintaining the required particle size, and by the use of surfactants.

[0321] These compositions may also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. in the compositions. Prolonged absorption of injectable pharmaceutical forms can also be achieved by the inclusion of substances that delay absorption, such as aluminum monostearate and gelatin.

[0322] In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. In this case, the absorption rate of the drug depends on its dissolution rate, which may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0323] Injectable depot forms are prepared by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0324] For use in the methods of the present disclosure, the active compound can be administered alone or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient together with a pharmaceutically acceptable carrier.

[0325] The method of introduction can be by rechargeable or biodegradable device. A variety of slow-release polymeric devices have been developed in recent years and tested in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, have been used to form implants for the sustained release of compounds at specific target sites.

[0326] Actual dosage levels of the active ingredient in the pharmaceutical compositions can be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0327] The selected dosage level will depend on a variety of factors, including the activity of the individual compound or combination of compounds or their esters, salts, or amides used, the route of administration, the time of administration, the rate of excretion of the individual compounds used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the individual compounds used, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and similar factors well known in the medical arts.

[0328] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian may start with a lower dose of the pharmaceutical composition or compound than is necessary to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of a compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, other types of therapeutic agents administered together with the compounds of the present disclosure. A larger total dose may be delivered by multiple administrations of the agent. Numerous methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0329] Generally, a suitable daily dose of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective amount will generally depend upon the factors described above.

[0330] If desired, the effective daily amount of the active compound may be administered as one, two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments of the present disclosure, the active compound may be administered two or three times daily. In a preferred embodiment, the active compound is administered once daily.

[0331] Patients for this treatment may be any animal in need thereof, such as primates, especially humans; and other mammals, such as horses, cattle, pigs, sheep, cats and dogs; poultry; and pets in general.

[0332] In certain embodiments, the compounds of the present disclosure can be administered alone or in combination with another type of therapeutic agent.

[0333] The present disclosure includes the use of pharmaceutically acceptable salts of the disclosed compounds in the compositions and methods of the present disclosure. In some embodiments, contemplated salts of the present disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In some embodiments, contemplated salts of the present disclosure include, but are not limited to, L-arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In some embodiments, contemplated salts of the present disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In certain embodiments, contemplated salts of the present disclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, and the like. Acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid , sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid and undecylenate.

[0334] Pharmaceutically acceptable acid addition salts may also exist as various solvates, for example with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be that of the solvent of crystallization, that which is inherent in the solvent of preparation or crystallization, or that which is incidental to such solvent.

[0335] Wetting agents, emulsifying agents and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0336] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0337] The composition can be prepared in an injectable form, either as a liquid solution or suspension.Solid forms suitable for injection can also be prepared, for example, as emulsions or by encapsulating the antibody-drug conjugate in liposomes.The antibody-drug conjugate can be combined with a pharmaceutically acceptable carrier, including any carrier that does not induce the production of antibodies harmful to the subject to which the carrier is administered.Suitable carriers typically include large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0338] The composition may also contain diluents, such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present. The composition can be administered parenterally by injection, where such injection may be either subcutaneous or intramuscular. In some embodiments, the composition can be administered intratumorally. The composition can be inserted (e.g., injected) into the tumor. Additional formulations are suitable for other administration forms, such as suppositories or oral administration. Oral compositions can be administered as solutions, suspensions, tablets, pills, capsules, or sustained-release formulations.

[0339] The composition may be administered in a manner compatible with the dosage and formulation. The composition preferably contains a therapeutically effective amount of the antibody-drug conjugate. The dosage may vary depending on the subject being treated, the subject's health and physical condition, the degree of protection desired, and other relevant factors. The precise amount of the active ingredient (e.g., antibody-drug conjugate) may depend on the judgment of the physician. For example, a therapeutically effective amount of the antibody-drug conjugate or a composition containing it may be administered to a patient suffering from cancer or tumor to treat the cancer or tumor.

[0340] The antibody-drug conjugate according to the present disclosure or a composition containing the same may be administered in the form of a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the antibody-drug conjugate according to the present disclosure or a composition containing the same may be administered together with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable additive. The effective amount and type of pharmaceutically acceptable salt or solvate, excipient, and additive may be determined using standard methods (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th Edition, 1990).

[0341] Exemplary solvates that may be used for the pharmaceutically acceptable solvates of the antibody-drug conjugates described herein include water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.

[0342] Exemplary solvates that may be used for the pharmaceutically acceptable solvates of the antibody-drug conjugates described herein include water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.

[0343] In some embodiments, the present 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, felines, canines, porcines, ovines, bovines, equines, and primates. In certain preferred embodiments, the subject is a human.

[0344] The conjugates (also referred to herein as "active compounds") of the present disclosure, and their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the conjugate and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0345] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfite; a chelating agent, e.g., ethylenediaminetetraacetic acid (EDTA); a buffer, e.g., acetate, citrate, or phosphate; and an agent for adjusting tonicity, e.g., sodium chloride or dextrose. pH may be adjusted with acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampoules, disposable syringes, or multiple-dose vials (glass or plastic).

[0346] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, and, in the case of dispersions, by the maintenance of the required particle size and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0347] Sterile injectable solution can be prepared by incorporating the required amount of active compound in a suitable solvent, together with one or a combination of the above-listed components as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound in a sterile vehicle that contains a basic dispersion medium and other required components from the above-listed components.In the case of sterile powders for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired component from the corresponding solution that has been previously sterilized and filtered.

[0348] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be formulated with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared by using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is orally applied, gargled, expectorated or swallowed. Pharmaceutically compatible binding agents and / or auxiliary substances can be formulated as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterothes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavor.

[0349] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0350] Systemic administration can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the active compound can be formulated into ointments, salves, gels, or creams generally known in the art.

[0351] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0352] In one embodiment, the active compounds are prepared using carriers that will protect the compound against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Such materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by suitable methods, for example, as described in U.S. Pat. No. 4,522,811.

[0353] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a single dose for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with necessary pharmaceutical carrier.The details of dosage unit form of the present disclosure are determined and directly depend on the specific characteristics of active compound and the individual therapeutic effect to be achieved, and the inherent limitations of the technology of compounding such active compound for individual treatment.

[0354] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0355] All of the above and any other publications, patents and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including specific definitions, will control.

[0356] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0357] The present disclosure will be described in detail below with reference to examples. However, the following examples are merely intended to aid in the understanding of the present disclosure. The scope of the present disclosure is not limited thereto. Furthermore, unless otherwise specified, the reagents, solvents, and starting materials described herein are readily available from commercial suppliers.

[0358] Example The following table shows the abbreviations used throughout the following examples.

[0359] [table] TIFF0007771235000060.tif225157

[0360] (Example) Example 1: Synthesis of antibody-drug conjugates Example 2: The antibody-drug conjugates disclosed herein can be produced by any suitable method, including methods known in the art. See, for example, i) WO 2018 / 182341, which describes the production of pyrrolobenzodiazepine dimers and antibody-drug conjugates therefor; ii) WO 2015 / 182984, which describes the production of antibody-drug conjugates containing glucuronic acid moieties; and iii) WO 2018 / 083535, which describes the production of certain anti-CD19 antibodies. The entire contents of each of the foregoing publications are incorporated herein by reference.

[0361] Lymphocyte binding analysis of anti-CD19 antibodies The ability of various anti-CD19 antibodies of the present disclosure to bind to various human B lymphocyte cell lines was evaluated.

[0362] Specifically, the human IgG1 5F5, 7F11, 9G8, F6, 7F1, and 10D8 anti-CD19 antibodies were evaluated for their ability to bind to (i) six human B lymphocyte cell lines: Raji, Ramos, Nalm6, Su-DHL6, Su-DHL4, and Mec2; and (ii) CD19-silenced B cell lines: Raji siRNA and a negative cell line (Jurkat). All incubations were prepared in FACS buffer (PBS, 2% BSA) at 4°C. Fc receptors on B cells were blocked with 10% mouse serum. Four doses of hIgG1 were tested: 10, 1, 0.1, and 0.01 μg / ml. Cell surface-bound hIgG1 was detected with a mouse anti-human IgG Fc-PE mAb. The results of this study are shown in Figures 1A–1F.

[0363] As shown in Figures 1A-1F, all of the tested anti-CD19 antibodies bind to all of the six different B lymphocytes, although they show different profiles and / or different affinities.For example, 7F1 and 10D8 bind better to Nalm6 cells than to Raji cells, while other antibodies show opposite binding profiles.All of the tested antibodies are clearly specific to CD19, and all of the tested antibodies lose their ability to bind to CD19 in the CD19-silenced cell line Raji siRNA.None of these cell lines bind to the negative cell line.

[0364] Example 3: Cross-reactivity analysis of anti-CD19 antibodies The ability of anti-CD19 antibodies to bind to human and / or cynomolgus CD19 was assessed. Specifically, the human IgG1 5F5, 7F11, 9G8, F6, 7F1, and 10D8 anti-CD19 antibodies were evaluated for their ability to bind to a CHO cell line transfected with cynomolgus monkey CD19 and a negative control cell line (CHO). All incubations were prepared in FACS buffer (PBS, 2% BSA) at 4°C. Fc receptors on B cells were blocked with 10% mouse serum. Four doses of hIgG1 were tested: 10, 1, 0.1, and 0.01 μg / ml. The results of this study are shown in Figure 2.

[0365] The 9G8 anti-CD19 antibody is clearly cross-reactive with cynomolgus monkey CD19. The anti-CD19 antibodies 5F5, 7F11, and F6 are also slightly cross-reactive with cynomolgus monkey, as seen in the FACS overlay in Figure 2. The anti-CD19 antibodies 7F1 and 10D8 did not bind to transfected CHO cynomolgus monkey CD19 cells.

[0366] Example 4: Peripheral blood mononuclear cell (PBMC) binding analysis of anti-CD19 antibodies The ability of various anti-CD19 antibodies of the present disclosure to bind to various peripheral blood mononuclear cells (PBMCs) was examined. Human PBMCs and cynomolgus monkey PBMCs from frozen aliquots in citrate buffer were tested. Two doses of the following human IgG1 anti-CD19 antibodies were tested at 30 μg / mL and 3 μg / mL: 5F5, 7F11, 9G8, F6, 10D8, 7F1, and MDX as positive controls, and an anti-IP-10 antibody (designated NI-0801) as a negative control. PBMCs were labeled with anti-CD20-PE monoclonal antibody (mAb), anti-CD14-FITC mAb, and anti-CD3-PerCP mAb (which is cross-reactive with both human and cynomolgus monkey species). Cell surface-bound hIgG1 was detected with a mouse anti-human IgG Fc-APC mAb. FACS gating was performed using anti-CD20 mAb for the B lymphocyte population, anti-CD3 for the T lymphocyte population, and anti-CD14 for the monocyte population. The results of these studies are shown in Figures 3A-3F.

[0367] As shown in Figure 3C, none of the tested anti-CD19 antibodies bound to human PBMC CD3+ at the high dose of 30 μg / mL. All of the tested antibodies exhibited the same binding levels to monocytes as seen with the negative control NI-0801, which binds via cell surface Fc receptors. As shown in Figure 3A, all of the tested anti-CD19 antibodies bound to human PBMC CD20+, with small affinity differences observed at 3 μg / mL. Figure 3C demonstrates the cross-reactivity of all anti-CD19 antibodies. Binding was up to 10-fold higher in human PBMC than in cynomolgus PBMC, which is in the same range as seen with the positive control Mdx.

[0368] Example 5: Exemplary xenograft study using anti-CD19 antibody 9G8 Antitumor efficacy of anti-CD19 ADC (9G8) in the Raji non-Hodgkin's lymphoma xenograft model The anti-tumor activity of the anti-CD19 (9G8) antibody-drug conjugate was evaluated in immunodeficient CB17 SCID mice subcutaneously implanted with Raji Burkitt's lymphoma cells.

[0369] 5 × 10 into the right flank of 7-week-old CB17-SCID mice (Charles River) 6 Raji cells (obtained from ATCC) were subcutaneously (sc) implanted. Four days after implantation, tumor volume (TV = (0.5 × [length × width]) 2 The tumor volume (calculated using the formula: [D4]) was measured with a digital caliper and mice were assigned to different groups (see table below) to obtain a uniform mean tumor volume across cohorts (n = 7-8 mice per group. Mean tumor volume at D4: hIgG1 control 6 mg / kg: 180 mm 3 ± 63; anti-CD19 ADC 0.3mg / kg:187mm 3 ± 61; anti-CD19 ADC 1 mg / kg:200 mm 3 ± 49; Rituximab 6 mg / kg: 194 mm 3On the same day, mice were treated intravenously in the tail vein with a single dose of hIgG1 control (6 mg / kg), 0.3 or 1 mg / kg of anti-CD19 ADC, or rituximab (6 mg / kg). The endpoint of the experiment (tumor volume = 1500 mm 3 Mice were monitored twice weekly for body weight and three times weekly for tumor growth until tumor growth was achieved. The animal facilities and experiments were approved by the Geneva Cantonal Animal Research Committee. Experiments were performed in accordance with the guidelines of the Swiss Federal Veterinary Office. Mice treated with the 9G8 anti-CD19 ADC showed regression of tumor growth (Figures 4A-4H).

[0370] Antitumor efficacy of anti-CD19 ADC (9G8) in the Ramos non-Hodgkin's lymphoma xenograft model The antitumor activity of the anti-CD19 (9G8) antibody-drug conjugate was evaluated in immunodeficient CB-17 scid mice subcutaneously implanted with Ramos Burkitt's lymphoma cells.

[0371] 5 × 10 into the right flank of 7-week-old CB17-SCID mice (Charles River) 6 Ramos cells (obtained from ATCC) were implanted subcutaneously (sc). Tumor volume (TV) = (0.5 × [length × width]) 2 The tumor volume was measured three times a week using a digital caliper and was calculated using the formula: 3 Mice were recruited for treatment once the tumor volume reached 100 μg / kg. Mice were assigned to different groups (see table below) to obtain uniform mean tumor volumes across cohorts (n = 7-8 mice per group. Mean tumor volume: hIgG1 control 1 mg / kg, single injection: 243 mm 3 ± 26; anti-CD19 CaaX 1 mg / kg, 1 injection: 233 mm 3 ± 18; hIgG1 ADC 1 mg / kg, 1 injection: 224 mm 3 ± 20; anti-CD19 ADC 0.33 mg / kg, 1 injection: 242 mm 3± 28; anti-CD19 ADC 0.66 mg / kg, single injection: 234 mm 3 ± 23; anti-CD19 ADC 1 mg / kg, 1 injection: 234 mm 3 ± 31; anti-CD19 ADC 0.33 mg / kg once weekly for 3 weeks: 251 mm 3 ± 25; rituximab 6 mg / kg, 1 injection: 242 mm 3 ± 31). Single-dose or multiple-dose (cohort anti-CD19 ADC 0.33 mg / kg 1× / week for 3 weeks only) treatments were administered by intravenous injection into the tail vein. The experimental endpoint (tumor volume = 1500 mm 3 Mice were monitored three times a week for body weight and three times a week for tumor growth until 24 h of age. The animal facilities and experiments were approved by the Geneva Cantonal Animal Research Committee. Experiments were performed in accordance with the guidelines of the Swiss Federal Veterinary Office.

[0372] [table] TIFF0007771235000061.tif63156

[0373] The 9G8 anti-CD19 ADC group showed tumor regression by day 70, and all mice survived to day 70 (Figures 5A-5B). Groups receiving the hIgG1 isotype control, anti-CD19 9G8-Caax Ab, and non-tumor-specific ADC (anti-Her2) showed tumor growth, and all mice in these groups reached the tumor size endpoint. None survived to day 70. In the rituximab-treated group, 4 of 7 mice reached the endpoint, and 3 survived to day 70. No negative side effects associated with administration of the 9G8 anti-CD19 ADC were observed.

[0374] Example 6: Exemplary in vitro studies using anti-CD19 antibody 9G8 The in vitro antitumor activity of anti-CD19 (9G8) antibody-drug conjugates was evaluated using an antiproliferation assay. Six commercially available B-cell lymphoma cancer cell lines were used: DOHH-2 (DSMZ, #ACC 47), NALM-6 (DSMZ, #ACC 58), Ramos (ECACC, #85030802), SU-DHL-6 (ATCC, #CRL-2959), WSU-DLCL2 (DSMZ, #ACC 575), and WSU-NHL (DSMZ, #ACC 58). SG2057 was used as a drug-free toxin. Each of the cancer cell lines was seeded at 2,000–10,000 cells per well in a 96-well plate for 96-hour treatment groups, incubated for 2 hours, and then treated with the drug and ADC at concentrations ranging from 0.00025–100 nM (5-fold serial dilutions). After 96 hours, the number of viable cells was quantified using the CellTiter-Glo Luminescent Cell Viability Assay (Promega-G7573). The inhibition rates (IR) of drugs and ADCs were determined by the following formula: IR (%) = (1 - (RLU compound - RLU blank) / (RLU control - RLU blank)). * 100%. The inhibition of the samples at various doses was calculated in an Excel file, which was then used to plot the inhibition curves and evaluate relevant parameters such as lower (%), upper (%) and relative IC50. The data were interpreted by GraphPad Prism and are shown in Figure 6. The IC50 for each experiment and treatment 50 was calculated (see table below). The inhibition rate of the 9G8 anti-CD19 ADC was comparable to that of the dPBD-free toxin (SG2057).

[0375] [table] TIFF0007771235000062.tif32160

[0376] INCORPORATION BY REFERENCE All publications and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0377] equivalent While specific embodiments of the present disclosure have been discussed above, the foregoing description is illustrative and not limiting. Numerous variations of the present disclosure will become apparent to those skilled in the art upon review of the specification and claims below. The full scope of the present disclosure should be determined by reference to the claims, along with their full scope of equivalents, and by reference to the specification, along with such variations. The present disclosure includes the following embodiments. [1] Formula I: Ab-(X) y Formula I [In the formula, An Ab is an anti-CD19 antibody or antigen-binding fragment thereof, or a bispecific antibody, which comprises a first arm that binds to CD19, wherein the Ab comprises variable heavy chain complementarity determining region 1 (CDRH1), variable heavy chain complementarity determining region 2 (CDRH2), variable heavy chain complementarity determining region 3 (CDRH3), variable light chain complementarity determining region 1 (CDRL1), variable light chain complementarity determining region 2 (CDRL2), and variable light chain complementarity determining region 3 (CDRL3); wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 23 or 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 24 or 30; CDRH3 comprises the amino acid sequence of SEQ ID NO: 25, 26, 27, 28, or 31; CDRL1 comprises the amino acid sequence of SEQ ID NO: 32, 37, 41, or 44; CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, 38, 42, or 45; CDRL3 comprises the amino acid sequence of SEQ ID NO: 34, 35, 36, 40, 43, or 46; each X is independently a chemical moiety comprising one or more active agents and a linker, where the linker connects the Ab to the active agent; and y is an integer between 1 and 20. or a pharmaceutically acceptable salt or solvate thereof. [2] The antibody conjugate of embodiment 1, wherein the Ab further comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 2, 6, 12, 16 or 20 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 4, 8, 10, 14, 18 or 22. [3] (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 4; (b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8; (c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10; (d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 14; (e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18, and (f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 22 2. The antibody conjugate of embodiment 1, further comprising a combination of variable heavy and light chain sequences selected from: [4] The antibody conjugate of embodiment 1, wherein the anti-CD19 antibody is 5F5, 7F11, 9G8, F6, 7F1, or 10D8. [5] The antibody conjugate of any one of embodiments 1 to 4, wherein CD19 is human CD19. [6] The antibody conjugate of any one of embodiments 1 to 5, wherein the Ab is a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), 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. [7] The antibody conjugate of any one of embodiments 1 to 6, wherein the Ab is a rabbit, mouse, chimeric, humanized, or fully human monoclonal antibody. [8] The antibody conjugate of any one of embodiments 1 to 7, wherein the Ab is an IgG isotype. [9] The antibody conjugate of any one of embodiments 1 to 8, wherein the Ab is of the IgG1 isotype.

[10] The antibody conjugate of any one of embodiments 1 to 9, wherein the link between the Ab and the active substance is cleavable.

[11] The linker has Formula II: [C1] JPEG0007771235000063.jpg38165[In the formula, G is a glucuronic acid moiety or [Case 2] JPEG0007771235000064.jpg42165, where R 3 is hydrogen or a carboxyl protecting group, and each R 4 are independently hydrogen or a hydroxyl protecting group; B is the active substance; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl; or W is -C(O)-, -C(O)NR'-, -C(O)O-, SO2NR'-, -P(O)R''NR'-, -SONR'-, or -PO2NR'-, where C, S, or P is directly attached to the phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 is aryl; Each Z is independently C 1-8 is alkyl, halogen, cyano or nitro; n is an integer from 0 to 3; L is a linker connecting Ab and W. The antibody conjugate according to any one of embodiments 1 to 10, wherein the antibody conjugate is represented by:

[12] L is C1-50 The antibody conjugate of embodiment 11, wherein the alkylene is an alkylene or a heteroalkylene of 1 to 50 atoms.

[13] L, (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the atoms they bridge, form a heteroarylene; (iii) L is heteroalkylene of 1 to 50 atoms; or (iv) Alkylene is 1 or more C 1-20 substituted by alkyl; 13. The antibody conjugate of embodiment 11 or 12, which satisfies at least one of the following:

[14] A compound of formula III, wherein L is recognized by an isoprenoid transferase: [C3] 14. The antibody conjugate of any of embodiments 11 to 13, comprising at least one isoprenyl derivative unit represented by JPEG0007771235000065.jpg28165.

[15] The linker has Formula II: [C4] JPEG0007771235000066.jpg40165[In the formula, G is a glucuronic acid moiety or [5] JPEG0007771235000067.jpg43160, where R 3 is hydrogen or a carboxyl protecting group, and each R 4 are independently hydrogen or a hydroxyl protecting group; B is the active substance; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl; or W is -C(O)-, -C(O)NR'-, -C(O)O-, SO2NR'-, -P(O)R''NR'-, -SONR'-, or -PO2NR'-, where C, S, or P is directly attached to the phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 is aryl; Each Z is independently C 1-8 is alkyl, halogen, cyano or nitro; n is an integer from 0 to 3; where: A) L is C 1-50 alkylene or heteroalkylene of 1 to 50 atoms; (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the atoms they bridge, form a heteroarylene; (iii) L is heteroalkylene of 1 to 50 atoms; (iv) Alkylene is 1 or more C 1-20 substituted by alkyl; At least one of the following is satisfied: B) L is a compound of formula III that can be recognized by an isoprenoid transferase. [6] JPEG0007771235000068.jpg27161] 15. The antibody conjugate according to any one of embodiments 1 to 14, wherein the antibody conjugate is represented by:

[16] G [7] JPEG0007771235000069.jpg43161; R 3 is hydrogen or a carboxyl protecting group; and Each R 4 16. The antibody conjugate of any of embodiments 11-15, wherein are independently hydrogen or hydroxyl protecting groups.

[17] R 3 is hydrogen, and each R 4 The antibody conjugate of any of embodiments 11 to 16, wherein is hydrogen.

[18] Each R 1 and R 2 The antibody conjugate of any of embodiments 11 to 17, wherein is hydrogen.

[19] Each Z independently represents C 1-8 The antibody conjugate of any of embodiments 11 to 18, wherein the aryl group is alkyl, halogen, cyano, or nitro.

[20] The antibody conjugate of any one of embodiments 11 to 19, wherein n is 0.

[21] W is -C(O)-, -C(O)NR'-, -C(O)O-, SO2NR'-, -P(O)R''NR'-, -SONR'-, or -PO2NR'-, where C, S, or P is directly attached to the phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 The antibody conjugate of any one of embodiments 11 to 20, wherein the antibody conjugate is aryl.

[22] The antibody conjugate of any one of embodiments 11 to 21, wherein W is —C(O)—, —C(O)NR′—, or —C(O)O—.

[23] The antibody conjugate of embodiment 22, wherein W is -C(O)NR'-, where C(O) is attached to the phenyl ring and NR' is attached to L.

[24] G. [8] JPEG0007771235000070.jpg43161; W is —C(O)NR′—, where C(O) is attached to the phenyl ring and NR′ is attached to L; and R 1 and R 2 The antibody conjugate of any of embodiments 11 to 23, wherein each represents hydrogen.

[25] L is C 1-50 alkylene or heteroalkylene of 1 to 50 atoms; (i) L contains at least one unsaturated bond; (ii) two atoms in L are substituted with divalent substituents which, together with the atoms they bridge, form a heteroarylene; (iii) L is heteroalkylene of 1 to 50 atoms; and (iv) Alkylene is 1 or more C 1-20 substituted by alkyl; The antibody conjugate of any one of embodiments 11 to 24, which satisfies at least one of the above.

[26] The antibody conjugate of any of embodiments 11-25, wherein L is a nitrogen-containing heteroalkylene of 1 to 50 atoms, and the linker comprises at least two atoms of a hydrophilic amino acid, the nitrogen forming a peptide bond with a carbonyl of the hydrophilic amino acid.

[27] The antibody conjugate of any one of embodiments 11 to 26, wherein W is —C(O)NR′— and the nitrogen of W is a nitrogen atom of a hydrophilic amino acid.

[28] The antibody conjugate of embodiment 26 or 27, wherein the amino acid covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker.

[29] The antibody conjugate of any one of embodiments 26 to 28, wherein the hydrophilic amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

[30] The antibody conjugate of any one of embodiments 26 to 29, wherein the hydrophilic amino acid is an amino acid containing a side chain having a moiety that is charged at neutral pH in aqueous solution.

[31] The antibody conjugate of embodiment 30, wherein the hydrophilic amino acid is aspartate or glutamate.

[32] The antibody conjugate of embodiment 30, wherein the hydrophilic amino acid is ornithine or lysine.

[33] The antibody conjugate of embodiment 30, wherein the hydrophilic amino acid is arginine.

[34] The antibody conjugate of any one of embodiments 1 to 33, wherein the linker comprises a peptide, and the peptide comprises at least one hydrophilic amino acid, preferably an amino acid having a side chain with a moiety (e.g., an amine, guanidine, or carboxyl moiety) that is charged at neutral pH in aqueous solution.

[35] The antibody conjugate of embodiment 34, wherein each amino acid of the peptide is independently selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, and threonine.

[36] The antibody conjugate of embodiment 34 or 35, wherein the peptide comprises at least one aspartate or glutamate.

[37] The antibody conjugate of any of embodiments 34 to 36, wherein W represents -C(O)NR'- and the nitrogen of W is the nitrogen of the N-terminal amino acid in the peptide.

[38] An antibody conjugate according to any one of embodiments 34 to 37, wherein the peptide covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker.

[39] An antibody conjugate according to any one of embodiments 34 to 38, wherein the peptide comprises 2 to 20 amino acids.

[40] The antibody conjugate of any one of embodiments 1 to 39, wherein the linker is covalently attached to the Ab via a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine ​​of the Ab.

[41] The antibody conjugate of embodiment 40, wherein the Ab comprises an amino acid motif recognized by an isoprenoid transferase, preferably at the C-terminus of the Ab, and the thioether bond comprises a sulfur atom of a cysteine ​​in the amino acid motif.

[42] The amino acid motif is the sequence CYYX; C represents cysteine; Y represents, independently for each occurrence, an aliphatic amino acid; X represents, independently for each occurrence, glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine; and The antibody conjugate of embodiment 41, wherein the thioether bond comprises the sulfur atom of a cysteine ​​of said amino acid motif.

[43] the amino acid motif is the sequence CYYX; and 43. The antibody conjugate of embodiment 41 or 42, wherein Y represents, independently for each occurrence, alanine, isoleucine, leucine, methionine, or valine.

[44] An antibody conjugate according to any one of embodiments 41 to 43, wherein the amino acid motif is the sequence CVIM or CVLL.

[45] An antibody conjugate according to any one of embodiments 41 to 44, wherein at least one of the seven amino acids preceding the amino acid motif is glycine.

[46] An antibody conjugate according to any one of embodiments 41 to 45, wherein at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine, aspartic acid, arginine, and serine.

[47] An antibody conjugate according to any one of embodiments 41 to 46, wherein at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine and proline.

[48] ​​An antibody conjugate according to any one of embodiments 41 to 47, wherein each of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif is glycine.

[49] The antibody conjugate of any of embodiments 41 to 48, wherein L comprises the amino acid sequence GGGGGGCVIM, preferably at the C-terminus.

[50] Formula III, in which L is recognized by isoprenoid transferase. [9] 50. The antibody conjugate of any of embodiments 11 to 49, comprising at least one isoprenyl derivative unit represented by JPEG0007771235000071.jpg29161.

[51] L is a heteroalkylene having 3 to 50 carbon atoms, including oxime, wherein: the oxygen atom of the oxime is on the side of L that is attached to W and the carbon atom of the oxime is on the side of L that is attached to Ab, or The antibody conjugate of any of embodiments 11-50, wherein the carbon atom of the oxime is on the side of L that is linked to W and the oxygen atom of the oxime is on the side of L that is linked to Ab.

[52] The antibody conjugate of embodiment 50 or 51, wherein L comprises an oxime and said at least one isoprenyl unit covalently links the oxime to Ab.

[53] L [C10] The antibody conjugate of any of embodiments 11 to 52, comprising JPEG0007771235000072.jpg25160.

[54] L [C11] The antibody conjugate of any of embodiments 11 to 53, comprising JPEG0007771235000073.jpg25160.

[55] L [C12] The antibody conjugate of any of embodiments 11 to 54, comprising JPEG0007771235000074.jpg25160.

[56] L is a group of formula VIII: -(CH2) r (V(CH2) p ) q - or Formula IX: -(CH2CH2X) w - [In the formula, V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO2- or -SO2NR25 -is; X is -O-, C 1-8 Alkylene or -NR 21 -is; R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl C 3-20 is heteroaryl; r is an integer between 1 and 10; p is an integer between 0 and 12; q is an integer from 1 to 20; and and w is an integer between 1 and 20. 56. The antibody conjugate of any of embodiments 11 to 55, further comprising a linking unit represented by:

[57] The antibody conjugate of embodiment 56, wherein q is an integer of 4 to 20.

[58] The antibody conjugate of embodiment 56, wherein q is an integer of 2 to 12.

[59] The antibody conjugate of any of embodiments 56 to 58, wherein q is an integer between 6 and 20.

[60] The antibody conjugate of any of embodiments 56 to 59, wherein q is 2, 5, or 11.

[61] The antibody conjugate of any of embodiments 56 to 60, wherein r is 2.

[62] The antibody conjugate of any of embodiments 56 to 61, wherein p is 2.

[63] The antibody conjugate of any of embodiments 56 to 62, wherein V is -O-.

[64] r is 2; p is 2; q is 2, 5, or 11; and The antibody conjugate of any of embodiments 56 to 63, wherein V is -O-.

[65] The antibody conjugate of any one of embodiments 56 to 64, wherein X is -O-.

[66] The antibody conjugate of any of embodiments 56 to 65, wherein w is an integer of 6 to 20.

[67] The antibody conjugate of any of embodiments 56-66, wherein X is -O- and w is an integer between 6 and 20.

[68] L, [C13] 68. The antibody conjugate of any of embodiments 11 to 67, comprising at least one polyethylene glycol unit represented by JPEG0007771235000075.jpg14161.

[69] The antibody conjugate of any of embodiments 11 to 68, wherein L comprises 1 to 12 -OCH2CH2- units.

[70] The antibody conjugate of any one of embodiments 11 to 69, wherein L further comprises 3 to 12 -OCH2CH2- units.

[71] The antibody conjugate of any of embodiments 11 to 70, wherein L further comprises 5 to 12 -OCH2CH2- units.

[72] The antibody conjugate of any of embodiments 11 to 71, wherein L further comprises 6 to 12 -OCH2CH2- units.

[73] The antibody conjugate of any of embodiments 11 to 70, wherein L further comprises three -OCH2CH2- units.

[74] The antibody conjugate of any of embodiments 11-73, wherein L comprises an oxime and at least one polyethylene glycol unit covalently attaches the oxime to the active agent.

[75] The antibody conjugate of any of embodiments 11-74, wherein L comprises a linking unit formed by a 1,3-dipolar cycloaddition reaction, a hetero-Diethel-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, an addition to a carbon-carbon multiple bond, an oxidation reaction, or a click reaction.

[76] The antibody conjugate of embodiment 75, wherein the linking unit is formed by the reaction of an acetylene with an azide, or by the reaction of an aldehyde or ketone group with a hydrazine or an alkoxyamine.

[77] L is a compound of general formula IV, V, VI or VII: [C14] JPEG0007771235000076.jpg28161 (in the formula, L 1 is a single bond, or C 1-30 is an alkylene of the formula: R 11 is hydrogen or C 1-10 (is an alkyl of 77. The antibody conjugate of any of embodiments 11 to 76, further comprising a binding unit represented by:

[78] L 1 The antibody conjugate of embodiment 77, wherein is a single bond.

[79] L 1 C 11 The antibody conjugate of embodiment 77, which is alkylene.

[80] L 1 C 12 The antibody conjugate of embodiment 77, which is alkylene.

[81] L, [C15] JPEG0007771235000077.jpg19161[In the formula, V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 -, preferably -O-; R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl, or C 1-6 Alkyl C 3-20 is heteroaryl; r is an integer between 1 and 10; p is an integer between 0 and 10; q is an integer from 1 to 20; and L1 is a single bond. 78. The antibody conjugate of any one of embodiments 11 to 77, comprising:

[82] The antibody conjugate of embodiment 81, wherein r is 2 or 3.

[83] The antibody conjugate of embodiment 81 or 82, wherein p is 1 or 2.

[84] The antibody conjugate of any of embodiments 81 to 83, wherein q is 1 to 6.

[85] The antibody conjugate of any of embodiments 81-84, wherein r is 2 or 3; p is 1 or 2; and q is 1 to 6.

[86] Structure: [C16] JPEG0007771235000078.jpg52161 (wherein Ab represents an anti-CD19 antibody, B represents an active agent, and n is an integer from 1 to 20).

[87] Structure: [C17] JPEG0007771235000079.jpg63161 (wherein Ab represents an anti-CD19 antibody, B represents an active agent, and n is an integer from 1 to 20).

[88] Structure: [C18] JPEG0007771235000080.jpg38161 (wherein Ab represents an anti-CD19 antibody, B represents an active agent, and n is an integer from 1 to 20).

[89] Structure: [C19] JPEG0007771235000081.jpg52161 (wherein Ab represents an anti-CD19 antibody, B represents an active agent, and n is an integer from 1 to 20).

[90] The antibody conjugate of any of embodiments 11 to 89, wherein the isoprenoid transferase is farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase).

[91] L comprises one or more branched linkers covalently attached to Ab, wherein: i) each branched linker comprises a branching unit (BR) covalently linked to an Ab by a first linker (PL); ii) each branched linker comprises a first branch (B1), which covalently attaches a first active agent to the branching unit, and comprises a second linker (SL) and a cleavage group (CG); iii) each branched linker further comprises a second branch (B2), wherein a) a second active agent is covalently attached to the branched unit by a second linker (SL) and a cleavage group (CG), or b) a polyethylene glycol moiety is covalently attached to the branched unit; 86. The antibody conjugate of any of embodiments 11 to 85, wherein each cleavage group can be hydrolyzed to release the active agent from the antibody conjugate.

[92] At least one branching unit has the structure

[20] JPEG0007771235000082.jpg38161[In the formula, L 2 , L 3 and L 4 are each independently a direct bond or -C n H 2n - is; where n is an integer from 1 to 30; G 1 , G 2 and G 3 are each independently a direct bond,

[21] JPEG0007771235000083.jpg25161; R 30 is hydrogen or C 1-30 is alkyl; and where R 405 -COOR 50 , where L5 is a direct bond or C 1-10 alkylene; and R 50 is hydrogen or C 1-30 alkyl] 92. The antibody conjugate of embodiment 91, having:

[93] Structure:

[22] JPEG0007771235000084.jpg77161 (in the formula, B and B' are active substances and may be the same or different; n, independently for each occurrence, represents an integer from 0 to 30; f, independently for each occurrence, represents an integer from 0 to 30; and L represents linkage to Ab) 92. The antibody conjugate of embodiment 91, comprising:

[94] The antibody conjugate of embodiment 93, wherein n is an integer of 1 to 10.

[95] The antibody conjugate of embodiment 93, wherein n is an integer of 4 to 20.

[96] The antibody conjugate of any of embodiments 11-95, wherein L comprises an oxime and at least one polyethylene glycol unit covalently attaches the oxime to the active agent.

[97] The antibody conjugate of any one of embodiments 1 to 96, wherein the cleavable group is cleavable within the target cell.

[98] The antibody conjugate of any one of embodiments 1 to 97, wherein the cleavable group is capable of releasing one or more active agents.

[99] The antibody conjugate of any of embodiments 11-98, wherein the antibody conjugate comprises an Ab, at least one branched linker covalently attached to the Ab, and at least two active agents covalently attached to the branched linker.

[0100] An antibody conjugate described in any of embodiments 91 to 99, wherein at least two branched linkers are attached to the Ab, and each branched linker is attached to at least two active substances.

[0101] An antibody conjugate according to embodiment 100, wherein three branched linkers are attached to the Ab.

[0102] An antibody conjugate according to embodiment 100, wherein four branched linkers are attached to the Ab.

[0103] An antibody conjugate according to embodiment 91 or 92, wherein exactly one branched linker is attached to the Ab.

[0104] An antibody conjugate described in any of embodiments 91 to 103, wherein each branched linker is bound to exactly two active substances.

[0105] An antibody conjugate described in any of embodiments 11 to 104, wherein the conjugate comprises at least two different active substances.

[0106] An antibody conjugate described in any of embodiments 91 to 105, wherein at least one branched linker is attached to two different active substances.

[0107] An antibody conjugate according to any of embodiments 91 to 106, wherein each active agent is attached to the branched linker by a cleavable (eg, hydrolyzable) bond.

[0108] An antibody conjugate described in any of embodiments 91 to 107, wherein each branched linker comprises a branched unit, each active substance is attached to the branched unit via a second linker, and the branched unit is attached to the anti-CD19 antibody by a first linker.

[0109] An antibody conjugate according to embodiment 108, wherein the branching unit is a nitrogen atom, for example of an amine or amide.

[0110] An antibody conjugate according to embodiment 108 or 109, wherein the branching unit is an amide and the first linker comprises a carbonyl of the amide.

[0111] An antibody conjugate according to embodiment 108 or 109, wherein the branching unit is an amide and the second linker comprises a carbonyl of the amide.

[0112] An antibody conjugate according to any one of embodiments 108 to 111, wherein the branching unit is a lysine unit.

[0113] An antibody conjugate according to any one of embodiments 11 to 112, wherein the active substances are independently selected from chemotherapeutic agents and toxins.

[0114] An antibody conjugate described in embodiment 113, wherein the active substance is a chemotherapeutic agent.

[0115] An antibody conjugate according to embodiment 113 or 114, wherein the active substance is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent or a combination thereof.

[0116] The activator is: (a) Erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, Tothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, melphalan, nobembitine, fenesterine, prednimustine, trofosfamide, u Rasil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antormicin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorub cin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex,Zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, tiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, Bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, velaculin A, Roridin A and Anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-modified nanoparticle formulation of paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vindesine ... blastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid of any of the foregoing, (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic factor, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16), interleukin-17 (IL-14), interleukin-18 (IL-14), interleukin-19 (IL-15), interleukin-20 (IL-15), interleukin-21 (IL-15), interleukin-22 (IL-15), interleukin-33 (IL-15), interleukin-34 (IL-15), interleukin-16). interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factors ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing; (c) diphtheria toxin, botulium toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, azonafide, aplidine, toxoids, or any combination of the foregoing; (d) an affinity ligand, which is a substrate, an inhibitor, a stimulator, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) radioactive label; 32 P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, an incomplete antigen, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; (f) immunomodulatory compounds, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents, and anti-parasitic agents, or a combination of any of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxacitabine, (j) aromatase inhibitors, (k) protein kinase inhibitors, (l) lipid kinase inhibitors, (m) antisense oligonucleotides; (n) ribozyme, (o) vaccines; and (p) anti-angiogenic agent 116. The antibody conjugate of any one of embodiments 11 to 115, independently selected from:

[0117] Ab is an anti-CD19 antibody; The active substance is a pyrrolobenzodiazepine dimer; 117. The antibody conjugate of any of embodiments 1-116, wherein the linker connects the Ab to the N10 or N'10 position of the pyrrolobenzodiazepine dimer; and y is an integer from 1 to 20.

[0118] The active substance is a pyrrolobenzodiazepine dimer; the pyrrolobenzodiazepine dimer is substituted at the N10 position by X or at the N'10 position by X', where X or X' connects the pyrrolobenzodiazepine dimer to a linker; X and X' are each independently -C(O)O * , -S(O)O- *, -C(O)- * , -C(O)NR X - * , -S(O)NR X - * , -(P(O)R')NR X - * , -S(O)NR X - * or -PO2NR X - * is; R X H, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 3-20 Heteroaryl or C 5-20 is aryl; R X 'OH, N3, CN, SH, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 is aryl or amino; * 117. The antibody conjugate of any of embodiments 1-116, wherein is the attachment site between the pyrrolobenzodiazepine dimer and the linker.

[0119] X and X' are each independently -C(O)O- * , -C(O)- * or -C(O)NR X - * 119. The antibody conjugate of embodiment 118, wherein: The pyrrolobenzodiazepine dimer may be represented by formula X or formula XI:

[23] JPEG0007771235000085.jpg97146[In the formula, The dotted lines indicate possible double bonds between C1 and C2 or between C2 and C3 and between C'1 and C'2 or between C'2 and C'3; R X1 and R X1’ are independently H, OH, =O, =CH2, CN, R m , OR m, =CH-R m’ , =C(R m ')2, O-SO2-R m , CO2R m , C.O.R. m , selected from halo and dihalo; R m’ are independently m , CO2R m , C.O.R. m , selected from CHO, COH and halo; Each R m independently, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R X2 , R X2’ , R X3 , R X3’ , R X5 and R X5’ are independently H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3Sn and halo; R X4 and R X4’ are independently H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3Sn, Halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n, -OC(O)NR n R n ',-OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n ', -S(O)2NR n R n ',-OS(O)NR n R n ',-OS(O)2NR n R n ', -NR n R n ', -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o ', -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o ', -NR n S(O)NR o R o ', -C(O)R n , -C(O)OR n and -C(O)NR n R n ' selected from; R X and R X ' is independently H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C 1-8 Alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, C 3-20 Heteroaryl, C 5-20 Aryl or mono- or di-C 1-8 selected from alkylamino; Y and Y' are independently selected from O, S and N(H); R x6 is C 3-12 Alkylene, C 3-12 Alkenylene or C 3-12 selected from heteroalkylene; R X7 and R X7’ are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r ',-OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r ', -S(O)2NR r R r ',-OS(O)NR r R r ',-OS(O)2NR r R r ', -NR r R r ', -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s ', -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s ', -NR r S(O)NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r Rr ' selected from; Each R r 、 R r’ , R s and R s’ are independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 selected from aryl and 5- to 7-membered heteroaryl; Each R X8 and R X8’ are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Heteroalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -S(O)R m , -S(O)2R m , -S(O)NR m R m ', -S(O)2NR m R m ', -NR m R m ', -NR m C(O)R m , -NR m C(O)OR n , -NR m C(O)NR n R n ', -NR m S(O)R n , -NR m S(O)2R n , -NR m S(O)NR n R n ', -NR m S(O)NR n R n ', -C(O)R m , -C(O)OR m and -C(O)NR m R m ' selected from; Z a is ORX12a , N.R. X12a R X12a or SR X12a Selected from; Z b is OR X13a , N.R. X13a R X13a or SR X13a Selected from; Z a’ is OR X12a , N.R. X12a R X12a or SR X12a Selected from; Z b’ is OR X13a’ , N.R. X13a’ R X13a’ or SR X13a’ Selected from; R X12a , R X12a’ , R X13a’ and R x13a’ Each of the is independently non-existent, H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -C(O)R X15a , -C(O)OR X15a and -C(O)NR X15a R X15a’ selected from; and Each R X15a and R x15a’ independently, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; where R X13a and R X14aoptionally, together with the atom to which they are attached, form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; and R X13a’ and R X14a’ optionally, together with the atom to which they are attached, form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; and Here, each R n , R n’ , R o , R o’ , R p and R p’ are independently H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 aryl and 5- to 7-membered heteroaryl] 120. The antibody conjugate of embodiment 118 or 119, represented by:

[0121] Each R m But independently, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; where R m optionally, one or more C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 5-20 Aryl, C 5-20 Heteroaryl, C 3-6 The antibody conjugate of embodiment 120, which is optionally substituted with cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl.

[0122] R X4 and R X4’However, independently, H, R m , OH, OR m , S.H., S.R. m , NH2, NHR m , N.R. m R m ', NO2, Me3Sn, Halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 Aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -OR n , -OC(O)R n , -OC(O)NR n R n ',-OS(O)R n , -OS(O)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n ', -S(O)2NR n R n ',-OS(O)NR n R n ',-OS(O)2NR n R n ', -NR n R n ', -NR n C(O)R o , -NR n C(O)OR o , -NR n C(O)NR o R o ', -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o ', -NR n S(O)NR o R o ', -C(O)R n , -C(O)OR n and -C(O)NR n Rn ' is selected from where R X4 or R X4’ C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-12 When it is aryl, 5- to 7-membered heteroaryl, it may optionally contain one or more C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 Aryl, 5- to 7-membered heteroaryl, -OR p , -OC(O)R p , -C(O)NR p R p ',-OS(O)R p , -OS(O)2R p , -SR p ,-S(O)R p , -S(O)2R p , -S(O)NR p R p ', -S(O)2NR p R p ',-OS(O)NR p R p ',-OS(O)2NR p R p ', -NR p R p ', -NR p C(O)R q , -NR p C(O)OR q , -NR p C(O)NR q R q ', -NR p S(O)R q , -NR p S(O)2R q , -NR p S(O)NR q R q ', -NR pS(O)NR q R q ', -C(O)R p , -C(O)OR p or -C(O)NR p R p 122. The antibody conjugate of embodiment 120 or 121, optionally substituted by:

[0123] R X7 and R X7’ However, independently, H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r ',-OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r ', -S(O)2NR r R r ',-OS(O)NR r R r ',-OS(O)2NR r R r ', -NR r R r ', -NR r C(O)R s , -NR r C(O)OR s , -NR r C(O)NR s R s ', -NR r S(O)R s , -NR r S(O)2R s , -NR r S(O)NR s R s ', -NR r S(O)NR s R s , -C(O)Rr , -C(O)OR s or -C(O)NR r R r ' is selected from where R X7 and R X7’ C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 When it is aryl, 5- to 7-membered heteroaryl, it may optionally contain one or more C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6-10 Aryl, 5- to 7-membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t ',-OS(O)R t , -OS(O)2R t , -SR t , -S(O)R t , -S(O)2R t , -S(O)NR t R t ', -S(O)2NR t R t ',-OS(O)NR t R t ',-OS(O)2NR t R t ', -NR t R t ', -NR t C(O)R u , -NR t C(O)OR u ,-NR t C(O)NR u R u ', -NR t S(O)R u , -NR t S(O)2R u , -NR t S(O)NR u R u', -NR t S(O)NR u R u ', -C(O)R t , -C(O)OR t or -C(O)NR t R t may be replaced by '; where R r , R r’ , R s , R s’ , R t , R t’ , R u and R u’ Each of these independently represents H, C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkynyl, C 3-13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5-10 The antibody conjugate of any of embodiments 120-122, wherein the aryl is selected from aryl and 5- to 7-membered heteroaryl.

[0124] R X1 and R X1’ But independently, R m Selected from R m C 1-6 Alkyl, C 2-6 Alkenyl, C 5-7 Aryl and C 3-6...

Claims

1. Formula I: Ab-(X) y Formula I [In the formula, An Ab is an anti-CD19 antibody or antigen-binding fragment thereof, or a bispecific antibody, which comprises a first arm that binds to CD19, wherein the Ab comprises variable heavy chain complementarity determining region 1 (CDRH1), variable heavy chain complementarity determining region 2 (CDRH2), variable heavy chain complementarity determining region 3 (CDRH3), variable light chain complementarity determining region 1 (CDRL1), variable light chain complementarity determining region 2 (CDRL2), and variable light chain complementarity determining region 3 (CDRL3); (a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 23; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 24; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 25; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34; (b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 23; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 24; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 26; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 35; (c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 23; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 24; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 26; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 36; (d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 23; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 24; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 27; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 37; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 38, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 40; (e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 23; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 24; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 28; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 41; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 42, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 43; Or, (f) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 44; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 45, and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 46; each X is independently a chemical moiety comprising one or more active agents and a linker, wherein the linker connects the Ab to the one or more active agents, and the linker is cleavable by enzyme-mediated hydrolysis; and and y is an integer from 1 to 20. or a pharmaceutically acceptable salt thereof, the Ab comprises an amino acid motif and a spacer at its C-terminus; the amino acid motif comprises a sequence selected from CXX, CXC, XCXC, XXCC, and CYYX, where C represents cysteine, each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine, and X is independently glutamine, glutamic acid, serine, cysteine, methionine, alanine, or leucine; the spacer comprises 1 to 20 amino acids, and at least one of the 1 to 20 amino acids is glycine; The antibody conjugate or a pharmaceutically acceptable salt thereof.

2. 2. The antibody conjugate of claim 1, wherein the Ab comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 2, 6, 12, 16 or 20 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 4, 8, 10, 14, 18 or 22.

3. (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 4; (b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8; (c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10; (d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 14; (e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18; and (f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 22 2. The antibody conjugate of claim 1, comprising a combination of variable heavy and light chain sequences selected from:

4. Ab can be a monoclonal antibody, single-chain antibody (scAb), Fab fragment, or F(ab') 2 The antibody conjugate of any one of claims 1 to 3, which is a fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a variant antibody, a multimeric antibody or a bispecific antibody.

5. The antibody conjugate has Formula II: 【Chemistry 1】 Formula II [In the formula, G is 【Chemistry 2】 where R 3 is hydrogen or a carboxyl protecting group, and each R 4 are independently hydrogen or a hydroxyl protecting group; B is an active substance; R 1 and R 2 are each independently hydrogen, C 1-8 Alkyl or C 3-8 is cycloalkyl; W is -C(O)-, -C(O)NR'-, -C(O)O-, SO 2 NR'-, -P(O)R''NR'-, -SONR'- or -PO 2 NR'-, where C, S, or P is directly attached to the phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylthio, mono- or di-C 1-8 Alkylamino, C 3-20 Heteroaryl or C 6-20 is aryl; Each Z is independently C 1-8 alkyl, halogen, cyano or nitro; n is an integer from 0 to 3; L is a linker connecting Ab and W. The antibody conjugate of any one of claims 1 to 4, comprising a structure represented by:

6. L, (i) at least one unsaturated bond; (ii) heteroarylene; (iii) heteroalkylene of 1 to 50 atoms; or (iv) C equal to or greater than 1 1-20 alkylene substituted by alkyl; The antibody conjugate of claim 5, comprising at least one of:

7. Formula III, wherein L is recognized by an isoprenoid transferase: 【Transformation 3】 7. The antibody conjugate of claim 5, comprising at least one isoprenyl derivative unit represented by:

8. R 3 is hydrogen, and each R 4 The antibody conjugate of any one of claims 5 to 7, wherein is hydrogen.

9. Each Z is independently C 1-8 The antibody conjugate of any one of claims 5 to 8, wherein the aryl group is alkyl, halogen, cyano, or nitro.

10. The antibody conjugate of any one of claims 5 to 9, wherein n is 0.

11. The antibody conjugate of any one of claims 5 to 10, wherein W is -C(O)NR'-.

12. 12. The antibody conjugate of any one of claims 5 to 11, wherein L comprises a peptide comprising at least one hydrophilic amino acid, preferably an amino acid having a side chain with a moiety that carries a charge in aqueous solution at neutral pH.

13. The antibody conjugate of claim 12, wherein the peptide comprises 2 to 20 amino acids.

14. The antibody conjugate of any one of claims 1 to 13, wherein the linker is covalently bound to the Ab by a thioether bond, the thioether bond comprising a sulfur atom of a cysteine ​​of the Ab.

15. The antibody conjugate described in claim 14, wherein the thioether bond comprises the sulfur atom of a cysteine ​​of the amino acid motif.

16. The antibody conjugate of any one of claims 1 to 15, wherein the amino acid motif is the sequence CVIM or CVLL.

17. An antibody conjugate described in any one of claims 1 to 16, wherein the spacer comprises 7 to 20 amino acids.

18. An antibody conjugate described in any one of claims 1 to 17, wherein each amino acid of the spacer is glycine.

19. L is 3 to 50 heteroalkylene, including oxime, where: the oxygen atom of the oxime is on the side of L that is attached to W and the carbon atom of the oxime is on the side of L that is attached to Ab, or 19. The antibody conjugate of any one of claims 5 to 18, wherein the carbon atom of the oxime is on the side of L that is linked to W and the oxygen atom of the oxime is on the side of L that is linked to Ab.

20. L is a group of formula VIII: -(CH 2 ) r (V(CH 2 ) p ) q - or Formula IX: -(CH 2 CH 2 X) w - [In the formula, V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO 2 -or- SO 2 NR 25 -is; X is -O-, C 1-8 Alkylene or -NR 21 -is; R 21 ~R 25 are each independently hydrogen, C 1-6 Alkyl, C 1-6 Alkyl C 6-20 Aryl or C 1-6 Alkyl C 3-20 is heteroaryl; r is an integer from 1 to 10; p is an integer from 0 to 12; q is an integer from 1 to 20; and and w is an integer from 1 to 20.

20. The antibody conjugate of any one of claims 5 to 19, comprising a linking unit represented by:

21. L is a member of general formula IV, V, VI or VII: 【Chemistry 4】 (In the formula, L 1 is a single bond, or C 1-30 is an alkylene of the formula: R 11 is hydrogen or C 1-10 is an alkyl of 21. The antibody conjugate of claim 5, comprising a binding unit represented by:

22. structure: 【Transformation 5】 6. The antibody conjugate of claim 5, comprising: (wherein B represents an active agent and n is an integer from 1 to 20).

23. L is branched, and L is i) a branching unit (BR) covalently linked to the Ab by a first linker (PL); ii) a first branch (B1), covalently linking a first active substance to the branching unit and comprising a second linker (SL) and a cleavage group (CG); and iii) a second branch (B2), wherein a) a second active agent is covalently attached to said branching unit by a second linker (SL) and a cleavage group (CG), or b) a polyethylene glycol moiety is covalently attached to said branching unit; Including, 22. The antibody conjugate of any one of claims 5 to 21, wherein each cleavage group can be hydrolyzed to release the active agent from the antibody conjugate.

24. At least one branching unit has the structure 【Transformation 6】 [In the formula, L 2 , L 3 and L 4 are each independently a direct bond or -C n H 2n - is; where n is an integer from 1 to 30; G 1 , G 2 and G 3 are each independently a bond, 【Transformation 7】 is; R 30 is hydrogen or C 1-30 is alkyl; and R 40 is L 5 -COOR 50 , where L 5 is a bond or C 1-10 alkylene, and R 50 is hydrogen or C 1-30 alkyl.

24. The antibody conjugate of claim 23, having:

25. structure: 【Transformation 8】 (In the formula, B and B' are active agents, which may be the same or different; n, independently for each occurrence, represents an integer from 0 to 30; f, independently for each occurrence, represents an integer from 0 to 30; and L' represents the linkage to Ab) 24. The antibody conjugate of claim 23, comprising:

26. 26. The antibody conjugate of claim 25, wherein n is an integer from 1 to 10.

27. The antibody conjugate of any one of claims 23 to 26, wherein one, two, three, or four branched linkers are attached to the Ab.

28. 28. The antibody conjugate of any one of claims 23 to 27, wherein each branched linker is bound to two active substances.

29. The antibody conjugate of any one of claims 5 to 28, wherein the active substance is a chemotherapeutic agent or a toxin.

30. 30. The antibody conjugate of claim 29, wherein the active agent is a chemotherapeutic agent.

31. 29. The antibody conjugate of any one of claims 5 to 28, wherein the active agent is an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof.

32. The activator is: (a) Erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, topotecan, bryostatin, kallistatin, adzelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, duocarmycin, KW-2189, CB1 -TM1, eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomycin, actinomycin, antimycin, azaserine, bleomycin, carubicin, Carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin , olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, Cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, demecolcine, diaziconazole, etanercept Irfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidamol, pentostatin, phenamt, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziconazole, 2,2',2''-trichlorotriethylamine, T-2 toxin, veraculin A, roridin A and anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside, paclitaxel, albumin-modified nanoparticle formulations of paclitaxel, doxetaxel, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, vincristine, vinorelbine, teniposide, edatrexate, aminopterin, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate, or acid of any of the foregoing. (b) Monokines, lymphokines, conventional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic factor, interferon, interferon-like growth factor interferon-α, interferon-β, interferon-γ, colony stimulating factors ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukins ("IL"), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing; (c) diphtheria toxin, botulium toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, SG2285, dolastatin, camptothecin, rhizoxin, CC-1065, enediyne antibiotic, epothilone, azonafide, aplidine, toxoid, or any combination of the foregoing; (d) an affinity ligand, which is a substrate, an inhibitor, a stimulator, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) radioactive label; 32 P, 35 S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, an incomplete antigen, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; (f) immunomodulatory compounds, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents, and anti-parasitic agents, or a combination of any of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, megestrol acetate, exemestane, or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine, (j) aromatase inhibitors, (k) protein kinase inhibitors, (l) lipid kinase inhibitors, (m) antisense oligonucleotides; (n) ribozyme, (o) vaccines; and (p) anti-angiogenic agent The antibody conjugate of any one of claims 5 to 31, independently selected from:

33. The active substance is a pyrrolobenzodiazepine dimer; and The linker connects the Ab to the N10 or N'10 position of the pyrrolobenzodiazepine dimer; The antibody conjugate according to any one of claims 5 to 32.

34. X is 【Chemistry 9】 【change】 【change】 【change】 The antibody conjugate of any one of claims 1 to 4, comprising: 【Request Item 35】 【Chemistry 10】 where: 【Chemistry 11】 2. The antibody conjugate of claim 1, wherein: is an Ab, and the Ab comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

10.

36. A pharmaceutical composition comprising the antibody conjugate of any one of claims 1 to 35 and an excipient.

37. A composition comprising the antibody conjugate of any one of claims 1 to 35 for treating cancer, an autoimmune disease or an inflammatory disease.

38. 38. The composition of claim 37, wherein the composition is for use in treating cancer, and the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, or melanoma.

39. 38. The composition of claim 37, wherein the composition is for use in treating an autoimmune disease or an inflammatory disease, wherein the autoimmune disease or inflammatory disease is selected from B-cell mediated autoimmune disease, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and lupus nephritis.

40. 36. Use of the antibody conjugate of any one of claims 1 to 35 in the manufacture of a medicament for treating cancer, an autoimmune disease or an inflammatory disease.

Images