Tubulysin and Protein-Tubulysin Conjugates

Abstract

The present invention provides compounds, compositions, and methods for treating diseases and disorders associated with cancer, including tubulysin and its protein (e.g., antibody) drug conjugates.

Description

[0001] Cross-reference

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 043,771, filed on June 24, 2020, the content of which is hereby incorporated by reference in its entirety into this application. Field of the Invention

[0003] The present invention provides novel tubulysins and their protein conjugates, as well as methods for treating a variety of diseases, disorders, and conditions, including administering the tubulysins and their protein conjugates. Background of the Invention

[0005] Although the use of antibody-drug conjugates (ADCs) in cancer treatment regimens is increasing, de novo or acquired resistance mechanisms may compromise clinical benefits. Two resistance mechanisms that emerge under continuous ADC exposure in vitro both involve upregulation of transporters that cause multi-drug resistance (MDR) and loss of homologous antigen expression. New technologies to bypass these resistance mechanisms may help to expand the utility of the next generation of ADCs.

[0006] Tubulysin, first isolated from the culture broth of myxobacteria, is a group of extremely potent tubulin polymerization inhibitors that can rapidly disassemble the cytoskeleton of dividing cells and induce apoptosis. Tubulysin is composed of N-methyl-D-pipecolic acid (Mep), L-isoleucine (Ile), and tubuvaline (Tuv), which contains unusual N,O-acetals and secondary alcohol or acetoxy groups. Tubulysin A, B, C, G, and I contain a C-terminal tubutyrosine (Tut) γ-amino acid, while tubulysin D, E, F, and H have a tubuphenylalanine (Tup) at this position (Angew. Chem. Int. Ed. Engl. 43, 4888–4892).

[0007] Due to the strong activity of laulimalide in drug-resistant cells through a validated mechanism of action, laulimalide has become a very promising anti-cancer drug. The average cell growth inhibitory activity is more than 10 to 1000 times better than that of the well-known epothilones, vinblastine, and paclitaxel, including the activity against multi-drug resistant cancers (Biochem. J. 2006, 396, 235-242; Nat. Prod. Rep. 2015, 32, 654-662). Laulimalide has extremely strong anti-proliferative activity against cancer cells (including multi-drug resistant KB-V1 cervical cancer cells). (Angew. Chem. Int. Ed. 2004, 43, 4888-4892; and Biochemical Journal 2006, 396, 235-242). Abstract of the Invention

[0008] The present invention provides compounds for, for example, anti-cancer and anti-angiogenic therapies.

[0009] In one embodiment, the present invention provides a compound having the structure shown by the following formula:

[0010]

[0011] or a pharmaceutically acceptable salt thereof, wherein,

[0012] BA is a binder;

[0013] L is a linker covalently connected to both BA and T;

[0014] T is wherein,

[0015] R 1 is a bond, H, C1-C 10 alkyl, a first N-terminal amino acid residue, a first amino acid residue, –C1-C 10 alkyl-NR 3a R 3b 、or –C1-C 10 alkyl-OH;

[0016] R 3 is a hydroxyl group, –O–, –O-C1-C5 alkyl, –OC(O)C1-C5 alkyl, –OC(O)N(H)C1-C 10 alkyl, –OC(O)N(H)C1-C 10 alkyl-NR 3a R 3b 、–NHC(O)C1-C5 alkyl, or –OC(O)N(H)(CH2CH2O) n C1-C 10 alkyl-NR 3a R3b ,

[0017] wherein R 3a and R 3b , in each case, are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0018] R 4 and R 5 , in each case, are independently H or C1-C5 alkyl;

[0019] R 6 is –OH, –O–, –NHNH2, –NHNH–, –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ,

[0020] wherein the aryl is substituted or unsubstituted; and

[0021] R 6a and R 6b , in each case, are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0022] R 7 in each case, is independently H, –OH, –O–, halogen, or –NR 7a R 7b ,

[0023] wherein R 7a and R 7b , in each case, are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first amino acid residue, a first N-terminal peptide residue, a first peptide residue, –CH2CH2NH2, and –CH2CH2NH–; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0024] R 8 in each case, is independently H, –NHR 9 , or halogen,

[0025] wherein R 9 is H, –C1-C5 alkyl, or –C(O)C1-C5 alkyl; and

[0026] m is 1 or 2;

[0027] R 10 , when present, is -C1-C5 alkyl;

[0028] Q is –CH2– or –O–, where,

[0029] R 2 is alkyl, alkylene, alkynyl, alkynylene, regioisomeric triazole, or regioisomeric triazolyl;

[0030] wherein the regioisomeric triazole or regioisomeric triazolyl is unsubstituted or substituted by alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or acyl;

[0031] where n is an integer from 1 to 10;

[0032] where r is an integer from 1 to 6;

[0033] where a, a1 and a2 are each independently 0 or 1; and

[0034] k is an integer from 1 to 30;

[0035] where T is not: compound IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c, which are covalently linked to L, or a pharmaceutically acceptable salt thereof.

[0036] In one embodiment, the present invention provides a compound having the structure shown in formula I:

[0037]

[0038] or a pharmaceutically acceptable salt thereof, wherein,

[0039] R 1 is H, C1-C 10 alkyl, the first N-terminal amino acid residue, –C1-C 10 alkyl-NR 3a R 3b 、or –C1-C 10 alkyl-OH;

[0040] R3 is hydroxy, –O-C1-C5 alkyl, –OC(O)C1-C5 alkyl, –OC(O)N(H)C1-C 10 alkyl, –OC(O)N(H)C1-C 10 alkyl-NR 3a R 3b 、–NHC(O)C1-C5 alkyl, or –OC(O)N(H)(CH2CH2O) n C1-C 10 alkyl-NR 3a R 3b ,

[0041] wherein R 3a and R 3b , in each case, are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0042] R 4 and R 5 , in each case, are independently H or C1-C5 alkyl;

[0043] R 6 is –OH, –NHNH2, –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ,

[0044] wherein the aryl is substituted or unsubstituted; and

[0045] R 6a and R 6b , in each case, are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0046] R 7 in each case, are independently H, –OH, halogen, or –NR 7a R 7b ,

[0047] wherein R 7a and R 7b , in each case, are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, –C(O)CH2OH, first N-terminal amino acid residue, first N-terminal peptide residue, and –CH2CH2NH2; wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0048] R 8 In each case, independently for H, –NHR 9 , or halogen,

[0049] wherein R 9 is H, –C1-C5 alkyl, or –C(O)C1-C5 alkyl; and

[0050] m is 1 or 2;

[0051] R 10 , when present, is -C1-C5 alkyl;

[0052] Q is –CH2– or –O–, wherein,

[0053] R 2 is alkyl, alkynyl, or regioisomeric triazole;

[0054] wherein the regioisomeric triazole is unsubstituted or substituted by alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl;

[0055] wherein n is an integer from 1 to 10;

[0056] wherein r is an integer from 1 to 6;

[0057] wherein a, a1 and a2 are independently 0 or 1; and

[0058] wherein T is not: compound IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va ′ , Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, D-5c, tubulysin A-I, U-X, or Z, pretubulysin D, or N 14 -deacetoxytubulysin H (N 14 -desacetoxytubulysin H).

[0059] In another embodiment, the present invention provides a method for treating a tumor that expresses an antigen selected from the group consisting of PRLR and STEAP2.

[0060] In another embodiment, the present invention provides a linker - payload having the structure shown by the following formula:

[0061] L - T

[0062] or a pharmaceutically acceptable salt thereof, wherein,

[0063] L is a linker covalently linked to T;

[0064] T is wherein,

[0065] R 1 is a bond, H, C1 - C 10 alkyl, a first N - terminal amino acid residue, a first amino acid residue, –C1 - C 10 alkyl - NR 3a R 3b 、or –C1 - C 10 alkyl - OH;

[0066] R 3 is a hydroxyl group, –O–, –O - C1 - C5 alkyl, –OC(O)C1 - C5 alkyl, –OC(O)N(H)C1 - C 10 alkyl, –OC(O)N(H)C1 - C 10 alkyl - NR 3a R 3b 、–NHC(O)C1 - C5 alkyl, or –OC(O)N(H)(CH2CH2O) n C1 - C 10 alkyl - NR 3a R 3b ;

[0067] wherein R 3a and R 3b , in each case, are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted;

[0068] R 4 and R 5 , in each case, are independently H or C1 - C5 alkyl;

[0069] R 6 is –OH, –O–, –NHNH2, –NHNH–, –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ;

[0070] wherein the aryl is substituted or unsubstituted; and

[0071] R 6a and R 6b , in each case, independently of one another, is a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0072] R 7 , in each case, independently of one another, is H, –OH, –O–, halogen, or –NR 7a R 7b ,

[0073] wherein R 7a and R 7b , in each case, independently of one another, is a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first amino acid residue, a first N-terminal peptide residue, a first peptide residue, –CH2CH2NH2, and –CH2CH2NH–; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and acyl are each optionally substituted;

[0074] R 8 , in each case, independently of one another, is H, –NHR 9 , or halogen,

[0075] wherein R 9 is H, –C1-C5 alkyl, or –C(O)C1-C5 alkyl; and

[0076] m is 1 or 2;

[0077] R 10 , when present, is -C1-C5 alkyl;

[0078] Q is –CH2– or –O–, wherein,

[0079] R 2 is alkyl, alkylene, alkynyl, alkynylene, regioisomeric triazole, or regioisomeric triazolyl;

[0080] wherein the regioisomeric triazole or regioisomeric triazolyl is unsubstituted or substituted by alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or acyl;

[0081] wherein n is an integer from 1 to 10;

[0082] wherein r is an integer from 1 to 6;

[0083] wherein a, a1 and a2 are each independently 0 or 1; and ​

[0084] Wherein the linker-payload is not: LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve, or a pharmaceutically acceptable salt thereof.

[0085] In another embodiment, the present invention provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is conjugated to a compound as described in the present invention.

[0086] In another embodiment, the present invention provides a method for preparing a compound, a linker-payload, or an antibody-drug conjugate, and a composition as described in the present invention.

[0087] Brief Description of the Drawings

[0088] Figures 1-11, 12A, 12B, 13A, 13B, 14, 15A, 15B, 15C and 16 show synthetic chemical schemes of tubulysin payloads and tubulysin linker-payloads, wherein each compound is capable of being conjugated to or conjugated with an antibody or an antigen-binding fragment thereof. Detailed Description

[0089] The present invention provides compounds, compositions, and methods for treating, for example, cancer in a subject.

[0090] Definitions

[0091] When referring to the compounds provided by the present invention, unless otherwise specified, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In the case where there are multiple definitions for the terms provided in the present invention, the said definitions shall prevail unless otherwise specified.

[0092] "Alkyl" as used in the present invention refers to a monovalent and saturated hydrocarbon-based group moiety. The alkyl is optionally substituted and can be straight-chain, branched-chain, or cyclic, i.e., cycloalkyl. Alkyl includes, but is not limited to, having 1-20 carbon atoms, i.e., C 1-20 alkyl; 1-12 carbon atoms, i.e., C 1-12 alkyl; 1-10 carbon atoms, i.e., C 1-10Alkyl; having 1 - 8 carbon atoms, i.e., C 1-8 Alkyl; having 5 - 10 carbon atoms, i.e., C 5-10 Alkyl; having 1 - 5 carbon atoms, i.e., C 1-5 Alkyl; having 1 - 6 carbon atoms, i.e., C 1-6 Alkyl; and having 1 - 3 carbon atoms, i.e., C 1-3 Those groups of alkyl. Examples of the alkyl group moiety include, but are not limited to, methyl, ethyl, n - propyl, isopropyl, n - butyl, sec - butyl, tert - butyl, isobutyl, pentyl group moiety, hexyl group moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The pentyl group moiety includes, but is not limited to, n - pentyl and isopentyl. The hexyl group moiety includes, but is not limited to, n - hexyl.

[0093] "Alkylene" as used in the present invention refers to a divalent alkyl group. Unless otherwise specified, alkylene includes, but is not limited to, having 1 - 20 carbon atoms. The alkylene group is optionally substituted as described for alkyl in the present invention. In some embodiments, the alkylene is unsubstituted.

[0094] In the case where the stereochemistry is not specified, the name of an amino acid or amino acid residue is intended to cover L - amino acids, D - amino acids, or their racemic mixtures.

[0095] "Haloalkyl" as used in the present invention refers to an alkyl as defined above, wherein said alkyl includes at least one substituent selected from halogens such as fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, but are not limited to, - CF3, - CH2CF3, –CCl2F, and –CCl3.

[0096] "Alkenyl" as used in the present invention refers to a monovalent hydrocarbon group moiety containing at least two carbon atoms and one or more non - aromatic carbon - carbon double bonds. The alkenyl is optionally substituted and can be straight - chain, branched - chain, or cyclic. Alkenyl includes, but is not limited to, having 2 - 20 carbon atoms, i.e., C 2-20 Alkenyl; having 2 - 12 carbon atoms, i.e., C 2-12 Alkenyl; having 2 - 8 carbon atoms, i.e., C 2-8 Alkenyl; having 2 - 6 carbon atoms, i.e., C 2-6 Alkenyl; and having 2 - 4 carbon atoms, i.e., C 2-4 Those groups of alkenyl. Examples of the alkenyl group moiety include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl.

[0097] "Alkynyl" as used in the present invention refers to a monovalent hydrocarbon group moiety containing at least two carbon atoms and one or more carbon - carbon triple bonds. The alkynyl is optionally substituted and can be straight - chain, branched - chain, or cyclic. Alkynyl includes, but is not limited to, having 2 - 20 carbon atoms, i.e., C 2-20 Alkynyl; having 2 - 12 carbon atoms, i.e., C 2-12Alkynyl; having 2 to 8 carbon atoms, i.e., C 2-8 Alkynyl; having 2 to 6 carbon atoms, i.e., C 2-6 Alkynyl; and having 2 to 4 carbon atoms, i.e., C 2-4 Those groups of alkynyl. Examples of the alkynyl group moiety include, but are not limited to, ethynyl, propynyl, and butynyl.

[0098] As used in the present invention, "alkoxy" refers to a monovalent and saturated hydrocarbon group moiety, wherein said hydrocarbon includes a single bond connected to an oxygen atom, and wherein said radical is located on the oxygen atom, such as ethoxy CH3CH2–O·. The alkoxy substituent is connected to the compound by substitution through the oxygen atom of the alkoxy substituent. The alkoxy is optionally substituted and can be straight-chain, branched-chain, or cyclic, i.e., cycloalkoxy. The alkoxy includes, but is not limited to, having 1 to 20 carbon atoms, i.e., C 1-20 Alkoxy; having 1 to 12 carbon atoms, i.e., C 1-12 Alkoxy; having 1 to 8 carbon atoms, i.e., C 1-8 Alkoxy; having 1 to 6 carbon atoms, i.e., C 1-6 Alkoxy; and having 1 to 3 carbon atoms, i.e., C 1-3 Those of alkoxy. Examples of the alkoxy group moiety include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, isobutoxy, pentyloxy group moiety, hexyloxy group moiety, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.

[0099] As used in the present invention, "haloalkoxy" refers to an alkoxy as defined above, wherein said alkoxy includes at least one substituent selected from halogens such as F, Cl, Br, or I.

[0100] As used in the present invention, "aryl" refers to a monovalent group moiety, which is an atomic group of an aromatic compound, wherein all of the ring atoms are carbon atoms. The aryl is optionally substituted and can be monocyclic or polycyclic, such as bicyclic or tricyclic. Examples of the aryl group moiety include, but are not limited to, having 6 to 20 ring carbon atoms, i.e., C 6-20 Aryl; having 6 to 15 ring carbon atoms, i.e., C 6-15 Aryl, and having 6 to 10 ring carbon atoms, i.e., C 6-10 Those of aryl. Examples of the aryl group moiety include, but are not limited to, phenyl, naphthyl, fluorenyl, azulyl, anthryl, phenanthryl, and pyrenyl.

[0101] The "arylalkyl" or "aralkyl" used in the present invention refers to the monovalent group portion of an alkyl compound moiety, wherein the alkyl compound is substituted by an aromatic substituent, i.e., the aromatic compound includes a single bond connecting to the alkyl group, and wherein the radical is located on the alkyl group. The arylalkyl group is connected to the shown chemical structure through the alkyl group. The arylalkyl can be represented by the following structure, for example, wherein B is an aromatic group portion, such as an aryl or phenyl group. The arylalkyl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed in the present invention. Examples of arylalkyl include but are not limited to benzyl.

[0102] The "alkylaryl" used in the present invention refers to the monovalent group portion of an aryl compound moiety, wherein the aryl compound is substituted by an alkyl substituent, i.e., the aryl compound includes a single bond connecting to the alkyl group, and wherein the radical is located on the aryl group. The alkylaryl group is connected to the shown chemical structure through the aryl group. The alkylaryl can be represented by the following structure, for example, wherein B is an aromatic group portion, such as a phenyl group. The alkylaryl is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed in the present invention. Examples of alkylaryl include but are not limited to toluoyl.

[0103] The "aryloxy / aryl-oxy" used in the present invention refers to the monovalent group portion of an aromatic compound moiety, wherein all of the ring atoms are carbon atoms, and wherein the ring is substituted by an oxy group, i.e., the aromatic compound includes a single bond connecting to an oxygen atom, and wherein the radical is located on the oxygen atom, such as phenoxy The aryloxy substituent is connected to the compound through this oxygen atom substitution. The aryloxy is optionally substituted. The aryloxy includes but is not limited to those having 6 to 20 ring carbon atoms, i.e., C 6-20 aryloxy; 6 to 15 ring carbon atoms, i.e., C 6-15 aryloxy, and 6 to 10 ring carbon atoms, i.e., C 6-10 aryloxy groups. Examples of the aryloxy group portion include but are not limited to phenoxy, naphthyloxy, and anthryloxy.

[0104] The "arylene" used in the present invention refers to the divalent group portion of an aromatic compound, wherein all of the ring atoms are only carbon atoms. The aryene is optionally substituted and can be monocyclic or polycyclic, such as bicyclic or tricyclic. Examples of the aryene group portion include but are not limited to those having 6 to 20 ring carbon atoms, i.e., C 6-20 arylene; 6 to 15 ring carbon atoms, i.e., C 6-15 arylene; and 6 to 10 ring carbon atoms, i.e., C 6-10 arylene.

[0105] As used herein, "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by heteroatoms. "Heteroalkenyl" as used herein refers to an alkenyl group in which one or more carbon atoms are replaced by heteroatoms. "Heteroalkynyl" as used herein refers to an alkynyl group in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. The heteroalkyl, heteroalkenyl, and heteroalkynyl are each optionally substituted. Examples of heteroalkyl group moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl group moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.

[0106] As used herein, "heteroaryl" refers to a monovalent group moiety of an aromatic compound radical, wherein the ring atoms include carbon atoms and at least one oxygen, sulfur, nitrogen, or phosphorus atom. Examples of heteroaryl group moieties include, but are not limited to, those having 5 to 20 ring atoms, 5 to 15 ring atoms, and 5 to 10 ring atoms. The heteroaryl is optionally substituted.

[0107] As used herein, "heteroarylene" refers to a divalent heteroaryl in which one or more ring atoms of the aromatic ring are replaced by oxygen, sulfur, nitrogen, or phosphorus atoms. The heteroarylene is optionally substituted.

[0108] As used herein, "heterocycloalkyl" refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. The heterocycloalkyl is optionally substituted. Examples of heterocycloalkyl group moieties include, but are not limited to, morpholinyl, piperidinyl, tetrahydropyranyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, dioxolanyl, dithiolanyl, tetrahydropyranyl (oxanyl), or tetrahydrothianyl (thianyl).

[0109] As used herein, "Lewis acid" refers to a molecule or ion that accepts a lone pair of electrons. The Lewis acids used in the methods of the present invention are those other than protons. Lewis acids include, but are not limited to, non-metal acids, metal acids, hard Lewis acids, and soft Lewis acids. Lewis acids include, but are not limited to, Lewis acids of aluminum, boron, iron, tin, titanium, magnesium, copper, antimony, phosphorus, silver, ytterbium, scandium, nickel, and zinc. Exemplary Lewis acids include, but are not limited to: AlBr3, AlCl3, BCl3, boron trichloride methyl sulfide, BF3, boron trifluoride methyl ether complex, boron trifluoride methyl sulfide, boron trifluoride tetrahydrofuran, dicyclohexylboron trifluoromethanesulfonate, iron(III) bromide, iron(III) chloride, tin(IV) chloride, titanium(IV) chloride, titanium(IV) isopropoxide, Cu(OTf)2, CuCl2, CuBr2, zinc chloride, alkylaluminum halides (R n AlX 3-n, where R is a hydrocarbon group), Zn(OTf)2, ZnCl2, Yb(OTf)3, Sc(OTf)3, MgBr2, NiCl2, Sn(OTf)2, Ni(OTf)2, and Mg(OTf)2.

[0110] As used herein, "N-containing heterocycloalkyl" refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms and in which at least one of the replacing heteroatoms is a nitrogen atom. Suitable heteroatoms include, but are not limited to, oxygen and sulfur atoms in addition to nitrogen atoms. The N-containing heterocycloalkyl is optionally substituted. Examples of the N-containing heterocycloalkyl group moiety include, but are not limited to, morpholinyl, piperidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, or thiazolidinyl.

[0111] As used herein, "optionally substituted" or "optionally substituted" when used to describe a group moiety, such as an optionally substituted alkyl group, means that this group moiety is optionally linked to one or more substituents. Examples of such substituents include, but are not limited to, halogen, cyano, nitro, amino, hydroxy, optionally substituted haloalkyl, aminoalkyl, hydroxyalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, where R A 、R B and R C each independently represents a hydrogen atom, alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heteroaryl, or heterocycloalkyl at each occurrence, or R A and R B together with the atoms to which they are attached form a saturated or unsaturated carbocycle, where the ring is optionally substituted and where one or more of the ring atoms are optionally replaced by heteroatoms. In certain embodiments, when a group moiety is optionally substituted by an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocycle, the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocycle, if they are substituted, are not further substituted by substituents that are optionally substituted by additional substituents. In some embodiments, when a group as described herein is optionally substituted, unless otherwise indicated, the substituents attached to the group are unsubstituted.

[0112] As used herein, "binding agent" refers to any molecule, such as a protein, antibody, or fragment thereof, that is capable of specifically binding to a given binding partner (such as an antigen).

[0113] The "linker" used in the present invention is a divalent, trivalent, or polyvalent group moiety that covalently links or is capable of covalently linking (e.g., via a reactive group at one end; and in certain embodiments, via an amino acid and / or spacer group at the other end) the binder to one or more compounds described in the present invention (e.g., a payload compound, an enhancer, and / or a prodrug payload compound). The "payload" or "payload" used in the present invention refers to tubulysin or a tubulysin derivative. The "prodrug payload compound" or "prodrug" used in the present invention refers to a payload terminated with one or more amino acid residues or another chemical residue, as described elsewhere in the present invention. Thus, in certain embodiments, the linker may ultimately be cleaved to release the payload compound in the form of a tubulysin derivative. In other embodiments, the linker may ultimately be cleaved to release a prodrug payload compound in the form of a tubulysin derivative that retains one or more terminal amino acid residues. Such a prodrug payload compound may be further processed via recognized biological processes (e.g., amide bond hydrolysis) to ultimately yield the payload compound in the form of a tubulysin payload compound without terminal amino acid residues.

[0114] The "amide synthesis conditions" used in the present invention refer to reaction conditions suitable for promoting the formation of amides, for example, by reacting carboxylic acids, activated carboxylic acids, or acyl halides with amines. In some embodiments, the "amide synthesis conditions" refer to reaction conditions suitable for promoting the formation of amide bonds between carboxylic acids and amines. In some of these embodiments, the carboxylic acid is first converted to an activated carboxylic acid before reacting with the amine to form an amide. Suitable conditions for achieving amide formation include, but are not limited to, those using reagents to effect the reaction between carboxylic acids and amines, including but not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), tripyrrolidinophosphonium bromide hexafluorophosphate (PyBrOP), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), and carbonyldiimidazole (CDI). In some embodiments, the carboxylic acid is first converted to an activated carboxylic acid ester, and then the activated carboxylic acid ester is treated with an amine to form an amide bond. In some embodiments, the carboxylic acid is treated with a reagent. The reagent activates the carboxylic acid by deprotonating it, and then a product complex is formed with the deprotonated carboxylic acid due to the nucleophilic attack of the deprotonated carboxylic acid on the protonated reagent. For certain carboxylic acids, the activated carboxylic acid ester is more susceptible to nucleophilic attack by amines than the carboxylic acid before it is converted to the activated ester. This leads to the formation of an amide bond. Thus, the carboxylic acid is described as being activated. Exemplary reagents include DCC and DIC.

[0115] The "regioisomers" or "mixtures of regioisomers" used in the present invention refer to 1,3-cycloaddition products or strain-promoted alkyne-azide cycloaddition (SPAAC) (also known as click reaction) products obtained by treating suitable azides (e.g., -N3 or –PEG-N3 conjugated antibodies) with suitable alkyne compounds. In certain embodiments, for example, regioisomers and mixtures of regioisomers are characterized by click reaction products as shown below:

[0116]

[0117] In certain embodiments, more than one suitable azide and more than one suitable alkyne can be used in a synthetic scheme to generate a product, wherein each pair of azide-alkyne can participate in one or more independent click reactions to generate a mixture of regioisomeric click reaction products. For example, one of ordinary skill in the art will recognize that a first suitable azide can react independently with a first suitable alkyne, and a second suitable azide can react independently with a second suitable alkyne during the course of generating the product, thereby generating four possible click reaction regioisomers or a mixture of said four possible click reaction regioisomers.

[0118] As used herein, the term "residue" refers to the portion of a chemical group that remains within a compound after a chemical reaction. For example, the terms "amino acid residue", "N-alkyl amino acid residue", or "N-terminal amino acid residue" refer to the product of an amide coupling or peptide coupling of an amino acid, N-alkyl amino acid, or N-terminal amino acid with a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide coupling or peptide coupling of an amino acid or N-alkyl amino acid, thereby obtaining a product incorporating an amino acid residue, N-alkyl amino acid residue, or N-terminal amino acid residue. The term "amino acid" refers to natural and synthetic α, β, γ, or δ amino acids, including but not limited to the amino acids present in proteins, namely glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In certain embodiments, the amino acids are in the L-configuration. Alternatively, the amino acid can be a derivative of alanyl, valyl, leucyl, isoleucyl, prolyl, phenylalanyl, tryptophanyl, methionyl, glycyl, seryl, threonyl, cysteyl, tyrosyl, asparaginyl, glutaminyl, aspartyl, glutaryl, lysyl, arginyl, histidyl, β-alanyl, β-valyl, β-leucyl, β-isoleucyl, β-prolyl, β-phenylalanyl, β-tryptophanyl, β-methionyl, β-glycyl, β-seryl, β-threonyl, β-cysteyl, β-tyrosyl, β-asparaginyl, β-glutaminyl, β-aspartyl, β-glutaryl, β-lysyl, β-arginyl, or β-histidyl. The term "amino acid derivative" refers to a group that can be derived from a natural or non-natural amino acid, as described and exemplified herein. Amino acid derivatives will be apparent to those of ordinary skill in the art and include but are not limited to esters, amino alcohols, amino aldehydes, amino lactones, and N-methyl derivatives of natural and non-natural amino acids. In certain embodiments, the amino acid residue is wherein S cis a side chain or bond of a naturally occurring or non-naturally occurring amino acid (e.g., H in glycine; –CH2OH in serine; –CH2SH in cysteine; –CH2CH2CH2CH2NH2 in lysine; –CH2CH2COOH in glutamic acid; –CH2CH2C(O)NH2 in glutamine; or –CH2C6H5OH in tyrosine; etc.); and represents a linkage to another chemical entity (including but not limited to, another amino acid residue or N-alkyl amino acid residue that gives rise to a peptide or peptide residue). In certain embodiments, S c is selected from the group consisting of H, alkyl, heteroalkyl, arylalkyl, and heteroarylalkyl.

[0119] As used herein, "therapeutically effective amount" means an amount of a (compound) sufficient to provide a therapeutic benefit when treating or controlling a disease or disorder in a patient, or delaying or minimizing one or more symptoms associated with the disease or disorder.

[0120] As used herein, "constitutional isomer" means a compound having the same molecular formula but a different chemical structure due to the way the atoms are arranged. Exemplary constitutional isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, etc.

[0121] Certain groups, molecular / group moieties, substituents, and atoms are depicted with a wavy line intersecting a bond to indicate the atom through which the group, molecular / group moiety, substituent, atom is attached. For example, a phenyl group substituted with a propyl group can be represented as:

[0122] having the following structure:

[0123] As used herein, the illustrative description of a substituent attached to a cyclic group (e.g., an aromatic ring, a heteroaromatic ring, a fused ring, and a saturated or unsaturated cycloalkyl or heterocycloalkyl) by a bond between ring atoms is intended to mean that, unless otherwise specified, the cyclic group can be substituted with a substituent at any ring position of the cyclic group or on any ring of a fused ring group according to the techniques described herein or techniques known in the art as disclosed herein. For example, the group where the subscript q is an integer from 0 to 4, and where the substituent R 1 is generally described in terms of its position, i.e., not directly attached to any vertex of the bond-line structure, i.e., a particular ring carbon atom, including the following, where the substituent R 1 non-limiting examples of groups attached to a particular ring carbon atom:

[0124] The phrase "reactive linker" or the abbreviation "RL" used in the present invention refers to a monovalent group containing a reactive group ("RG") and a spacer group ("SP"), as shown, where RG is the reactive group and SP is the spacer group. As described in the present invention, the reactive linker may contain more than one reactive group and more than one spacer group. The spacer group is any divalent group moiety that bridges the reactive group to another group (e.g., a payload or a prodrug payload). The reactive linker (RL), together with the payload or prodrug payload to which they are attached, forms an intermediate ("linker-payload" or LP; or linker-prodrug payload) that can be used as a synthetic precursor for preparing the antibody conjugate described in the present invention. The reactive linker contains a reactive group, which is a functional group or group moiety capable of reacting with the reactive moiety of another group (e.g., an antibody or an antigen-binding fragment thereof, a modified antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof modified by transglutaminase, or an enhancing group). The group moiety generated by the reaction of the reactive group with the antibody or an antigen-binding fragment thereof, a modified antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof modified by transglutaminase, together with the linking group, forms the "binding agent linker" ("BL") portion of the conjugate described in the present invention. In certain embodiments, the "reactive group" is a functional group or group moiety that reacts with a cysteine or lysine residue of an antibody or an antigen-binding fragment thereof (e.g., maleimide or N-hydroxysuccinimide (NHS) ester). In certain embodiments, the "reactive group" is a functional group or group moiety capable of performing a click chemical reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc. 1961, Wang et al. J. Am. Chem. Soc. 2003, and Agard et al. J. Am. Chem. Soc. 2004). In some embodiments of the click chemical reaction, the reactive group is an alkyne capable of performing a 1,3-cycloaddition reaction with an azide. Such suitable reactive groups include, but are not limited to, strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, benzocyclized alkynes, and alkynes capable of performing a 1,3-cycloaddition reaction with an alkyne compound in the absence of a copper catalyst. Suitable alkynes also include, but are not limited to, dibenzocyclooctyne or (DIBAC), dibenzocyclooctyne or (DIBO), diarylazacyclooctynone or (BARAC), difluorocyclooctyne or (DIFO), substituted, such as fluorinated alkynes, azacycloalkynes, bicyclo[6.1.0]nonyne or (BCN, where R is alkyl, alkoxy, or acyl), and their derivatives. Particularly useful alkynes include Linker-payload or linker-prodrug payloads containing such reactive groups can be used to conjugate antibodies functionalized with azide. The "transglutaminase-modified antibody or antigen-binding fragment thereof" used in the present invention refers to an antibody or antigen-binding fragment thereof having one or more glutamine (Gln or Q) residues, which residues are capable of reacting with a compound bearing a primary or secondary amino functional group in the presence of transglutaminase. Such transglutaminase-modified antibodies or antigen-binding fragments thereof include the conjugation of an antibody or antigen-binding fragment thereof with a primary amine of an azide-polyethylene glycol group moiety via transglutaminase-mediated reaction, and antibodies or antigen-binding fragments thereof functionalized with an azide-polyethylene glycol group moiety. In certain embodiments, such transglutaminase-modified antibodies or antigen-binding fragments thereof are derived by treating an antibody or antigen-binding fragment thereof having at least one glutamine residue (such as Gln295 in the heavy chain) with a compound bearing an amino group and an azide in the presence of transglutaminase, as further described elsewhere in the present invention.

[0125] In some embodiments, the reactive group is an alkyne, such as which can react with an azide (such as ) via click chemistry to form a click chemistry product, such as In some embodiments, the reactive group reacts with an azide on a modified antibody or antigen-binding fragment thereof. In some embodiments, the reactive group is an alkyne, such as which can react with an azide (such as ) via click chemistry to form a click chemistry product, such as In some embodiments, the reactive group is an alkyne, such as which can react with an azide (such as ) via click chemistry to form a click chemistry product, such as In some embodiments, the reactive group is a functional group, such as which reacts with a cysteine residue on an antibody or antigen-binding fragment thereof to form a C-S bond therewith, such as where Ab refers to an antibody or antigen-binding fragment thereof, and S refers to the sulfur (S) atom on the cysteine residue, and the functional group binds to Ab through the S atom on this cysteine residue. In some embodiments, the reactive group is a functional group, such as It reacts with a lysine residue on an antibody or an antigen-binding fragment thereof to form an amide bond therewith, such as wherein Ab refers to an antibody or an antigen-binding fragment thereof, and –NH– refers to the –NH– atom on the lysine side chain residue, and the functional group binds to Ab through the –NH– atom on this lysine side chain residue.

[0126] The phrase “biodegradable group moiety” as used in the present invention refers to a group moiety that degrades in vivo into non-toxic, biocompatible components that can be cleared from the body by normal biological processes. In some embodiments, the biodegradable group moiety degrades completely or substantially in vivo within about 90 days or less, about 60 days or less, or about 30 days or less, where the degree of degradation is based on the percentage mass loss of the biodegradable group moiety, and complete degradation corresponds to 100% mass loss. Exemplary biodegradable group moieties include, but are not limited to, aliphatic polyesters such as poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and copolymers thereof with glycolic acid (i.e., poly(D,L-lactide-co-glycolide) (PLGA) (Vert M, Schwach G, Engel R and Coudane J (1998) J Control Release 53(1-3):85-92; Jain R A (2000) Biomaterials 21(23):2475-2490; Uhrich K E, Cannizzaro S M, Langer R S and Shakesheff K M (1999) Chemical Reviews 99(11):3181-3198; and Park T G (1995) Biomaterials 16(15):1123-1130, each of which is incorporated herein by reference in its entirety).

[0127] The phrase "linker conjugate" or "BL" as used in the present invention refers to any divalent, trivalent, or multivalent group or group moiety that links, binds, or bonds a binder (e.g., an antibody or an antigen-binding fragment thereof) to a payload compound (e.g., tubulin) as described in the present invention, and optionally to one or more side chain compounds. Generally, suitable linker conjugates for the antibody conjugates described in the present invention are those that are sufficient to stabilize and utilize the circulating half-life of the antibody conjugate, and at the same time are capable of releasing their payload after antigen-mediated conjugate internalization. The linker can be cleavable or non-cleavable. A cleavable linker is a linker that is cleaved by intracellular metabolism after internalization, for example, by hydrolysis, reduction, or enzymatic reaction. A non-cleavable linker is a linker that releases the attached payload by lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically labile linkers, enzymatically cleavable linkers, reductively labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or contain peptides, glucuronides, succinimide-sulfides, polyethylene glycol (PEG) units, hydrazones, maleimide (mal)-hexanoyl units, dipeptide units, valine-citrulline units, and para-aminobenzyloxycarbonyl (PABC) units, para-aminobenzyl (PAB) units. In some embodiments, the binder linker (BL) comprises a group moiety formed by reacting an active group (RG) of an active linker (RL) with an active moiety of a binder (e.g., an antibody, a modified antibody, or an antigen-binding fragment thereof).

[0128] In some embodiments, the BL comprises the following group moiety: wherein is a bond linking to the binder. In some embodiments, the BL comprises the following group moiety: wherein is a bond linking to the binder. In some embodiments, the BL comprises the following group moiety: wherein is a bond linking to the binder. In some embodiments, the BL comprises the following group moiety: wherein is a bond linking to the cysteine of the antibody or its antigen-binding fragment. In some embodiments, the BL comprises the following group moiety: wherein is a bond linking to the lysine of the antibody or its antigen-binding fragment.

[0129] When applied to polypeptides, the phrase "substantially similar" or "substantially similarity" means that two peptide sequences, when optimally aligned, e.g., by the programs GAP or BESTFIT, using default gap weights, share at least 95% sequence identity, or at least 98% or 99% sequence identity. The BLAST algorithm described in Altschul et al. J. Mol. Biol. 215:403-10 (using the published default settings) can also be used to determine sequence similarity, or available at blast.ncbi.nlm.nih.gov / Blast.cgi. In certain embodiments, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage of sequence identity or similarity can be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains having similar chemical properties include (1) aliphatic side chain groups: glycine, alanine, valine, leucine, and isoleucine; (2) hydroxy aliphatic side chain groups: serine and threonine; (3) amide-containing side chain groups: asparagine and glutamine; (4) aromatic side chain groups: phenylalanine, tyrosine, and tryptophan; (5) basic side chain groups: lysine, arginine, and histidine; (6) acidic side chain groups: aspartic acid and glutamic acid; and (7) sulfur-containing side chain groups: cysteine and methionine. Particularly useful conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0130] The "enantiomeric excess (ee)" used in the present invention refers to a dimensionless molar ratio describing the purity of a chiral substance containing, for example, a single stereogenic center. For example, an enantiomeric excess of zero indicates a racemate (e.g., a 50:50 mixture of enantiomers, or one enantiomer is not in excess relative to the other enantiomer). Further by way of example, an enantiomeric excess of 99 indicates an almost stereopure enantiomeric compound (i.e., one enantiomer is in large excess relative to the other enantiomer). The enantiomeric excess percentage, %ee = ([(R)-compound] - [(S)-compound]) / ([(R)-compound] + [(S)-compound]) x 100, where the (R)-compound > (S)-compound; or %ee = ([(S)-compound] - [(R)-compound]) / ([(S)-compound] + [(R)-compound]) x 100, where the (S)-compound > (R)-compound. In addition, the "diastereomeric excess (de)" used in the present invention refers to a dimensionless molar ratio describing the purity of a chiral substance containing more than one stereogenic center. For example, a diastereomeric excess of zero indicates an equimolar mixture of diastereomers. Further by way of example, a diastereomeric excess of 99 indicates an almost stereopure diastereomeric compound (i.e., one diastereomer is in large excess relative to the other diastereomer). The diastereomeric excess can be calculated by a method similar to that for ee. As understood by those skilled in the art, de is usually reported as a percentage de (%de). %de can be calculated in a manner similar to %ee.

[0131] In certain embodiments, certain compounds or payloads listed in Table P below are excluded from the subject matter of the present invention.

[0132] In certain embodiments, the compounds provided by the present invention include any one or all of Compounds IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c listed in Table P. In certain embodiments, the compounds provided by the present invention exclude any one or all of Compounds IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c listed in Table P. For example, in certain embodiments, the compounds provided by the present invention include residues of any one or all of Compounds IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c linked to the linker and / or binder described in the present invention.In certain embodiments, the compounds provided by the present invention exclude residues of any one or all of compounds IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c that are linked to the linkers and / or binders described in the present invention.

[0133] Table P

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In certain embodiments, certain compounds or linker-payloads listed in Table P1 below are excluded from the subject matter described in the present invention.

[0147] In certain embodiments, the compounds provided by the present invention include any one or all of the compounds LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve shown in Table P1. In certain embodiments, the compounds provided by the present invention exclude any one or all of the compounds LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve shown in Table P1. For example, in certain embodiments, the compounds provided by the present invention include residues of any one or all of the compounds LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve linked to the binder described in the present invention. In certain embodiments, the compounds provided by the present invention exclude residues of any one or all of the compounds LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve linked to the binder described in the present invention.

[0148] Table P1

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] Compound, payload, or prodrug payload

[0156] The present invention provides compounds, bioactive compounds, or payloads. Without being bound by any particular theory of operation, the compounds include tubulin and tubulin derivatives, e.g., prodrugs thereof. The terms or phrases "compound", "bioactive compound", "prodrug", "prodrug payload", and "payload" are used interchangeably in the present invention.

[0157] In certain embodiments, the bioactive compound (D*) or its residue includes, e.g., amino, hydroxyl, carboxylic acid, and / or amide functional groups (e.g., D*–NH2 or D*–NH–R; D*–OH or D*–O–R; D*–COOH or D*–C(O)O–R; and / or D*–CONH2, D*–CONH–R, or D*–NHC(O)–R). In certain embodiments of the present invention, for purposes of illustration and convenience, heterocyclic nitrogen, R 2 、R 3 、R 6 、and / or R 7 represent the amino, hydroxyl, carboxylic acid, and amide functional groups within the bioactive compounds of the present invention, as understood by those skilled in the art. In other words, one skilled in the art will recognize that heterocyclic nitrogen, R 2 、R 3 、R 6 、and / or R 7It can be part of the bioactive compound (such as D*) of the present invention and can be used as a functional group for coupling purposes. In one embodiment, the hydroxyl functional group is a primary hydroxyl group moiety (for example, D*–CH2OH or D*–CH2O–R; or D*–C(O)CH2OH or D*–C(O)CH2O–R). In another embodiment, the hydroxyl functional group is a secondary hydroxyl group moiety (for example, D*–CH(OH)R or D*–CH(O–R)R; or D*–C(O)CH(R)(OH) or D*–C(O)CH(R)(O–R)). In another embodiment, the hydroxyl functional group is a tertiary hydroxyl group moiety (for example, D*–C(R1)(R2)(OH) or D*–C(R1)(R2)(O–R); or D*–C(O)C(R1)(R2)(OH) or D*–C(O)C(R1)(R2)(O–R)). In certain embodiments, the bioactive compound (D*) or its residue includes an amino functional group (for example, D*–NR2 or D*–N(R)–R). In one embodiment, the amino functional group is a primary amino group moiety (for example, D*–CH2NR2 or D*–CH2N(R)–R; or D*–C(O)CH2NR2 or D*–C(O)CH2N(R)–R). In another embodiment, the amino functional group is a secondary amino group moiety (for example, D*–CH(NR2)R or D*–CH(NR–R)R; or D*–C(O)CH(R)(NR2) or D*–C(O)CH(R)(NR–R)). In another embodiment, the amino functional group is a tertiary amino group moiety (for example, D*–C(R1)(R2)(NR2) or D*–C(R1)(R2)(N(R)–R); or D*–C(O)C(R1)(R2)(NR2) or D*–C(O)C(R1)(R2)(N(R)–R)). In another embodiment, the amino functional group is a quaternary ammonium salt, as understood by those skilled in the art. In another embodiment, D* including the amino functional group is an arylamine (for example, D*–Ar–NR2, D*–Ar–N(R)–R). Those skilled in the art will recognize that each of the functional groups in the foregoing sentences can be part of the bioactive compound D* and is shown in the general formula for clarity, convenience, and / or emphasis. In another embodiment, D* including the hydroxyl functional group is an aryl hydroxyl or phenolic hydroxyl (for example, D*–Ar–OH, D*–Ar–O–R). In another embodiment, D* including the amide functional group is a tubulin prodrug residue, which is produced by reacting a tubulin compound or derivative (for example, at the position R described in the present invention) with an amino acid compound as also described in the present invention. For example, in certain embodiments, D*–NHC(O)C(S 7 at) and c )(H)NH2 represents a tubulin prodrug with an N-terminal amino acid residue, where Sc represents an amino acid side chain. By way of further example, in certain embodiments, D*–NH[C(O)C(S c )(H)NH] aa C(O)C(S c )(H)NH2 represents a tubulin prodrug with an N-terminal peptide residue, where S c represents an amino acid side chain, and aa is an integer from 1 to 100. In certain embodiments, aa is 1. In certain embodiments, aa is 2. In certain embodiments, aa is 3. In certain embodiments, aa is 4. In certain embodiments, aa is 5. As used herein, "amino acid side chain" refers to the additional chemical group moiety on the same carbon that bears a primary or secondary amine and the carboxylic acid of the amino acid. As will be understood by those skilled in the art, there are 21 "standard" amino acids. Exemplary "standard" amino acids include, but are not limited to, alanine, serine, proline, arginine, and aspartic acid. Other amino acids include cysteine, selenocysteine, and glycine (e.g., where the additional chemical group moiety on the same carbon bearing the primary amine and the carboxylic acid of glycine is H). Exemplary amino acid side chains include, but are not limited to, methyl (i.e., alanine), sec-butyl (i.e., isoleucine), isobutyl (i.e., leucine), –CH2CH2SCH3 (i.e., methionine), –CH2Ph (i.e., phenylalanine), (i.e., tryptophan), (i.e., tyrosine), isopropyl (i.e., valine), hydroxymethyl (i.e., serine), –CH(OH)CH3 (i.e., threonine), –CH2C(O)NH2 (i.e., asparagine), –CH2CH2C(O)NH2 (i.e., glutamine), –CH2SH (i.e., cysteine), –CH2SeH (i.e., selenocysteine), –CH2NH2 (i.e., glycine), propylene or -CH2CH2CH2- (i.e., proline), –CH2CH2CH2NHC(=NH)NH2 (i.e., arginine), (i.e., histidine), –CH2CH2CH2CH2NH2 (i.e., lysine), –CH2COOH (i.e., aspartic acid), and –CH2CH2COOH (i.e., glutamic acid).

[0158] In certain embodiments, a bioactive compound (D*) comprising an amide functional group (D*–NHC(O)–R) (e.g., located at R 7 ) is a prodrug compound of formula Ia:

[0159]

[0160] In certain embodiments, the prodrug of formula Iaa can be linked to a linker or binder, as described elsewhere in the present invention, where Denotes the attachment point to the linker and / or conjugate, as described elsewhere in the present invention.

[0161]

[0162] In certain embodiments, the compound can be delivered to a cell as part of a conjugate. In certain embodiments, the compound is capable of effecting any activity of tubulin or a tubulin derivative at or in a target (e.g., a target cell). Certain compounds can have one or more additional activities. In certain embodiments, the compound is capable of modulating the activity of a folate receptor, a somatostatin receptor, and / or a bombesin receptor.

[0163] Compound, payload, or prodrug payload—Q is C

[0164] In certain embodiments, the present invention provides compounds having the structure shown in Formula I, wherein r is 4.

[0165] In certain embodiments of Formula I above, useful R 3 groups include hydroxy, –O-C1-C5 alkyl, –OC(O)C1-C5 alkyl, –OC(O)N(H)C1-C 10 alkyl, –OC(O)N(H)C1-C 10 alkyl-NR 3a R 3b 、–NHC(O)C1-C5 alkyl, or –OC(O)N(H)(CH2CH2O) n C1-C 10 alkyl-NR 3a R 3b , wherein R 3a and R 3b , in each case, are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl; wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted. In one embodiment, R 3 is hydroxy. In one embodiment, R 3 is –O-C1-C5 alkyl. In one embodiment, R 3 is –OMe. In one embodiment, R 3 is –OEt. In one embodiment, R 3 is –O-propyl, and its constitutional isomers. And its constitutional isomers. In one embodiment, R 3 is –O-butyl, and its constitutional isomers. In one embodiment, R 3 is –O-pentyl, and its constitutional isomers. In one embodiment, R 3is –OC(O)C1-C5 alkyl. In one embodiment, R 3 is –OC(O)Me. In one embodiment, R 3 is –OC(O)Et. In one embodiment, R 3 is –OC(O)-propyl and its structural isomers. In one embodiment, R 3 is –OC(O)-butyl and its structural isomers. In one embodiment, R 3 is –OC(O)-pentyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)C1-C 10 alkyl. In one embodiment, R 3 is –OC(O)N(H)Me. In one embodiment, R 3 is –OC(O)N(H)Et. In one embodiment, R 3 is –OC(O)N(H)-propyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-butyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-pentyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-hexyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-heptyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-octyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-nonyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)-decyl and its structural isomers. In one embodiment, R 3 is –OC(O)N(H)C1-C 10 alkyl-NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3is –OC(O)N(H)CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b 。In one embodiment, R 3 is –OC(O)N(H)CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b 。In any one of the eleven immediately preceding embodiments, R 3a and R 3b are both H. In one embodiment, R 3 is –NHC(O)C1-C5 alkyl. In one embodiment, R 3 is –NHC(O)Me. In one embodiment, R 3 is –NHC(O)Et. In one embodiment, R 3 is –NHC(O)-propyl and its constitutional isomers. In one embodiment, R 3 is –NHC(O)-butyl and its constitutional isomers. In one embodiment, R 3 is –NHC(O)-pentyl and its constitutional isomers. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n C1-C 10 alkyl-NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3is –OC(O)N(H)(CH2CH2O) n CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2NR 3a R 3b where n is 3. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b where n is an integer from 1 to 10. In one embodiment, R 3is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2CH2CH2CH2CH2NR 3a R 3b , where n is an integer from 1 to 10. In one embodiment, R 3 is –OC(O)N(H)(CH2CH2O) n CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2NR 3 a R 3b , where n is an integer from 1 to 10. In any one of the immediately preceding twelve embodiments, R 3a and R 3b are both H.

[0166] In certain embodiments of Formula I above, useful R 7 groups independently include H, –OH, fluorine, chlorine, bromine, iodine, and –NR 7a R 7b . In one embodiment, R 7 is H. In one embodiment, R 7 is –OH. In one embodiment, R 7 is fluorine. In another embodiment, R 7 is chlorine. In another embodiment, R 7 is bromine. In another embodiment, R 7 is iodine. In one embodiment, R 7 is –NR 7a R 7b . In one embodiment, R 7a and R 7b are both H. In one embodiment, R 7a is H, and R 7b is –C(O)CH2OH. In one embodiment, R 7a is H, and R 7b is the first N-terminal amino acid residue. R 7b As the first N-terminal amino acid residue, these amino acid residues are distinguished from the second amino acid residue in the linker, as described elsewhere in the present invention. In one embodiment, R 7a is H, and R 7b is the first N-terminal peptide residue. R 7b As the first N-terminal peptide residue, these peptide residues are distinguished from the second peptide residue in the linker, as described elsewhere in the present invention. In one embodiment, R 7a is H, and R 7b is –CH2CH2NH2.

[0167] In certain embodiments of the above formula I, useful R 8 groups independently include H, –NHR 9 , and halogen. In one embodiment, R 8 is H. In one embodiment, R 8 is –NHR 9 , where R 9 is H. In one embodiment, R 8 is fluorine. In another embodiment, R 8 is chlorine. In another embodiment, R 8 is bromine. In another embodiment, R 8 is iodine. In one embodiment, m is 1. In one embodiment, m is 2.

[0168] In certain embodiments, the present invention provides a compound having the structure shown in formula I:

[0169]

[0170] or a pharmaceutically acceptable salt or prodrug thereof, where Q is –CH2–; R 1 is C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10 is absent; where r is 4; and where a is 1. In formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their constitutional isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its constitutional isomers. In one embodiment, R 1 is butyl and its constitutional isomers. In one embodiment, R 1 is pentyl and its constitutional isomers. In one embodiment, R 1 is hexyl and its constitutional isomers. In one embodiment, R 1 is heptyl and its constitutional isomers. In one embodiment, R 1 is octyl and its constitutional isomers. In one embodiment, R 1 is nonyl and its constitutional isomers. In one embodiment, R 1is decyl, and its structural isomers. In Formula I, in certain of the above embodiments, useful R 2 groups include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In one embodiment, R 2 is n-pentyl, or its structural isomer. In another embodiment, R 2 is n-hexyl, or its structural isomer. In another embodiment, R 2 is n-heptyl, or its structural isomer. In another embodiment, R 2 is n-octyl, or its structural isomer. In another embodiment, R 2 is n-nonyl, or its structural isomer. In another embodiment, R 2 is n-decyl, or its structural isomer. In one embodiment, Q-R 2 is n-hexyl. In Formula I, in certain embodiments, useful R 3 groups are all as described above. In certain embodiments of Formula I above, useful R 4 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl, and its structural isomers. In another embodiment, R 4 is butyl, and its structural isomers. In another embodiment, R 4 is pentyl, and its structural isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl, and its structural isomers. In another embodiment, R 5 is butyl, and its structural isomers. In another embodiment, R 5 is pentyl, and its structural isomers. In certain embodiments of Formula I above, the present invention encompasses independent combinations of R 4 and R 5 . For example, in one embodiment, R 4 and R 5 are both methyl. In one embodiment, R 4 and R 5 are both ethyl. In one embodiment, R 4 and R 5 are independently propyl and its structural isomer, respectively. In one embodiment, R 4 and R 5Independently of each other, they are butyl and structural isomers. In one embodiment, R 4 and R 5 Independently of each other, they are pentyl and structural isomers. In one embodiment, R 4 is ethyl, and R 5 is methyl. In one embodiment, R 4 is ethyl, and R 5 Independently is propyl and its structural isomers. In one embodiment, R 4 Independently is propyl and its structural isomers; and R 5 Independently is butyl and its structural isomers. In one embodiment, R 4 Independently is butyl and its structural isomers; and R 5 Independently is pentyl and its structural isomers.

[0171] In certain embodiments, the present invention provides a compound having the structure shown in Formula II:

[0172]

[0173] Or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 5 、R 7 、R 8 and m are as described in the context of Formula I above. In certain embodiments, R 3 is hydroxyl, –OEt, –OC(O)N(H)CH2CH2NH2, –NHC(O)Me, or –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH2. In one embodiment, R 3 is hydroxyl. In one embodiment, R 3 is –OEt. In one embodiment, R 3 is –OC(O)N(H)CH2CH2NH2. In one embodiment, R 3 is –NHC(O)Me. In one embodiment, R 3 is –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH2.

[0174] In certain embodiments, the present invention provides a compound shown in Formula II, which is selected from the group consisting of:

[0175]

[0176]

[0177] its pharmaceutically acceptable salts.

[0178] In certain embodiments, the present invention provides a compound having the structure shown in Formula I:

[0179]

[0180] or a pharmaceutically acceptable salt or prodrug thereof, wherein Q is –CH2–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; wherein r is 3 or 4; and wherein a is 1. In Formula I, in one embodiment, R 1 is H. In Formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its structural isomers. In one embodiment, R 1 is butyl and its structural isomers. In one embodiment, R 1 is pentyl and its structural isomers. In one embodiment, R 1 is hexyl and its structural isomers. In one embodiment, R 1 is heptyl and its structural isomers. In one embodiment, R 1 is octyl and its structural isomers. In one embodiment, R 1 is nonyl and its structural isomers. In one embodiment, R 1 is decyl and its structural isomers. In Formula I, in certain of the above embodiments, useful R 2 groups include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In one embodiment, R 2 is n-pentyl or its structural isomers. In another embodiment, R 2 is n-hexyl or its structural isomers. In another embodiment, R 2 is n-heptyl or its structural isomers. In another embodiment, R 2 is n-octyl or its structural isomers. In another embodiment, R 2is n-nonyl, or a structural isomer thereof. In another embodiment, R 2 is n-decyl, or a structural isomer thereof. In one embodiment, Q-R 2 is n-hexyl. In Formula I, in certain embodiments, useful R 3 groups are all as described above. In certain embodiments of Formula I above, useful R 4 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl, and its structural isomers. In another embodiment, R 4 is butyl, and its structural isomers. In another embodiment, R 4 is pentyl, and its structural isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl, and its structural isomers. In another embodiment, R 5 is butyl, and its structural isomers. In another embodiment, R 5 is pentyl, and its structural isomers. In certain embodiments of Formula I above, the present invention encompasses independent combinations of R 4 and R 5 . For example, in one embodiment, R 4 and R 5 are both methyl. In one embodiment, R 4 and R 5 are both ethyl. In one embodiment, R 4 and R 5 are independently propyl, and structural isomers, respectively. In one embodiment, R 4 and R 5 are independently butyl, and structural isomers, respectively. In one embodiment, R 4 and R 5 are independently pentyl, and structural isomers, respectively. In one embodiment, R 4 is ethyl, and R 5 is methyl. In one embodiment, R 4 is ethyl, and R 5 is independently propyl, and its structural isomers. In one embodiment, R 4 is independently propyl, and its structural isomers; and R 5Independently butyl and its structural isomers. In one embodiment, R 4 is independently butyl and its structural isomers; and R 5 is independently pentyl and its structural isomers. In Formula I, in certain embodiments, useful R 7 and R 8 groups are all as described above. In certain embodiments of Formula I, R 10 is -C1-C5 alkyl. In certain embodiments, useful R 10 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 10 is methyl. In one embodiment, R 10 is ethyl. In one embodiment, R 10 is propyl and its structural isomers. In one embodiment, R 10 is butyl and its structural isomers. In one embodiment, R 10 is pentyl and its structural isomers. In one embodiment, R 10 is hexyl and its structural isomers. In one embodiment, R 10 is heptyl and its structural isomers. In one embodiment, R 10 is octyl and its structural isomers. In one embodiment, R 10 is nonyl and its structural isomers. In one embodiment, R 10 is decyl and its structural isomers. In one embodiment, r is 3. In one embodiment, r is 4.

[0181] In certain embodiments, the present invention provides a compound having the structure shown in Formula III:

[0182]

[0183] or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 5 、R 7 、R 8 、R 10 and m are all as described in the context of Formula I above. In certain embodiments, R 1 is H or methyl; and R 10 is methyl. In one embodiment, R 1 is H; and R 10 is methyl. In one embodiment, R 1 is methyl; and R 10 is methyl.

[0184] In certain embodiments, the present invention provides a compound of formula III, which is selected from the group consisting of:

[0185]

[0186]

[0187]

[0188] its pharmaceutically acceptable salts.

[0189] In certain embodiments, the present invention provides a compound having the structure of formula I:

[0190]

[0191] or a pharmaceutically acceptable salt or prodrug thereof, wherein Q is –CH2–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10 is absent; wherein r is 4; and wherein a is 1. In formula I, in one embodiment, R 1 is H. In formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its structural isomers. In one embodiment, R 1 is butyl and its structural isomers. In one embodiment, R 1 is pentyl and its structural isomers. In one embodiment, R 1 is hexyl and its structural isomers. In one embodiment, R 1 is heptyl and its structural isomers. In one embodiment, R 1 is octyl and its structural isomers. In one embodiment, R 1 is nonyl and its structural isomers. In one embodiment, R 1 is decyl and its structural isomers. In formula I, in certain of the above embodiments, useful R 2The groups include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In one embodiment, R 2 is n-pentyl, or a constitutional isomer thereof. In another embodiment, R 2 is n-hexyl, or a constitutional isomer thereof. In another embodiment, R 2 is n-heptyl, or a constitutional isomer thereof. In another embodiment, R 2 is n-octyl, or a constitutional isomer thereof. In another embodiment, R 2 is n-nonyl, or a constitutional isomer thereof. In another embodiment, R 2 is n-decyl, or a constitutional isomer thereof. In one embodiment, Q-R 2 is n-hexyl. In Formula I, in certain embodiments, useful R 3 groups are all as described above. In certain embodiments of Formula I above, useful R 4 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl, and its constitutional isomers. In another embodiment, R 4 is butyl, and its constitutional isomers. In another embodiment, R 4 is pentyl, and its constitutional isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl, and its constitutional isomers. In another embodiment, R 5 is butyl, and its constitutional isomers. In another embodiment, R 5 is pentyl, and its constitutional isomers. In certain embodiments of Formula I above, the present invention encompasses independent combinations of R 4 and R 5 . For example, in one embodiment, R 4 and R 5 are both methyl. In one embodiment, R 4 and R 5 are both ethyl. In one embodiment, R 4 and R 5 are independently propyl and its constitutional isomers, respectively. In one embodiment, R 4 and R 5 are independently butyl and its constitutional isomers, respectively. In one embodiment, R 4 and R 5Independently of each other, they are pentyl and constitutional isomers. In one embodiment, R 4 is ethyl, and R 5 is methyl. In one embodiment, R 4 is ethyl, and R 5 independently of each other are propyl and its constitutional isomers. In one embodiment, R 4 independently of each other are propyl and its constitutional isomers; and R 5 independently of each other are butyl and its constitutional isomers. In one embodiment, R 4 independently of each other are butyl and its constitutional isomers; and R 5 independently of each other are pentyl and its constitutional isomers. In formula I, in certain embodiments, useful R 7 and R 8 groups are as described above.

[0192] In certain embodiments, the present invention provides a compound having the structure shown in formula II:

[0193]

[0194]

[0195] or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 and m are as described in the context of formula I above. In certain embodiments, R 7 is hydroxyl, –N(H)C(O)CH2NH2, –N(H)C(O)CH2OH, or –N(H)CH2CH2NH2; and R 8 is H or fluorine. In one embodiment, R 7 is –N(H)C(O)CH2NH2; and R 8 is fluorine. In one embodiment, R 7 is –N(H)C(O)CH2NH2; and R 8 is H. In one embodiment, R 7 is –N(H)C(O)CH2OH; and R 8 is H. In one embodiment, R 7 is –N(H)CH2CH2NH2; and R 8 is H.

[0196] In certain embodiments, the present invention provides a compound shown in formula II, which is selected from the group consisting of:

[0197]

[0198]

[0199] Its pharmaceutically acceptable salts.

[0200] Compound, payload, or prodrug payload—Q is O

[0201] In certain embodiments, the present invention provides compounds having the structure shown in Formula I:

[0202]

[0203] or a pharmaceutically acceptable salt or prodrug thereof, wherein Q is –O–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 3 is hydroxy or –OC(O)C1-C5 alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; R 10 , when present, is -C1-C5 alkyl; where r is 3 or 4; and where a is 1. In Formula I, in one embodiment, R 1 is H. In Formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its structural isomers. In one embodiment, R 1 is butyl and its structural isomers. In one embodiment, R 1 is pentyl and its structural isomers. In one embodiment, R 1 is hexyl and its structural isomers. In one embodiment, R 1 is heptyl and its structural isomers. In one embodiment, R 1 is octyl and its structural isomers. In one embodiment, R 1 is nonyl and its structural isomers. In one embodiment, R 1 is decyl and its structural isomers. In Formula I, in certain of the above embodiments, useful R 2The groups include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In one embodiment, R 2 is n-pentyl, or a structural isomer thereof. In another embodiment, R 2 is n-hexyl, or a structural isomer thereof. In another embodiment, R 2 is n-heptyl, or a structural isomer thereof. In another embodiment, R 2 is n-octyl, or a structural isomer thereof. In another embodiment, R 2 is n-nonyl, or a structural isomer thereof. In another embodiment, R 2 is n-decyl, or a structural isomer thereof. In one embodiment of Formula I, R 2 is –CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 3 is a hydroxyl group. In certain embodiments of Formula I above, useful R 3 groups include –C(O)Me, –C(O)Et, –C(O)propyl, –C(O)butyl, and –C(O)pentyl. In one embodiment, R 3 is –C(O)Me. In another embodiment, R 3 is –C(O)Et. In another embodiment, R 3 is –C(O)propyl, and its structural isomers. In another embodiment, R 3 is –C(O)butyl, and its structural isomers. In another embodiment, R 3is –C(O) pentyl, and its structural isomers. In certain embodiments of Formula I above, useful R 4 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl, and its structural isomers. In another embodiment, R 4 is butyl, and its structural isomers. In another embodiment, R 4 is pentyl, and its structural isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl, and its structural isomers. In another embodiment, R 5 is butyl, and its structural isomers. In another embodiment, R 5 is pentyl, and its structural isomers. In certain embodiments of Formula I above, the present invention encompasses independent combinations of R 4 and R 5 For example, in one embodiment, both R 4 and R 5 are methyl. In one embodiment, both R 4 and R 5 are ethyl. In one embodiment, R 4 and R 5 are independently propyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently butyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently pentyl and its structural isomers, respectively. In one embodiment, R 4 is ethyl, and R 5 is methyl. In one embodiment, R 4 is ethyl, and R 5 is independently propyl and its structural isomers. In one embodiment, R 4 is independently propyl and its structural isomers; and R 5 is independently butyl and its structural isomers. In one embodiment, R 4 is independently butyl and its structural isomers; and R 5 is independently pentyl and its structural isomers. In Formula I, in certain embodiments, useful R 7 and R8 The groups are as described above. In certain embodiments of Formula I, R 10 is absent. In certain embodiments of Formula I, R 10 is -C1-C5 alkyl. In certain embodiments, useful R 10 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 10 is methyl. In one embodiment, R 10 is ethyl. In one embodiment, R 10 is propyl and its structural isomers. In one embodiment, R 10 is butyl and its structural isomers. In one embodiment, R 10 is pentyl and its structural isomers. In one embodiment, R 10 is hexyl and its structural isomers. In one embodiment, R 10 is heptyl and its structural isomers. In one embodiment, R 10 is octyl and its structural isomers. In one embodiment, R 10 is nonyl and its structural isomers. In one embodiment, R 10 is decyl and its structural isomers. In one embodiment, r is 3. In one embodiment, r is 4.

[0204] In certain embodiments, the present invention provides a compound having the structure shown in Formula IV:

[0205]

[0206] or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 10 and m are as described in the context of Formula I above. In certain embodiments, R 7 is H or –NH2; and R 8 is H or fluorine. In one embodiment, R 7 is –NH2; and R 8 is H. In one embodiment, R 7 is –NH2; and R 8 is fluorine.

[0207] In certain embodiments, the present invention provides a compound of Formula IV selected from the group consisting of:

[0208]

[0209] Its pharmaceutically acceptable salts.

[0210] In certain embodiments, the present invention provides a compound having the structure shown in Formula I:

[0211]

[0212] or its pharmaceutically acceptable salts or prodrugs, wherein Q is –O–; R 1 is C1-C 10 alkyl; R 2 is alkynyl; R 3 is –OC(O)C1-C5 alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10 is absent; wherein r is 4; and wherein a is 1. In Formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its structural isomers. In one embodiment, R 1 is butyl and its structural isomers. In one embodiment, R 1 is pentyl and its structural isomers. In one embodiment, R 1 is hexyl and its structural isomers. In one embodiment, R 1 is heptyl and its structural isomers. In one embodiment, R 1 is octyl and its structural isomers. In one embodiment, R 1 is nonyl and its structural isomers. In one embodiment, R 1 is decyl and its structural isomers. In one embodiment of Formula I, R 2 is –CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R2 is –CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 3 is hydroxy. In certain embodiments of Formula I above, useful R 3 groups include –C(O)Me, –C(O)Et, –C(O)propyl, –C(O)butyl, and –C(O)pentyl. In one embodiment, R 3 is –C(O)Me. In another embodiment, R 3 is –C(O)Et. In another embodiment, R 3 is –C(O)propyl and its constitutional isomers. In another embodiment, R 3 is –C(O)butyl and its constitutional isomers. In another embodiment, R 3 is –C(O)pentyl and its constitutional isomers. In certain embodiments of Formula I above, useful R 4 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl and its constitutional isomers. In another embodiment, R 4 is butyl and its constitutional isomers. In another embodiment, R 4 is pentyl and its constitutional isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl and its constitutional isomers. In another embodiment, R 5 is butyl and its constitutional isomers. In another embodiment, R 5 is pentyl and its constitutional isomers. In certain embodiments of Formula I above, the present invention encompasses R 4 and R5 Independent combinations. For example, in one embodiment, R 4 and R 5 are both methyl. In one embodiment, R 4 and R 5 are both ethyl. In one embodiment, R 4 and R 5 are independently propyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently butyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently pentyl and its structural isomers, respectively. In one embodiment, R 4 is ethyl and R 5 is methyl. In one embodiment, R 4 is ethyl and R 5 is independently propyl and its structural isomers. In one embodiment, R 4 is independently propyl and its structural isomers; and R 5 is independently butyl and its structural isomers. In one embodiment, R 4 is independently butyl and its structural isomers; and R 5 is independently pentyl and its structural isomers. In Formula I, in certain embodiments, useful R 7 and R 8 groups are as described above.

[0213] In certain embodiments, the present invention provides compounds having the structure shown in Formula V:

[0214]

[0215] or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 and m are as described in the context of Formula I above. In certain embodiments, R 7 is H or –N(H)C(O)CH2OH, –N(H)C(O)CH2NHC(O)CH2NH2, or and R 8 is H. In one embodiment, R 7 is –N(H)C(O)CH2OH; and R 8 is H. In one embodiment, R 7is –N(H)C(O)CH2NHC(O)CH2NH2; and R 8 is H. In one embodiment, R 7 is and R 8 is H.

[0216] In certain embodiments, the present invention provides a compound of formula V, which is selected from the group consisting of:

[0217]

[0218]

[0219] its pharmaceutically acceptable salts.

[0220] Compound, payload, or prodrug payload—Q is C or O

[0221] In certain embodiments, the present invention provides a compound having the structure shown in formula I:

[0222]

[0223] or its pharmaceutically acceptable salt or prodrug, wherein Q is –CH2– or –O–; R 1 is C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 3 ; R 4 and R 5 are each independently C1-C5 alkyl; R 6 is –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ; R 10 is absent; where r is 4; and where a, a1 and a2 are each independently 0 or 1. In formula I, in one embodiment, Q is –CH2–. In formula I, in one embodiment, Q is –O–. In formula I, in certain embodiments, useful R 1 groups include methyl and ethyl. In certain embodiments, useful R 1 groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and their structural isomers. In one embodiment, R 1 is methyl. In one embodiment, R 1 is ethyl. In one embodiment, R 1 is propyl and its structural isomers. In one embodiment, R 1 is butyl and its structural isomers. In one embodiment, R 1is pentyl, and its structural isomers. In one embodiment, R 1 is hexyl, and its structural isomers. In one embodiment, R 1 is heptyl, and its structural isomers. In one embodiment, R 1 is octyl, and its structural isomers. In one embodiment, R 1 is nonyl, and its structural isomers. In one embodiment, R 1 is decyl, and its structural isomers. In Formula I, in certain of the above embodiments, useful R 2 groups include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In one embodiment, R 2 is n-pentyl, or its structural isomer. In another embodiment, R 2 is n-hexyl, or its structural isomer. In another embodiment, R 2 is n-heptyl, or its structural isomer. In another embodiment, R 2 is n-octyl, or its structural isomer. In another embodiment, R 2 is n-nonyl, or its structural isomer. In another embodiment, R 2 is n-decyl, or its structural isomer. In one embodiment of Formula I, R 2 is –CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In one embodiment of Formula I, R 2 is –CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CCH. In Formula I, in certain embodiments, useful R 3 groups are all as described above. In certain of the above embodiments of Formula I, useful R4 The groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 4 is methyl. In another embodiment, R 4 is ethyl. In another embodiment, R 4 is propyl and its structural isomers. In another embodiment, R 4 is butyl and its structural isomers. In another embodiment, R 4 is pentyl and its structural isomers. In certain embodiments of Formula I above, useful R 5 groups include methyl, ethyl, propyl, butyl, and pentyl. In one embodiment, R 5 is methyl. In another embodiment, R 5 is ethyl. In another embodiment, R 5 is propyl and its structural isomers. In another embodiment, R 5 is butyl and its structural isomers. In another embodiment, R 5 is pentyl and its structural isomers. In certain embodiments of Formula I above, the present invention encompasses independent combinations of R 4 and R 5 . For example, in one embodiment, both R 4 and R 5 are methyl. In one embodiment, both R 4 and R 5 are ethyl. In one embodiment, R 4 and R 5 are independently propyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently butyl and its structural isomers, respectively. In one embodiment, R 4 and R 5 are independently pentyl and its structural isomers, respectively. In one embodiment, R 4 is ethyl, and R 5 is methyl. In one embodiment, R 4 is ethyl, and R 5 is independently propyl and its structural isomers. In one embodiment, R 4 is independently propyl and its structural isomers; and R 5 is independently butyl and its structural isomers. In one embodiment, R 4 is independently butyl and its structural isomers; and R 5 is independently pentyl and its structural isomers. In Formula I, in certain embodiments, useful R 6a and R 6bThe groups are all H. In Formula I, in certain embodiments, a is 0. In Formula I, in certain embodiments, a is 1. In Formula I, in certain embodiments, a1 is 0 and a2 is 1. In Formula I, in certain embodiments, a1 is 0 and a2 is 0. In Formula I, in certain embodiments, a1 is 1 and a2 is 0. In Formula I, in certain embodiments, a is 0, a1 is 0, and a2 is 1. In Formula I, in certain embodiments, a is 0, a1 is 0, and a2 is 0. In Formula I, in certain embodiments, a is 0, a1 is 1, and a2 is 0. In Formula I, in certain embodiments, a is 1, a1 is 0, and a2 is 1. In Formula I, in certain embodiments, a is 1, a1 is 0, and a2 is 0. In Formula I, in certain embodiments, a is 1, a1 is 1, and a2 is 0.

[0224] In certain embodiments, the present invention provides a compound having the structure shown in Formula VI:

[0225]

[0226]

[0227] or a pharmaceutically acceptable salt or prodrug thereof. In certain embodiments, Q, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are all as described in the context of Formula I above. In one embodiment, R 6 is In one embodiment, R 6 is In one embodiment, R 6 is In one embodiment, R 6 is In one embodiment, a is 0; and R 6 is In one embodiment, a is 0; and R 6 is In one embodiment, a is 0; and R 6 is In one embodiment, a is 0; and R 6 is In one embodiment, a is 1; and R 6 is In one embodiment, a is 1; and R 6 is In one embodiment, a is 1; and R6 is In one embodiment, a is 1; and R 6 is

[0228] In certain embodiments, the present invention provides compounds of formula VI, which are selected from the group consisting of:

[0229]

[0230]

[0231]

[0232] their pharmaceutically acceptable salts.

[0233] Binding agent

[0234] Suitable binding agents for any conjugate provided by the present invention include, but are not limited to, antibodies, lymphokines (e.g., IL-2 or IL-3), hormones (e.g., insulin and glucocorticoids), growth factors (e.g., EGF, transferrin, and fibronectin type III), virus receptors, interleukins, or any other class of cell-binding molecules or substances or peptide-binding molecules or substances. Binding agents also include, but are not limited to, ankyrin repeat proteins and interferons.

[0235] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody can be in any form known to those skilled in the art. As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy-chain variable region (abbreviated herein as HCVR or V H ) and a heavy-chain constant region. The heavy-chain constant region contains three domains, C H 1, C H 2, and C H 3. Each light chain contains a light-chain variable region (abbreviated herein as LCVR or V L ) and a light-chain constant region. The light-chain constant region contains one domain (C L 1). The V H region and the V L region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each V H and V LComposed of three CDRs and four FRs respectively, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed in the present invention, the FRs of the antibody (or its antigen-binding portion) applicable to the compounds of the present invention may be the same as the human germline sequence, or may be natural or artificially modified. The amino acid consensus sequence can be defined based on the juxtaposition analysis of two or more CDRs. The term "antibody" as used in the present invention also includes antigen-binding fragments of the intact antibody molecule. The term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. as used in the present invention include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The antigen-binding fragment of an antibody can be derived, for example, from the intact antibody molecule using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable domains and optionally the constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical methods or by using molecular biology techniques, for example, arranging one or more variable domains and / or constant domains in a suitable configuration, or introducing codons to generate cysteine residues, modifying, adding, or deleting amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues of the hypervariable regions of mimetic antibodies (e.g., isolated CDRs, such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibody classes, single-domain antibody classes, domain-deleted antibody classes, chimeric antibody classes, CDR-grafted antibody classes, diabody classes, triabody classes, tetrabody classes, microantibody classes, nanobody classes (e.g., monovalent nanobody classes, bivalent nanobody classes, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included within the expression "antigen-binding fragment" as used in the present invention. The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can have any size or amino acid composition and generally contains at least one CDR, and the at least one CDR is adjacent to or within one or more framework sequences. In an antigen-binding fragment having a V L domain-related V H domain, the V H and V LThe domains can be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimeric and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, the antigen-binding fragment of the antibody can comprise a monomeric V H or V L domain. In certain embodiments, the antigen-binding fragment of the antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L. In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly connected to each other or can be connected through a full or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Like a full antibody molecule, an antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of an antibody generally contain at least two different variable domains, where each variable domain is capable of specifically binding a separate antigen or binding different epitopes on the same antigen. Any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed in the present invention, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art. In certain embodiments of the present invention, the antibodies of the present invention are human antibodies. The term "human antibody" as used in the present invention is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), such as in the CDRs, particularly in CDR3. However, the term "human antibody" as used in the present invention is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto a human framework sequence. The term "human antibody" does not include naturally occurring molecules that are typically present without modification or human intervention / manipulation in a naturally occurring, unmodified organism. In some embodiments, the antibodies of the present invention can be recombinant human antibodies. The term "recombinant human antibody" as used in the present invention is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below); antibodies isolated from a recombinant, combinatorial human antibody library (described further below); antibodies isolated from an animal (e.g., mouse) that is a transgenic animal for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies prepared, expressed, produced or isolated by any other method involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are mutagenized in vitro (or, when using an animal with transgenic human Ig sequences, by in vivo somatic mutagenesis), and thus the V H regions and V LThe amino acid sequences of the regions are derived from and related to human germline V H and V L sequences but may not be naturally present in the human antibody germline repertoire in vivo. Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150 - 160 kDa, wherein the dimers are linked together by inter-chain heavy chain disulfide bonds. In the second form, the dimers are not linked by inter-chain disulfide bonds and form a molecule of approximately 75 - 80 kDa, which consists of covalently coupled light and heavy chains (half-antibodies). These forms are extremely difficult to separate even after affinity purification. The frequency of occurrence of the second form among the various intact IgG isotypes is due to, but not limited to, structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the frequency of occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. The present disclosure includes in the hinge region, C H 2 region or C HAntibodies having one or more mutations in region 3, said mutations may be, for example, required in production to increase the yield of the desired antibody form. The antibodies of the present invention may be isolated antibodies. The "isolated antibody" used in the present invention refers to an antibody that has been identified, isolated, and / or recovered from at least one component of its natural environment. For example, for the purposes of the present invention, an antibody that has been isolated or removed from at least one component of an organism or from the tissue or cells in which the antibody naturally exists or is naturally produced is an "isolated antibody". Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular materials and / or chemicals. Compared with the corresponding germline sequences of the derived antibodies, the antibodies used in the present invention may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be easily determined by comparing the amino acid sequences disclosed in the present invention with germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies and their antigen-binding fragments derived from any amino acid sequence disclosed in the present invention, wherein one or more amino acids in one or more frameworks and / or CDRs are mutated to the corresponding residues of the germline sequence of the derived antibody, or to the corresponding residues of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to as "germline mutations" in the present invention). Those of ordinary skill in the art can easily generate many antibodies and antigen-binding fragments starting from the heavy and light chain variable region sequences disclosed in the present invention, said antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., mutated residues found only within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence of the originally derived antibody). Additionally, the antibodies of the invention can comprise any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residues of a specific germline sequence while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, the antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced agonistic or antagonistic biological properties (as appropriate), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are all included in the invention. Antibodies useful for the compounds of the invention also include such antibodies that comprise variants of any one of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by amino acids from different segments of a linear polypeptide chain juxtaposed in space. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain embodiments, an epitope can include a carbohydrate, phosphoryl, or sulfonyl group moiety on an antigen.

[0236] In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a κ light chain. In certain embodiments, the light chain is a λ light chain. In certain embodiments, the antibody comprises a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2.

[0237] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is an F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is a scFv (sFv) fragment. In some embodiments, the antibody fragment is a scFv-Fc fragment.

[0238] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a bispecific antibody that includes a first antigen-binding domain (also referred to herein as "D1") and a second antigen-binding domain (also referred to herein as "D2").

[0239] As used herein, the term "antigen-binding domain" refers to any peptide, polypeptide, nucleic acid molecule, scaffold molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding to a particular antigen of interest (e.g., PRLR or STEAP2). As used herein, terms such as "specifically binds" mean that the antigen-binding domain forms a complex with a particular antigen, characterized by a dissociation constant (K D ) of 1 μM or less and does not bind to other unrelated antigens under typical assay conditions. "Unrelated antigens" are proteins, peptides, or polypeptides that have less than 95% amino acid identity to each other.

[0240] Exemplary classes of antigen-binding domains that can be used in the context of the present invention include antibody classes, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptidomimetics), receptor molecules that specifically interact with a particular antigen, proteins that contain the ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPin classes, HEAT repeat proteins, ARM repeat proteins, triangular tetrapeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamer classes or portions thereof.

[0241] Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, antigen-binding domains used in the context of the present invention include polypeptides that bind to a particular antigen (e.g., target molecule [T] or internalizing effector protein [E]) or a portion thereof, as determined by surface plasmon resonance assay, with a K DLess than about 1 μM, less than about 500 nM, less than about 250 nM, less than about 125 nM, less than about 60 nM, less than about 30 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM.

[0242] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0243] In some embodiments, the antibody is an anti-PSMA, anti-PRLR, anti-MUC16, anti-HER2, anti-EGFRvIII, anti-MET, or anti-STEAP2 antibody. In some embodiments, the antibody or antigen-binding fragment is anti-PSMA. In some embodiments, the antibody or antigen-binding fragment is anti-MUC16. In some embodiments, the antibody or antigen-binding fragment is anti-HER2. In some embodiments, the antibody or antigen-binding fragment is anti-EGFRvIII. In some embodiments, the antibody or antigen-binding fragment is anti-MET. In some embodiments, the antibody or antigen-binding fragment is anti-PRLR or anti-STEAP2. In some embodiments, the antibody is an anti-PRLR or anti-HER2 antibody. In some embodiments, the antibody or its antigen-binding fragment is anti-STEAP2. In some embodiments, the antibody or its antigen-binding fragment is anti-PRLR.

[0244] The antibody can have binding specificity for any antigen that would be considered suitable by those skilled in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor). In one embodiment, the antigen is expressed on a tumor. In some embodiments, the binding agent interacts with or binds to a tumor antigen, including an antigen specific for one type of tumor or an antigen that is common, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on a solid tumor. Exemplary antigens include, but are not limited to, lipoproteins; α1 - antitrypsin; cytotoxic T lymphocyte - associated antigen (CTLA), such as CTLA - 4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or protein D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto (teratocarcinoma - derived growth factor antigen), αvβ6 (alphavbeta6), integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins, such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, or antibodies that bind to one or more tumor - associated antigens or cell - surface receptors, which are disclosed in U.S. Patent Publication No. 2008 / 0171040 or U.S. Patent Publication No. 2008 / 0305044, each of which is incorporated herein by reference in its entirety; erythropoietin; osteogenic inductive factors; immunotoxins; bone morphogenetic protein (BMP); T - cell receptors; surface membrane proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA - 4, and VCAM;Tumor-associated antigens, such as AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, endoglin, Epcam, EphA2, ErbB2 / Her2, ErbB3 / Her3, ErbB4 / Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / EBNA1, HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins, MART-1, mesothelin, ML-IAP, Muc1, Muc16, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1 (prostate transmembrane epithelial antigen-1), Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, and fragments of any of the polypeptides listed above; antigen classes expressed on the cell surface; MUC16; c-MET;Molecule classes, such as class A scavenger receptor classes (including scavenger receptor A (SR-A)), and other membrane protein classes, such as B7 family-related members (including V-set and Ig domain-containing protein 4 (VSIG4)), colony-stimulating factor 1 receptor (CSF1R), asialoglycoprotein receptor (ASGPR), and amyloid beta precursor-like protein 2 (APLP-2). In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antigen is STEAP2. In some embodiments, the antigen is human STEAP2. In some embodiments, the MAGE protein classes are selected from MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12. In some embodiments, the GAGE protein classes are selected from GAGE-1 and GAGE-2.;

[0245] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, GPNMB, and UPK3A. Exemplary antigens also include, but are not limited to, MUC16, STEAP2, and HER2.

[0246] In some embodiments, the antigen includes MUC16. In some embodiments, the antigen includes STEAP2. In some embodiments, the antigen includes PSMA. In some embodiments, the antigen includes HER2. In some embodiments, the antigen is prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA). In some embodiments, the antigen is MUC16. In some embodiments, the antigen includes PSMA. In some embodiments, the antigen is HER2. In some embodiments, the antigen is STEAP2.

[0247] In certain embodiments, the antibody contains a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present invention, this position is referred to as glutamine 295, or Gln295, or Q295. Those skilled in the art will recognize that it is a conserved glutamine residue in the wild-type sequences of many antibodies. In other useful embodiments, the antibody can be designed to contain a glutamine residue. In certain embodiments, the antibody contains one or more N297Q mutations. Techniques for modifying the antibody sequence to include a glutamine residue are all within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol.).

[0248] In some embodiments, the antibody or antigen-binding fragment thereof conjugated to a linker-payload or payload can be an antibody targeting STEAP2. Suitable anti-STEAP2 antibodies or antigen-binding fragments thereof include, for example, those in International Publication No. WO2018 / 058001 A1, including those comprising the amino acid sequences disclosed in Table 1 on page 75 thereof. In some embodiments, the anti-STEAP2 antibody is H1H7814N of WO 2018 / 058001 A1, which comprises the CDRs of H1M7814N in the same publication. In some embodiments, the anti-STEAP2 antibody comprises: heavy chain complementarity-determining region (HCDR)-1, which comprises SEQ ID NO: 2; HCDR2, which comprises SEQ ID NO: 3; HCDR3, which comprises SEQ ID NO: 4; light chain complementarity-determining region (LCDR)-1, which comprises SEQ ID NO: 6; LCDR2, which comprises SEQ ID NO: 7; and LCDR3, which comprises SEQ ID NO: 8. In some embodiments, the anti-STEAP2 antibody comprises: a heavy chain variable region (HCVR) comprising SEQ ID NO: 1 and a light chain variable region (LCVR) comprising SEQ ID NO: 5. In any of the foregoing embodiments, the anti-STEAP2 antibody can be prepared by site-directed mutagenesis to insert a glutamine residue in situ without causing antibody function or binding failure. For example, in any of the foregoing embodiments, the anti-STEAP2 antibody can comprise an Asn297Gln (N297Q) mutation. Such antibodies with the N297Q mutation can also comprise one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to transglutaminase and thus capable of conjugating to a payload or linker-payload (Table A). In certain embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 1; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the amino acid sequence of the light chain variable region (LCVR) of SEQ ID NO: 5. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR amino acid sequence of SEQID NO: 1; and the LCVR amino acid sequence of SEQ ID NO: 5. International Publication No. WO2018 / 058001A1 is hereby incorporated by reference in its entirety into the present invention.

[0249] In some embodiments, the antibody or antigen-binding fragment thereof conjugated to a linker-payload or payload can be an antibody that targets the human prolactin receptor (PRLR). Suitable anti-PRLR antibodies or antigen-binding fragments thereof include, for example, those in International Publication No. WO2015 / 026907A1, including those comprising the amino acid sequences disclosed in Table 1 on page 36 thereof. In some embodiments, the anti-PRLR antibody is H1H6958N2 of WO2015 / 026907A1, which comprises the CDRs of H2M6958N2 in the same publication. In some embodiments, the anti-PRLR antibody comprises: heavy chain complementarity determining region (HCDR)-1, which comprises SEQ ID NO: 10; HCDR2, which comprises SEQ ID NO: 11; HCDR3, which comprises SEQ ID NO: 12; light chain complementarity determining region (LCDR)-1, which comprises SEQ ID NO: 14; LCDR2, which comprises SEQ ID NO: 15; and LCDR3, which comprises SEQ ID NO: 16. In some embodiments, the anti-PRLR antibody comprises: a heavy chain variable region (HCVR) comprising SEQ ID NO: 9 and a light chain variable region (LCVR) comprising SEQ ID NO: 13. In any of the foregoing embodiments, the anti-PRLR antibody can be prepared by site-directed mutagenesis to insert a glutamine residue in situ without causing antibody dysfunction or binding failure. For example, in any of the foregoing embodiments, the anti-PRLR antibody can comprise an Asn297Gln (N297Q) mutation. Such antibodies with an N297Q mutation can also comprise one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to transglutaminase and thus capable of conjugating to a payload or linker-payload (Table A). In certain embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 9; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the amino acid sequence of the light chain variable region (LCVR) of SEQ ID NO: 13. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of the HCVR of SEQ ID NO: 9; and the amino acid sequence of the LCVR of SEQ ID NO: 13. International Publication No. WO2015 / 026907A1 is hereby incorporated by reference in its entirety into the present invention.

[0250] Table A. Sequences of exemplary antibodies H1H7814N (anti-STEAP2) and H1H6958N2 (anti-PRLR)

[0251]

[0252]

[0253] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises an HCVR, and the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0254] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises an LCVR, and the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0255] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), and the HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprises any HCVR amino acid sequence listed in Table A paired with any LCVR amino acid sequence listed in Table A. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises an HCVR / LCVR amino acid sequence pair, and the HCVR / LCVR amino acid sequence pair is comprised in any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of 250 / 258; as described in International Publication No. WO2018 / 058001A1, the content of which is incorporated herein by reference in its entirety.

[0256] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a heavy chain CDR1 (HCDR1), and the heavy chain CDR1 (HCDR1) comprises an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0257] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a heavy chain CDR2 (HCDR2), and the heavy chain CDR2 (HCDR2) comprises an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0258] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a heavy chain CDR3 (HCDR3), and the heavy chain CDR3 (HCDR3) comprises an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0259] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a light chain CDR1 (LCDR1), and the light chain CDR1 (LCDR1) comprises an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0260] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a light chain CDR2 (LCDR2), and the light chain CDR2 (LCDR2) comprises an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0261] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a light chain CDR3 (LCDR3), and the light chain CDR3 (LCDR3) comprises an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0262] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), and the pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3) comprises any HCDR3 amino acid sequence listed in Table A paired with any LCDR3 amino acid sequence listed in Table A. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises a pair of HCDR3 / LCDR3 amino acid sequences, and the pair of HCDR3 / LCDR3 amino acid sequences is comprised in any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the pair of HCDR3 / LCDR3 amino acid sequences is selected from the group consisting of 256 / 254; as described in International Publication No. WO2018 / 058001A1, the content of which is incorporated herein by reference in its entirety.

[0263] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a collection of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), and the collection of the six CDRs is comprised in any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the collection of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is selected from the group consisting of: 252-254-256-260-262-264; as described in International Publication No. WO2018 / 058001A1, the content of which is incorporated herein by reference in its entirety.

[0264] In related embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises a collection of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), and the collection of six CDRs is contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-STEAP2 antibodies listed in Table A. For example, the present invention includes an antibody or an antigen-binding fragment thereof that specifically binds to STEAP2, which comprises the collection of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences, and the collection of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of 250 / 258; as described in International Publication No. WO2018 / 058001A1, the content of which is incorporated herein by reference in its entirety. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify the CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed in the present invention. Exemplary conventions or practices for identifying CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0265] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises an HCVR, and the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table A, or a substantially similar sequence, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0266] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises an LCVR, and the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0267] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises an amino acid sequence pair of HCVR and LCVR (HCVR / LCVR), and the amino acid sequence pair of HCVR and LCVR (HCVR / LCVR) comprises any HCVR amino acid sequence listed in Table A paired with any LCVR amino acid sequence listed in Table A. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises an HCVR / LCVR amino acid sequence pair, and the HCVR / LCVR amino acid sequence pair is comprised in any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of 18 / 26, 66 / 74, 274 / 282, 290 / 298, and 370 / 378; as described in International Publication No. WO2015 / 026907A1, the content of which is incorporated herein by reference in its entirety.

[0268] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a heavy chain CDR1 (HCDR1), and the heavy chain CDR1 (HCDR1) comprises an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0269] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a heavy chain CDR2 (HCDR2), and the heavy chain CDR2 (HCDR2) comprises an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0270] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a heavy chain CDR3 (HCDR3), and the heavy chain CDR3 (HCDR3) comprises an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table A, or a substantially similar sequence thereto, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0271] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a light chain CDR1 (LCDR1), and the light chain CDR1 (LCDR1) comprises an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table A, or a substantially similar sequence thereof, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0272] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a light chain CDR2 (LCDR2), and the light chain CDR2 (LCDR2) comprises an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table A, or a substantially similar sequence thereof, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0273] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a light chain CDR3 (LCDR3), and the light chain CDR3 (LCDR3) comprises an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table A, or a substantially similar sequence thereof, and the substantially similar sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0274] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to PRLR, which comprises a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), and the pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3) comprises any HCDR3 amino acid sequence listed in Table A paired with any LCDR3 amino acid sequence listed in Table A. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that comprises an HCDR3 / LCDR3 amino acid sequence pair, and the HCDR3 / LCDR3 amino acid sequence pair is comprised in any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of 24 / 32, 72 / 80, 280 / 288, 296 / 304, and 376 / 384; as described in International Publication No. WO2015 / 026907A1, the content of which is incorporated herein by reference in its entirety.

[0275] The present invention also provides an antibody or antigen-binding fragment thereof that specifically binds to PRLR, which comprises a collection of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), and the collection of six CDRs is comprised in any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the collection of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is selected from the group consisting of: 20-22-24-28-30-32, 68-70-72-76-78-80, 276-278-280-284-286-288, 292-294-296-300-302-304, and 372-374-376-380-382-384; as described in International Publication No. WO2015 / 026907A1, the content of which is incorporated herein by reference in its entirety.

[0276] In related embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to PRLR and comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), wherein the set of six CDRs is comprised within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-PRLR antibodies listed in Table A. For example, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to PRLR and comprises the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences, wherein the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is comprised within an HCVR / LCVR amino acid sequence pair selected from the group consisting of 18 / 26, 66 / 74, 274 / 282, 290 / 298, and 370 / 378; as described in International Publication No. WO2015 / 026907A1, the contents of which are incorporated herein by reference in their entirety. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify the CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed by the present invention. Exemplary conventions or practices for identifying CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0277] The linker can be linked to the binding agent (e.g., an antibody or antigen-binding molecule) by attachment at a specific amino acid within the antibody or antigen-binding molecule. Exemplary amino acid attachments that can be used in the context of this embodiment of the invention include, for example, lysine (see, e.g., US 5,208,020; US 2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358–361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / 0585592); cysteine (see, e.g., US 2007 / 0258987; WO 2013 / 055993; WO 2013 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 0101546; and US 7,750,116); selenocysteine (see, e.g., WO 2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451–12456); formylglycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321–322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46–51, and Rabuka et al., Nat. Protocols, 2012, 10:1052–1067); unnatural amino acids (see, e.g., WO 2013 / 068874, and WO 2012 / 166559), and acidic amino acids (see, e.g., WO 2012 / 05982). The linker can also be conjugated to an antigen-binding protein by attachment to a carbohydrate (see, e.g., US 2008 / 0305497, WO 2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127–130).

[0278] In some embodiments, the binding agent is an antibody or antigen-binding molecule, and the antibody is linked to the linker by a lysine residue. In some embodiments, the antibody or antigen-binding molecule is linked to the linker by a cysteine residue.

[0279] The linker can also be conjugated to one or more glutamine residues by transglutaminase-based chemoenzymatic conjugation (see, e.g., Dennler et al., Bioconjugate Chem. 2014, 25, 569–578). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be conjugated to a primary amine compound. Primary amine compounds include, for example, a payload or a linker-payload, which directly provides a transglutaminase-modified antibody-drug conjugate by transglutaminase-mediated conjugation. Primary amine compounds also include linkers and spacers functionalized with reactive groups, which can then be reacted with further compounds to synthesize an antibody-drug conjugate (e.g., in certain embodiments, a transglutaminase-modified antibody-drug conjugate). Antibodies containing glutamine residues can be isolated from natural sources or engineered to contain one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain (a glutaminyl-modified antibody or antigen-binding molecule) are within the skill of the art. In certain embodiments, the antibody is aglycosylated.

[0280] In certain embodiments, the antibody, glutaminyl-modified antibody, or transglutaminase-modified antibody, or an antigen-binding fragment thereof, contains at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody, glutaminyl-modified antibody, or transglutaminase-modified antibody, or an antigen-binding fragment thereof, contains two heavy chain polypeptides, each having a Gln295 or Q295 residue. In further embodiments, the antibody, glutaminyl-modified antibody, or transglutaminase-modified antibody, or an antigen-binding fragment thereof, contains one or more glutamine residues at sites other than heavy chain 295. The invention includes the antibodies of this section having the N297Q mutation described in the present invention.

[0281] Primary amine compounds

[0282] In certain embodiments, the primary amine compounds useful for transglutaminase-mediated conjugation of an antibody (or antigen-binding compound) containing one or more glutamine residues (i.e., to produce a transglutaminase-modified antibody or an antigen-binding fragment thereof) can be any primary amine compound that would be considered useful by one of ordinary skill in the art. Generally, a primary amine compound has the structure shown by the formula H2N-R, where R can be any group compatible with the antibody and the reaction conditions. In certain embodiments, R is an alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl.

[0283] In some embodiments, the primary amine compound comprises an active group or a protected active group. Useful active groups include azides, alkynes, cycloalkynes, thiols, alcohols, ketones, aldehydes, carboxylic acids, esters, amides, hydrazides, anilines, and amines. In certain embodiments, the active group is selected from the group consisting of azide, alkyne, thiol, cycloalkyne, aldehyde, and carboxyl.

[0284] In certain embodiments, the primary amine compound has the structure shown by the formula H2N-LL-X, where LL is a divalent spacer group, and X is an active group or a protected active group. In a particular embodiment, LL is a divalent polyethylene glycol (PEG) group. In certain embodiments, X is selected from the group consisting of -SH, –N3, alkyne, aldehyde, and tetrazole. In a particular embodiment, X is –N3.

[0285] In certain embodiments, the primary amine compound is a structure having one of the following general formulas:

[0286] H2N-(CH2) n -X;

[0287] H2N-(CH2CH2O) n -(CH2) p -X;

[0288] H2N-(CH2) n -N(H)C(O)-(CH2) m -X;

[0289] H2N-(CH2CH2O) n -N(H)C(O)-(CH2CH2O) m -(CH2) p -X;

[0290] H2N-(CH2) n -C(O)N(H)-(CH2) m -X;

[0291] H2N-(CH2CH2O) n -C(O)N(H)-(CH2CH2O) m -(CH2) p -X;

[0292] H2N-(CH2) n -N(H)C(O)-(CH2CH2O) m -(CH2) p -X;

[0293] H2N-(CH2CH2O) n-N(H)C(O)-(CH2) m -X;

[0294] H2N-(CH2) n -C(O)N(H)-(CH2CH2O) m -(CH2) p -X; and

[0295] H2N-(CH2CH2O) n -C(O)N(H)-(CH2) m -X;

[0296] where n is an integer selected from 1 to 12;

[0297] m is an integer selected from 0 to 12;

[0298] p is an integer selected from 0 to 2;

[0299] and X is selected from the group consisting of: –SH, –N3, –C≡CH, –C(O)H, tetrazole, and one of the following:

[0300]

[0301] In the foregoing, any of the alkyl or alkylene (i.e., -CH2-) groups may optionally be substituted, for example, with C 1-8 alkyl, methylformyl, or –SO3H. In certain embodiments, the alkyl groups are unsubstituted.

[0302] In certain embodiments, the primary amine compound is selected from the group consisting of:

[0303]

[0304] In a particular embodiment, the primary amine compound is:

[0305]

[0306] Exemplary conditions for the above reaction are provided in the following examples.

[0307] Linker

[0308] In certain embodiments, the linker L portion of the conjugate of the present invention is a group portion, such as a divalent group portion, which covalently links the binder to the payload compound of the present invention. In other embodiments, the linker L is a trivalent or polyvalent group portion that covalently links the binder to the payload compound of the present invention. Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins; Phillips, G.L., Ed.; Springer-Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, J.L., Eds.; Springer International Publishing, 2015, the contents of each of which are incorporated herein by reference in their entirety. In certain embodiments, the linker L portion of the linker-payload or linker-prodrug payload of the present invention is a group portion covalently linked to the payload or prodrug payload compound of the present invention and capable of covalently linking the binder divalently to the payload or prodrug payload compound of the present invention. In other embodiments, the linker L portion of the linker-payload of the present invention is a group portion covalently linked to the payload or prodrug payload compound of the present invention and capable of covalently linking the binder to the payload or prodrug payload compound of the present invention as a trivalent or polyvalent group portion. The payload or prodrug payload compounds include the compounds of formulas I, Ia, Iaa, II, III, IV, V, and VI above, and the residues after their connection or incorporation with the linker L are linker-payload or linker-prodrug payloads. The linker-payload can be further linked to a binder such as an antibody or an antigen-binding fragment thereof to form an antibody-drug conjugate. Those skilled in the art will recognize that certain functional groups of the payload group portion facilitate connection to the linker and / or the binder. For example, in certain embodiments, there is no linker, and the payload or prodrug payload is directly linked to the binder. In one embodiment, the payload or prodrug payload includes terminal alkynes, and the binder includes azides, where each alkyne and azide participate in regioselective click chemistry to directly link the payload residue or prodrug payload residue to the binder residue. In another embodiment, the payload or prodrug payload includes carboxylic acids, and the binder includes lysine, where each carboxylic acid and lysine participate in amide bond formation to directly link the payload residue or prodrug payload residue to the binder residue.The payload functional groups further include amines (e.g., of formulae C, D, E, LPc, LPd, and LPe), quaternary ammonium ions (e.g., of formulae A and LPa), hydroxyl groups (e.g., of formulae C, D, E, LPc, LPd, and LPe), phosphate esters, carboxylic acids (e.g., in the form of esters when linked to L, as shown in formulae B, D, LPb, and LPd), hydrazides (e.g., of formulae B and LPb), amides (e.g., anilines derived from formula C and LPc, or amines derived from formulae D, E, LPd, and LPe), and saccharides.

[0309] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, e.g., capable of releasing at least the payload moiety in the presence of an enzyme or within a specific pH range or pH value. In some embodiments, the linker comprises an enzyme-cleavable group moiety. Exemplary enzyme-cleavable group moieties include, but are not limited to, peptide bonds (i.e., distinct from prodrug payloads having peptide bonds, as described elsewhere in the present invention), ester bonds, hydrazones, β-glucuronide bonds, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker. In some embodiments, the linker comprises a β-glucuronidase (GUSB)-cleavable linker (see, e.g., GUSB linkers from Creative Biolabs, creative-biolabs.com / adc / beta-glucuronide-linker.htm, or ACS Med. Chem. Lett. 2010, 1:277–280).

[0310] In some embodiments, the linker comprises a non-cleavable group moiety. In some embodiments, the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is or a regioisomer thereof. In some embodiments, the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is or a regioisomer thereof. In one embodiment, the linker is maleimidocyclohexanecarboxylate or 4-(N-maleimidomethyl)cyclohexanecarboxylic acid (MCC). In the structure, represents the bond linked to the binder. In the structure, in some embodiments, represents a click chemistry residue generated by the reaction of a binder, e.g., having an azide or alkyne functional group, with a linker-payload having a complementary alkyne or azide functional group. In the structure, in other embodiments, represents a divalent sulfide produced by the reaction of, for example, one or more binder cysteines with one or more linkers or linker-payloads having maleimide functional groups via a Michael addition reaction. In said structure, in other embodiments, represents an amide bond produced by the reaction of, for example, one or more binder lysines with one or more linkers or linker-payloads having activated or unactivated carboxyl functional groups, as understood by those skilled in the art. In one embodiment, represents an amide bond produced by the reaction of, for example, one or more binder lysines with one or more linkers or linker-payloads having activated carboxyl functional groups, as understood by those skilled in the art.

[0311] In some embodiments, suitable linkers include, but are not limited to, those that chemically bond to two cysteine residues of a single binder (e.g., an antibody). Such linkers can be used to mimic the disulfide bonds of the antibody that are disrupted due to the coupling process.

[0312] In some embodiments, the linker comprises one or more amino acids (i.e., distinct from a prodrug payload that comprises peptide bonds derived from distinguishable amino acids, as described elsewhere in the present invention). Suitable amino acids include natural, unnatural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-type α-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, or derivatives thereof, or any combination thereof (e.g., dipeptides, tripeptides, oligopeptides, polypeptides, etc.). In certain embodiments, one or more side chains of the amino acids are linked to side chain groups as described below. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: valine and citrulline (e.g., divalent –Val-Cit– or divalent –VCit–). In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: alanine and alanine, or divalent –AA–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: glutamic acid and alanine, or –EA–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: glutamic acid and glycine, or –EG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: glycine and glycine, or –GG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: glutamine, valine, and citrulline, or –Q-V-Cit– or –QVCit–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: glutamic acid, valine, and citrulline, or –E-V-Cit– or –EVCit–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGGGS–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGGGG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGGGK–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GFGG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGGG–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –GGFG–.In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: lysine, valine, and citrulline, or –KVCit–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –KVA–. In some embodiments, the linker is a peptide comprising or consisting of the following amino acids: –VA–. As will be understood by those skilled in the art, in any of the embodiments of this paragraph, and throughout the present invention, standard three-letter or single-letter amino acid nomenclature is used. Exemplary single-letter amino acid names include G for glycine, K for lysine, S for serine, V for valine, A for alanine, and F for phenylalanine.

[0313] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can be any such group known to those skilled in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group is capable of undergoing a chemical reaction that releases the remaining atoms of the linker from the payload.

[0314] In some embodiments, the linker is:

[0315]

[0316] wherein:

[0317] SP 1 is a spacer group;

[0318] SP 2 is a spacer group;

[0319] are one or more bonds connecting to the binder;

[0320] are one or more bonds connecting to the payload;

[0321] each AA is an amino acid residue respectively; and

[0322] p is an integer from 0 to 10.

[0323] In certain embodiments, each AA within linker L of the present invention can be characterized as a second amino acid residue, in contrast to the first amino acid residue within a payload or prodrug payload described elsewhere in the present invention. As will be understood by those skilled in the art, in certain embodiments, more than one AA within linker L of the present invention can be characterized as a second peptide residue, in contrast to the first peptide residue within a payload or prodrug payload described elsewhere in the present invention.

[0324] SP 1 A spacer group is a group that links an (AA) p moiety or residue to a binder (BA) or to a group moiety of an active group residue linked to the BA. Suitable SP 1 spacer groups include, but are not limited to, those groups containing an alkylene group or a polyether or both an alkylene group and a polyether. The terminus of the spacer group, e.g., the portion of the spacer group that links to the BA or AA, can be a group moiety derived from the active group moiety that is used for the purpose of coupling an antibody or AA to the spacer group during the chemical synthesis of the conjugate. In certain embodiments, p is 0, 1, 2, 3, or 4. In a particular embodiment, p is 2. In a particular embodiment, p is 3. In a particular embodiment, p is 4.

[0325] In some embodiments, SP 1 the spacer group contains an alkylene group. In some embodiments, SP 1 the spacer group contains a C 5-7 alkylene group. In some embodiments, SP 1 the spacer group contains a polyether. In some embodiments, SP 1 the spacer group contains a polymer of ethylene oxide, such as polyethylene glycol.

[0326] In some embodiments, SP 1 the spacer group is:

[0327]

[0328] wherein:

[0329] RG' is the active group residue after the reaction of the active group RG with the binder;

[0330] is a bond linked to the binder;

[0331] is a bond linked to the (AA) p where p is an integer from 0 to 10; and

[0332] b is an integer from 2 to 8.

[0333] The reactive group RG can be any reactive group known to those skilled in the art that is capable of forming one or more bonds with a binder. The reactive group RG is a group moiety that contains, in its structure, a moiety capable of reacting with a binder (e.g., reacting with an antibody at a cysteine or lysine residue or azide group portion of the antibody, e.g., reacting with a PEG-N3 functionalized antibody at one or more glutamine residues) to form a compound represented by formula A, A′, B, B′, C, C′, D, D′, E, or E′. After coupling with the binder, the reactive group becomes a reactive group residue (RG′). Exemplary reactive groups include, but are not limited to, those containing a haloacetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, or maleimide moiety capable of reacting with a binder.

[0334] In certain embodiments, the reactive group includes, but is not limited to, alkynes. In certain embodiments, the alkyne is an alkyne capable of undergoing a 1,3-cycloaddition reaction with an azide in the absence of a copper catalyst, such as strained alkynes. Strained alkynes are those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, and benzocyclized alkynes. Suitable alkynes include, but are not limited to, dibenzocyclooctyne or (DIBAC); dibenzocyclooctyne or (DIBO); diarylazacyclooctynone or (BARAC); difluorocyclooctyne or (DIFO); substituted, e.g., fluorinated alkynes, azacycloalkynes, bicyclo[6.1.0]nonyne or (BCN); and derivatives thereof. Particularly useful alkynes include

[0335] In certain embodiments, the binder is directly linked to RG′. In certain embodiments, the binder is linked to RG′ through a spacer such as SP 4 (located and RG′). In a particular embodiment, the binder is linked to RG′ through SP 4(e.g., a PEG spacer group) is indirectly linked to RG'. As discussed in detail below, in certain embodiments, the binder is prepared by functionalizing with one or more azide groups. Each azide group is capable of reacting with RG to form RG' separately. In a particular embodiment, the binder is derived from –PEG-N3 linked to a glutamine residue (e.g., a binder modified by transglutaminase). The present invention provides exemplary –N3-derived binders, methods for their preparation, and methods for their use in reacting with RG. In certain embodiments, RG is an alkyne suitable for participating in a 1,3-cycloaddition, and RG' is a regioisomeric 1,2,3-triazolyl group moiety formed by the reaction of RG with an azide-functionalized binder. As a further example, in certain embodiments, RG' is linked to the binder as shown in or a mixture of the regioisomers. Each R and R' has the definitions described in the present invention or as exemplified in the present invention.

[0336] SP 2 The spacer group, when present, is the group moiety that links the (AA) p portion to the payload. Suitable spacer groups include, but are not limited to, those described above as the SP 1 spacer group. Other suitable SP 2 spacer groups include, but are not limited to, those containing an alkylene or a polyether or both an alkylene and a polyether. The terminus of the SP 2 spacer group (e.g., the portion of the spacer group directly linked to the payload, prodrug payload, or AA) can be a group moiety derived from an active group moiety for the purpose of coupling the payload, prodrug payload, or AA to the SP 2 spacer group during the process of chemically synthesizing the conjugate. In some embodiments, the terminus of the SP 2 spacer group (e.g., the portion of the SP 2 spacer group directly linked to the payload, prodrug payload, or AA) can be a residue of an active group moiety for the purpose of coupling the payload, prodrug payload, or AA to the spacer group during the process of chemically synthesizing the conjugate.

[0337] In some embodiments, the SP 2 spacer group, when present, is selected from the group consisting of: –NH-(p-C6H4)-CH2–, –NH-(p-C6H4)-CH2OC(O)–, amino acids, dipeptides, tripeptides, oligopeptides, –O–, –N(H)–,

[0338] and any combination thereof. In certain embodiments, each are a bond connected to the payload or prodrug payload, respectively, and each are respectively a bond connected to (AA) p bond.

[0339] In the above general formula, each (AA) p is respectively an amino acid, or optionally a p-aminobenzyloxycarbonyl residue (PABC),

[0340] If PABC is present, only one PABC is present in certain embodiments. In some embodiments, if a PABC residue is present, it is connected to the terminal AA in the (AA) p group and is proximal to the payload or prodrug payload. If is present, only is present. In some embodiments, the residue, if present, is connected to the payload or prodrug payload through a benzyloxycarbonyl group moiety and no AA is present. In some embodiments, the residue, if present, is connected to the payload or prodrug payload via –O–. Suitable amino acids for each AA include natural, unnatural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-type α-amino acids. In some embodiments, the AA comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or any combination thereof (e.g., dipeptides, tripeptides, and oligopeptides, etc.). In certain embodiments, one or more side chains of the amino acid are connected to a side chain group as described below. In some embodiments, p is 2. In some embodiments, the (AA) p is valine-citrulline. In some embodiments, (AA) p is citrulline-valine. In some embodiments, (AA) p is valine-alanine. In some embodiments, (AA) p is alanine-valine. In some embodiments, (AA) p is valine-glycine. In some embodiments, (AA) p is glycine-valine. In some embodiments, p is 3. In some embodiments, the (AA) p is valine-citrulline-PABC. In some embodiments, (AA) p is citrulline-valine-PABC. In some embodiments, (AA)p is Glutamate - Valine - Citrulline. In some embodiments, (AA) p is Glutamine - Valine - Citrulline. In some embodiments, (AA) p is Lysine - Valine - Alanine. In some embodiments, (AA) p is Lysine - Valine - Citrulline. In some embodiments, p is 4. In some embodiments, (AA) p is Glutamate - Valine - Citrulline - PAB. In some embodiments, (AA) p is Glutamine - Valine - Citrulline - PABC. One of ordinary skill in the art will recognize that PABC is the residue of p - aminobenzyloxycarbonyl having the following structure:

[0341] The PABC residue has been shown to facilitate cleavage of certain linkers in vitro and in vivo. One of ordinary skill in the art will recognize that PAB is the divalent residue of p - aminobenzyl or –NH-(p - C6H4)-CH2–.

[0342] In some embodiments, the linker is:

[0343]

[0344]

[0345]

[0346] wherein:

[0347] each is a bond connecting to the transglutaminase - modified binder;

[0348] each is a bond connecting to the payload;

[0349] each R 9 is independently –CH3 or –(CH2)3N(H)C(O)NH2; and

[0350] each A is independently –O–, –NH–, where ZZ is H, or the side chain of an amino acid, as discussed elsewhere in this invention. As another example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6Heteroalkyl. In certain embodiments of this paragraph, A may be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction between an azide and an alkyne or terminal acetylene of a compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond linking to the binder may be a direct bond or a bond via a spacer group. In certain embodiments, the bond linking to the binder is linked to the glutamine residue of the binder via a PEG spacer group.

[0351] In some embodiments, the linker is:

[0352]

[0353] wherein:

[0354] each is a bond linking to the transglutaminase-modified binder;

[0355] each is a bond linking to the payload;

[0356] each R 9 is independently –CH3 or –(CH2)3N(H)C(O)NH2; and

[0357] each A is independently –O–, –N(H)–, where ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A may be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction between an azide and an alkyne or terminal acetylene of a compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond connecting to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond connecting to the binder is a bond connecting to the glutamine residue of the binder via a PEG spacer group.

[0358] In any of the above embodiments, the (AA) p group can be modified with one or more enhancing groups. Advantageously, the enhancing group can be connected to the (AA) pThe side chain of any amino acid. Useful amino acids for attaching an enhancing group include lysine, asparagine, aspartic acid, glutamine, glutamic acid, and citrulline. The attachment to the enhancing group can be a direct attachment to the amino acid side chain, or the attachment can be an indirect attachment via a spacer and / or a reactive group. Useful spacer groups and reactive groups include any of the above groups. The enhancing group can be any group considered useful by those skilled in the art. For example, the enhancing group can be any group that confers a beneficial effect on a compound, payload, linker payload, or antibody conjugate, including but not limited to biological, biochemical, synthetic, solubilizing, imaging, detecting, and reactivity / activity aspects. In certain embodiments, the enhancing group is a hydrophilic group. In certain embodiments, the enhancing group is a cyclodextrin. In certain embodiments, the enhancing group is an alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, heteroalkylene sulfonic acid, heteroalkyl taurine, heteroalkyl phosphoric acid or phosphate / ester, heteroalkyl amine (e.g., quaternary amine), or heteroalkyl sugar. In certain embodiments, sugars include but are not limited to monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, etc. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugate forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, etc. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, etc. Cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the enhancing group is capable of improving the solubility of the remainder of the conjugate. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is substituted or unsubstituted. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) 1-5 SO3H, –(CH2) n –NH-(CH2) 1- 5SO3H, –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, –(CH2)n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkylsulfonic acid, or alkylene sulfonic acid, is –(CH2) 1-5 SO3H. In another embodiment, the heteroalkylsulfonic acid, or heteroalkylene sulfonic acid, is –(CH2) n –NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid, is –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid, is –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid, is –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid, is –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid, is –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where m is 1, 2, 3, 4, or 5. In some embodiments, the linker is:

[0359]

[0360] Where:

[0361] SP1 is a spacer group;

[0362] SP 2 is a spacer group;

[0363] SP 3 is a spacer group, connected to one of the AAs in (AA) p ;

[0364] is one or more bonds connecting to the binder;

[0365] is one or more bonds connecting to the payload or prodrug payload;

[0366] is one or more bonds connecting to the enhancing group EG;

[0367] each AA is an amino acid respectively; and

[0368] p is an integer from 0 to 10.

[0369] As discussed above, the bond connecting to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond connecting to the binder is a bond via a PEG spacer group to the glutamine residue of the binder.

[0370] SP 1 The spacer group has the definition as described above. SP 2 The spacer group has the definition as described above. Each (AA) p group has the definition as described above respectively.

[0371] SP 3 The spacer group is the group part that connects the (AA) p part to the enhancing group (EG). Suitable SP 3 spacer groups include but are not limited to those containing an alkylene group or a polyether, or both an alkylene group and a polyether. SP 3 The end of the spacer group (i.e., the part of the SP 3 spacer group directly connecting to the enhancing group or AA) can be a group part derived from an active group part for the purpose of coupling the enhancing group or AA with the SP 3 spacer group during the chemical synthesis of the conjugate. In some embodiments, SP 3 the end of the spacer group (i.e., the part of the spacer group directly connecting to the enhancing group or AA) can be a residue of the active group part for the purpose of coupling the enhancing group or AA with the spacer group during the chemical synthesis of the conjugate. In certain embodiments, SP3 is a spacer group that is attached to one and only one AA of (AA) p . In certain embodiments, SP 3 the spacer group is attached to the side chain of a lysine residue of (AA) p .

[0372] In some embodiments, SP 3 the spacer group is:

[0373]

[0374] wherein:

[0375] RG' is the residue of the active group RG after reaction of the active group RG with the enhancer EG;

[0376] is the bond attached to the enhancer;

[0377] is the bond attached to (AA) p ;

[0378] a is an integer from 2 to 8; and

[0379] p is an integer from 0 to 4.

[0380] The active group RG can be any active group known to those skilled in the art that is capable of forming one or more bonds with an enhancer. The active group RG is a group moiety that contains, in its structure, a moiety capable of reacting with an enhancing group to form a compound represented by formula LPa, LPb, LPc, LPd, LPe, LPa′, LPb′, LPc′, LPd′, LPe′, A, B, C, D, E, A′, B′, C′, D′, or E′. After coupling with the enhancing group, the active group becomes the active group residue (RG'). The active group RG can be any of the above active groups. Exemplary active groups include, but are not limited to, those containing a haloacetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, or maleimide moiety capable of reacting with a binder.

[0381] In certain embodiments, the active group includes, but is not limited to, alkynes. In certain embodiments, the alkyne is an alkyne capable of undergoing a 1,3-cycloaddition reaction with an azide in the absence of a copper catalyst, such as strained alkynes. Strained alkynes are those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, and benzocyclized alkynes. Suitable alkynes include, but are not limited to, dibenzoazacyclooctyne or (DIBAC), dibenzocyclooctyne or (DIBO), diarylazacyclooctynone or (BARAC), cyclooctyne difluoride or (DIFO), substituted, such as fluorinated alkynes, azacycloalkynes, bicyclo[6.1.0]nonyne or (BCN), and its derivatives. Particularly useful alkynes include

[0382] In some embodiments, the linker is:

[0383]

[0384] Wherein:

[0385] RG' is the residue of the active group RG after reaction with the binder;

[0386] PEG is –NH–PEG4–C(O)–;

[0387] SP 2 is a spacer group;

[0388] SP 3 is a spacer group, connected to one AA residue in (AA) p in;

[0389] is one or more bonds connected to the binder;

[0390] is one or more bonds connected to the payload;

[0391] is one or more bonds connected to the enhancing group EG;

[0392] Each AA is an amino acid residue respectively; and

[0393] p is an integer from 0 to 10.

[0394] As discussed above, the bond connected to the binder can be a direct connection or a connection via a spacer group. In certain embodiments, the bond connected to the binder is connected to the glutamine residue of the binder via a PEG spacer group.

[0395] In certain embodiments, the linker is:

[0396]

[0397] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof, wherein:

[0398] Each is a bond linking to the transglutaminase-modified binder as described above;

[0399] Each is a bond linking to the payload as described above;

[0400] Each is a bond linking to the enhancer as described above;

[0401] Each R 9 is independently –CH3 or –(CH2)3N(H)C(O)NH2; and

[0402] Each A is independently –O–, –N(H)–, where ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction between an azide and an alkyne or terminal acetylene of a compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In certain embodiments, the 1,3-cycloaddition or SPAAC regioisomers, or a mixture of regioisomers, are derived from a PEG-N3-derivatized antibody treated with a suitable alkyne. For example, in one embodiment, the linker is:

[0403] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers. As another example, in one embodiment, the linker is:

[0404] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers. As another example, the linker is:

[0405] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof. As another example, in one embodiment, the linker is:

[0406] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof. As described above, the bond connecting to the binder can be a direct bond or can be via a spacer group. In certain embodiments, the bond connecting to the binder is connected to the glutamine residue of the binder via a PEG spacer group. In certain embodiments, the enhancer is a hydrophilic group. In certain embodiments, the enhancer is a cyclodextrin. In certain embodiments, the enhancing group is an alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, heteroalkylene sulfonic acid, heteroalkyl taurine, heteroalkyl phosphoric acid or phosphate / ester, heteroalkylamine (e.g., quaternary amine), or heteroalkyl sugar. In certain embodiments, sugars include but are not limited to monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, etc. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, etc. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, etc. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) 1-5 SO3H, –(CH2) n –NH-(CH2) 1-5 SO3H, –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1- 5SO3H)2, –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or –(CH2CH2O)m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkylsulfonic acid or alkylene sulfonic acid is –(CH2) 1-5 SO3H. In another embodiment, the heteroalkylsulfonic acid or heteroalkylene sulfonic acid is –(CH2) n –NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1- 5SO3H)2, where m is 1, 2, 3, 4, or 5.

[0407] In some embodiments, the linker is:

[0408]

[0409] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers, wherein:

[0410] each Bonds respectively linked to the transglutaminase-modified binder;

[0411] Each Bonds respectively linked to the enhancer;

[0412] Each Bonds respectively linked to the payload;

[0413] Each R 9 Is respectively –CH3 or –(CH2)3N(H)C(O)NH2; and

[0414] Each A is respectively –O–, –N(H)–, Where ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 Alkyl. As another example, in one embodiment, ZZ is C 1-6 Heteroalkyl. In a specific embodiment of this paragraph, A can be derived from a primary amine compound or its residue, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product after the azide participates in a click chemical reaction with the alkyne or terminal acetylene of the compound or payload described in the present invention, as described elsewhere in the present invention. Therefore, in a non-limiting example, A is Or a mixture thereof. Alternatively, in another embodiment, A is Or a mixture thereof. In another embodiment, A is Or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond linking to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond linking to the binder is a bond linking to the glutamine residue of the binder via a PEG spacer group. In certain embodiments, the enhancer is a hydrophilic group. In certain embodiments, the enhancer is a cyclodextrin. In certain embodiments, the enhancing group is an alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, heteroalkylene sulfonic acid, heteroalkyltaurine, heteroalkyl phosphate or phosphate / ester, heteroalkylamine (e.g., quaternary amine), or heteroalkyl sugar. In certain embodiments, sugars include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, etc. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugate forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, etc. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, etc. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) 1-5 SO3H, –(CH2) n –NH-(CH2) 1-5 SO3H, –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkylsulfonic acid, or alkylene sulfonic acid is –(CH2) 1-5SO3H. In another embodiment, the heteroalkylsulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1- 5SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where m is 1, 2, 3, 4, or 5.

[0415] In some embodiments, the linker is:

[0416]

[0417]

[0418] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof, wherein:

[0419] each is respectively a bond linked to the transglutaminase-modified binder;

[0420] each is respectively a bond linked to the payload;

[0421] R 9 is –CH3 or –(CH2)3N(H)C(O)NH2; and

[0422] A is –O–, –N(H)–, wherein ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction involving an azide and an alkyne or terminal acetylene of the compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in a non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond connecting to the binder can be a direct connection or a connection via a spacer group. In certain embodiments, the bond connecting to the binder is connected to the glutamine residue of the binder via a PEG spacer group.

[0423] In some embodiments, the linker is:

[0424]

[0425]

[0426] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof, wherein:

[0427] each is respectively the bond connecting to the transglutaminase-modified binder;

[0428] each is respectively the bond connecting to the payload;

[0429] R 9 is –CH3 or –(CH2)3N(H)C(O)NH2; and

[0430] A is –O–, –N(H)–, wherein ZZ is H, or a side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction between an azide and an alkyne or terminal acetylene of a compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond connecting to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond connecting to the binder is connected to the glutamine residue of the binder via a PEG spacer group.

[0431] In some embodiments, the linker is:

[0432]

[0433] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof, wherein:

[0434] each is a bond connecting to the transglutaminase-modified binder, respectively;

[0435] each is a bond connecting to the payload, respectively;

[0436] each is a bond connecting to the enhancing group, respectively;

[0437] each R 9 is –CH3 or –(CH2)3N(H)C(O)NH2, respectively; and

[0438] each A is –O–, –N(H)–, wherein ZZ is H, or a side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6Heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction between an azide and an alkyne or terminal acetylene of the compounds or payloads described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond connecting to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond connecting to the binder is connected to the glutamine residue of the binder via a PEG spacer group. In certain embodiments, the enhancer is a hydrophilic group. In certain embodiments, the enhancer is a cyclodextrin. In certain embodiments, the enhancing group is an alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, heteroalkylene sulfonic acid, heteroalkyltaurine, heteroalkyl phosphoric acid or phosphate / ester, heteroalkylamine (e.g., quaternary amine), or heteroalkyl sugar. In certain embodiments, sugars include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, etc. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, etc. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, etc. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) 1-5 SO3H, –(CH2) n –NH-(CH2) 1-5 SO3H, –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, –(CH2) n –N((CH2) 1-5 C(O)NH(CH2)1-5 (SO3H)2, –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 (SO3H)2, or –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 (SO3H)2, where n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkylsulfonic acid, or alkylene sulfonic acid, is –(CH2) 1-5 (SO3H). In another embodiment, the heteroalkylsulfonic acid, or heteroalkylene sulfonic acid, is –(CH2) n –NH-(CH2) 1-5 (SO3H), where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –C(O)NH-(CH2) 1-5 (SO3H), where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2CH2O) m –C(O)NH-(CH2) 1-5 (SO3H), where m is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 (SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1- (5SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylene sulfonic acid, or heteroalkylene sulfonic acid is –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 (SO3H)2, where m is 1, 2, 3, 4, or 5.

[0439] In some embodiments, the linker is:

[0440]

[0441] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers, wherein:

[0442] each is respectively a bond linked to the transglutaminase-modified binder;

[0443] each is respectively a bond linked to the payload;

[0444] each R 9 is respectively –CH3 or –(CH2)3N(H)C(O)NH2; and

[0445] each A is respectively –O–, –N(H)–, wherein ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In a particular embodiment of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction involving an azide and an alkyne or terminal acetylene of the compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in a non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond linking to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond linking to the binder is a bond linking to the glutamine residue of the binder via a PEG spacer group. In certain embodiments, the enhancer is a hydrophilic group. In certain embodiments, the enhancer is a cyclodextrin. In certain embodiments, the enhancing group is an alkylsulfonic acid, heteroalkylsulfonic acid, alkanesulfonic acid, heteroalkanesulfonic acid, heteroalkyltaurine, heteroalkylphosphoric acid or phosphate / ester, heteroalkylamine (e.g., quaternary amine), or heteroalkyl sugar. In certain embodiments, sugars include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, etc. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugate forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, etc. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, etc. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the alkylsulfonic acid, heteroalkylsulfonic acid, alkanesulfonic acid, or heteroalkanesulfonic acid is –(CH2) 1-5 SO3H, –(CH2) n –NH-(CH2) 1-5 SO3H, –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkylsulfonic acid, or alkanesulfonic acid is –(CH2) 1-5SO3H. In another embodiment, the heteroalkylsulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –C(O)NH-(CH2) 1-5 SO3H, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1- 5SO3H)2, where n is 1, 2, 3, 4, or 5. In another embodiment, the alkylsulfonic acid, heteroalkylsulfonic acid, alkylenesulfonic acid, or heteroalkylenesulfonic acid is –(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where m is 1, 2, 3, 4, or 5.

[0446] In some embodiments, the linker is:

[0447]

[0448]

[0449] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers thereof, wherein:

[0450] each is respectively a bond linked to the transglutaminase-modified binder;

[0451] each is respectively a bond linked to the payload;

[0452] R 9 is –CH3 or –(CH2)3N(H)C(O)NH2; and

[0453] A is –O–, –N(H)–, wherein ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction involving an azide and an alkyne or terminal acetylene of the compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in a non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond connecting to the binder can be a direct bond or a bond via a spacer group. In certain embodiments, the bond connecting to the binder is a bond via a PEG spacer group to the glutamine residue of the binder.

[0454] In some embodiments, the linker is:

[0455]

[0456]

[0457] or a pharmaceutically acceptable salt, solvate, or stereoisomeric form thereof, or a regioisomer thereof, or a mixture of regioisomers, wherein:

[0458] each is respectively a bond connecting to the transglutaminase-modified binder;

[0459] each is respectively a bond connecting to the payload;

[0460] R 9 is –CH3 or –(CH2)3N(H)C(O)NH2; and

[0461] A is –O–, –N(H)–, wherein ZZ is H, or the side chain of an amino acid, as discussed elsewhere in the present invention. For example, in one embodiment, ZZ is C 1-6 alkyl. As another example, in one embodiment, ZZ is C 1-6 heteroalkyl. In certain embodiments of this paragraph, A can be derived from a primary amine compound or a residue thereof, where X is –N3, as described elsewhere in the present invention. In these embodiments, the 1,2,3-triazole residue is derived from the product of a click chemical reaction involving an azide and an alkyne or terminal acetylene of a compound or payload described in the present invention, as described elsewhere in the present invention. Thus, in one non-limiting example, A is or a mixture thereof. Alternatively, in another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. In another embodiment, A is or a mixture thereof. As discussed above, the bond linking to the binder can be a direct bond or via a spacer group. In certain embodiments, the bond linking to the binder is linked to the glutamine residue of the binder via a PEG spacer group.

[0462] In certain embodiments, a compound, payload or prodrug payload having an alkyne or terminal acetylene disclosed in the present invention can be linked to a binder, the binder being derived from –PEG-N3 linked to a glutamine residue (i.e., a binder modified by transglutaminase). The present invention provides exemplary –N3-derived binders (i.e., binders modified by transglutaminase), methods for their preparation and methods for their use. In certain embodiments, a compound or payload having an alkyne as described in the present invention is suitable for participating in a 1,3-cycloaddition with a binder derived from –PEG-N3 to provide a regioselective 1,2,3-triazolyl linking group moiety. For example, in certain embodiments, the compound or payload linked to the binder can be or a mixture thereof, where each is a bond linking to the binder, respectively.

[0463] Linker-payload

[0464] In certain embodiments, the linker-payload or linker-prodrug payload (i.e., these descriptors are used interchangeably throughout the specification) includes any particular compound linked to the linker and encompassed by any one or more of the above formulas I, Ia, II, III, IV, V, or VI, wherein the linker of the present invention includes a moiety that reacts with the antibody of the present invention or an antigen-binding fragment thereof. In a particular embodiment, the linker is heterocyclically linked to one or more of N, R 1 、R 2 、R 3 、R 6 、or R 7 in the above formulas I, Ia, II, III, IV, V, or VI.

[0465] In one embodiment, the linker-payload has a structure represented by formula LPa, LPb, LPc, LPd, or LPe:

[0466]

[0467]

[0468] wherein L is a linker.

[0469] In one embodiment, the linker-payload has a structure represented by formula LPa, LPb, LPc, LPd, or LPe, wherein:

[0470] L is a linker; and R 7 in each case is independently H, –OH, –O–, halogen, or –NR 7a R 7b wherein R 7a and R 7b in each case are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl,

[0471] –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first N-terminal peptide residue, –CH2CH2NH2, and –CH2CH2NH–, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted.

[0472] In one embodiment, the linker-payload has a structure represented by formula LPa′:

[0473]

[0474] wherein SP 1 、(AA) p 、SP 2, R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , r and a are as described in any of the embodiments disclosed in the present invention. In one embodiment, the linker-payload has the structure shown by formula LPb':

[0475]

[0476] wherein SP 1 , (AA) p , SP 2 , R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , r and a are as described in any of the embodiments disclosed in the present invention. In one embodiment, the linker-payload has the structure shown by formula LPc':

[0477]

[0478] wherein SP 1 , (AA) p , SP 2 , R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , r and a are as described in any of the embodiments disclosed in the present invention. In one embodiment, the linker-payload has the structure shown by formula LPd':

[0479]

[0480] wherein SP 1 , (AA) p , SP 2 , R 1 , Q, R 2 , R 3 , R 4 , R 5 , R6 、R 7 、R 8 、R 10 、r and a are as described in any of the embodiments disclosed in the present invention. In one embodiment, the linker-payload has the structure shown by LPe':

[0481]

[0482] wherein SP 1 、(AA) p 、SP 2 、R 1 、Q, R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 10 、r and a are as described in any of the embodiments disclosed in the present invention. In any of the embodiments of this paragraph, LPa', LPb', LPc', LPd', or LPe' can be a pharmaceutically acceptable salt or prodrug thereof. In any of the embodiments of this paragraph, p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker-payload has the structure shown by LPa', LPb', LPc', LPd', or LPe', wherein the –SP 2 – spacer group, when present, is the second –(AA) p – is the –SP 1 – spacer group is wherein RG is a reactive group; and b is an integer from 1 to 4. In one embodiment, the linker-payload has the structure shown by LPa', LPb', LPc', LPd', or LPe', wherein Q is –O–. In one embodiment, the linker-payload has the structure shown by LPa', LPb', LPc', LPd', or LPe', wherein Q is –CH2–; R 1 is C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10Absent; where r is 4; and where a is 1. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H or fluorine. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is fluorine. In one embodiment, the linker-payload has the structure shown as LPe′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown as LPe′, or a pharmaceutically acceptable salt thereof, where R 3 is –OC(O)N(H)CH2CH2NH– or –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH–. In one embodiment, the linker-payload has the structure shown as LPe′, or a pharmaceutically acceptable salt thereof, where R 3 is –OC(O)N(H)CH2CH2NH–. In one embodiment, the linker-payload has the structure shown as LPe′, or a pharmaceutically acceptable salt thereof, where R 3 is –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH–. In one embodiment, the linker-payload has the structure shown as LPa′, LPb′, LPc′, LPd′, or LPe′, where Q is –CH2–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; where r is 3 or 4; and where a is 1. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H. In one embodiment, the linker-payload has the structure shown as LPa′, LPb′, LPc′, LPd′, or LPe′, where Q is –CH2–; R1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10 is absent; where r is 4; and where a is 1. In one embodiment, the linker-payload has the structure shown by LPc′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown by LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H. In one embodiment, the linker-payload has the structure shown by LPa′, LPb′, LPc′, LPd′, or LPe′, where Q is –O–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 3 is hydroxy or –OC(O)C1-C5 alkyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –OH; R 10 , when present, is -C1-C5 alkyl; where r is 3 or 4; and where a is 1. In one embodiment, the linker-payload has the structure shown by LPc′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown by LPc′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H. In one embodiment, the linker-payload has the structure shown by LPa′, LPb′, LPc′, LPd′, or LPe′, where Q is –CH2– or –O–; R 1 is C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 4 and R 5 are both C1-C5 alkyl; R 6 is –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ; R 10Absent; where r is 4; and where a, a1, and a2 are each independently 0 or 1. In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof. In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 1; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 1; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 1; and R 6 is In one embodiment, the linker-payload has the structure shown as LPb′, or a pharmaceutically acceptable salt thereof, where a is 1; and R 6 is In one embodiment, the linker-payload has the structure shown as LPc′, or a pharmaceutically acceptable salt thereof, wherein R 7 is –O–; and R 8 is H.

[0483] In any of the foregoing embodiments, aryl includes phenyl, naphthyl, fluorenyl, azulyl, anthryl, phenanthryl, and pyrenyl; heteroaryl includes furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, benzofuranyl, dibenzofuranyl, benzothienyl, benzoxazolyl, benzothiazolyl, dibenzothienyl, indolyl, indolinyl, benzimidazolyl, indazolyl, and benzotriazolyl; nitrogen-containing heterocycles include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, and azocanyl; and acyl includes –C(O)R 3c wherein R 3cInclude alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl. In one embodiment, the aryl is phenyl. In one embodiment, the aryl is naphthyl. In one embodiment, the aryl is fluorenyl. In one embodiment, the aryl is azulyl. In one embodiment, the aryl is anthryl. In one embodiment, the aryl is phenanthryl. In one embodiment, the aryl is pyrenyl. In one embodiment, the heteroaryl is furyl. In one embodiment, the heteroaryl is thienyl. In one embodiment, the heteroaryl is pyrrolyl. In one embodiment, the heteroaryl is oxazolyl. In one embodiment, the heteroaryl is thiazolyl. In one embodiment, the heteroaryl is imidazolyl. In one embodiment, the heteroaryl is pyrazolyl. In one embodiment, the heteroaryl is isoxazolyl. In one embodiment, the heteroaryl is isothiazolyl. In one embodiment, the heteroaryl is pyridyl. In one embodiment, the heteroaryl is pyrazinyl. In one embodiment, the heteroaryl is pyrimidinyl. In one embodiment, the heteroaryl is pyridazinyl. In one embodiment, the heteroaryl is quinolinyl. In one embodiment, the heteroaryl is isoquinolinyl. In one embodiment, the heteroaryl is cinnolinyl. In one embodiment, the heteroaryl is quinazolinyl. In one embodiment, the heteroaryl is quinoxalinyl. In one embodiment, the heteroaryl is phthalazinyl. In one embodiment, the heteroaryl is pteridinyl. In one embodiment, the heteroaryl is benzofuryl. In one embodiment, the heteroaryl is dibenzofuryl. In one embodiment, the heteroaryl is benzothienyl. In one embodiment, the heteroaryl is benzoxazolyl. In one embodiment, the heteroaryl is benzothiazolyl. In one embodiment, the heteroaryl is dibenzothienyl. In one embodiment, the heteroaryl is indolyl. In one embodiment, the heteroaryl is indolinyl. In one embodiment, the heteroaryl is benzimidazolyl. In one embodiment, the heteroaryl is indazolyl. In one embodiment, the heteroaryl is benzotriazolyl. In one embodiment, the nitrogen-containing heterocycle is aziridinyl. In one embodiment, the nitrogen-containing heterocycle is azetidinyl. In one embodiment, the nitrogen-containing heterocycle is pyrrolidinyl. In one embodiment, the nitrogen-containing heterocycle is piperidinyl. In one embodiment, the nitrogen-containing heterocycle is azepanyl. In one embodiment, the nitrogen-containing heterocycle is azocanyl. In one embodiment, the acyl group is –C(O)R 3c , and R 3c is alkyl. In one embodiment, the acyl group is –C(O)R 3c , and R 3c is alkenyl. In one embodiment, the acyl group is –C(O)R 3c , and R 3c is alkynyl. In one embodiment, the acyl group is –C(O)R 3c , and R 3c is cycloalkyl. In one embodiment, the acyl group is –C(O)R 3c , and R3c is an aryl. In one embodiment, the acyl group is –C(O)R 3c , and R 3c is a heteroaryl.

[0484] In any of the foregoing embodiments of this section, R 7 is –O– or –NR 7a R 7b , where R 7a and R 7b in each case are independently a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, a first N-terminal amino acid residue, or a first N-terminal peptide residue, where the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted. In certain embodiments, R 7a is H, and R 7b is a bond. In certain embodiments, R 7 is –O–. In certain embodiments, R 7a is H, and R 7b is a first N-terminal amino acid residue.

[0485] Conjugate / antibody-drug conjugate (ADC)

[0486] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody is conjugated to one or more compounds of Formula I, Ia, II, III, IV, V, or VI described herein.

[0487] The present invention provides a conjugate having a structure shown by Formula A, B, C, D, or E:

[0488]

[0489]

[0490] wherein L is a linker. In certain embodiments, R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , m, r, and a are as described above in the context of Formula I, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4.

[0491] The present invention provides Formula The conjugate shown in A, B, C, D, or E, where T is as described elsewhere in the present invention, or a pharmaceutically acceptable salt, solvate, regioisomeric form, or stereoisomeric form thereof, where R 7 In each case, independently of one another, is H, –OH, –O–, halogen, or –NR 7a R 7b ,

[0492] where R 7a and R 7b , in each case, independently of one another, is a bond, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first N-terminal peptide residue, –CH2CH2NH2, and –CH2CH2NH–, where the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted. In certain embodiments, R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , m, r, and a are each as described above in the context of formula I, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4.

[0493] The present invention provides the conjugate shown in A′, B′, C′, D′, or E′:

[0494]

[0495]

[0496] or a pharmaceutically acceptable salt, prodrug, solvate, regioisomeric form, or stereoisomeric form thereof, where SP 1 and SP 2 , when present, are each a spacer group; each AA, when present, is a second amino acid residue; and p is an integer from 0 to 10. In certain embodiments, R 1 , Q, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10, m, r, and a are as described above in the context of Formula I, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1 - 2, 1 - 3, 2 - 3, 2 - 4, 3 - 4, or 1 - 4. In certain embodiments, the –SP 2 – spacer group, when present, is the second –(AA) p – is the –SP 1 – spacer group is where RG′ is the residue of the reactive group RG after reaction with the binder; is a bond that is directly or indirectly linked to the binder; and b is an integer from 1 to 4. In certain embodiments, p has the definition as described above. In certain embodiments, b is 1. In certain embodiments, b is 2. In certain embodiments, b is 3. In certain embodiments, b is 4. In certain embodiments, Q is –O–. In certain embodiments, the conjugate has the structure shown in Formula A′, B′, C′, D′, or E′, where Q is –CH2–; R 1 is C1 - C 10 alkyl; R 2 is alkyl; R 4 and R 5 are both C1 - C5 alkyl; R 6 is –OH; R 10 is absent; where r is 4; and where a is 1. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H or fluorine. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is H. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, where R 7 is –NH–; and R 8 is fluorine. In one embodiment, the conjugate has the structure shown in Formula E′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula E′, or a pharmaceutically acceptable salt thereof, where R 3 is –OC(O)N(H)CH2CH2NH– or –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH–. In one embodiment, the conjugate has the structure shown in Formula E′, or a pharmaceutically acceptable salt thereof, where R 3is –OC(O)N(H)CH2CH2NH–. In one embodiment, the conjugate has the structure shown in Formula E′, or a pharmaceutically acceptable salt thereof, wherein R 3 is –OC(O)N(H)CH2CH2OCH2CH2OCH2CH2OCH2CH2NH–. In certain embodiments, the conjugate has the structure shown in Formula A′, B′, C′, D′, or E′, wherein Q is –CH2–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; wherein r is 3 or 4; and wherein a is 1. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, wherein R 7 is –NH–; and R 8 is H. In certain embodiments, the conjugate has the structure shown in Formula A′, B′, C′, D′, or E′, wherein Q is –CH2–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; R 10 is absent; wherein r is 4; and wherein a is 1. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, wherein R 7 is –NH–; and R 8 is H. In certain embodiments, the conjugate has the structure shown in Formula A′, B′, C′, D′, or E′, wherein Q is –O–; R 1 is H or C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 3 is hydroxy or –OC(O)C1-C5 alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; R 10 , when present, is -C1-C5 alkyl; wherein r is 3 or 4; and wherein a is 1. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, R7 is –NH–; and R 8 is H. In certain embodiments, the conjugate has the structure shown in Formula A′, B′, C′, D′, or E′, where Q is –CH2– or –O–; R 1 is C1-C 10 alkyl; R 2 is alkyl or alkynyl; R 4 and R 5 are each independently C1-C5 alkyl; R 6 is –NHSO2(CH2) a1 -aryl-(CH2) a2 NR 6a R 6b ; R 10 is absent; where r is 4; and where a, a1, and a2 are each independently 0 or 1. In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof. In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where a is 0; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, where a is 1; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, wherein a is 1; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, wherein a is 1; and R 6 is In one embodiment, the conjugate has the structure shown in Formula B′, or a pharmaceutically acceptable salt thereof, wherein a is 1; and R 6 is In one embodiment, the conjugate has the structure shown in Formula C′, or a pharmaceutically acceptable salt thereof, wherein R 7 is –O–; and R 8 is H.

[0497] The present invention provides a conjugate of Formula A. In certain embodiments, the compound conjugated to —L—BA in Formula A comprises one or more compounds of Formula I, Ia, II, III, IV, V, and / or VI above, wherein BA is a binder; L is a linker; and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula I, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula Ia, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula II, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula III, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula IV, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula V, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of Formula VI, as described above. In any of the embodiments of this paragraph, any one or more of the compounds of Formula I, Ia, II, III, IV, V, and / or VI conjugated to —L—BA in Formula A are conjugated through a nitrogen-containing heterocycle, as described elsewhere in the present invention. In certain embodiments, when Q is –O–, then R 2 is C1-C 10 alkyl, C1-C 10 alkynyl, regioisomeric triazole, –C1-C 10Alkylene-(heteroaryl consisting of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn Aryl, C1-C3 hydroxyalkyl, or C1-C 10 Alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–. In certain embodiments, when Q is –CH2–, then R 2 is C5-C 10 Alkyl, C1-C 10 Alkynyl, –C1-C 10 Alkylene-(heteroaryl consisting of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn Aryl, C1-C3 hydroxyalkyl, or C1-C 10 Alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–.

[0498] The present invention provides a conjugate of formula B. In certain embodiments, the compound conjugated to —L—BA in formula B comprises one or more compounds of formula I, Ia, II, III, IV, V, and / or VI as described above, wherein BA is a binding agent; L is a linker; and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula I as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula Ia as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula II as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula III as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula IV as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula V as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula VI as described above. In any of the embodiments of this paragraph, any one or more compounds of formula I, Ia, II, III, IV, V, and / or VI conjugated to —L—BA in formula B are conjugated via a divalent R 6 For coupling. In certain embodiments, when Q is –O–, then R 2 is C1-C 10 alkyl, C1-C 10 alkynyl, regioisomeric triazole, –C1-C 10 alkylene-(heteroaryl composed of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1is –O–. In certain embodiments, when Q is –CH2–, then R 2 is C5-C 10 alkyl, C1-C 10 alkynyl, –C1-C 10 alkylene-(heteroaryl consisting of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–.

[0499] The present invention provides a conjugate of formula C. In certain embodiments, the compound conjugated to —L—BA in formula C comprises one or more compounds of formula I, Ia, II, III, IV, V, and / or VI as described above, wherein BA is a binding agent; L is a linker; and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula I, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula Ia, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula II, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula III, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula IV, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula V, as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula VI, as described above. In any of the embodiments of this paragraph, any one or more compounds of formula I, Ia, II, III, IV, V, and / or VI conjugated to —L—BA in formula C are conjugated via a divalent R 7 For coupling. In certain embodiments, when Q is –O–, then R 2 is C1-C 10 alkyl, C1-C 10 alkynyl, regioisomeric triazole, –C1-C 10 alkylene-(heteroaryl consisting of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1is –O–. In certain embodiments, when Q is –CH2–, then R 2 is C5-C 10 alkyl, C1-C 10 alkynyl, –C1-C 10 alkylene-(heteroaryl consisting of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–.

[0500] The present invention provides a conjugate of formula D. In certain embodiments, the compound conjugated to —L—BA in formula D comprises one or more compounds of formula I, Ia, II, III, IV, V, and / or VI as described above, wherein BA is a binder; L is a linker; and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula I as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula Ia as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula II as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula III as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula IV as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula V as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula VI as described above. In any of the embodiments of this paragraph, any one or more compounds of formula I, Ia, II, III, IV, V, and / or VI conjugated to —L—BA in formula D are conjugated via a divalent R 2 For coupling. In certain embodiments, when Q is –O–, then R 2 is C1-C 10 alkylene, C1-C 10 alkynylene, regioisomeric C1-C 10 triazolylene, regioisomeric –C1-C 10 alkylene-(5-atom heteroarylene), or –C1-C3 alkylene–Q 1 –(CH2) nn arylene. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1is –O–. In certain embodiments, when Q is –CH2–, then R 2 is C5-C 10 alkylene, C1-C 10 alkynylene, regioisomeric C1-C 10 triazolylene, regioisomeric –C1-C 10 alkylene-(5-atom heteroarylene), or –C1-C3 alkylene–Q 1 –(CH2) nn arylene. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–.

[0501] The present invention provides a conjugate of formula E. In certain embodiments, the compound conjugated to —L—BA in formula E comprises one or more compounds of formula I, Ia, II, III, IV, V, and / or VI as described above, wherein BA is a binder; L is a linker; and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, k ranges from 1-2, 1-3, 2-3, 2-4, 3-4 or 1-4. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula I as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula Ia as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula II as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula III as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula IV as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula V as described above. In any of the embodiments of this paragraph, BA is an antibody, or an antigen-binding fragment thereof, wherein the antibody is conjugated to a compound of formula VI as described above. In any of the embodiments of this paragraph, any one or more compounds of formula I, Ia, II, III, IV, V, and / or VI conjugated to —L—BA in formula E are conjugated via a divalent R 3 For coupling. In certain embodiments, when Q is –O–, then R 2 is C1-C 10 alkyl, C1-C 10 alkynyl, regioisomeric triazole, –C1-C 10 alkylene-(heteroaryl composed of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1is –O–. In certain embodiments, when Q is –CH2–, then R 2 is C5-C 10 alkyl, C1-C 10 alkynyl, –C1-C 10 alkylene-(heteroaryl composed of 5 atoms), –C1-C3 alkylene–Q 1 –(CH2) nn aryl, C1-C3 hydroxyalkyl, or C1-C 10 alkyl ether. In certain embodiments of this paragraph, nn is 1. In certain embodiments of this paragraph, nn is 2. In certain embodiments of this paragraph, nn is 3. In certain embodiments of this paragraph, nn is 4. In certain embodiments of this paragraph, nn is 5. In certain embodiments of this paragraph, nn is 6. In certain embodiments of this paragraph, nn is 7. In certain embodiments of this paragraph, nn is 8. In certain embodiments of this paragraph, nn is 9. In certain embodiments of this paragraph, nn is 10. In certain embodiments of this paragraph, Q 1 is –CH2–. In certain embodiments of this paragraph, Q 1 is –O–.

[0502] In certain embodiments, the compounds represented by formula A′, B′, C′, D′, or E′ are selected from the group consisting of:

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520] or a pharmaceutically acceptable salt thereof, wherein BA is a binder; and k is 1, 2, 3, or 4.

[0521] In certain embodiments, the antibody or antigen-binding fragment thereof can be directly conjugated via a linker to any one or more of Formulas I, Ia, II, III, IV, V, and / or VI described herein. In one embodiment, the antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof conjugated to any one or more of Formulas I, Ia, II, III, IV, V, and / or VI selected from the group consisting of, as described herein:

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534] In any of the provided compound or conjugate embodiments, the BA is an antibody that binds to PRLR or an antigen-binding fragment thereof. In any of the provided compound or conjugate embodiments, the BA is an antibody that binds to STEAP2 or an antigen-binding fragment thereof. In any of the provided compound or conjugate embodiments, the BA is an antibody or an antigen-binding fragment thereof and is conjugated via at least one Q295 residue. In any of the provided compound or conjugate embodiments, the BA is an antibody or an antigen-binding fragment thereof and is conjugated via two Q295 residues. In any of the provided compound or conjugate embodiments, the BA is an N297Q antibody or an antigen-binding fragment thereof. In any of the provided compound or conjugate embodiments, the BA is an N297Q antibody or an antigen-binding fragment thereof and is conjugated via at least one Q295 residue and at least one Q297 residue. In any of the provided compound or conjugate embodiments, the BA is an N297Q antibody or an antigen-binding fragment thereof and is conjugated via two Q295 residues and two Q297 residues. In certain embodiments, the numbering is according to the EU numbering system.

[0535] In any of the above embodiments, BA is an anti-STEAP2 antibody. In certain embodiments, BA is the anti-STEAP2 antibody H1H7814N described in the following examples. In certain embodiments, BA is the anti-STEAP2 antibody H1H7814N N297Q described in the following examples. In certain embodiments, BA is an anti-STEAP2 antibody comprising an HCVR according to SEQ ID NO: 1 and an LCVR according to SEQ ID NO: 5. In certain embodiments, BA is an N297Q antibody comprising an HCVR according to SEQ ID NO: 1 and an LCVR according to SEQ ID NO: 5. In certain embodiments, BA is an anti-STEAP2 antibody comprising 1, 2, 3, 4, 5, or 6 of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In certain embodiments, BA is an N297Q antibody comprising 1, 2, 3, 4, 5, or 6 of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. N297Q means that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). In certain embodiments, each residue 297 is mutated to Q separately. In certain embodiments, the numbering is according to the EU numbering system. In certain embodiments of this paragraph, k is from 1 to 4. In certain embodiments, k is 1, 2, 3, or 4. In certain embodiments, k is 4.

[0536] In any of the foregoing embodiments, BA is an anti-PRLR antibody. In certain embodiments, BA is the anti-PRLR antibody H1H6958N2 described in the following examples. In certain embodiments, BA is the anti-PRLR antibody H1H6958N2N297Q described in the following examples. In certain embodiments, BA is an anti-PRLR antibody comprising an HCVR according to SEQ ID NO: 9 and an LCVR according to SEQ ID NO: 13. In certain embodiments, BA is an N297Q antibody comprising an HCVR according to SEQ ID NO: 9 and an LCVR according to SEQ ID NO: 13. In certain embodiments, BA is an anti-PRLR antibody comprising 1, 2, 3, 4, 5, or 6 of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. In certain embodiments, BA is an N297Q antibody comprising 1, 2, 3, 4, 5, or 6 of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. N297Q means that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). In certain embodiments, each residue 297 is mutated to Q separately. In certain embodiments, the numbering is according to the EU numbering system. In certain embodiments of this paragraph, k is from 1 to 4. In certain embodiments, k is 1, 2, 3, or 4. In certain embodiments, k is 4.

[0537] In any of the foregoing embodiments of this section, R 7 is –NR 7a R 7b wherein R 7a and R 7b in each case are independently, respectively, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl, and amino acid residue, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and acyl are each optionally substituted. In certain embodiments, R 7a is H, and R 7b is an amino acid residue.

[0538] Methods for preparing compounds or payloads and linker-payloads

[0539] The compounds provided by the present invention can all be prepared, separated or obtained by any method obvious to those skilled in the art. Exemplary preparation methods are described in detail in the following examples.

[0540] In certain embodiments, the present invention provides compounds (such as linker-payload or linker-prodrug payload) selected from the group consisting of:

[0541]

[0542]

[0543]

[0544]

[0545]

[0546] or a pharmaceutically acceptable salt thereof. In certain embodiments of this paragraph, all diastereoisomers are contemplated. For example, in one embodiment, the stereochemistry within is undefined or racemic. As another example, in one embodiment, the stereochemistry within is (R)-. As another example, in one embodiment, the stereochemistry within is (S)-. As another example, in one embodiment, the stereochemistry within is (R)- in excess relative to (S)-. As another example, in one embodiment, the stereochemistry within is (S)- in excess relative to (R)-.

[0547] The conjugate of the present invention can be synthesized by conjugating the linker-payload of the present invention with a binding agent (such as an antibody) under standard conjugation conditions (see, for example, Doronina et al. Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference in its entirety). When the binding agent is an antibody, the antibody can be conjugated to the linker-payload through one or more cysteine or lysine residues of the antibody. The linker-payload can, for example, conjugate to cysteine residues by subjecting the antibody to a reducing agent (such as dithiothreitol) to cleave the disulfide bonds of the antibody, purifying the reduced antibody, for example, by gel filtration, and then treating the antibody with a linker-payload containing a suitable reactive group moiety (such as a maleimide group). Suitable solvents include, but are not limited to, water, DMA, DMF, and DMSO. A linker-payload or linker-prodrug payload containing a reactive group such as an activated ester or acyl halide group can be conjugated to the lysine residues of the antibody. Suitable solvents include, but are not limited to, water, DMA, DMF, and DMSO. The conjugate can be purified using known protein techniques, including, for example, size exclusion chromatography (gel filtration chromatography), dialysis, and ultrafiltration / diafiltration.

[0548] A binding agent, such as an antibody, can also be conjugated by click chemistry. In some embodiments of the click chemistry, the linker-payload includes a reactive group capable of regioselective 1,3-cycloaddition reaction with an azide, such as an alkyne. Such suitable reactive groups are as described above. The antibody includes one or more azide groups. Such antibodies include antibodies functionalized with, for example, azido-polyethylene glycol groups. In certain embodiments, such functionalized antibodies are derived by treating an antibody having at least one glutamine residue (such as heavy chain Gln295) with a primary amine compound in the presence of transglutaminase. In certain embodiments, such functionalized antibodies are derived by treating an antibody having at least one glutamine residue (such as heavy chain Gln297) with a primary amine compound in the presence of transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, such functionalized antibodies are derived by treating an antibody having at least two glutamine residues (such as heavy chain Gln295 and heavy chain Gln297) with a primary amine compound in the presence of transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, the antibody has two heavy chains as described in this paragraph for a total of two or a total of four glutamine residues.

[0549] In certain embodiments, the antibody comprises two glutamine residues, one in each heavy chain. In a particular embodiment, the antibody comprises the Q295 residue in each heavy chain. In a further embodiment, the antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, or more glutamine residues. These glutamine residues can be located in the heavy chain, the light chain, or in both the heavy and light chains. These glutamine residues can be wild-type residues or engineered residues. The antibody can be prepared according to standard techniques.

[0550] Those skilled in the art will recognize that antibodies are typically glycosylated at residue N297 near residue Q295 in the heavy chain sequence. Glycosylation at residue N297 can interfere with transglutaminase at residue Q295 (Dennler et al., supra). Thus, in a preferred embodiment, the antibody is not glycosylated. In certain embodiments, the antibody is deglycosylated or non-glycosylated. In a particular embodiment, the heavy chain of the antibody has an N297 mutation. In other words, the antibody is mutated such that it no longer has an asparagine residue at position 297. In a particular embodiment, the heavy chain of the antibody has an N297Q mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or inactivate the glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at any site outside of the glycosylation site or any other interfering structure. Such antibodies can also be isolated from natural or artificial sources.

[0551] The antibody that does not interfere with glycosylation is then reacted or treated with a primary amine compound. In certain embodiments, the non-glycosylated antibody is reacted or treated with a primary amine compound to generate a glutaminyl-modified antibody or a transglutaminase-modified antibody. In certain embodiments, the deglycosylated antibody is reacted or treated with a primary amine compound to generate a glutaminyl-modified antibody or a transglutaminase-modified antibody.

[0552] A primary amine can be any primary amine that is capable of forming a covalent bond with a glutamine residue in the presence of transglutaminase. Useful primary amines are described below. The transglutaminase can be any transglutaminase considered suitable by those skilled in the art. In certain embodiments, the transglutaminase is an enzyme that catalyzes the formation of an isopeptide bond between a free amine group on a primary amine compound and an acyl group on the side chain of a glutamine residue. Transglutaminase is also known as protein-glutamine-γ-glutamyltransferase. In a particular embodiment, the transglutaminase is classified as EC2.3.2.13. The transglutaminase can be from any source considered suitable. In certain embodiments, the transglutaminase is a microorganism. Useful transglutaminases have been isolated from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces griseo-carneum, Streptomyces lavendulae, and Bacillus subtilis. Non-microbial transglutaminases can also be used, including mammalian transglutaminases. In certain embodiments, the transglutaminase can be generated by any technique or obtained from any source considered suitable by those skilled in the art. In a particular embodiment, the transglutaminase is obtained from a commercial source.

[0553] In a particular embodiment, the primary amine compound contains a reactive group that is capable of further reacting after transglutamination. In these embodiments, the glutaminyl-modified antibody or the transglutaminase-modified antibody can be reacted with or treated with an active payload compound or an active prodrug payload compound, or an active linker-payload compound or an active linker-prodrug payload compound to form an antibody-payload conjugate or an antibody-linker-payload conjugate. In certain embodiments, the primary amine compound contains an azide.

[0554] In certain embodiments, a glutaminyl-modified antibody or a transglutaminase-modified antibody is reacted with or treated with an active linker-payload to form an antibody-linker-payload conjugate. The reaction can be carried out under conditions considered suitable by those skilled in the art. In certain embodiments, a glutaminyl-modified antibody or a transglutaminase-modified antibody is contacted with an active linker-payload compound or an active linker-prodrug payload compound under conditions suitable for forming a bond between the glutaminyl-modified antibody or the transglutaminase-modified antibody and the linker-payload compound or the linker-prodrug payload compound. Suitable reaction conditions are well known to those skilled in the art. Exemplary reactions are provided in the following examples.

[0555] Drug Compositions and Therapeutic Methods

[0556] The present invention provides methods of treating and preventing diseases, conditions, or disorders, which comprise administering a therapeutically or prophylactically effective amount of one or more compounds disclosed herein, such as one or more compounds of the general formula provided herein. Diseases, disorders, and / or conditions include, but are not limited to, those associated with the antigens listed herein.

[0557] The compounds described herein can be administered alone or in combination with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered before, simultaneously with, or shortly after the administration of the compounds described herein. The present invention also includes pharmaceutical compositions comprising any compound described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to a subject in need thereof.

[0558] Suitable additional therapeutic agents include, but are not limited to: a second microtubule lysin, an autoimmune therapeutic agent, a hormone, a biologic, or a monoclonal antibody. Suitable therapeutic agents also include, but are not limited to, any pharmaceutically acceptable salts, acids, or derivatives of the compounds described herein.

[0559] In some embodiments of the methods of the present invention, multiple doses of the compounds of the present invention (or pharmaceutical compositions comprising a combination of the compounds of the present invention and any additional therapeutic agents mentioned in the present invention) can be administered to a subject over a defined time course. The method according to this embodiment of the present disclosure includes sequentially administering multiple doses of the compounds of the present invention to a subject. As used herein, "sequentially administering" means that each dose of the compound is administered to the subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention encompasses methods that include sequentially administering a single initial dose of the compounds of the present invention to a patient, followed by administering one or more secondary doses of the compound, and optionally subsequently administering one or more tertiary doses of the compound.

[0560] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the chronological order of administration of the compounds of the present invention. Thus, an "initial dose" is the dose administered at the start of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is the dose administered after the initial dose; and a "tertiary dose" is the dose administered after the secondary dose. The initial, secondary, and tertiary doses can all contain the same amount of the compound of the present invention, but typically may differ from each other in terms of dosing frequency. In certain embodiments, during the course of treatment, the amounts of the compound contained in the initial, secondary, and / or tertiary doses vary from each other (e.g., upregulated or downregulated as appropriate). In certain embodiments, at the start of a treatment regimen, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose", followed by subsequent doses administered in a less frequent manner (e.g., a "maintenance dose").

[0561] In some exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 171 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2, or more). The phrase "immediately following the foregoing dose" as used in the present invention refers to a dose of a compound of the present invention administered to a patient immediately prior to the administration of the next adjacent dose in a sequence of multiple administrations, with no intervening dose in said administration sequence.

[0562] The method of this embodiment of the present invention may comprise administering to a patient any number of secondary and / or tertiary doses of a compound of the present invention. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient. The dosing regimen may be carried out indefinitely during the lifetime of a particular subject or until such treatment is no longer therapeutically required or beneficial.

[0563] In embodiments involving multiple secondary doses, each secondary dose may be administered / dosed at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after immediately following the foregoing dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered / dosed at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after immediately following the foregoing dose. In certain embodiments of the present invention, the frequency of administering secondary and / or tertiary doses to a patient may vary during the course of the treatment regimen. The dosing frequency may also be adjusted by a physician during the course of treatment according to the needs of an individual patient after a clinical examination.

[0564] The present invention includes a dosing regimen in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), and then two or more maintenance doses are administered to the patient in a less frequent manner. For example, according to this embodiment of the present invention, if the loading dose is administered at a monthly frequency, then the maintenance dose may be administered once every 6 weeks, once every two months, once every three months, etc.

[0565] The present invention includes a pharmaceutical composition of the compound and / or conjugate (such as the compounds shown in Formula I, Ia, II, III, IV, V and VI) of the present invention. For example, the composition comprises the compound of the present invention, its salts, stereoisomers, regioisomers, polymorphs, and pharmaceutically acceptable carriers, diluents, and / or excipients. Examples of suitable carriers, diluents, and excipients include, but are not limited to: buffering agents for maintaining the appropriate pH value of the composition (e.g., citrate buffer, succinate buffer, acetate buffer, phosphate buffer, lactate buffer, oxalate buffer, etc.), carrier proteins (such as human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, etc.), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, etc.), antimicrobial agents, and antioxidants.

[0566] In some embodiments, the present invention provides a method for treating cancer, which comprises administering a therapeutically effective amount of a compound of Formula I, Ia, II, III, IV, V and VI, or its pharmaceutical composition, to a patient suffering from the cancer. In some embodiments, the present invention provides a method for treating cancer, which comprises administering a therapeutically effective amount of the antibody-microtubulin conjugate of the present invention, or its pharmaceutical composition, to a patient suffering from the cancer. In some embodiments, the binder (e.g., antibody) of the conjugate (e.g., the antibody-drug conjugate of the present invention) interacts with or binds to a tumor antigen, including an antigen specific to one type of tumor, or an antigen shared, overexpressed, or modified on a specific type of tumor. Examples include, but are not limited to, α-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B-RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma, and the following shared tumor-specific antigens, such as, BAGE-1, GAGE, GnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2. Other examples of tumor antigens include, but are not limited to, PSMA, PRLR, MUC16, HER2, EGFRvIII and anti-STEAP2, and MET.

[0567] The compounds disclosed in the present invention can be used for treating primary and / or metastatic tumors occurring in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tracts, muscle, bone, skin and its appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sense organs (such as the eyes). In certain embodiments, the compounds provided by the present invention can be used for treating one or more of the following cancers: renal cell carcinoma, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), prostate cancer, castration-resistant prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), mesothelioma, malignant mesothelioma, multiple myeloma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, PRLR-positive (PRLR+) breast cancer, melanoma, acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, glioblastoma, Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lymphoma, MFH / fibrosarcoma, nasopharyngeal cancer, rhabdomyosarcoma, colon cancer, gastric cancer, uterine cancer, residual cancer (wherein "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with anti-cancer therapy), and Wilms' tumor. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer.

[0568] In some embodiments, the present invention provides a method for preventing prostate cancer, which comprises administering to a patient suffering from the disease a prophylactically effective amount of a compound of formula I, Ia, II, III, IV, V, and VI, or a pharmaceutical composition thereof.

[0569] Examples

[0570] The present invention provides novel tubulins, their protein conjugates, and methods for treating diseases, disorders, and conditions, including administering the tubulins and conjugates.

[0571] Certain embodiments of the present invention are illustrated by the following non-limiting examples. As used in the present invention, whether or not a specific abbreviation is specifically defined, the symbols and conventions used in the methods, protocols, and examples are consistent with those used in contemporary scientific literature (e.g., Journal of the American Chemical Society or Journal of Biological Chemistry). Specifically but not limited to, the following abbreviations can be used in the examples and throughout the specification:

[0572]

[0573]

[0574]

[0575]

[0576] Unless otherwise specified, reagents and solvents were obtained from commercial sources such as Sinopharm Chemical Reagent Co. (SCRC), Sigma-Aldrich, Alfa or other suppliers. Recorded on Bruker AVIII 400 or Bruker AVIII 500 1 1H NMR and other NMR spectra. The data were processed using Nuts software or MestReNova software, and the proton displacement parts per million (ppm) of the internal standard tetramethylsilane (TMS) at low magnetic field was determined.

[0577] HPLC-MS determinations were performed on an Agilent 1200 HPLC / 6100 SQ system using the following conditions: Method A for HPLC-MS determination included as mobile phases: A: water (0.01% trifluoroacetic acid (TFA)) and B: acetonitrile (0.01% TFA); gradient phase: from 5% B to 95% B in 15 minutes (min); flow rate: 1.0 mL / min; column: SunFire C18, 4.6 x 50 mm, 3.5 μm; column temperature was 50 °C. Detectors: analog-to-digital converter (ADC) evaporative light scattering detector (ELSD), diode array detector (DAD) (214 nm and 254 nm), and electrospray ionization-atmospheric pressure ionization (ES-API). Method B for HPLC-MS determination included as mobile phases: A: water (10 mM NH4HCO3), B: acetonitrile; gradient phase: from 5% B to 95% B in 15 minutes (min); flow rate: 1.0 mL / min; column: XBridge C18, 4.6 x 50 mm, 3.5 μm; column temperature: 50 °C. Detectors: ADC ELSD, DAD (214 nm and 254 nm), and mass selective detector (MSD) (ES-API).

[0578] The LC-MS determination was carried out on an Agilent 1200 HPLC / 6100 SQ system using the following conditions: Method A for LC-MS determination included the instrument WATERS 2767; Column: Shimadzu Shim-Pack, PRC-ODS, 20x250 mm, 15 μm, two connected in series; Mobile phase: A: water (0.01% TFA), B: acetonitrile (0.01% TFA); Gradient phase: from 5% B to 95% B in 3 minutes; Flow rate: 1.8 - 2.3 mL / min; Column: SunFire C18, 4.6x50 mm, 3.5 μm; Column temperature: 50 °C. Detectors: ADC ELSD, DAD (214 nm and 254 nm), ES-API. Method B for LC-MS determination included the instrument Gilson GX-281; Column: Xbridge preparative C18 10um OBD, 19x250 mm; Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile; Gradient phase: from 5% B to 95% B in 3 minutes; Flow rate: 1.8 - 2.3 mL / min; Column: XBridge C18, 4.6x50 mm, 3.5 μm; Column temperature: 50 °C. Detectors: ADC ELSD, DAD (214 nm and 254 nm), and MSD (ES-API).

[0579] Preparative high performance liquid chromatography (Preparative HPLC) was carried out on a Gilson GX-281 instrument using an acidic or basic solvent system. The acidic solvent system included a Waters SunFire 10μm C18 column ( 250x19 mm), and for Preparative HPLC, solvent A was water / 0.05% TFA, and solvent B was acetonitrile. The elution conditions were a linear gradient that increased from 5% solvent B to 100% solvent B at a flow rate of 30 mL / min over a 20-minute period. The basic solvent system included a Waters Xbridge 10μm C18 column ( 250x19 mm), and for Preparative HPLC, solvent A was water / 10 mM ammonium bicarbonate (NH4HCO3), and solvent B was acetonitrile. The elution conditions were a linear gradient that increased from 5% solvent B to 100% solvent B at a flow rate of 30 mL / min over a 20-minute period.

[0580] Flash chromatography was carried out on a Biotage instrument using an Agela Flash Column silica-CS column. Reverse-phase flash chromatography was carried out on a Biotage instrument using a Boston ODS or Agela C18 column.

[0581] Analytical Chiral HPLC Method – SFC Conditions

[0582] a) Instrument: SFC Method Station (Thar, Waters)

[0583] b) Column: CHIRALPAK AD-H / AS-H / OJ-H / OD-H 4.6×100 mm, 5 μm (Daicel)

[0584] c) Column temperature: 40 °C

[0585] d) Mobile phase: CO2 / IPA (0.1% DEA) = 55 / 45

[0586] e) Flow rate: 4.0 mL / min

[0587] f) Back pressure: 120 Bar

[0588] g) Injection volume: 2 μL

[0589] Preparative Chiral HPLC Method – SFC Conditions

[0590] a) Instrument: SFC-80 (Thar, Waters)

[0591] b) Column: CHIRALPAK AD-H / AS-H / OJ-H / OD-H 20×250 mm, 10 μm (Daicel)

[0592] c) Column temperature: 35 °C

[0593] d) Mobile phase: CO2 / IPA (0.2% methanol-ammonia) = 30 / 70

[0594] e) Flow rate: 80 g / min

[0595] f) Back pressure: 100 bar

[0596] g) Detection wavelength: 214 nm

[0597] h) Cycle time: 6.0 min

[0598] i) Sample solution: 1500 mg dissolved in 70 mL of methanol

[0599] j) Injection volume: 2 mL (loading: 42.86 mg / injection)

[0600] Preparation Method

[0601] Intermediate 1A was synthesized as shown in Figure 1.

[0602] Compound 1A-1 (Figure 1) was synthesized according to Organic & Biomolecular Chemistry (2013), 11(14), 2273-2287, and compound 1A-7 (Figure 1) was synthesized according to WO2008 / 138561A1. The ketone 1A-1 was subjected to enantioselective reduction using the (R,R)-Ru-catalyst to give the (R,R)-isomer 1A-2 (Figure 1). The ketone 1A-1 was subjected to enantioselective reduction using the (S,S)-Ru-catalyst to give the (S,S)-isomer 1C-2 (Figure 3).

[0603] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl]amino}-1-hydroxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A-2)

[0604]

[0605] To a solution of compound 1A-1 (0.30 kg, 0.81 mol) in ethanol (4.5 L) were added the R,R-Ru-catalyst (CAS: 192139-92-7, 26 g, 41 mmol) and potassium hydroxide (4.5 g, 81 mmol). After stirring at room temperature for 3 h, the reaction mixture was quenched with saturated aqueous ammonium chloride (1.5 L) as monitored by LCMS. The volatiles were removed in vacuo and the residue was diluted with water (1.2 L). The aqueous mixture was extracted with ethyl acetate (2.0 L x 2), the combined organic extracts were washed with brine (0.50 L), dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (9-15% ethyl acetate / petroleum ether) to give compound 1A-2 (0.13 kg, 42% yield) as a white solid. ESI m / z: 373 (M+H) + , 395 (M+Na) + . TLC (silica gel): R f = 0.4 (33% ethyl acetate / petroleum ether; R of the other diastereomer f value was 0.2); 11H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 5.20 (d, J = 4.4 Hz, 1H), 5.05 - 4.97 (m, 1H), 4.55 (d, J = 10 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.81 - 3.66 (m, 1H), 2.14 - 2.03 (m, 1H), 1.82 - 1.69 (m, 2H), 1.44 (s, 9H), 1.40 (t, J = 7.2 Hz, 3H), 0.96 (d, J = 2.0 Hz, 3H), 0.95 (d, J = 2.4 Hz, 3H) ppm. After AS, AD, OD and OJ column chromatography, >99.9% ee.

[0606] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl]amino}-1-[(tert-butyldimethylsilyl)oxy]-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A-3)

[0607]

[0608] Under nitrogen, imidazole (0.12 kg, 1.8 mol) was added portionwise to a solution of compound 1A-2 (0.11 kg, 0.30 mol) in DCM (1.1 L) over 15 minutes, and tert-butyldimethylchlorosilane (TBSCl) (0.14 kg, 0.90 mol) was added dropwise. The reaction mixture was refluxed (35 °C) for 4 h until 1A-2 was completely consumed according to LCMS. After cooling to room temperature, the reaction mixture was quenched with saturated aqueous ammonium chloride (0.40 L), then extracted with DCM (0.40 L x 2). The combined organic solutions were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in ethyl acetate (0.40 L) and concentrated in vacuo 10 times to give crude 1A-3 (0.14 kg, crude) as a yellow oil. The crude 1A-3 was used in the next step without further purification. ESI m / z: 487 (M+H) + , 509 (M+Na) + . 11H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 5.18 (dd, J = 9.2 and 2.0 Hz, 1H), 4.64 (d, J = 9.2 Hz, 1H), 4.41 (q, J = 7.2 Hz, 2H), 3.81 - 3.66 (m, 1H), 1.89 - 1.77 (m, 2H), 1.71 - 1.61 (m, 1H), 1.44 (s, 9H), 1.39 (t, J = 7.2 Hz, 3H), 0.92 (s, 9H), 0.85 - 0.81 (m, 6H), 0.13 (s, 3H), -0.10 (s, 3H) ppm.

[0609] Ethyl 2-[(1R,3R)-3-amino-1-[(tert-butyldimethylsilyl)oxy]-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A-4)

[0610]

[0611] A solution of crude 1A-3 (0.14 kg, 0.29 mol) in DCM (1.4 L) was cooled to 0 °C. Over 30 minutes, TFA (0.24 L) was added dropwise to the cooled solution. The resulting mixture was stirred at room temperature for 16 h until 1A-3 was completely consumed according to LCMS. The mixture was then cooled to 0 °C and quenched with saturated aqueous sodium bicarbonate (2.8 L). The organic layer was washed with water (0.28 L x 2) and brine (0.28 L), dried over anhydrous sodium sulfate, and concentrated in vacuo to give crude compound 1A-4 (0.14 kg, crude) as a semi-solid. The crude 1A-4 was used in the next step without further purification. ESI m / z: 387 (M+H) + 。 1 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 5.58 - 5.53 (m, 1H), 4.37 (q, J = 7.2 Hz, 2H), 3.15 - 3.02 (m, 1H), 2.32 - 2.20 (m, 1H), 2.16 - 1.95 (m, 2H), 1.38 (t, J = 7.2 Hz, 3H), 0.98 - 0.95 (m, 6H), 0.94 (s, 9H), 0.20 (s, 3H), 0.06 (s, 3H) ppm.

[0612] Ethyl 2-[(1R,3R)-1-[(tert-butyldimethylsilyl)oxy]-3-(hexylamino)-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A-6)

[0613]

[0614] Under nitrogen, hexanal (1A-5, 20 g, 0.20 mol) was added dropwise to a solution of crude compound 1A-4 (90 g, 0.23 mol) in DCM (0.12 L) within 10 minutes. The reaction mixture was stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (0.15 kg, 0.70 mol) was added portionwise to the reaction mixture at 0 °C under nitrogen. The reaction mixture was then stirred at room temperature for 1 hour and monitored by LCMS. The resulting mixture was quenched with saturated aqueous sodium bicarbonate (0.20 L) and diluted with water (0.20 L). The organic layer was washed with water (0.20 L) and brine (0.20 L), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (9 - 50% ethyl acetate / petroleum ether) to give compound 1A-6 (45 g, 41% yield over 3 steps) as a white solid. ESI m / z: 471 (M+H) + 。 1 H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 5.27 (t, J = 5.6 Hz, 1H), 4.46 - 4.36 (m, 2H), 3.00 - 2.87 (m, 2H), 2.80 - 2.68 (m, 1H), 2.20 - 2.06 (m, 3H), 1.75 - 1.62 (m, 1H), 1.40 (t, J = 7.2 Hz, 3H), 1.34 - 1.21 (m, 8H), 0.94 (s, 9H), 0.93 - 0.85 (m, 9H), 0.20 (s, 3H), 0.06 (s, 3H) ppm.

[0615] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-azido-N-hexyl-3-methylpentanamido]-1-[(tert-butyldimethylsilyl)oxy]-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A-8)

[0616]

[0617] Under nitrogen, DIPEA (8.2 g, 64 mmol) was added dropwise to a cooled solution of compound 1A-6 (6.0 g, 13 mmol) in DCM (60 mL) within 2 minutes, and then compound 1A-7 (7.9 g, 45 mmol) was added dropwise within 5 minutes. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour until 1A-6 was completely consumed according to LCMS. Brine (12 mL) was added to the resulting mixture. The aqueous layer was extracted with DCM (18 mL), and the combined DCM solutions were dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (10% ethyl acetate / petroleum ether) to give compound 1A-8 (5.0 g, 64% yield) as a yellow oil. ESI m / z: 610 (M+H)+ ,632 (M+Na) + 。 1 H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 4.99 - 4.91 (m, 1H), 4.47 - 4.32 (m, 3H), 3.32 - 3.16 (m, 2H), 2.88 - 3.02 (m, 1H), 2.29 - 2.19 (m, 1H), 2.10 - 2.06 (m, 1H), 1.88 - 1.73 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H), 1.35 - 1.20 (m, 10H), 1.03 - 0.95 (m, 6H), 0.94 (s, 9H), 0.93 - 0.85 (m, 9H), 0.16 (s, 3H), -0.10 (s, 3H) ppm. Optical rotation: +99.5° (temperature: 19.8 °C, concentration: 1.25 mg / mL methanol solution).

[0618] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-amino-N-hexyl-3-methylpentanamido]-1-[(tert-butyldimethylsilyl)oxy]-4-methylpentyl]-1,3-thiazole-4-carboxylate (1A)

[0619]

[0620] Under nitrogen, at room temperature, within 5 minutes, triphenylphosphine (15 g, 57 mmol) was added dropwise to a solution of compound 1A-8 (5.0 g, 8.2 mmol) in THF (50 mL) and water (2.5 mL). The reaction mixture was stirred at 35 °C for 16 hours and monitored by LCMS. Then the volatiles were removed in vacuo, and the residue was dissolved in ethyl acetate (10 mL). Zinc chloride (3.3 g, 25 mmol) was added to the mixture, and the suspension was stirred at room temperature for 2 hours. The resulting suspension was filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (50% ethyl acetate / petroleum ether) to give intermediate 1A (3.0 g, 63% yield) as a yellow solid. ESI m / z: 584 (M+H) + 。 11H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 4.86 - 4.77 (m, 1H), 4.39 - 4.23 (m, 2H), 3.74 - 3.64 (m, 1H), 3.29 - 3.16 (m, 1H), 3.12 - 2.99 (m, 2H), 2.19 - 2.03 (m, 2H), 1.98 - 1.88 (m, 1H), 1.86 - 1.74 (m, 1H), 1.68 - 1.54 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.35 - 1.20 (m, 10H), 1.03 - 0.94 (m, 6H), 0.90 (s, 9H), 0.88 - 0.77 (m, 9H), 0.13 (s, 3H), -0.11 (s, 3H) ppm. Optical rotation: +41.3° (temperature: 19.8 °C, concentration: 1.16 mg / mL methanol solution).

[0621] Intermediate 1B was synthesized as shown in Figure 2.

[0622] Compound 1B-1 was synthesized according to WO2008 / 138561A1.

[0623] Ethyl 2-(3-{[(tert-butoxy)carbonyl](hex-5-yn-1-yl)amino}-4-methylvaleryl)-1,3-thiazole-4-carboxylate (1B-3)

[0624]

[0625] To a -65 °C solution of compound 1B-2 (73 g, 0.37 mol) in dry THF (1.2 L) was added dropwise KHMDS (1 M THF solution, 0.37 L, 0.37 mol) over 30 minutes, and then a solution of compound 1B-1 (62 g, 0.25 mol) in THF (0.20 L) was added while maintaining the temperature below -60 °C over 30 minutes. The reaction mixture was stirred at -65 °C for 4 hours until 1B-1 was completely consumed according to TLC. The resulting mixture was quenched with saturated aqueous ammonium chloride (0.30 L). The aqueous layer was extracted with ethyl acetate (0.5 L x 3). All the organic layers were combined, washed with brine (0.5 L), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% ethyl acetate / petroleum ether) to give compound 1B-3 (55 g, 50% yield) as a yellow oil. ESI m / z: 351 (M–Boc + H) + 。 11H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 4.44 (q, J = 7.2 Hz, 2H), 4.09 (brs, 1H), 3.70 - 3.42 (m, 2H), 3.30 - 2.99 (m, 2H), 2.25 - 2.15 (m, 2H), 2.12 - 1.90 (m, 2H), 1.70 - 1.55 (m, 2H), 1.55 - 1.43 (m, 5H), 1.42 (s, 9H), 1.00 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H) ppm。

[0626] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl](hex-5-yn-1-yl)amino}-1-hydroxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B-4)

[0627]

[0628] To a solution of compound 1B-3 (54 g, 0.12 mol) in isopropanol (0.60 L) was added R,R-Ru-catalyst (CAS: 192139-92-7, 3.9 g, 6.0 mmol) and potassium hydroxide (0.73 g, 12 mmol). The mixture was stirred at room temperature for 6 h until 1B-3 was completely consumed according to TLC. The reaction mixture was quenched with saturated aqueous ammonium chloride (0.3 L). The mixture was extracted with ethyl acetate (0.5 L x 3), and the combined organic extracts were washed with brine (0.5 L), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (10 - 20% ethyl acetate / petroleum ether) to give compound 1B-4 (15 g, 28% yield) as a yellow oil. ESI m / z: 453 (M+H) + , 475 (M+Na) + 。

[0629] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl](hexyl)amino}-1-hydroxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B-5)

[0630]

[0631] Under nitrogen, 10% Pd / C (50 mg, 11 wt%) was added to a solution of compound 1B-4 (0.45 g, 1.0 mmol) in methanol (10 mL). The suspension was degassed and purged with hydrogen three times, and then stirred under a hydrogen balloon at room temperature for 1 hour. The reaction was monitored by LCMS. The resulting suspension was filtered through Celite, and the filtrate was concentrated in vacuo to give the crude product 1B-5 (0.45 g, crude) as a white solid. The crude 1B-5 was used in the next step without further purification. ESI m / z: 457 (M+H) + , 479 (M+Na) + .

[0632] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl](hexyl)amino}-1-ethoxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B-6)

[0633]

[0634] Under nitrogen at -78 °C, a solution of KHMDS in THF (1.0 M, 2.0 mL, 2.0 mmol) was added dropwise over 5 minutes to a solution of compound 1B-5 (0.44 g, 1.0 mmol) and 18-crown-6 (0.53 g, 2.0 mmol) in THF (10 mL). The reaction mixture was stirred at -78 °C for 30 minutes, and then iodoethane (0.78 g, 5.0 mmol) was added. The mixture was then slowly warmed to room temperature and stirred for 1 hour, monitored by LCMS. After cooling to -10 °C, the resulting mixture was quenched with water (20 mL), and then extracted with ethyl acetate (20 mL x 3). The combined organic solutions were washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by preparative HPLC (5 - 95% acetonitrile / aqueous ammonium bicarbonate solution (10 mM)) to give compound 1B-6 (0.29 g, 60% yield over 2 steps) as a white solid. ESI m / z: 485 (M+H), 507 (M+Na) + .

[0635] Ethyl 2-[(1R,3R)-1-ethoxy-3-(hexylamino)-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B-7)

[0636]

[0637] At room temperature, TFA (1.0 mL) was added dropwise to a solution of compound 1B-6 (0.20 g, 0.41 mmol) in DCM (5.0 mL). The mixture was stirred at room temperature for 2 hours until LCMS showed complete removal of Boc. The volatiles were removed in vacuo to give the crude product 1B-7 (0.12 g, crude) as a white solid. The crude 1B-7 was used in the next step without further purification. ESI m / z: 385 (M+H) + .

[0638] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-azido-N-hexyl-3-methylpentanamido]-1-ethoxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B-8)

[0639]

[0640] Following a similar method to 1A-8, except using 1B-6 (0.15 g, 0.39 mmol) instead of 1A-6, compound 1B-8 (0.12 g, 60% yield) was obtained as a white solid. ESI m / z: 520 (M+H) + , 542 (M+Na) + .

[0641] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-amino-N-hexyl-3-methylpentanamido]-1-ethoxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1B)

[0642]

[0643] Under nitrogen, 10% Pd / C (50 mg, 50 wt%) was added to a solution of compound 1B-8 (0.10 g, 0.19 mmol) in methanol (10 mL). The suspension was degassed and purged with hydrogen three times. Then the reaction was stirred at room temperature under a hydrogen balloon for 1 hour and monitored by LCMS. The resulting suspension was filtered through Celite and the filtrate was concentrated in vacuo to give the intermediate 1B (0.16 g, 85% yield) as a white solid. The intermediate 1B was used in the next step without purification. ESI m / z: 498 (M+H) + .

[0644] The intermediate 1C was synthesized as shown in Figure 3

[0645] Ethyl 2-[(1S,3R)-3-{[(tert-butoxy)carbonyl]amino}-1-hydroxy-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-2)

[0646]

[0647] Following a similar method to 1A-2, except using S,S-Ru-catalyst (CAS: 192139-90-5) instead of R,R-Ru-catalyst, compound 1C-2 (1.7 g, 45% yield, 80 e.e%) was obtained as a colorless oil. ESI m / z: 373 (M+H) + . TLC (silica gel): R f = 0.3 (33% ethyl acetate / petroleum ether; the R value of the other diastereomer was 0.4). f

[0648] A small amount of the product was separated by chiral HPLC (column: R'RWHELK20*250 mm, 10 μm (Daicel), mobile phase: CO2 / MeOH (0.2% methanol-ammonia) = 90 / 10) to obtain the enantiomerically pure product 1C-2 (>99.9% ee). Using AS, AD, OD, and OJ columns, chiral HPLC: >99.9%. 1 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 6.53 (d, J = 9.3 Hz, 1H), 6.25 (d, J = 4.7 Hz, 1H), 4.81 (d, J = 4.8 Hz, 1H), 4.30 - 4.27 (m, 2H), 3.53 (s, 1H), 2.06 - 1.89 (m, 1H), 1.77 - 1.70 (m, 2H), 1.34 (s, 9H), 1.30 (t, J = 7.2 Hz, 3H), 0.81 (d, J = 3.4 Hz, 3H), 0.78 (d, J = 3.4 Hz, 3H) ppm.

[0649] Ethyl 2-[(1S,3R)-3-{[(tert-butoxy)carbonyl]amino}-1-(methanesulfonyloxy)-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-3)

[0650]

[0651] ​At 0 °C, triethylamine (0.60 g, 6.0 mmol) and methanesulfonyl chloride (0.55 g, 4.8 mmol) were successively added dropwise to a suspension of compound 1C-2 (1.4 g, 4.0 mmol, 80% ee) in DCM (50 mL). After the reaction became clear, the reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 30 min, monitored by TLC. The solution was washed successively with aqueous hydrochloric acid (1 N, 50 mL), water (50 mL), aqueous sodium carbonate (10%, 50 mL), and brine (50 mL). The resulting organic solution was dried over anhydrous sodium sulfate and concentrated in vacuo to give crude compound 1C-3 (1.6 g, crude) as a yellow oil. The crude 1C-3 was used in the next step without further purification. ESI m / z: 451 (M+H) + 。

[0652] Ethyl 2-[(1R,3R)-1-azido-3-{[(tert-butoxy)carbonyl]amino}-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-4)

[0653]

[0654] At room temperature, sodium azide (1.2 g, 18 mmol) was added to a stirred mixture of compound 1C-3 (1.6 g, crude) in DMF (10 mL). The reaction mixture was stirred at room temperature for 1 h, monitored by LCMS. The mixture was then diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic solutions were washed with water (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give crude compound 1C-4 (1.3 g, crude) as a yellow oil. ESI m / z: 398 (M+H) + 。

[0655] Ethyl 2-[(1R,3R)-1-amino-3-{[(tert-butoxy)carbonyl]amino}-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-5)

[0656]

[0657] Under nitrogen, 10% Pd / C (0.12 g, 10 wt%) was added to a solution of compound 1C-4 (1.3 g, crude) in methanol (50 mL). The suspension was degassed and purged with hydrogen three times. Then the reaction was stirred at room temperature under a hydrogen balloon for 1 hour and monitored by LCMS. The resulting suspension was filtered through Celite, and the filtrate was concentrated in vacuo to give crude compound 1C-5 (1.0 g, crude) as a yellow oil. The crude 1C-5 was used in the next step without further purification. ESI m / z: 371 (M+H) + 。

[0658] Ethyl 2-[(1R,3R)-3-{[(tert-butoxy)carbonyl]amino}-1-acetamido-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-6)

[0659]

[0660] At 0 °C, triethylamine (0.45 g, 4.5 mmol) and acetyl chloride (0.28 g, 3.6 mmol) were successively added to a stirred suspension of compound 1C-5 (1.0 g, crude) in DCM (50 mL). After the reaction became clear, the reaction mixture was stirred at room temperature for 1.5 hours and monitored by LCMS. Then the resulting solution was washed with aqueous hydrochloric acid (1 N, 50 mL), water (50 mL), aqueous sodium carbonate (10%, 50 mL), and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (15 - 20% ethyl acetate / petroleum ether) to give compound 1C-6 (1.0 g, 66% yield over 4 steps) as a yellow oil. ESI m / z: 413 (M+H) + 。

[0661] Ethyl 2-[(1R,3R)-3-amino-1-acetamido-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-7)

[0662]

[0663] At 0 °C, TFA (4 mL) was added to a solution of compound 1C-6 (1.3 g, 3.0 mmol) in DCM (20 mL). The mixture was stirred at room temperature for 1 hour and monitored by LCMS. The volatiles were removed in vacuo to give crude compound 1C-7 (1.0 g, crude) as a yellow solid. The crude 1C-7 was used in the next step without further purification. ESI m / z: 314 (M+H) + 。

[0664] Ethyl 2-[(1R,3R)-1-acetamido-3-(hexylamino)-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-8)

[0665]

[0666] Under nitrogen, to a solution of crude compound 1C-7 (0.70 g, 2.2 mmol) in DCM (30 mL) were added successively n-hexanal (1A-5, 0.26 g, 2.6 mmol), sodium triacetoxyborohydride (0.70 g, 3.3 mmol), and 2 drops of TFA over 5 minutes. The reaction mixture was stirred at room temperature for 1 hour and monitored by LCMS. The resulting mixture was washed with water (20 mL), aqueous sodium carbonate (10%, 20 mL), brine (20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by chiral HPLC (column: IG 20*250 mm, 10 μm, mobile phase: CO2 / methanol (0.2% methanol-ammonia) = 80 / 20) to give compound 1C-8 (0.52 g, 60% yield over 2 steps) as a colorless oil. ESI m / z: 398 (M+H) + 。 1 H NMR (400 MHz, DMSO d6 ) δ 8.77 (d, J = 7.8 Hz, 1H), 8.39 (s, 1H), 5.33 - 5.26 (m, 1H), 4.38 - 4.18 (m, 2H), 2.56 - 2.50 (m, 1H), 2.39 - 2.30 (m, 2H), 1.89 (s, 3H), 1.83 - 1.70 (m, 2H), 1.37 - 1.19 (m, 12H), 0.85 - 0.79 (m, 9H) ppm. Using an IG column, >99.9% ee.

[0667] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-azido-N-hexyl-3-methylpentanamido]-1-acetamido-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C-9)

[0668]

[0669] To a mixture of compound 1C-8 (0.20 g, 0.50 mmol) in DCM (5 mL) were successively added DIPEA (0.13 g, 1.0 mmol) and compound 1A-7 (0.18 g, 1.0 mmol). The mixture was stirred at room temperature for 2 hours and monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by silica gel column chromatography (15 - 20% ethyl acetate / petroleum ether) to give compound 1C-9 (0.19 g, 70% yield) as a yellow oil. ESI m / z: 537 (M+H) + 。

[0670] Ethyl 2-[(1R,3R)-3-[(2S,3S)-2-amino-N-hexyl-3-methylpentanamido]-1-acetamido-4-methylpentyl]-1,3-thiazole-4-carboxylate (1C)

[0671]

[0672] Under nitrogen, to a solution of compound 1C-9 (0.19 g, 0.35 mmol) in methanol (10 mL) was added 10% Pd / C (20 mg, 10 wt%). The suspension was degassed and purged with hydrogen three times. Then the reaction was stirred at room temperature under a hydrogen balloon for 2 hours and monitored by LCMS. The resulting suspension was filtered through Celite, and the filtrate was concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (50% ethyl acetate / petroleum ether) to give intermediate 1C (0.15 g, 90% yield) as a yellow oil. ESI m / z: 511 (M+H) + 。

[0673] Intermediate 1G was synthesized as shown in Figure 4 and as shown in U.S. Patent Application No. 16 / 724,164 filed on December 20, 2019. The synthesis of the corresponding compounds in U.S. Patent Application No. 16 / 724,164 are incorporated herein by reference.

[0674] Intermediate: MEP

[0675] Intermediates MEPa - MEPe are all commercially available. The CAS numbers and structural formulas are shown below.

[0676]

[0677] Intermediate: TUP

[0678] The intermediate TUPa-l was synthesized as shown in Figure 5. The intermediates TUPa-TUPe were all synthesized as shown in ...

Claims

1. A compound having the structure shown by the following formula: or a pharmaceutically acceptable salt thereof, wherein, BA is a binder; L is a linker covalently linked to both BA and T; T is Among them, R 1 is H or C1-C 10 alkyl; R 3 is hydroxy, –O-C1-C5 alkyl, –OC(O)C1-C5 alkyl, –OC(O)N(H)C1-C 10 alkyl, –OC(O)N(H)C1-C 10 alkylene-NR 3a R 3b or –OC(O)N(H)(CH2CH2O) n C1-C 10 alkylene-NR 3a R 3b , wherein R 3a and R 3b , in each case independently, is H or a C1-C5 alkyl group; R 4 and R 5 , in each case independently being H or a C1-C5 alkyl group; R 6 is –OH or –NHSO2(CH2) a1 -phenyl-(CH2) a2 NR 6a R 6b ; and R 6a and R 6b , in each case independently, is H or a C1-C5 alkyl group; R 7 In each case, independently for H, –OH, –O–, halogen, or –NR 7a R 7b , wherein R 7a and R 7b , in each case independently of one another, is a key, H, C1-C5 alkyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first amino acid residue, a first N-terminal peptide residue, a first peptide residue, –CH2CH2NH2, and –CH2CH2NH–; R 8 , in each case independently of one another, is H, –NHR 9 , or a halogen, wherein R 9 is H or –C1-C5 alkyl; and m is 1 or 2; R 10 , when present, is -C1-C5 alkyl; Q is –O–, where R 2 is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, or regiomeric triazole; wherein the regioisomeric triazole is unsubstituted or substituted with a C1-C5 alkyl group; wherein n is an integer from 1 to 10; wherein r is an integer from 1 to 6; wherein a, a1 and a2 are each independently 0 or 1; and k is an integer from 1 to 30; wherein T is not: a compound IVa, IVa′, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IVl, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va′, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, VIa, IVb, VIc, VId, VIe, VIf, VIg, VIh, Vl, VIi, VII, VIII, IX, X, D-5a, and D-5c covalently linked to L, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, having the structure shown by formula C: wherein L is a linker.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, R 7 In each case, independently for H, –OH, –O–, halogen, or –NR 7a R 7b , wherein R 7a and R 7b , in each case independently, is a key, H, C1-C5 alkyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first N-terminal peptide residue, –CH2CH2NH2, and –CH2CH2NH–.

4. The compound according to claim 2, wherein the compound is a compound shown by formula C′: wherein SP 1 and SP 2 , when present, are each a spacer group; each AA, when present, is a second amino acid residue; and p is an integer from 0 to 10.

5. The compound according to claim 4, wherein, said –SP 2 – a spacer group, when present, is –(AA) p – is said –SP 1 – spacer group is wherein RG′ is the residue of the active group after the reaction of the active group RG with the binder; is a bond directly or indirectly connected to the binder; and b is an integer from 1 to 4.

6. The compound according to claim 5, wherein the binder is an antibody modified with a primary amine compound according to formula H2N-LL-X, wherein LL is a divalent linker selected from the group consisting of: a divalent polyethylene glycol (PEG) group; –(CH2) n– ; –(CH2CH2O) n -(CH2) p– ; –(CH2) n -N(H)C(O)-(CH2) m– ; –(CH2CH2O) n -N(H)C(O)-(CH2CH2O) m -(CH2) p– ; –(CH2) n -C(O)N(H)-(CH2) m– ; –(CH2CH2O) n -C(O)N(H)-(CH2CH2O) m -(CH2) p– ; –(CH2) n -N(H)C(O)-(CH2CH2O) m -(CH2) p– ; –(CH2CH2O) n -N(H)C(O)-(CH2) m– ; –(CH2) n -C(O)N(H)-(CH2CH2O) m -(CH2) p– ; and –(CH2CH2O) n -C(O)N(H)-(CH2) m –, wherein: n is an integer selected from 1 to 12; m is an integer selected from 0 to 12; p is an integer selected from 0 to 2; and X is selected from the group consisting of: –SH, –N3, –C≡CH, –C(O)H, tetrazole, 7. The compound according to claim 6, wherein the binder is an antibody modified with a primary amine having the structure represented by the following formula:

8. The compound according to claim 4, wherein, Q is –O–; R 1 is H or C1-C 10 alkyl; R 2 is C1-C 20 alkyl or C2-C 20 alkynyl; R 3 is a hydroxyl group or –OC(O)C1-C5 alkyl; R 4 and R 5 are each C1-C5 alkyl; R 6 is –OH; R 10 , when present, is a C1-C5 alkyl group; wherein r is 3 or 4; and wherein a is 1.

9. The compound according to claim 8, having the structure shown by C′, or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 9, wherein R 7 is –NH–; and R 8 is H.

11. The compound according to claim 4, wherein, Q is –O–; R 1 is C1-C 10 alkyl; R 2 is a C1-C 20 alkyl or a C2-C 20 alkynyl; R 4 and R 5 are each a C1-C5 alkyl group; R 6 is –NHSO2(CH2) a1 -phenyl-(CH2) a2 NR 6a R 6b ; R 10 Does not exist; wherein r is 4; and wherein a, a1 and a2 are each independently 0 or 1.

12. The compound according to claim 9, wherein R 7 is –O–; and R 8 is H.

13. The compound according to claim 4, selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein BA is a binder; and k is 1, 2, 3, or 4.

14. The compound according to claim 13, wherein BA is an antibody, or an antigen-binding fragment thereof.

15. The compound according to claim 13, wherein BA is an antibody modified with transglutaminase, or an antigen-binding fragment thereof, which contains at least one glutamine residue for conjugation.

16. The compound according to claim 13, wherein BA is a transglutaminase-modified antibody, or an antigen-binding fragment thereof, which comprises at least two glutamine residues for conjugation.

17. The compound according to claim 13, wherein BA is a transglutaminase-modified antibody, or an antigen-binding fragment thereof, which comprises at least four glutamine residues for conjugation.

18. The compound according to claim 17, wherein BA is a transglutaminase-modified antibody, or an antigen-binding fragment thereof, wherein the conjugation is carried out at two Q295 residues; and k is 2.

19. The compound according to claim 17, wherein BA is a transglutaminase-modified antibody, or an antigen-binding fragment thereof, wherein the conjugation is carried out at two Q295 residues and two N297Q residues; and k is 4.

20. The compound according to claim 1, wherein the compound is an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof is conjugated to a compound selected from the group consisting of:

21. The compound according to claim 14, wherein the antibody or the antigen-binding fragment thereof is selected from the group consisting of: anti-MUC16, anti-PSMA, anti-EGFRvIII, anti-HER2, and anti-MET or an antigen-binding fragment thereof.

22. The compound according to claim 14, wherein the antibody or the antigen-binding fragment thereof is anti-PRLR, or anti-STEAP2 or an antigen-binding fragment thereof.

23. The compound according to claim 14, wherein the antigen of the antibody or its antigen-binding fragment is selected from the group consisting of: lipoproteins; α1-antitrypsin; cytotoxic T lymphocyte-associated antigen (CTLA); vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or protein D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FLT3, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto (teratocarcinoma-derived growth factor antigen), αvβ6 (alphavbeta6), VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152; erythropoietin; osteogenic induction factors; immunotoxins; bone morphogenetic protein (BMP); T cell receptors; surface membrane proteins; integrins CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM;Tumor-associated antigens AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD123, CDK4, CLEC12A, c-kit, cMET, MET, cyclin-B1, CYP1B1, EGFRvIII, endoglin, EphA2, ErbB2 / Her2, ErbB3 / Her3, ErbB4 / Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins GAGE-1 and GAGE-2, GD2, GloboH, glypican-3, GM3, gp100, HER2, HLA / B-raf, HLA / EBNA1, HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins, MART-1, ML-IAP, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSGR, PSMA (FOLH1), RAGE proteins Ras, RGS5, Rho, SART-1, SART-3, Steap-1 (prostate transmembrane epithelial antigen-1), STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, uroplakin-3; antigens expressed on the cell surface; class A scavenger receptors; B7 family-related members including V-set and Ig domain-containing protein 4 (VSIG4), colony-stimulating factor 1 receptor (CSF1R), asialoglycoprotein receptor (ASGPR), and amyloid-β precursor-like protein 2 (APLP-2); BCMA; SLAMF7; GPNMB; and UPK3A.; 24. A pharmaceutical composition, which comprises the compound according to claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent.

25. Use of the compound according to claim 1 in the preparation of a medicament for the treatment of cancer.

26. Use of the compound according to claim 1 in the preparation of a medicament for the treatment of cancer, wherein the cancer is selected from the group consisting of: renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, castration-resistant prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, mesothelioma, multiple myeloma, ovarian cancer, lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, glioblastoma, Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lymphoma, MFH / fibrosarcoma, nasopharyngeal cancer, rhabdomyosarcoma, colon cancer, gastric cancer, uterine cancer, residual cancer, and Wilms' tumor.

27. The use according to claim 26, wherein the cancer is small cell lung cancer or non-small cell lung cancer.

28. Use of the compound according to claim 1 in the preparation of a medicament for the treatment of a tumor that expresses an antigen selected from the group consisting of PRLR and STEAP2.

29. A linker-payload, which has a structure represented by the following formula: L-T or a pharmaceutically acceptable salt thereof, wherein, L is a linker covalently linked to T; T is Among them, R 1 is H or C1-C 10 alkyl; R 3 is hydroxy, –O-C1-C5 alkyl, –OC(O)C1-C5 alkyl, –OC(O)N(H)C1-C 10 alkyl, –OC(O)N(H)C1-C 10 alkylene-NR 3a R 3b 、or –OC(O)N(H)(CH2CH2O) n C1-C 10 alkylene-NR 3a R 3b , wherein R 3a and R 3b , in each case independently of one another, are H or C1-C5 alkyl; R 4 and R 5 , in each case, independently of one another, is H or C1-C5 alkyl; R 6 is –OH or –NHSO2(CH2) a1 -phenyl-(CH2) a2 NR 6a R 6b ; and R 6a and R 6b is H or a C1-C5 alkyl group; R 7 In each case, independently for H, –OH, –O–, halogen, or –NR 7a R 7b , wherein R 7a and R 7b , in each case independently, is a key, H, C1-C5 alkyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first amino acid residue, a first N-terminal peptide residue, a first peptide residue, –CH2CH2NH2, and –CH2CH2NH–; R 8 in each case independently is H, –NHR 9 , or a halogen, wherein R 9 is H or a C1-C5 alkyl; and m is 1 or 2; R 10 , when present, is -C1-C5 alkyl; Q is –O–, where R 2 is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl or regiomeric triazole; wherein the regioisomeric triazole is unsubstituted or substituted with a C1-C5 alkyl group; wherein n is an integer from 1 to 10; wherein r is an integer from 1 to 6; wherein a, a1 and a2 are each independently 0 or 1; and wherein the linker-payload is not: LP1-IVa, LP2-Va, LP3-IVd, LP4-Ve, LP5-IVd, LP6-Vb, LP7-IVd, LP9-IVvB, LP10-VIh, LP11-IVvB, LP12-VIi, LP13-Ve, LP14-Ve, LP15-VIh, LP16-Ve, LP17-Ve, LP18-Ve, LP19-Ve, LP20-Ve, LP21-Ve, LP22-Ve, LP23-Vb, LP24-Vb, LP25-Ve, and LP26-Ve, or a pharmaceutically acceptable salt thereof.

30. The linker-payload according to claim 29, having the structure shown in formula LPc: where L is a linker.

31. The linker-payload according to claim 30, wherein, R 7 In each case, independently for H, –OH, –O–, halogen, or –NR 7a R 7b , wherein R 7a and R 7b , in each case independently, is a key, H, C1-C5 alkyl, –C(O)CH2OH, –C(O)CH2O–, a first N-terminal amino acid residue, a first N-terminal peptide residue, –CH2CH2NH2, and –CH2CH2NH–.

32. The linker-payload according to claim 31, having the structure shown in formula LPc': wherein, SP 1 and SP 2 which, when present, are each a spacer group; each AA, when present, is a second amino acid residue; and p is an integer from 0 to 10.

33. The linker-payload according to claim 32, wherein, The said –SP 2 – a spacer group, when present, is –(AA) p – is The said –SP 1 – the spacer group is wherein RG is a reactive group; and b is an integer from 1 to 4.

34. The linker-payload according to claim 32, wherein, Q is –O–; R 1 is H or C1-C 10 alkyl; R 2 is a C1-C 20 alkyl or a C2-C 10 alkynyl; R 3 is a hydroxyl group or –OC(O)C1-C5 alkyl; R 4 and R 5 are each independently a C1-C5 alkyl group; R 6 is –OH; R 10 , when present, is a C1-C5 alkyl group; wherein r is 3 or 4; and wherein a is 1.

35. The linker-payload according to claim 34, having the structure shown in LPc' or a pharmaceutically acceptable salt thereof.

36. The linker-payload according to claim 35, wherein R 7 is –NH–; and R 8 is H.

37. The linker-payload according to claim 32, wherein, Q is –O–; R 1 is a C1-C 10 alkyl group; R 2 is a C1-C 20 alkyl or C2-C 20 alkynyl; R 4 and R 5 are each a C1-C5 alkyl group; R 6 is –NHSO2(CH2) a1 -phenyl-(CH2) a2 NR 6a R 6b ; R 10 Does not exist; wherein r is 4; and wherein a, a1 and a2 are each independently 0 or 1.

38. The linker-payload according to claim 32, wherein the linker-payload is selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

39. Use of the pharmaceutical composition according to claim 24 for the preparation of a medicament for the treatment of cancer.

40. Use of the pharmaceutical composition according to claim 24 for the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of: renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, castration-resistant prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, mesothelioma, multiple myeloma, ovarian cancer, lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, glioblastoma, Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lymphoma, MFH / fibrosarcoma, nasopharyngeal cancer, rhabdomyosarcoma, colon cancer, gastric cancer, uterine cancer, residual cancer, and Wilms' tumor.

41. Use of the pharmaceutical composition according to claim 24 for the preparation of a medicament for treating a tumor that expresses an antigen selected from the group consisting of PRLR and STEAP2.

42. The compound according to claim 14, wherein the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: CTLA4; MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and / or MAGE-12; and scavenger receptor A (SR-A).

43. Use of the pharmaceutical composition according to claim 24 for the preparation of a medicament for treating cancer, wherein the cancer is selected from malignant mesothelioma, small cell lung cancer, non-small cell lung cancer, and PRLR-positive (PRLR+) breast cancer.

44. A compound selected from: or a pharmaceutically acceptable salt thereof, wherein BA is a binder; and k is 1, 2, 3, or 4.

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