Branched linkers for antibody-drug conjugates and methods of use thereof

Abstract

The present disclosure provides an antibody-drug conjugate structure. The antibody-drug conjugate structure comprises a branched linker, where two or more payloads per branched linker are attached to a polypeptide (e.g., an antibody). In addition, the present disclosure also includes compounds and methods for producing such conjugates. In addition, the present disclosure also includes methods of using the conjugates.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 237,450, filed August 26, 2021, U.S. Provisional Patent Application No. 63 / 156,156, filed March 3, 2021, U.S. Provisional Patent Application No. 63 / 186,581, filed May 10, 2021, and U.S. Provisional Patent Application No. 63 / 214,540, filed June 24, 2021, the disclosures of which are incorporated herein by reference.

[0002] introduction Antibody-drug conjugates (ADCs) have emerged over the past two decades as a new class of targeted delivery therapy. A typical ADC contains an antibody-based targeting element attached to a highly potent pharmaceutical (payload) via a chemical linker using available bioconjugation methods. The molar ratio of the targeting element (e.g., antibody) to the attached payload can vary and is referred to as the drug-to-antibody ratio (DAR). Commonly used bioconjugation methods either utilize endogenous amino acid residues in proteins (i.e., lysine and cysteine) or rely on the selective engagement of bioorthogonal functional groups intentionally introduced into proteins. As an example of the latter approach, the hydrazino-iso-pictet-spengler (HIPS) conjugation method (Figure 1) utilizes an aldehyde functional group ("aldehyde tag") that can be introduced into proteins, such as antibodies, via various means (e.g., by the action of formyl-generating enzymes (FGEs)) to serve as a conjugation handle. The aldehyde group reacts cleanly with the HIPS indole moiety to form a stable carbon-carbon bond that permanently attaches the payload of choice to the protein in a single chemical step. Summary of the Invention

[0003] Traditionally, HIPS conjugation methods have been used to generate conjugates with one HIPS-moiety payload per aldehyde tag, producing antibody conjugates with DAR values ​​of up to 4. The present disclosure provides the use of branched HIPS linkers that carry two (or more) molecules of the same or different payload per HIPS moiety, thus enabling the conjugation of two (or more) small molecule payloads per aldehyde group in a protein in a single conjugation step (Figure 2). Thus, the use of such branched linkers allows the generation of higher DAR site-specific conjugates (e.g., DARs of up to 8) with controlled payload placement, which, in the context of therapeutic ADCs, would result in greater amounts of drug delivered to target tissues.

[0004] The present disclosure provides antibody-drug conjugate (ADC) structures that include a branched HIPS linker. The present disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using the conjugates.

[0005] An embodiment of the present disclosure is a conjugate of formula (I): [ka] During the ceremony, Z 1 , Z 2 , Z 3 , and Z 4 However, each independently, CR 4 , N, and CL B -W 2 and at least one Z 1 , Z 2 , Z 3 , and Z 4 But CL B -W 2 and R 1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker, L B is the second linker, W 1 is the first drug, W 2 is the second drug, W 3 is a polypeptide.

[0006] In some embodiments, Z1 is CR 4 is.

[0007] In some embodiments, Z 1 is N.

[0008] In some embodiments, Z 3 is CL B -W 2 is.

[0009] In some embodiments, L A teeth, -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f - Including, During the ceremony, a, b, c, d, e, and f are each independently 0 or 1; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each x is an integer of 1 to 12; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0010] L AIn some embodiments of T 1 However, (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from the group consisting of SO2-, and -P(O)OH-; During the ceremony, (PEG) n but, [ka] and n is an integer from 1 to 30; EDA, structure, [ka] wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring.

[0011] In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside.

[0012] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0013] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0014] In some embodiments, L A is a linker, wherein T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 But it doesn't exist, T 3 But PABC, V 3But it doesn't exist, d, e, and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 However, PABA and V 4 is -CO-, T 5 However, (C1-C 12 ) alkyl, and V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -O-, T 4 However, (C1-C 12 ) alkyl, and V 4 is -CO-, T 5 But AA and V 5 But it doesn't exist, T 6 But PABC, V 6 does not exist, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 But it doesn't exist, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG)n and V 2 is -CONH-, T 3 is substituted (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 is PAP, and V 4 is -C(O)O-, e and f are each 0, or During the ceremony, T 1 However, (C1-C12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0.

[0015] In some embodiments, L B teeth, -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) m - Including, During the ceremony, g, h, i, j, k, 1, and m are each independently 0 or 1; T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each x is an integer of 1 to 12; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0016] In some embodiments, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside.

[0017] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0018] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0019] In some embodiments, L B is a linker, wherein T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10But it doesn't exist, T 11 However, PABA and V 11 is -CO-, T 12 However, (C1-C 12 ) alkyl, and V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -O-, T 11 However, (C1-C 12 ) alkyl, and V 11 is -CO-, T12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 But it doesn't exist, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CONH-, T 10 is substituted (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 However, (PEG) n and V 11 is -CO-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PAB, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 But it doesn't exist.

[0020] In some embodiments, the conjugate is selected from the following: [ka] , [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0021] In some embodiments, the conjugate is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka]

[0022] An aspect of the present disclosure is a compound of formula (II): [ka] During the ceremony, Z 1 , Z 2 , Z 3 , and Z 4 However, each independently, CR 4 , N, and CL B -W 2 and at least one Z 1 , Z 2 , Z 3 , and Z 4 But CL B -W 2 and R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker, L B is the second linker, W 1 is the first drug, W 2 is a second drug, a compound.

[0023] In some embodiments, Z 1 is CR 4 is.

[0024] In some embodiments, Z 1 is N.

[0025] In some embodiments, Z 3 is CL B -W 2 is.

[0026] In some embodiments, L A teeth, -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 )f - Including, During the ceremony, a, b, c, d, e, and f are each independently 0 or 1; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each x is an integer of 1 to 12; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0027] L A In some embodiments of T 1 However, (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from the group consisting of SO2-, and -P(O)OH-; During the ceremony, (PEG) n but, [ka] and n is an integer from 1 to 30; EDA, structure, [ka] wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups may be cyclically linked to form a piperazinyl ring.

[0028] In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside.

[0029] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0030] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0031] In some embodiments, L A is a linker, wherein T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 But it doesn't exist, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 However, PABA and V 4 is -CO-, T 5 However, (C1-C 12 ) alkyl, and V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -O-, T 4 However, (C1-C 12 ) alkyl, and V 4 is -CO-, T 5 But AA and V 5 But it doesn't exist, T 6 But PABC, V6 does not exist, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 But it doesn't exist, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CONH-, T 3 is substituted (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 is PAP, and V 4 is -C(O)O-, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0.

[0032] In some embodiments, L B teeth, -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) m - Including, During the ceremony, g, h, i, j, k, and l are each independently 0 or 1; T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each independently a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal group, hydrazine, disulfide, and ester; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer of 1 to 20; each n is an integer of 1 to 30; each p is an integer of 1 to 20; and each x is an integer of 1 to 12; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0033] In some embodiments, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside.

[0034] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0035] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0036] In some embodiments, L B is a linker, wherein T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 However, PABA and V 11 is -CO-, T 12 However, (C1-C 12 ) alkyl, and V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10However, (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -O-, T 11 However, (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 But it doesn't exist, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CONH-, T 10 is substituted (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 However, (PEG) n and V 11 is -CO-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PAB, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 But it doesn't exist.

[0037] In some embodiments, the compound is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0038] In some embodiments, the compound is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka]

[0039] Aspects of the present disclosure include pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable excipient.

[0040] Aspects of the present disclosure include methods of administering a conjugate to a subject, the methods comprising administering to a subject a conjugate described herein.

[0041] Aspects of the present disclosure include methods of treating cancer, the method comprising administering to a subject a therapeutically effective amount of a conjugate described herein, wherein the administering is effective to treat cancer in the subject. [Brief explanation of the drawings]

[0042] [Figure 1] Schematic of HIPS ligation for the synthesis of ADCs. An antibody bearing an aldehyde moiety is reacted with a hydrazino-iso-pictet-Spengler (HIPS) linker and a payload to generate a site-specifically conjugated ADC bearing a stable azacarboline bond. [Figure 2] 1 shows a schematic diagram of branched HIPS ligation for the synthesis of ADCs. An antibody bearing four aldehyde moieties is reacted with a branched HIPS linker to generate ADCs with drug-to-antibody (DAR) values ​​of up to 8, according to embodiments of the present disclosure. [Figure 3] 1 shows a graph illustrating that a CT-tagged polatuzumab conjugate of construct 15 resulted in a DAR of 3.57 as determined by hydrophobic interaction chromatography (HIC), according to an embodiment of the present disclosure. [Figure 4] 1 shows a graph demonstrating that the CT-tagged polatuzumab conjugate of construct 15 was 97.5% monomeric as determined by analytical size exclusion chromatography (SEC), according to an embodiment of the present disclosure. [Figure 5] 1 shows a graph showing that the CH1-3 / CT tagged polatuzumab conjugate of construct 15 resulted in a DAR of 7.19 as determined by polymer reverse phase (PLRP) according to embodiments of the present disclosure. [Figure 6] 1 shows a graph demonstrating that the CH1-3 / CT tagged polatuzumab conjugate of construct 15 was 97.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 7] 1 shows a graph showing that the LC-9 / CT dual-tagged polatuzumab conjugate of construct 15 resulted in a DAR of 7.06 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 8] 1 shows a graph demonstrating that the LC-9 / CT dual-tagged polatuzumab conjugate of construct 15 was 99.1% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 9] 1 shows a graph showing that the LC-9 / CH2-3 dual-tagged polatuzumab conjugate of construct 15 resulted in a DAR of 7.36 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 10] 1 shows a graph demonstrating that the LC-9 / CH2-3 dual-tagged polatuzumab conjugate of construct 15 was 87.3% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 11] 1 shows a graph showing that the CH1-5 / CT dual-tagged polatuzumab conjugate of Construct 15 resulted in a DAR of 5.78 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 12] 1 shows a graph demonstrating that the CH1-5 / CT dual-tagged polatuzumab conjugate of construct 15 was 99.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 13] 1 shows a graph showing that the CH1-6 / CT dual-tagged polatuzumab conjugate of Construct 15 resulted in a DAR of 6.86 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 14] 1 shows a graph demonstrating that the CH1-6 / CT dual-tagged polatuzumab conjugate of construct 15 was 99.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 15] 1 shows a graph showing that the CH1-5 / CH2-3 dual-tagged polatuzumab conjugate of Construct 15 resulted in a DAR of 7.06 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 16] 1 shows a graph demonstrating that the CH1-5 / CH2-3 dual-tagged polatuzumab conjugate of Construct 15 was 92.8% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 17]1 shows a graph showing that the CH1-6 / CH2-3 dual-tagged polatuzumab conjugate of Construct 15 resulted in a DAR of 7.14 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 18] 1 shows a graph demonstrating that the CH1-6 / CH2-3 dual-tagged polatuzumab conjugate of Construct 15 was 98.5% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 19] 1 shows a graph showing that the CH1-3 / CT dual-tagged polatuzumab conjugate of Construct 22 resulted in a DAR of 3.87 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 20] 1 shows a graph demonstrating that the CH1-3 / CT dual-tagged polatuzumab conjugate of construct 22 was 93.6% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 21] 1 shows a graph showing that a CT-tagged polatuzumab conjugate of construct 22 resulted in a DAR of 1.91 as determined by HIC, according to an embodiment of the present disclosure. [Figure 22] 1 shows a graph demonstrating that the CT-tagged polatuzumab conjugate of construct 22 was 95.0% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 23] 1 shows a graph showing that the CH1-3 / CT dual-tagged polatuzumab conjugate of construct 25 resulted in a DAR of 4.66 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 24] 1 shows a graph demonstrating that the CH1-3 / CT tagged polatuzumab conjugate of construct 25 was 96.9% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 25] 1 shows a graph showing that a CT-tagged polatuzumab conjugate of construct 25 resulted in a DAR of 2.27 as determined by HIC, according to an embodiment of the present disclosure. [Figure 26] 1 shows a graph demonstrating that the CT-tagged polatuzumab conjugate of construct 25 was 96.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 27] 1 shows a graph showing that a CT-tagged polatuzumab conjugate of construct 18 resulted in a DAR of 3.58 as determined by HIC, according to an embodiment of the present disclosure. [Figure 28] 1 shows a graph demonstrating that the CT-tagged polatuzumab conjugate of construct 18 was 96.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 29] 1 shows a graph showing that a CH1-3 / CT tagged anti-FITC conjugate of construct 15 resulted in a DAR of 7.0 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 30] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-FITC conjugate of construct 15 was 98.1% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 31] 1 shows a graph showing that a CH1-3 / CT tagged daclizumab conjugate of construct 15 resulted in a DAR of 7.07 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 32] 1 shows a graph demonstrating that the CH1-3 / CT tagged daclizumab conjugate of construct 15 was 98.6% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 33] 1 shows a graph showing that the CH1-3 / CT tagged anti-GPC-3 conjugate of construct 15 resulted in a DAR of 6.86 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 34] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-GPC-3 conjugate of construct 15 was 98.2% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 35]1 shows a graph showing that a CH2-3 / CT tagged anti-FITC conjugate of construct 15 resulted in a DAR of 4.60 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 36] 1 shows a graph demonstrating that the CH2-3 / CT tagged anti-FITC conjugate of construct 15 was 96.7% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 37] 1 shows a graph showing that a CH1-3 / CT tagged alemtuzumab conjugate of construct 15 resulted in a DAR of 7.07 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 38] 1 shows a graph demonstrating that the CH1-3 / CT tagged alemtuzumab conjugate of construct 15 was 98.0% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 39] 1 shows a graph showing that a CH1-3 / CT tagged daclizumab conjugate of construct 15 resulted in a DAR of 7.24 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 40] 1 shows a graph demonstrating that the CH1-3 / CT tagged daclizumab conjugate of construct 15 was 98.9% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 41] 1 shows a graph showing that a CH1-3 / CT tagged anti-CD70 conjugate of construct 15 resulted in a DAR of 6.97 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 42] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-CD70 conjugate of construct 15 was 99.4% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 43] 1 shows a graph showing that the CH1-3 / CT tagged anti-nectin-4 conjugate of construct 15 resulted in a DAR of 7.56 as determined by HIC, according to an embodiment of the present disclosure. [Figure 44] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-nectin-4 conjugate of construct 15 was 99.5% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 45] 1 shows a graph showing that a CH1-3 / CT tagged polatuzumab conjugate of construct 18 resulted in a DAR of 6.57 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 46] 1 shows a graph demonstrating that the CH1-3 / CT tagged polatuzumab conjugate of construct 18 was 98.1% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 47] 1 shows a graph showing that the CH1-3 / CT tagged anti-nectin-4 conjugate of construct 18 resulted in a DAR of 6.1 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 48] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-nectin-4 conjugate of construct 18 was 96.6% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 49] 1 shows a graph showing that a CH1-3 / CT tagged anti-FITC conjugate of construct 18 resulted in a DAR of 6.16 as determined by PLRP, according to an embodiment of the present disclosure. [Figure 50] 1 shows a graph demonstrating that the CH1-3 / CT tagged anti-FITC conjugate of construct 18 was 93.5% monomeric as determined by analytical SEC, according to an embodiment of the present disclosure. [Figure 51] 1 shows a graph of an in vitro cytotoxicity assay of free topoisomerase inhibitors on NCI-N87 gastric cancer cells. [Figure 52] 1 shows a graph of an in vitro cytotoxicity assay of free topoisomerase inhibitors on Sk-Br-3 breast cancer cells. [Figure 53] 1 shows a graph of an in vitro cytotoxicity assay of free topoisomerase inhibitors on Granta NHL cells. [Figure 54] 1 shows a graph of an in vitro cytotoxicity assay of free topoisomerase inhibitors on MDA-MB-468 breast cancer cells. [Figure 55] 1 shows a graph of an in vitro cytotoxicity assay of free topoisomerase inhibitors in MDA-PCa-2b prostate cancer cells. [Figure 56] 1 shows a graph of an in vitro cytotoxicity assay in MDA-MB-468 breast cancer cells of an ADC targeting TROP-2 made using compound 32, according to an embodiment of the disclosure. [Figure 57] 1 shows a graph of an in vitro cytotoxicity assay in NCI-N87 gastric cancer cells of a HER2-targeting ADC made using compound 32, according to an embodiment of the present disclosure. [Figure 58] 1 shows a graph of an in vitro cytotoxicity assay in SU-DHL-1 ALCL cells of a CD25-targeting ADC made using compound 32, according to an embodiment of the disclosure. [Figure 59] 1 shows a graph of an in vitro cytotoxicity assay in BxPC-3 pancreatic cancer cells of an ADC targeting TROP-2 made using compound 32, according to an embodiment of the present disclosure. [Figure 60] 1 shows a graph of an in vitro cytotoxicity assay in NCI-N87 gastric cancer cells of a HER2-targeting ADC made using compound 36, according to an embodiment of the present disclosure. [Figure 61] 1 shows a graph of an in vitro cytotoxicity assay in Sk-Br-3 breast cancer cells of a HER2-targeting ADC made using compound 36, according to an embodiment of the disclosure. [Figure 62] FIG. 1 shows a schematic diagram of the ELISA assays used to determine total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis. [Figure 63] 1 shows a graph of concentration (μg / mL) versus days after administration of trastuzumab antibody at 0.9 mg / kg. [Figure 64]1 shows a graph of concentration (μg / mL) versus days post-dose following administration of 0.9 mg / kg of polatuzumab antibody. [Figure 65] FIG. 1 shows a graph of concentration (μg / mL) versus days post-dose following a 0.9 mg / kg dose of a conventional HER2 topoisomerase inhibitor-conjugated ADC with a protease-cleavable linker according to an embodiment of the disclosure. [Figure 66] 1 shows a graph of concentration (μg / mL) versus days post-dosing following a 0.9 mg / kg dose of CH1-3 / CT-tagged trastuzumab conjugated to construct 32 according to an embodiment of the present disclosure. [Figure 67] 1 shows a graph of concentration (μg / mL) versus days post-dose following a 0.9 mg / kg dose of CH1-3 / CT-tagged trastuzumab conjugated to construct 36 according to an embodiment of the present disclosure. [Figure 68] 1 shows a graph of concentration (μg / mL) versus days post-dose following a 0.9 mg / kg dose of CT-tagged polatuzumab conjugated to construct 15 according to an embodiment of the present disclosure. [Figure 69] 1 shows a graph of concentration (μg / mL) versus days post-dose following a 0.9 mg / kg dose of CH2-3 tagged polatuzumab conjugated to construct 15 according to an embodiment of the present disclosure. [Figure 70] 1 shows a graph of concentration (μg / mL) versus days post-dose following a 0.9 mg / kg dose of CH1-2 tagged polatuzumab conjugated to construct 15 according to an embodiment of the present disclosure. [Figure 71] 1 shows a graph of mean tumor volume (mm) versus days demonstrating the in vivo efficacy of TROP-2-targeting ADCs with a topoisomerase inhibitor payload against NCI-H292 xenografts according to embodiments of the present disclosure. n=8 mice / group. Dosing is indicated by arrows. [Figure 72] 1 shows the structure of a dual payload construct with an MMAE payload and a second payload with an alternative mechanism of action (MOA), according to an embodiment of the present disclosure. [Figure 73]1 shows the structure of a dual payload construct with a belotecan payload and a second payload with an alternative MOA, according to an embodiment of the present disclosure. [Figure 74] 1 shows a graph of mean tumor volume (mm) versus days demonstrating the in vivo efficacy of TROP-2-targeting ADCs with a topoisomerase inhibitor payload against NCI-H292 xenografts according to embodiments of the present disclosure. n=7 mice / group. A single intravenous dose was administered on day 0. [Figure 75] 1 shows a graph demonstrating the in vivo efficacy of Nectin-4-targeting ADCs with a topoisomerase inhibitor payload against NCI-H1781 xenografts according to embodiments of the present disclosure. n=5 mice / group. A dose of 5 mg / kg was delivered intravenously on days 0 and 7. [Figure 76] 1 shows a graph of in vitro potency of HER2-targeting ADCs bearing (36) or (175), or isotype control ADCs, compared to free payload on NCI-N87 cells, according to embodiments of the present disclosure. [Figure 77] 1 shows a graph of in vitro potency of HER2-targeting ADCs bearing (36) or (175), or isotype control ADCs, compared to free payload on SK-BR-3 cells, according to embodiments of the present disclosure. [Figure 78] 1 shows a graph of the in vitro potency of TROP-2-targeting ADCs bearing (36) or (80) compared to (16) on MDA-MB-468 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 79] 1 shows a graph of the in vitro potency of TROP-2-targeting ADCs bearing (36) or (80) compared to (16) on BxPC-3 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 80] 1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (99), (103), or (110) compared to (16) on NCI-N87 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 81]1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (36), (86), or (92) compared to (16) on SK-BR-3 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 82] 1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (36), (86), or (92) compared to (16) on NCI-N87 cells, or an isotype control ADC, according to an embodiment of the present disclosure. [Figure 83] 1 shows a graph of the in vitro potency of a TROP-2-targeting ADC bearing (65), or an isotype control ADC, Trodelvy, or a CL2A-SN38 isotype control conjugate, compared to (1) or (2) on SK-BR-3 cells, according to embodiments of the disclosure. [Figure 84] 1 shows a graph of the in vitro potency of a HER2-targeting ADC bearing (65), an isotype control ADC, Enhertu, or an MC-GGFG-Dxd isotype control conjugate compared to (2) on NCI-N87 cells, according to embodiments of the present disclosure. [Figure 85] 1 shows a graph of the in vitro potency of TROP-2-targeting ADCs bearing (36) or (113) compared to (16) on MDA-MB-468 cells, or isotype control ADCs, according to embodiments of the present disclosure. [Figure 86] 1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (36), (136), or (142) compared to (16) on NCI-N87 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 87] 1 shows a graph of the in vitro potency of TROP-2-targeting ADCs bearing (36), (127), or (131) compared to (16) on MDA-MB-468 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 88]1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (36), (127), or (131) compared to (16) on NCI-N87 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 89] 1 shows a graph of the in vitro potency of TROP-2-targeting ADCs bearing (151), (147), or (131) compared to (16) on NCI-N87 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 90] 1 shows a graph of the in vitro potency of HER2-targeting ADCs bearing (151), (147), or (131) compared to (16) on SK-BR-3 cells, or an isotype control ADC, according to embodiments of the present disclosure. [Figure 91] 1 shows a graph demonstrating that compound 127 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 7.15 as determined by PLRP. [Figure 92] 1 shows a graph demonstrating that compound 131 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 6.80 as determined by PLRP. [Figure 93] 1 shows a graph demonstrating that the Compound 127 CH1-3 / CT tagged trastuzumab conjugate was 94.4% monomeric as determined by analytical SEC. [Figure 94] 1 shows a graph demonstrating that Compound 131 CH1-3 / CT tagged trastuzumab conjugate was 93.6% monomeric as determined by analytical SEC. [Figure 95] 1 shows a graph demonstrating that compound 80 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 5.86 as determined by PLRP. [Figure 96] 1 shows a graph demonstrating that Compound 80 CH1-3 / CT tagged trastuzumab conjugate was 97.4% monomeric as determined by analytical SEC. [Figure 97]1 shows a graph demonstrating that compound 80 CH1-3 / CT tagged sacituzumab conjugate resulted in a DAR of 6.19 as determined by PLRP. [Figure 98] 1 shows a graph demonstrating that the compound 80 CH1-3 / CT tagged sacituzumab conjugate was 97.1% monomeric as determined by analytical SEC. [Figure 99] 1 shows a graph demonstrating that Compound 86 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 5.46 as determined by PLRP. [Figure 100] 1 shows a graph demonstrating that Compound 86 CH1-3 / CT tagged anti-FITC conjugate was 98.0% monomeric as determined by analytical SEC. [Figure 101] 1 shows a graph demonstrating that Compound 92 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 1.58 as determined by PLRP. [Figure 102] 1 shows a graph demonstrating that Compound 92 CH1-3 / CT tagged anti-FITC conjugate was 96.1% monomeric as determined by analytical SEC. [Figure 103] 1 shows a graph demonstrating that Compound 99 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 3.07 as determined by PLRP. [Figure 104] 1 shows a graph demonstrating that Compound 99 CH1-3 / CT tagged anti-FITC conjugate was 97.9% monomeric as determined by analytical SEC. [Figure 105] 1 shows a graph demonstrating that compound 103 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 6.56 as determined by PLRP. [Figure 106] 1 shows a graph demonstrating that Compound 103 CH1-3 / CT tagged trastuzumab conjugate was 97.3% monomeric as determined by analytical SEC. [Figure 107]1 shows a graph demonstrating that compound 110 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 5.66 as determined by PLRP. [Figure 108] 1 shows a graph demonstrating that Compound 110 CH1-3 / CT tagged anti-FITC conjugate was 98.5% monomeric as determined by analytical SEC. [Figure 109] 1 shows a graph demonstrating that compound 113 CH1-3 / CT tagged sacituzumab conjugate resulted in a DAR of 6.41 as determined by PLRP. [Figure 110] 1 shows a graph demonstrating that the compound 113 CH1-3 / CT tagged sacituzumab conjugate was 97.4% monomeric as determined by analytical SEC. [Figure 111] 1 shows a graph demonstrating that compound 123 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 5.56 as determined by PLRP. [Figure 112] 1 shows a graph demonstrating that Compound 123 CH1-3 / CT tagged anti-FITC conjugate was 95.5% monomeric as determined by analytical SEC. [Figure 113] 1 shows a graph demonstrating that compound 123 CH1-3 / CT tagged sacituzumab conjugate resulted in a DAR of 6.27 as determined by PLRP. [Figure 114] 1 shows a graph demonstrating that the compound 123 CH1-3 / CT tagged sacituzumab conjugate was 98.0% monomeric as determined by analytical SEC. [Figure 115] 1 shows a graph demonstrating that compound 151 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 5.67 as determined by PLRP. [Figure 116] 1 shows a graph demonstrating that Compound 151 CH1-3 / CT tagged anti-FITC conjugate was 97.8% monomeric as determined by analytical SEC. [Figure 117] 1 shows a graph demonstrating that compound 147 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 6.47 as determined by PLRP. [Figure 118] 1 shows a graph demonstrating that Compound 147 CH1-3 / CT tagged anti-FITC conjugate was 96.4% monomeric as determined by analytical SEC. [Figure 119] 1 shows a graph demonstrating that compound 73 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 5.41 as determined by PLRP. [Figure 120] 1 shows a graph demonstrating that compound 67 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 4.02 as determined by PLRP. [Figure 121] 1 shows a graph demonstrating that compound 136 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 7.26 as determined by PLRP. [Figure 122] 1 shows a graph demonstrating that Compound 136 CH1-3 / CT tagged trastuzumab conjugate was 98.9% monomeric as determined by analytical SEC. [Figure 123] 1 shows a graph demonstrating that compound 142 CH1-3 / CT tagged trastuzumab conjugate resulted in a DAR of 6.9 as determined by PLRP. [Figure 124] 1 shows a graph demonstrating that compound 175 CH1-3 / CT tagged anti-FITC conjugate resulted in a DAR of 5.08 as determined by PLRP. [Figure 125] 1 shows a graph demonstrating that Compound 175 CH1-3 / CT tagged anti-FITC conjugate was 93.0% monomeric as determined by analytical SEC. [Figure 126] 1 shows a graph demonstrating that compound 181 CT-tagged trastuzumab conjugate resulted in a DAR of 3.45 as determined by HIC. [Figure 127] 1 shows a graph demonstrating that the Compound 181 CT-tagged trastuzumab conjugate was 97.6% monomeric as determined by analytical SEC. [Figure 128] 1 shows a graph demonstrating that compound 181 CT-tagged anti-MUC1 conjugate resulted in a DAR of 3.59 as determined by HIC. [Figure 129] 1 shows a graph demonstrating that the Compound 181 CT-tagged anti-MUC1 conjugate was 99.0% monomeric as determined by analytical SEC. [Figure 130] 1 shows a graph demonstrating that compound 181 CH1 / CT tagged anti-MUC1 conjugate resulted in a DAR of 2.13 as determined by PLRP. [Figure 131] 1 shows a graph demonstrating that the Compound 181 CH1 / CT tagged anti-MUC1 conjugate was 96.9% monomeric as determined by analytical SEC. [Figure 132] 1 shows a graph demonstrating that compound 181 CH1 / CT tagged trastuzumab conjugate resulted in a DAR of 3.28 as determined by PLRP. [Figure 133] 1 shows a graph demonstrating that Compound 181 CH1 / CT tagged trastuzumab conjugate was 92.2% monomeric as determined by analytical SEC. [Figure 134] 1 shows a graph demonstrating that Compound 194 CT-tagged trastuzumab conjugate resulted in a DAR of 3.04 as determined by HIC. [Figure 135] 1 shows a graph demonstrating that the Compound 194 CT-tagged trastuzumab conjugate was 97.2% monomeric as determined by analytical SEC. [Figure 136] 1 shows a graph demonstrating that Compound 194 CT-tagged anti-MUC1 conjugate resulted in a DAR of 3.01 as determined by HIC. [Figure 137]1 shows a graph demonstrating that the Compound 194 CT-tagged anti-MUC1 conjugate was 99.3% monomeric as determined by analytical SEC. [Figure 138] 1 shows a graph demonstrating that Compound 194 CH1 / CT tagged anti-MUC1 conjugate resulted in a DAR of 5.07 as determined by PLRP. [Figure 139] 1 shows a graph demonstrating that the Compound 194 CH1 / CT tagged anti-MUC1 conjugate was 97.0% monomeric as determined by analytical SEC. [Figure 140] 1 shows a graph demonstrating that Compound 194 CH1 / CT tagged trastuzumab conjugate resulted in a DAR of 6.77 as determined by PLRP. [Figure 141] 1 shows a graph demonstrating that Compound 194 CH1 / CT tagged trastuzumab conjugate was 96.6% monomeric as determined by analytical SEC. [Figure 142] 1 shows a graph demonstrating that Compound 200 CH1 / CT tagged trastuzumab conjugate resulted in a DAR of 6.49 as determined by PLRP. [Figure 143] 1 shows a graph demonstrating that Compound 200 CH1 / CT tagged trastuzumab conjugate was 94.2% monomeric as determined by analytical SEC. [Figure 144] 1 shows a graph demonstrating that compound 200 CH1 / CT tagged sacituzumab conjugate resulted in a DAR of 6.19 as determined by PLRP. [Figure 145] 1 shows a graph demonstrating that the compound 200 CH1 / CT tagged sacituzumab conjugate was 96.8% monomeric as determined by analytical SEC. [Figure 146] 1 shows a graph demonstrating that Compound 200 CH1 / CT tagged anti-FITC conjugate resulted in a DAR of 6.5 as determined by PLRP. [Figure 147]1 shows a graph demonstrating that Compound 200 CH1 / CT tagged anti-FITC conjugate was 94.4% monomeric as determined by analytical SEC.

[0043] definition The following terms have the following meanings unless otherwise indicated: Any term not defined has its art-recognized meaning.

[0044] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, e.g., 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, straight-chain and branched-chain hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0045] The term "substituted alkyl" refers to an alkyl group, as defined herein, wherein one or more carbon atoms in the alkyl chain (except the C1 carbon atom) are optionally substituted with -O-, -N-, -S-, -S(O) n- (wherein n is 0-2), -NR- (wherein R is hydrogen or alkyl), and includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic ring.

[0046] "Alkylene" refers to a divalent aliphatic hydrocarbyl group, preferably having 1 to 6, more preferably 1 to 3, carbon atoms, either straight or branched, and optionally including -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, by way of example, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), isopropylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0047] "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with a substituent, as described for carbon in the definition of "substituted" below.

[0048] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0049] The terms "alkylaminoalkyl," "alkylaminoalkenyl," and "alkylaminoalkynyl" refer to the group R'NHR"- where R' is an alkyl group as defined herein and R" is an alkylene, alkenylene, or alkynylene group as defined herein.

[0050] The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0051] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0052] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0053] The term "alkoxyamino" refers to the group --NH-alkoxy, where alkoxy is defined herein.

[0054] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group and includes, by way of example, groups such as trifluoromethoxy.

[0055] The term "haloalkyl" refers to a substituted alkyl group as defined above, in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

[0056] The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0057] The term "alkylthioalkoxy" refers to the groups -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0058] "Alkenyl" refers to straight or branched chain hydrocarbyl groups having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1, and preferably 1 to 2, sites of double bond unsaturation. This term includes, by way of example, bivinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0059] The term "substituted alkenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0060] "Alkynyl" refers to a straight- or branched-chain monovalent hydrocarbyl group having two to six carbon atoms, preferably two to three carbon atoms, and having at least one, and preferably one or two, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0061] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0062] "Alkynyloxy" refers to the group -O-alkynyl, where alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0063] "Acyl" means HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted ... "C(O)-," "heteroaryl-C(O)-," and "substituted heterocyclyl-C(O)-" groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0064] "Acylamino" is -NR 20 C(O) alkyl, -NR 20 C(O) substituted alkyl, NR 20 C(O)cycloalkyl, -NR 20 C(O)-substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O) substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O) substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O) substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O) heterocyclic ring, and -NR 20 C(O) refers to a group of substituted heterocyclic rings, R 20is hydrogen or alkyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0065] The term "aminocarbonyl" or "aminoacyl" refers to -C(O)NR 21 R 22 R refers to the group 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring; R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic ring or a substituted heterocyclic ring group, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0066] "Aminocarbonylamino" is -NR 21 C(O)NR 22 R 23 R refers to the group 21 , R 22 , and R 23 are independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups joined to form a heterocyclyl group.

[0067] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0068] The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O-, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0069] "Aminosulfonyl" is -SO2NR 21 R 22 R refers to the group 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring; R 21 and R 22 optionally, combined with the nitrogen bonded thereto, form a heterocyclic ring or a substituted heterocyclic ring group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0070] "Sulfonylamino" is -NR 21 SO2R 22 R refers to the group 21 and R 22is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring; R 21 and R 22 optionally, combined with the nitrogen bonded thereto, form a heterocyclic ring or a substituted heterocyclic ring group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0071] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group), or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), which may or may not be aromatic, provided that the point of attachment is through an aromatic ring atom. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition of the aryl substituent, such aryl groups may be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0072] "Aryloxy" refers to the group -O-aryl, where aryl is as defined herein, including optionally substituted aryl groups, also defined herein, including by way of example, phenoxy, naphthoxy, and the like.

[0073] "Amino" refers to the group -NH2.

[0074] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0075] The term "azido" refers to the group -N3.

[0076] "Carboxyl", "carboxy" or "carboxylate" refers to -CO2H or a salt thereof.

[0077] The terms "carboxyl ester" or "carboxy esters" or "carboxyalkyl" or "carboxylalkyl" refer to -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O) refers to the groups O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic ring, and -C(O)O-substituted heterocyclic ring, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0078] "(Carboxyl ester)oxy" or "carbonate" refers to -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cycloalkenyl, -OC(O)O- refers to the groups -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic ring, and -OC(O)O-substituted heterocyclic ring, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0079] "Cyano" or "nitrile" refers to the group --CN.

[0080] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0081] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0082] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having single or multiple rings and at least one double bond, preferably 1 to 2 double bonds.

[0083] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0084] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group of from 5 to 10 carbon atoms having single or multiple rings and at least one triple bond.

[0085] "Cycloalkoxy" refers to -O-cycloalkyl.

[0086] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0087] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0088] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0089] "Heteroaryl" refers to an aromatic group of 1 to 15 carbon atoms, e.g., 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or multiple condensed rings (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl) in the ring system, with at least one ring in the ring system being aromatic. To satisfy valency requirements, any heteroatom in such a heteroaryl ring may be bonded or unbonded to an H or a substituent, e.g., an alkyl group or other substituent described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of a heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0090] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0091] "Heteroaryloxy" refers to -O-heteroaryl.

[0092] "Heterocycle," "heterocyclic ring," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having 3 to 20 ring atoms, including 1 to 10 heteroatoms, having a single ring or multiple fused rings, including fused, bridged, and spiro ring systems. These ring atoms are selected from nitrogen, sulfur, or oxygen; in fused ring systems, one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of a heterocyclic ring group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valency requirements, any heteroatom in such a heterocyclic ring may be unbonded or bonded to one or more H or one or more substituents, such as alkyl groups or other substituents described herein.

[0093] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, These include, but are not limited to, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0094] Unless otherwise constrained by the definition of the heterocyclic substituent, such heterocyclic ring groups can be optionally substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0095] "Heterocyclyloxy" refers to the group --O-heterocyclyl.

[0096] The term "heterocyclylthio" refers to the group heterocyclic ring-S-.

[0097] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0098] The term "hydroxyamino" refers to the group --NHOH.

[0099] "Nitro" refers to the -NO2 group.

[0100] "Oxo" refers to the atom (=O).

[0101] "Sulfonyl" refers to the groups -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocycle, and -SO2-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein. Sulfonyl, by way of example, includes methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0102] "Sulfonyloxy" refers to the groups -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2-alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cycloalkyl, -OSO2-cycloalkenyl, -OSO2-substituted cycloalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2-heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocycle, and -OSO2-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

[0103] "Sulfate" or "sulfate ester" refers to -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O-substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O-SO2-O-substituted cycloalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cycloalkenyl, -O-SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O- refers to the groups heteroaryl, -O-SO2-O-substituted heteroaryl, -O-SO2-O-heterocycle, and -O-SO2-O-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

[0104] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic ring, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic ring are as defined herein.

[0105] "Thiol" refers to the group --SH.

[0106] The term "thioxo" or "thioketo" refers to the atom (=S).

[0107] The terms "alkylthio" or "thioalkoxy" refer to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, sulfur can be oxidized to -S(O)-. Sulfoxides can exist as one or more stereoisomers.

[0108] The term "substituted thioalkoxy" refers to the group --S-substituted alkyl.

[0109] The term "thioaryloxy" refers to an aryl-S- group, where aryl is as defined herein, including optionally substituted aryl groups, also defined herein.

[0110] The term "thioheteroaryloxy" refers to a heteroaryl-S- group, where heteroaryl is as defined herein, including optionally substituted aryl groups, also defined herein.

[0111] The term "thioheterocyclooxy" refers to a heterocyclyl-S- group where heterocyclyl is as defined herein, including optionally substituted heterocyclyl groups, also defined herein.

[0112] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are replaced, independently of each other, with the same or different substituents, as defined below.

[0113] In addition to the groups disclosed for individual terms herein, substituents (such as ═O, ═NR) to replace one or more hydrogens on a saturated carbon atom in a particular group or radical are also included. 70 , =N-OR 70 , =N2, or =S) is replaced by -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M+ , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 and R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 But independently R 70 or alternatively, two R 80 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, which may optionally contain 1 to 4 additional heteroatoms, which may be the same or different, selected from the group consisting of O, N, and S, wherein N may have —H or C1-C3 alkyl substitution; and each M + is a counterion with a net single positive charge. + are independently, for example, alkali ions, e.g., K + , Na + , Li + ammonium ions, e.g., + N(R 60 ) 4; or alkaline earth ions, e.g., [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5(The "0.5" subscript means that one of the counterions to such divalent alkaline earth ions may be the ionized form of a compound of the invention and the other may be a typical counterion such as chloride, or a two-ionized compound disclosed herein may serve as the counterion to such divalent alkaline earth ions, or a doubly-ionized compound of the invention may serve as the counterion to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0114] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkyne, aryl, and heteroaryl group is, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70, -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 , and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + provided that it is not.

[0115] In addition to the groups disclosed for each individual term herein, the substituents of the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR80 R 80 、 trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 、 -S(O)2O - M + 、 -S(O)2OR 70 、 -OS(O)2R 70 、 -OS(O)2O - M + 、 -OS(O)2OR 70 、 -P(O)(O - )2(M + )2、 -P(O)(OR 70 )O - M + 、 -P(O)(OR 70 )(OR 70 )、 -C(O)R 70 、 -C(S)R 70 、 -C(NR 70 )R 70 、 -C(O)OR 70 、 -C(S)OR 70 、 -C(O)NR 80 R 80 、 -C(NR 70 )NR 80 R 80 、 -OC(O)R 70 、 -OC(S)R 70 、 -OC(O)OR 70 、 -OC(S)OR 70 、 -NR 70 C(O)R 70 、 -NR 70 C(S)R 70 、 -NR 70 C(O)OR 70 、 -NR 70 C(S)OR 70 、 -NR 70 C(O)NR 80 R 80 、 -NR 70 C(NR 70 )R 70 、 and -NR 70 C(NR 70 )NR 80 R 80 and R 60 、 R 70 、 R 80 、 and M +is as previously defined.

[0116] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0117] For all of the substituents defined above, it is understood that polymers arrived at by defining a substituent that itself has further substituents (e.g., a substituted aryl group having as a substituent a substituted aryl group, which itself is substituted with a substituted aryl group, which is further substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0118] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is arrived at by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0119] With respect to any of the groups disclosed herein that contain one or more substituents, it is to be understood that such groups do not contain any substitutions or substitution patterns that are sterically infeasible and / or synthetically infeasible. Additionally, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0120] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, e.g., a mammal (a salt having a counterion that has acceptable mammalian safety for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, organic or inorganic acid salts such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0121] The term "salt thereof" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds not intended for administration to a patient. By way of example, salts of the compounds of the present invention include those in which the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid being the anionic component of the salt.

[0122] "Solvate" refers to a complex formed by the combination of solvent molecules with solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0123] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atomic connectivity but different atomic arrangements in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0124] "Tautomer" refers to alternative forms of molecules that differ only in the electronic bonding of the atoms and / or in the position of the protons, for example, enol keto and imine-enamine tautomers, or tautomers of heteroaryl groups containing the -N=C(H)-NH- ring atom configuration, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom configurations are possible.

[0125] It is to be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include salts, solvates, and all permutations of stereoisomers, for example, solvates of pharmaceutically acceptable salts of a stereoisomer of the subject compound.

[0126] "Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a particular disorder or disease, or one or more of its symptoms, and / or prevent the onset of the disease or disorder. With respect to tumorigenic proliferative disorders, a pharmaceutically effective amount or therapeutically effective amount specifically includes an amount sufficient to shrink a tumor or reduce the rate of tumor growth.

[0127] "Patient" refers to human and non-human subjects, particularly mammalian subjects.

[0128] The term "treating" or "treatment," as used herein, means treating or curing a disease or medical condition in a patient, such as a mammal (particularly a human), and includes (a) preventing the occurrence of a disease or medical condition, such as prophylactic treatment of a subject; (b) ameliorating a disease or medical condition, such as eliminating or reversing the disease or medical condition in a patient; (c) inhibiting a disease or medical condition, such as by delaying or halting the onset of the disease or medical condition in a patient; or (d) alleviating the symptoms of a disease or medical condition in a patient.

[0129] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless otherwise indicated, "polypeptide," "peptide," and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include, but are not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, fusion proteins including proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant bacterial host cells), immunologically tagged proteins, and the like. In certain embodiments, the polypeptide is an antibody.

[0130] "Native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein and refer to the amino acid sequence of a polypeptide before modification to include at least one modified amino acid residue.

[0131] The terms "amino acid analog," "unnatural amino acid," and the like, may be used interchangeably and include amino acid-like compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D-forms, as well as L-forms of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, the difference being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (e.g., N) with a related atom (e.g., S), addition of a group (e.g., methyl, or hydroxyl, etc.) or an atom (e.g., Cl, or Br, etc.), deletion of a group, replacement of a covalent bond (e.g., a single bond with a double bond, etc.), or a combination thereof. For example, amino acid analogs may include alpha-hydroxy acids, alpha-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfoalanine, etc.

[0132] Terms such as "amino acid side chain" or "side chain of an amino acid" can be used to refer to a substituent attached to the alpha carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. Amino acid side chains can also include those amino acid side chains described in the context of modified amino acids and / or conjugates described herein.

[0133] Terms such as "carbohydrate" may be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides, etc. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical), or absent (e.g., removed or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the subject compounds and conjugates.

[0134] The term "glycoside" or "glycosyl" refers to a sugar molecule or group attached to a moiety via a glycosidic bond. For example, the moiety to which a glycoside is attached can be a cleavable linker described herein. The glycosidic bond can link a glycoside to another moiety via various types of bonds, including, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, glycosides can be cleaved from the moiety to which they are attached, for example, by chemically or enzymatically mediated hydrolysis.

[0135] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like. Antibodies are capable of binding to target antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen may have one or more binding sites, also called epitopes, recognized by complementarity-determining regions (CDRs) formed by one or more variable regions of an antibody.

[0136] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody are produced and paired by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, and serum are examples of tissues that produce natural antibodies. For example, antibodies produced by antibody-producing cells isolated from a first animal immunized with an antigen are natural antibodies.

[0137] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody that contains one or more amino acids (e.g., in the framework region, constant region, or CDR) substituted with a correspondingly positioned amino acid from a human antibody. Generally, a humanized antibody generates a reduced immune response in a human host when compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400, PCT Publication No. WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, EP 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), Studnicka et al., Protein Engineering 7(6):805-814 (1994), Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, and to identify unusual framework residues at specific positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos. 5,750,078, 5,502,167, 5,705,154, 5,770,403, 5,698,417, 5,693,493, 5,558,864, 4,935,496, and 4,816,567, and PCT publications WO 98 / 45331 and WO 98 / 45332. In certain embodiments, the subject rabbit antibodies may be humanized according to the methods described in US2004 / 0086979 and US2005 / 0033031. Accordingly, the above-described antibodies may be humanized using methods well known in the art.

[0138] The term "chimeric antibody" refers to an antibody in which the antibody light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody may be linked to human constant segments such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of variable or antigen-binding domains from a mouse antibody and constant or effector domains from a human antibody, although domains from other mammalian species may also be used.

[0139] The immunoglobulin light or heavy chain variable region of an immunoglobulin polypeptide is composed of a framework region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., USDapartment of Health and Human Services, 1991). The framework region of an antibody, the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an antigen epitope.

[0140] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions).

[0141] As used herein, the term "isolated" is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. "Isolated" is meant to include compounds within a sample in which the compound of interest is substantially enriched and / or in which the compound of interest is partially or substantially purified.

[0142] As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free from other components with which it is naturally associated.

[0143] The term "physiological conditions" is meant to encompass those conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0144] "Reactive partner" refers to a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include the cysteine ​​or serine of a sulfatase motif and formylglycine generating enzyme (FGE), which react to form a reaction product of a converted aldehyde tag containing formylglycine (fGly) in place of the cysteine ​​or serine in the motif. Other exemplary reactive partners include the aldehyde (e.g., reactive aldehyde group) of the fGly residue of a converted aldehyde tag, and an "aldehyde-reactive reactive partner" comprising an aldehyde-reactive group and a moiety of interest, which react to form a reaction product of a polypeptide having a moiety of interest conjugated to the polypeptide via the fGly residue.

[0145] "N-terminus" refers to the terminal amino acid residue of a polypeptide having a free amine group; the amine group in the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0146] "C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group; the carboxyl group in the non-C-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0147] An "internal site" when used in reference to a polypeptide or the amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or C-terminus.

[0148] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0149] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed in the invention, subject to any specifically excluded boundary in that stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0150] It should also be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments of the invention are specifically embraced by the present invention and, indeed, to the extent that such combinations encompass subject matter that is, for example, a stable compound (i.e., a compound that can be made, isolated, characterized, and tested for biological activity), are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of various embodiments and elements thereof (e.g., elements of chemical groups listed in an embodiment describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination were individually and explicitly disclosed herein.

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described here. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the cited publications.

[0152] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0153] It is also to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0154] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION OF THE INVENTION

[0155] The present disclosure provides antibody-drug conjugate structures that include branched HIPS linkers. The present disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using them.

[0156] Antibody-drug conjugates The present disclosure provides conjugates, e.g., antibody-drug conjugates (ADCs). A "conjugate" refers to a polypeptide (e.g., an antibody) covalently linked to two or more other moieties (e.g., drugs or active agents). For example, an antibody-drug conjugate according to the present disclosure comprises two or more drugs or active agents covalently linked to an antibody. In certain embodiments, the polypeptide (e.g., an antibody) and the two or more drugs or active agents are linked to each other via one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds may comprise a branched linker, as described herein.

[0157] In certain embodiments, the conjugate is a polypeptide conjugate comprising a polypeptide (e.g., an antibody) conjugated to two or more other moieties. In certain embodiments, the two or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest, such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilizing the polypeptide to a membrane or surface. In certain embodiments, the conjugate is a drug conjugate in which the polypeptide is an antibody, thus providing an antibody-drug conjugate (ADC). For example, the conjugate can be a drug conjugate in which the polypeptide is conjugated to two or more drugs or active agents. Various types of drugs or active agents may be used in the conjugates, as described in more detail below.

[0158] A moiety of interest (e.g., a drug or active agent) can be conjugated to a polypeptide (e.g., an antibody) at any desired site on the polypeptide. Thus, the present disclosure provides polypeptides having moieties conjugated at two or more sites on the polypeptide, such as, for example, a site at or near the C-terminus of the polypeptide, a position at or near the N-terminus of the polypeptide, and a position between the C-terminus and N-terminus of the polypeptide (e.g., an internal site on the polypeptide). Combinations of the above conjugation sites are also possible.

[0159] In certain embodiments, a conjugate of the present disclosure comprises two (or more) drugs or active agents conjugated to amino acid residues of a polypeptide at the alpha carbons of the amino acid residues. Stated another way, a conjugate comprises a polypeptide in which the side chains of amino acid residues in the polypeptide are modified to attach two (or more) drugs or active agents (e.g., attached to the two drugs or active agents via a branched linker described herein). For example, a conjugate comprises a polypeptide in which the alpha carbons of amino acid residues in the polypeptide are modified to attach two drugs or active agents (e.g., attached to the two drugs or active agents via a branched linker described herein).

[0160] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to two or more moieties, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more moieties. Moieties may be conjugated to the polypeptide at multiple sites within the polypeptide. In some embodiments, two moieties may be conjugated to a single amino acid residue of the polypeptide. For example, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, two moieties are conjugated to a first amino acid residue of the polypeptide and two other moieties are conjugated to a second amino acid residue of the polypeptide. For example, a polypeptide may be conjugated to a first and second moiety at a first amino acid residue and to a third and fourth moiety at a second amino acid residue. In some cases, two or more amino acid residues in the polypeptide are each conjugated to a pair of moieties (i.e., two moieties), and each pair of moieties is conjugated to the polypeptide via a branched linker described herein. In some cases, one amino acid residue in the polypeptide is conjugated to a pair of moieties via a branched linker described herein. In other examples, two or more amino acid residues in the polypeptide, for example, three, four, five, six, seven, eight, nine, or ten or more amino acid residues, are each conjugated to a pair of moieties via a branched linker described herein.

[0161] One or more amino acid residues of a polypeptide conjugated to a moiety of interest can be naturally occurring amino acids, non-natural amino acids, or a combination thereof. For example, a conjugate can include a moiety of interest (e.g., a drug or active agent) conjugated to a naturally occurring amino acid residue of a polypeptide. In other cases, a conjugate can include a moiety of interest conjugated to a non-natural amino acid residue of a polypeptide. A moiety of interest can be conjugated to a polypeptide at a single natural or non-natural amino acid residue, as described above. One or more natural or non-natural amino acid residues in a polypeptide can be conjugated to a moiety of interest as described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in a polypeptide can each be conjugated to two moieties via a branched linker, resulting in multiple sites in the polypeptide being conjugated to a moiety of interest.

[0162] As described herein, a polypeptide can be conjugated to two or more moieties of interest. In certain embodiments, the moiety of interest is a payload, e.g., a chemical entity such as a drug, an active agent, or a detectable label. For example, a drug (or active agent) can be conjugated to the polypeptide, or in other embodiments, a detectable label can be conjugated to the polypeptide. In other embodiments, a combination of different payloads can be conjugated to the polypeptide. Thus, for example, embodiments of the present disclosure include, but are not limited to, conjugates of a polypeptide and two or more drugs, conjugates of a polypeptide and two or more active agents, conjugates of a polypeptide and two or more detectable labels, and combinations thereof.

[0163] In certain embodiments, the polypeptide (e.g., an antibody) and the moiety of interest (e.g., a drug or active agent) are conjugated via a conjugating moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bonded) to a conjugating moiety, thus indirectly linking the polypeptide and the moiety together via the conjugating moiety. In some cases, the conjugating moiety comprises a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. A general scheme for coupling a moiety of interest to a polypeptide, for example, via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety, is shown in the general reaction scheme below. Hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moieties are also referred to herein as hydrazino-iso-pictet-Spengler (HIPS) conjugated moieties and aza-hydrazino-iso-pictet-Spengler (azaHIPS) conjugated moieties, respectively. [ka]

[0164] In the above reaction scheme, each R independently comprises a moiety of interest (e.g., a drug or active agent) to be conjugated to a polypeptide (e.g., conjugated to a polypeptide via a linker described herein), where n is an integer from 1 to 4. As shown in the above reaction scheme, a conjugating moiety (e.g., a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety) is attached to two or more drugs or active agents R. A polypeptide containing a 2-formylglycine residue (fGly) reacts with the conjugating moiety to generate a polypeptide conjugate, thus attaching two or more drugs or active agents to the polypeptide via the conjugating moiety.

[0165] As described herein, this moiety can be any of a variety of moieties, including, but not limited to, a chemical entity, such as a detectable label, or a drug or active agent. R' and R" can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z is CR 21 , N.R. 22 , N, O, or S, wherein R 21 and R 22 are each independently selected from any of the substituents described for R' and R" above.

[0166] As shown in the conjugates and compounds described herein, other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also possible. For example, a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be attached (e.g., covalently attached) to two or more linkers. Thus, embodiments of the present disclosure include hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties connected to two or more drugs or active agents via corresponding linkers, respectively. Thus, conjugates of the present disclosure may include two or more linkers, each linker connecting a corresponding drug or active agent to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. Thus, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety and two or more linkers can be considered collectively as a "branched linker," where the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety connects to two of many "branches," each branch containing a linker that connects to a drug or active agent.

[0167] The same combination of different payloads can be conjugated to a polypeptide via a branched linker. In certain embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are the same payload (e.g., drugs, active agents, or detectable labels). For example, the first branch of the branched linker can be attached to a payload (e.g., a drug, active agent, or detectable label), and the second branch of the branched linker can be attached to the same payload (e.g., a drug, active agent, or detectable label) as the first branch.

[0168] In other embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are different payloads (e.g., drugs, active agents, or detectable labels). For example, a first branch of the branched linker can be attached to a first payload (e.g., a first drug, active agent, or detectable label), and a second branch of the branched linker can be attached to a second payload (e.g., a second drug, active agent, or detectable label) that is different from the first payload (e.g., the first drug, active agent, or detectable label) attached to the first branch.

[0169] In some embodiments, when two different drugs or active agents are attached to a branched linker, the drugs or active agents can be selected from drugs and active agents that have a synergistic therapeutic effect. "Synergistic effect," "synergistic effect," or "synergistic effect" refers to a therapeutic effect that is greater than the sum of the effects of the drugs or active agents taken separately. For example, in some cases, the use of two different drugs or active agents attached to a branched linker can provide a low therapeutically effective concentration at which both payloads act, thereby increasing the overall efficacy of the ADC.

[0170] In some embodiments, when two different drugs or active agents are attached to a branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit compared to the drugs or active agents used separately. For example, the drug or active agent may provide increased efficacy in drug delivery of the ADC (e.g., some payloads, such as iRGD peptides, may increase extravasation into tissues and enhance tumor infiltration).

[0171] In some embodiments, when two different drugs or active agents are attached to a branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may result in a reduction in tumor drug resistance by targeting multiple pathways. Examples of payload combinations include, but are not limited to, cytotoxic drugs, immunomodulatory molecules for activating or inhibiting immune cell populations, cytokines, hormones, radioisotopes, and the like.

[0172] In some embodiments, when two different payloads are attached to the branched linker, the payloads can be selected from a combination of a drug or active agent and a detectable label. For example, the first payload can be a detectable label used as an imaging agent or tracer to detect the location of the ADC in vivo, while the second payload can be a drug or active agent that provides therapeutic activity.

[0173] Various embodiments of linkers that can couple a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, such as a cleavable linker described herein.

[0174] In certain embodiments, a polypeptide may be conjugated to two or more moieties of interest, and one or more amino acids of the polypeptide are modified before conjugation to the moieties of interest. Modification of one or more amino acids of the polypeptide can produce a polypeptide containing one or more reactive groups suitable for conjugation to the moieties of interest. In some cases, the polypeptide can contain one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to the moieties of interest (e.g., when two or more moieties are attached to a conjugation moiety such as the hydrazinyl-indolyl conjugation moiety or hydrazinyl-pyrrolo-pyridinyl conjugation moiety described above). For example, an amino acid of the polypeptide can be modified to contain a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an "aldehyde tag" or "ald-tag," which, as used herein, refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR) that has been converted to contain 2-formylglycine residues (referred to herein as "fGly") by the action of formylglycine generating enzyme (FGE). The fGly residue generated by FGE may also be referred to as "formylglycine." In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence that includes a "converted" sulfatase motif (i.e., a sulfatase motif in which a cysteine ​​or serine residue has been converted to fGly by the action of FGE, e.g., L(fGly)TPSR). A converted sulfatase motif can be generated from an amino acid sequence that includes an "unconverted" sulfatase motif (i.e., a sulfatase motif in which the cysteine ​​or serine residue has not been converted to fGly by FGE but is capable of being converted, e.g., an unconverted sulfatase motif having the sequence L(C / S)TPSR)."Conversion," when used in the context of the action of formylglycine generating enzyme (FGE) on a sulfatase motif, refers to the biochemical modification of a cysteine ​​or serine residue in the sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly or Ser to fGly). Further aspects of aldehyde tags and their use in site-specific protein modification are described in U.S. Pat. Nos. 7,985,783 and 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0175] In some cases, a polypeptide containing an fGly residue can be conjugated to a moiety of interest by reacting the fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety, as described above) to produce a conjugate. For example, an fGly-containing polypeptide can be contacted with a reactive partner under conditions suitable to provide a conjugate of two or more drugs to the polypeptide. In some cases, the reactive partner can include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety described above. For example, two or more drugs or active agents can be attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moiety. In some cases, a drug or active agent is attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety, e.g., covalently attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, and each drug or active agent is attached to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety via a corresponding linker.

[0176] In certain embodiments, the conjugates of the present disclosure comprise a polypeptide (e.g., an antibody) having at least one amino acid residue attached to two or more moieties of interest (e.g., drugs or active agents). To create the conjugate, the amino acid residue of the polypeptide can be modified and then coupled to two or more drugs or active agents attached to the above-described hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moieties. In certain embodiments, the amino acid residue of the polypeptide (e.g., an antibody) is a cysteine ​​or serine residue that is modified to a fGly residue, as described above. In certain embodiments, the modified amino acid residue (e.g., an fGly residue) is conjugated to two or more drugs or active agents containing the above-described hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moieties, providing conjugates of the present disclosure in which the two or more drugs or active agents are conjugated to the polypeptide via the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugating moieties. As used herein, the term fGly' refers to the amino acid residue of a polypeptide (e.g., an antibody) that is coupled to a moiety of interest (e.g., a drug or active agent).

[0177] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one amino acid residue attached to a branched linker described herein, which is in turn attached to two or more drugs or active agents. For example, the conjugate may comprise a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') conjugated to a moiety of interest (e.g., a drug or active agent) as described above.

[0178] An embodiment of the present disclosure is a conjugate of formula (I): [ka] During the ceremony, Z 1 , Z 2 , Z3 , and Z 4 However, each independently, CR 4 , N, and CL B -W 2 and at least one Z 1 , Z 2 , Z 3 , and Z 4 But CL B -W 2 and R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker, L B is the second linker, W 1 is the first drug, W 2 is the second drug, W 3 is a polypeptide.

[0179] The substituents associated with the conjugates of formula (I) are described in more detail below.

[0180] In certain embodiments, Z 1 , Z 2 , Z 3 , and Z 4 However, each independently, CR 4 , N, and CL B -W 2 and at least one Z 1 , Z 2 , Z 3 , and Z 4 But CL B -W 2 In certain embodiments, Z 1 is CR 4 In certain embodiments, Z 1 is N. In certain embodiments, Z 1 is CL B -W 2 In certain embodiments, Z 2 is CR 4 In certain embodiments, Z 2 is N. In certain embodiments, Z 2 is CL B -W 2 In certain embodiments, Z 3 is CR 4 In certain embodiments, Z 3 is N. In certain embodiments, Z 3 is CL B -W 2 In certain embodiments, Z 4 is CR 4In certain embodiments, Z 4 is N. In certain embodiments, Z 4 is CL B -W 2 is.

[0181] Various Z 1 , Z 2 , Z 3 , and Z 4 For example, in some cases, Z 1 is CL B -W 2 and Z 2 is CR 4 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CL B -W 2 and Z 3 is CR 4 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CR 4 and Z 3 is CL B -W 2 and Z 4 is CR 4 In some cases, Z 1 is CR 4 and Z 2 is CR 4 and Z 3 is CR 4 and Z 4 is CL B -W 2 is.

[0182] In certain embodiments, R 1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 1 is hydrogen. In certain embodiments, R 1 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 1 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 1 is alkynyl or substituted alkynyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 1 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 1 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 1 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 1 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0183] In certain embodiments, R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl.

[0184] In certain embodiments, R 2is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 2 is alkynyl or substituted alkynyl. In certain embodiments, R 2 is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acylamino or aminoacyl. In certain embodiments, R 2is an alkylamide or a substituted alkylamide. In certain embodiments, R 2 is sulfonyl. In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 2 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 2 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 2 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 2 is heterocyclyl or substituted heterocyclyl, for example, C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0185] In certain embodiments, R 3is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 3 is hydrogen. In certain embodiments, R 3 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 3 is methyl. In certain embodiments, R 3 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 3 is alkynyl or substituted alkynyl. In certain embodiments, R 3 is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acylamino or aminoacyl. In certain embodiments, R 3is an alkylamide or a substituted alkylamide. In certain embodiments, R 3 is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 3 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 3 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 3 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 3 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0186] In certain embodiments, R 2 and R 3 Both of the groups are methyl.

[0187] In certain embodiments, R 2 and R 3are optionally cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5- or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 6-membered heterocyclyl.

[0188] In certain embodiments, each R 4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0189] Each R 4 Various possibilities for R are described in more detail below. In certain embodiments, R 4 is hydrogen. In certain embodiments, each R 4 is hydrogen. In certain embodiments, R 4 is a halogen, such as F, Cl, Br, or I. In certain embodiments, R 4 is F. In certain embodiments, R 4 is Cl. In certain embodiments, R 4 is Br. In certain embodiments, R 4 is I. In certain embodiments, R 4 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 4 is methyl. In certain embodiments, R 4 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 4 is alkynyl or substituted alkynyl. In certain embodiments, R 4 is alkoxy or substituted alkoxy. In certain embodiments, R 4 is amino or substituted amino. In certain embodiments, R 4 is carboxyl or carboxyl ester. In certain embodiments, R 4 is acyl or acyloxy. In certain embodiments, R 4 is acylamino or aminoacyl. In certain embodiments, R 4 is an alkylamide or a substituted alkylamide. In certain embodiments, R 4 is sulfonyl. In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 4 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C5 aryl or C5 substituted aryl, or C6 aryl or C6 substituted aryl (e.g., phenyl or substituted phenyl). 4 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 4 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 4 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0190] In certain embodiments, L A is the first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0191] In certain embodiments, L B is a second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0192] In certain embodiments, W 1 is the first drug (or first active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0193] In certain embodiments, W 2 is a second drug (or second active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0194] In certain embodiments, W 3 is a polypeptide (e.g., an antibody). In certain embodiments, W 3 comprises one or more fGly' residues as described herein. In certain embodiments, the polypeptide is connected to the remainder of the conjugate via an fGly' residue as described herein. Examples of polypeptides and antibodies that can be used in the conjugates of the present disclosure are described in more detail below.

[0195] In certain embodiments, the conjugate of Formula (I) comprises a first linker, L A The first linker, L A may be utilized to attach a first moiety of interest (e.g., a first drug or active agent) to a polypeptide (e.g., an antibody) via a conjugating moiety. A may be attached (e.g., covalently attached) to a conjugating moiety (e.g., as described herein). For example, a first linker, L A can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety to a first drug. A hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety can be used to attach a first linker, L A The (and thus the first drug) may be conjugated to a polypeptide, for example, an antibody.

[0196] For example, as shown in formula (I) above, L A is linked to W via a conjugation moiety 3 binds to and therefore W 3 is linked to the linker L via a hydrazinyl-indolyl conjugated moiety or a hydrazinyl-pyrrolo-pyridinyl conjugated moiety A As mentioned above, W 3 is a polypeptide (e.g., an antibody), and therefore L Ais attached to a polypeptide (e.g., an antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety, e.g., a linker L A is indirectly attached to a polypeptide (eg, an antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety.

[0197] Any convenient linker may be used as the first linker L in the subject conjugates and compounds. A In certain embodiments, the first linker L A can comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker L A may comprise an alkyl or substituted alkyl group. In certain embodiments, the first linker L A may comprise an alkenyl or substituted alkenyl group. In certain embodiments, the first linker L A may comprise an alkynyl or substituted alkynyl group. In certain embodiments, the first linker L A may comprise an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L A may comprise an amino or substituted amino group. In certain embodiments, the first linker L A may contain a carboxyl or carboxyl ester group. In certain embodiments, the first linker L A may comprise an acylamino group. In certain embodiments, the first linker L A may comprise an alkylamide or substituted alkylamide group. In certain embodiments, the first linker L Amay comprise an aryl or substituted aryl group. In certain embodiments, the first linker L A may comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L A may comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker, L A may include a heterocyclyl or substituted heterocyclyl group.

[0198] In certain embodiments, the first linker L A may comprise a polymer. For example, the polymer may comprise polyalkylene glycol and its derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated by the conjugates and compounds described in more detail below.

[0199] In some embodiments, L A is a first linker described by the formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, In the formula, L 1 , L 2 , L 3 , L 4 , L 5, and L 6 are each independently a linker subunit; and a, b, c, d, e, and f are each independently 0 or 1.

[0200] In certain embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 0. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0. In certain embodiments, a is 1, and b, c, d, e, and f are each 0.

[0201] In certain embodiments, the linker subunit L 1 is attached to a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 2 is the first drug or active agent W, if present 1 In certain embodiments, the linker subunit L 3 is the first drug or active agent W, if present 1 In certain embodiments, the linker subunit L 4 is the first drug or active agent W, if present 1In certain embodiments, the linker subunit L 5 is the first drug or active agent W, if present 1 In certain embodiments, the linker subunit L 6 is the first drug or active agent W, if present 1 Combine with.

[0202] Any suitable linker subunit may be used as the first linker L A Linker subunits of interest include, but are not limited to, polyethylene glycol, polyethylene and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues thereof and derivatives thereof, polynucleotides, polymeric units such as alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each (when present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0203] In some embodiments, L 1 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 In some embodiments, L 1 comprises an aryl group or a substituted aryl group. In some embodiments, L1 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0204] In some embodiments, L 2 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 In some embodiments, L 2 comprises an aryl group or a substituted aryl group. In some embodiments, L 2 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0205] In some embodiments, L 3 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 In some embodiments, L 3 comprises an aryl group or a substituted aryl group. In some embodiments, L 3 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0206] In some embodiments, L 4(when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 In some embodiments, L 4 comprises an aryl group or a substituted aryl group. In some embodiments, L 4 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0207] In some embodiments, L 5 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L 5 In some embodiments, L 5 comprises an aryl group or a substituted aryl group. In some embodiments, L 5 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0208] In some embodiments, L 6 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 6 In some embodiments, L 6In some embodiments, L 6 In some embodiments, L 6 In some embodiments, L 6 comprises an aryl group or a substituted aryl group. In some embodiments, L 6 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0209] In some embodiments, L A is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - a first linker comprising: -(L 1 ) a - is -(T 1 -V 1 ) a - and -(L 2 ) b - is -(T 2 -V 2 ) b - and -(L 3 ) c - is -(T 3 -V 3 ) c - and -(L 4 ) d - is -(T 4 -V 4 ) d - and -(L 5 ) e - is -(T 5 -V 5 ) e - and -(L 6 ) f - is -(T6 -V 6 ) f - and T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 is a tether group, if present, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is, when present, a covalent bond or linking functional group; a, b, c, d, e, and f are each independently 0 or 1.

[0210] In certain embodiments, the sum of a, b, c, d, e, and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 0. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0. In certain embodiments, a is 1, and b, c, d, e, and f are each 0.

[0211] As noted above, in certain embodiments, L 1is attached to a hydrazinyl-indolyl conjugated moiety or a hydrazinyl-pyrrolo-pyridinyl conjugated moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 1 is attached to a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety (e.g., as shown in formula (I) above). In certain embodiments, V 1 is attached to a first drug or active agent. In certain embodiments, L 2 When present, T binds to the first drug or active agent. 2 If present, binds to the first drug or active agent or V 2 When present, L binds to the first drug or active agent. In certain embodiments, L 3 When present, T binds to the first drug or active agent. 3 If present, binds to the first drug or active agent or V 3 When present, L binds to the first drug or active agent. In certain embodiments, L 4 When present, T binds to the first drug or active agent. 4 If present, binds to the first drug or active agent or V 4 When present, L binds to the first drug or active agent. In certain embodiments, L 5 When present, T binds to the first drug or active agent. 5 If present, binds to the first drug or active agent or V 5 When present, L binds to the first drug or active agent. In certain embodiments, L 6 When present, T binds to the first drug or active agent. 6 If present, binds to the first drug or active agent or V 6 When present, binds to the first drug or active agent.

[0212] In certain embodiments, the conjugate of Formula (I) comprises a second linker, L B A second linker, L B may be utilized to attach a second moiety of interest (e.g., a second drug or active agent) to the polypeptide (e.g., an antibody) via a conjugating moiety. B can be attached (e.g., covalently attached) to a conjugating moiety (e.g., as described herein). For example, a second linker, L B can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety to a second drug. A hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety can be used to attach a second linker, L B The (and thus the second drug) may be conjugated to a polypeptide, for example, an antibody.

[0213] For example, as shown in formula (I) above, L B is linked to W via a conjugation moiety 3 binds to and therefore W 3 is connected to a second linker L via a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety B As mentioned above, W 3 is a polypeptide (e.g., an antibody), and therefore L B is attached to a polypeptide (e.g., an antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety, e.g., a linker L B is indirectly attached to a polypeptide (eg, an antibody) via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety.

[0214] Any convenient linker may be used as the second linker L in the subject conjugates and compounds. BIn certain embodiments, the second linker L B can comprise a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamido, substituted alkylamido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker L B may comprise an alkyl or substituted alkyl group. In certain embodiments, the second linker, L B may comprise an alkenyl or substituted alkenyl group. In certain embodiments, the second linker, L B may comprise an alkynyl or substituted alkynyl group. In certain embodiments, the second linker, L B may comprise an alkoxy or substituted alkoxy group. In certain embodiments, the second linker, L B may comprise an amino or substituted amino group. In certain embodiments, the second linker, L B may contain a carboxyl or carboxyl ester group. In certain embodiments, the second linker L B may comprise an acylamino group. In certain embodiments, the second linker, L B may comprise an alkylamide or substituted alkylamide group. In certain embodiments, the second linker, L B may comprise an aryl or substituted aryl group. In certain embodiments, the second linker, L B may comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker, L B may comprise a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker, L B may include a heterocyclyl or substituted heterocyclyl group.

[0215] In certain embodiments, the second linker L Bmay comprise a polymer. For example, the polymer may comprise polyalkylene glycol and its derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are possible, as illustrated by the conjugates and compounds described in more detail below.

[0216] In some embodiments, L B is a second linker described by the formula: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m , In the formula, L 7 , L 8 , L 9 , L 10 , L 11 , L 12 , and L 13 are each independently a linker subunit; and g, h, i, j, k, l, and m are each independently 0 or 1.

[0217] In certain embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 0. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k, and l are each 1 and m is 0. In certain embodiments, g, h, i, j, and k are each 1 and l and m are each 0. In certain embodiments, g, h, i, and j are each 1 and k, l, and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l, and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l, and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0.

[0218] In certain embodiments, the linker subunit L 7 is attached to a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 8 If present, the second drug or active agent W 2 In certain embodiments, the linker subunit L 9 If present, the second drug or active agent W 2 In certain embodiments, the linker subunit L10 If present, the second drug or active agent W 2 In certain embodiments, the linker subunit L 11 If present, the second drug or active agent W 2 In certain embodiments, the linker subunit L 12 If present, the second drug or active agent W 2 In certain embodiments, the linker subunit L 13 If present, the second drug or active agent W 2 Combine with.

[0219] Any convenient linker subunit may be used as the second linker L B Linker subunits of interest include, but are not limited to, polymeric units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues thereof and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic ring groups, combinations thereof, and substituted versions thereof. In some embodiments, L 7 , L 8 , L 9 , L 10 , L 11 , L 12 , and L 13 Each (when present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0220] In some embodiments, L 7 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 7 In some embodiments, L 7In some embodiments, L 7 In some embodiments, L 7 In some embodiments, L 7 comprises an aryl group or a substituted aryl group. In some embodiments, L 7 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0221] In some embodiments, L 8 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 In some embodiments, L 8 comprises an aryl group or a substituted aryl group. In some embodiments, L 8 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0222] In some embodiments, L 9 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 In some embodiments, L 9 comprises an aryl group or a substituted aryl group. In some embodiments, L9 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0223] In some embodiments, L 10 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 In some embodiments, L 10 comprises an aryl group or a substituted aryl group. In some embodiments, L 10 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0224] In some embodiments, L 11 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 comprises an aryl group or a substituted aryl group. In some embodiments, L 11 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0225] In some embodiments, L 12(when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 comprises an aryl group or a substituted aryl group. In some embodiments, L 12 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0226] In some embodiments, L 13 (when present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 comprises an aryl group or a substituted aryl group. In some embodiments, L 13 comprises a diamine (eg, a linking group comprising an alkylenediamine).

[0227] In some embodiments, L B is -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L12 ) l -(L 13 ) m a second linker comprising: -(L 7 ) g - is -(T 7 -V 7 ) g - and -(L 8 ) h - is -(T 8 -V 8 ) h - and -(L 9 ) i - is -(T 9 -V 9 ) i - and -(L 10 ) j - is -(T 10 -V 10 ) j - and -(L 11 ) k - is -(T 11 -V 11 ) k - and -(L 12 ) l - is -(T 12 -V 12 ) l - and -(L 13 ) m - is -(T 13 -V 13 ) m - and T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 is a tether group, if present, V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13is, when present, a covalent bond or linking functional group; g, h, i, j, k, l, and m are each independently 0 or 1.

[0228] In certain embodiments, the sum of g, h, i, j, k, l, and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 0. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k, and l are each 1 and m is 0. In certain embodiments, g, h, i, j, and k are each 1 and l and m are each 0. In certain embodiments, g, h, i, and j are each 1 and k, l, and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l, and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l, and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0.

[0229] As noted above, in certain embodiments, L 7 is attached to a hydrazinyl-indolyl conjugated moiety or a hydrazinyl-pyrrolo-pyridinyl conjugated moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 7is attached to a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety (e.g., as shown in formula (I) above). In certain embodiments, V 7 is attached to a second drug or active agent. In certain embodiments, L 8 If present, T binds to a second drug or active agent. 8 If present, binds to a second drug or active agent or V 8 When present, L binds to a second drug or active agent. 9 If present, T binds to a second drug or active agent. 9 If present, binds to a second drug or active agent or V 9 When present, L binds to a second drug or active agent. 10 If present, T binds to a second drug or active agent. 10 If present, binds to a second drug or active agent or V10 4 When present, L binds to a second drug or active agent. 11 If present, T binds to a second drug or active agent. 11 If present, binds to a second drug or active agent or V 11 When present, L binds to a second drug or active agent. 12 If present, T binds to a second drug or active agent. 12 If present, binds to a second drug or active agent or V 12 When present, L binds to a second drug or active agent. 13 If present, T binds to a second drug or active agent. 13If present, binds to a second drug or active agent or V 13 If present, binds to a second drug or active agent.

[0230] Tether group, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 With respect to T, any convenient tether group may be utilized in the subject linkers. In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are covalent bonds, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) xand each w is an integer of 1 to 20, each n is an integer of 1 to 30, each p is an integer of 1 to 20, and each x is an integer of 1 to 12.

[0231] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (C1-C 12 ) alkyl or substituted (C1-C 12 ) alkyl. In certain embodiments, (C-C 12 ) Alkyl is a straight or branched chain alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. 12 ) Alkyl is alkyl or substituted alkyl, e.g., C-C 12 Alkyl, or C1-C 10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) alkyl is C2-alkyl. For example, (C1-C 12 ) Alkyl is alkylene or substituted alkylene, e.g., C-C 12 Alkylene, or C1-C10 It can be alkylene, or C1-C6 alkylene, or C1-C3 alkylene. 12 ) alkyl is C1-alkylene (e.g., CH2). In some cases, (C1-C 12 ) alkyl is C2-alkylene (e.g., CH2CH2). In some cases, (C1-C 12 ) Alkyl is C3-alkylene (e.g., CH2CH2CH2).

[0232] In certain embodiments, the substitution (C-C 12 )Alkyl is a straight or branched chain substituted alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. 12 ) alkyl is a substituted alkyl, e.g., a substituted C-C 12 Alkyl or substituted C1-C 10 It can be alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl. 12 ) alkyl is a substituted C2-alkyl. For example, substituted (C1-C 12 ) alkyl is a substituted alkylene, e.g., a substituted C-C 12 Alkylene or substituted C-C 10 It can be alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. 12 ) alkyl is substituted C1-alkylene (e.g., C1-alkylene substituted with —SO3H). 12 ) alkyl is a substituted C2-alkylene. 12 ) alkyl is a substituted C3-alkylene. For example, substituted (C1-C 12 ) alkyl is (PEG) as defined herein k groups (e.g., -CONH(PEG) such as -CONH(PEG) or -CONH(PEG) kor -NHCO(PEG) such as -NHCO(PEG) k ) C1-C substituted with 12 C1-C which may contain alkylene or substituted with CONHCH2CH2SO3H groups 12 It may contain alkylene (e.g., C3-alkylene) or C1-C substituted with -NHCOCH2SO3H groups. 12 It may include alkylene (eg, C5-alkylene).

[0233] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl is (C-C 12 ) alkyl, substituted (C1-C 12) may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Optionally, the substituted aryl is a substituted phenyl and the substituent comprises a cleavable moiety (e.g., an enzymatically cleavable moiety, e.g., a glycoside or glycoside derivative) as described herein.

[0234] In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes heteroaryl or substituted heteroaryl, such as triazolyl (e.g., 1,2,3-triazolyl). In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes cycloalkyl or substituted cycloalkyl. In some cases, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13) includes heterocyclyl or substituted heterocyclyl. In some cases, a substituent on a substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl includes a cleavable moiety (e.g., an enzymatically cleavable moiety, e.g., a glycoside or glycoside derivative) as described herein.

[0235] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) comprises an ethylenediamine (EDA) moiety, e.g., an EDA-containing tether group. In certain embodiments, (EDA) w comprises one or more EDA moieties, e.g., w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) moieties can optionally be substituted at one or more convenient positions with any convenient substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety is described by the following structure: [ka] In the formula, y is an integer of 1 to 6, or 0 or 1, and each R 12is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be linked cyclically to form, for example, a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 In certain embodiments, y is 1 and the adjacent R 12 The groups are selected from hydrogen, alkyl (eg, methyl), and substituted alkyl (eg, lower alkyl-OH, such as ethyl-OH, or propyl-OH).

[0236] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety can be optionally substituted at one or more convenient positions with any convenient substituent, for example, alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure: [ka] In the formula, R 12 is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxy-modified polyethylene glycol.

[0237] In certain embodiments, R 12 is a polyethylene glycol moiety (PEG) described by the formula k and may be represented by the following structure: [ka] wherein k is an integer from 1 to 20, e.g., from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, k is 2. In certain embodiments, R 17 is selected from OH, COOH, OR, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 17 is COOH. In certain embodiments, R 17 is OH. In certain embodiments, R 17 is OCH3.

[0238] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (PEG) n wherein (PEG) n is a polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG) n is described by the following structure: [ka] In the formula, n is an integer from 1 to 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0239] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is (AA) p where AA is an amino acid residue. Any convenient amino acid may be utilized. Amino acids of interest include, but are not limited to, L-amino acids and D-amino acids, naturally occurring amino acids, e.g., any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), e.g., non-naturally occurring alpha-amino acids, or non-naturally occurring beta-amino acids. In certain embodiments, p is an integer between 1 and 50, e.g., between 1 and 40, 1 and 30, 1 and 20, 1 and 12, or 1 and 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0240] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9, T 10 , T 11 , T 12 , and / or T 13 ) includes amino acid analogs. Amino acid analogs include compounds similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as the naturally occurring D-forms, as well as L-forms of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more naturally occurring amino acids, the difference being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (e.g., N) with a related atom (e.g., S), addition of a group (e.g., methyl, or hydroxyl, etc.) or an atom (e.g., Cl, or Br, etc.), deletion of a group, replacement of a covalent bond (e.g., a single bond with a double bond, etc.), or a combination thereof. For example, amino acid analogs may include alpha-hydroxy acids, alpha-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfoalanine, etc.

[0241] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) is the formula -(CR 13 OH) x-, where x is 0 or x is an integer from 1 to 50, e.g., from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12, or from 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 13 is hydrogen. In certain embodiments, R 13 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 13 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. In certain embodiments, R 13 is amino or substituted amino. In certain embodiments, R 13is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 13 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 13 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 13 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 13 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0242] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl are selected from R 13 As described above for

[0243] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises a hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

[0244] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 ) includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).

[0245] In some embodiments, the tether group comprises a MABO group described by the following structure: [ka]

[0246] In some embodiments, the tether group comprises an MABC group described by the following structure: [ka]

[0247] In some embodiments, the tether group comprises a PABO group described by the following structure: [ka]

[0248] In some embodiments, the tether group comprises a PABC group described by the following structure: [ka]

[0249] In some embodiments, the tether group comprises a PAB group described by the following structure: [ka]

[0250] In some embodiments, the tether group comprises a PABA group described by the following structure: [ka]

[0251] In some embodiments, the tether group comprises a PAP group described by the following structure: [ka]

[0252] In some embodiments, the tether group comprises a PHP group described by the following structure: [ka]

[0253] In certain embodiments, each R 14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0254] In certain embodiments, R 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 14 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 14is alkynyl or substituted alkynyl. In certain embodiments, R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14 is acyl or acyloxy. In certain embodiments, R 14 is acylamino or aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 14 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 14 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 14 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 14 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0255] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring can be substituted with one or more additional groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0256] In certain embodiments, the tether group T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and / or T 13 and wherein one or more of T are each optionally substituted with a glycoside or glycoside derivative. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside. 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13are each optionally substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0257] In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0258] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] may be selected from:

[0259] Linking functional group, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13With respect to V, any convenient linking functional group may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoridate, and the like. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 and -SO2-, and -P(O)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0260] In some embodiments, each R 15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0261] In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is alkyl or substituted alkyl, e.g., C 1-6 Alkyl or C 1-6 Substituted alkyl, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 In certain embodiments, R 15 is alkenyl or substituted alkenyl, for example, C 2-6 Alkenyl or C 2-6 substituted alkenyl, or C 2-4 Alkenyl or C 2-4 substituted alkenyl, or C 2-3 Alkenyl or C 2-3 In certain embodiments, R 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 15 is aryl or substituted aryl, for example, C 5-8 Aryl or C 5-8 substituted aryl, for example, C aryl or C substituted aryl, or C aryl or C substituted aryl. In certain embodiments, R 15 is heteroaryl or substituted heteroaryl, for example, C 5-8 Heteroaryl or C 5-8 In certain embodiments, R is a C heteroaryl or a C substituted heteroaryl, for example, a C heteroaryl or a C substituted heteroaryl. 15 is cycloalkyl or substituted cycloalkyl, for example, C 3-8 Cycloalkyl or C 3-8 Substituted cycloalkyl, e.g., C 3-6 Cycloalkyl or C 3-6 substituted cycloalkyl, or C 3-5 Cycloalkyl or C 3-5 In certain embodiments, R 15 is heterocyclyl or substituted heterocyclyl, for example, C 3-8 Heterocyclyl or C 3-8 Substituted heterocyclyl, e.g., C 3-6 Heterocyclyl or C 3-6 substituted heterocyclyl, or C 3-5 Heterocyclyl or C 3-5 It is a substituted heterocyclyl.

[0262] In certain embodiments, each R 15are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are independently selected from R 15 As described above for

[0263] As noted above, in some embodiments, L A is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f wherein a, b, c, d, e, and f are each independently 0 or 1.

[0264] In some embodiments, the first linker L A In T 1 However, (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 However, each independently, (C1-C 12) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 selected from -SO2- and -P(O)OH-, and q is an integer of 1 to 6; During the ceremony, (PEG) n but, [ka] and n is an integer from 1 to 30; EDA has the following structure: [ka] wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and AA is an amino acid residue, and p is an integer of 1 to 20; Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 the groups may be cyclically linked to form a piperazinyl ring; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0265] In certain embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is selected from the following: During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 But it doesn't exist, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 However, PABA and V 4 is -CO-, T 5 However, (C1-C 12 ) alkyl, and V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -O-, T 4 However, (C1-C 12 ) alkyl, and V 4 is -CO-, T 5 But AA and V 5 But it doesn't exist, T 6 But PABC, V 6 does not exist, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 But it doesn't exist, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CONH-, T 3 is substituted (C1-C 12 ) alkyl, and V 3is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 is PAP, and V 4 is -C(O)O-, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0.

[0266] In certain embodiments, the first linker L A The left side of the above linker structure for A The right side of the above linker structure is attached to a first drug or active agent.

[0267] As noted above, in some embodiments, L B is -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V13 ) m -, and g, h, i, j, k, l, and m are each independently 0 or 1.

[0268] In some embodiments, the second linker L B In T 7 However, (C1-C 12 ) alkyl and substituted (C1-C 12 ) alkyl; T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 However, each independently, (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 each independently represents a covalent bond, -CO-, or -NR 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15selected from -SO2- and -P(O)OH-, and q is an integer of 1 to 6; During the ceremony, (PEG) n but, [ka] and n is an integer from 1 to 30; EDA has the following structure: [ka] wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) [ka] and AA is an amino acid residue, and p is an integer of 1 to 20; Each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 the groups may be cyclically linked to form a piperazinyl ring; Each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0269] Any convenient tether group may be T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13For example, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Any of the tether groups described above in connection with the tether group T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 It can be used for:

[0270] Any convenient linking functional group may be V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 For example, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 Any of the linking functional groups described above in connection with the linking functional group V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 It can be used for:

[0271] In certain embodiments, each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are independently selected from R 13 As described above for

[0272] In certain embodiments, each R 15are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are independently selected from R 15 In these embodiments, the various possible substituents are as described above for R 15 As described above for

[0273] Second linker L B In certain embodiments, the tether group T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 wherein one or more of each is optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0274] Second linker L BIn certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0275] In certain embodiments, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 , and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 is selected from the following: During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 However, PABA and V 11 is -CO-, T 12 However, (C1-C 12 ) alkyl, and V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -O-, T 11 However, (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 But it doesn't exist, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CONH-, T 10 is substituted (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 However, (PEG) n and V 11 is -CO-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PAB, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 But it doesn't exist.

[0276] In certain embodiments, the second linker L B The left side of the above linker structure for is attached to a hydrazinyl-indolyl conjugated moiety or a hydrazinyl-pyrrolo-pyridinyl conjugated moiety and a second linker L B The right side of the above linker structure is attached to a second drug or active agent.

[0277] In certain embodiments, the conjugate is an antibody-drug conjugate, as described above, in which the antibody and drug are linked together by a linker. In some cases, a linker m (e.g., L A and / or L B) is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moieties include one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separable parts. For example, the cleavable moiety may include one or more covalent bonds that can dissociate or separate under certain conditions, separating the cleavable linker into two or more parts. Thus, such a linker included in an antibody-drug conjugate can be a cleavable linker, such that under appropriate conditions, the cleavable linker is cleaved, separating or releasing the drug from the antibody at the desired target site of action for the drug.

[0278] In some cases, the cleavable linker comprises two cleavable moieties, e.g., a first cleavable moiety and a second cleavable moiety. The cleavable moieties may be configured such that cleavage of both cleavable moieties is required to separate or release the drug from the antibody at the desired target site of action for the drug. For example, cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that prevents cleavage of the first cleavable moiety. "Prevents cleavage" means that the presence of the uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage, thereby substantially reducing the amount of cleavage of the cleavable linker or preventing cleavage. For example, the presence of the uncleaved second cleavable moiety can prevent cleavage of the first cleavable moiety. The prevention of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount of or prevents release of the drug from the antibody, e.g., can substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug.

[0279] In some cases, the second cleavable moiety interferes with the cleavage of the first cleavable moiety, so cleavage of the cleavable linker can be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the interference with cleavage of the first cleavable moiety, thus enabling cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety can dissociate or separate the cleavable linker into two or more moieties as described above, thereby releasing the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially, it is meant that in the presence of an uncleaved second cleavable portion, about 10% or less cleavage of the first cleavable portion occurs, for example, about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable portion occurs in the presence of an uncleaved second cleavable portion.

[0280] In other words, the second cleavable moiety can protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. Thus, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thereby enabling cleavage of the first cleavable moiety, cleavage of the cleavable linker occurs, and then separation or release of the drug from the antibody at the desired target site of action for the drug. In some cases, cleavage of the second cleavable moiety exposes the first cleavable moiety for subsequent cleavage, but the second cleavable moiety is not within the cleavable linker and does not itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

[0281] Each cleavable moiety included in the cleavable linker can be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety, and the second cleavable moiety can be a second enzymatically cleavable moiety. The enzymatically cleavable moiety can be separated into two or more moieties as described above by the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved by the enzymatic action of an enzyme, such as, but not limited to, an ester, a peptide, a glycoside, etc. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at the desired target site of action, such as the desired target site of action of a drug released from an antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in significant amounts in other regions, such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety can be controlled so that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0282] For example, as described herein, the antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, e.g., for the delivery of a cancer therapeutic drug to a desired site of action where cancer cells are present. In some cases, enzymes, e.g., esterases that cleave ester bonds, can be biomarkers for cancer that are overexpressed in cancer cells. The overexpression, and therefore localization, of specific enzymes in cancer can be used in the context of an enzymatically cleavable moiety included in the cleavable linker of an antibody-drug conjugate of the present disclosure to specifically release a drug at a desired site of action (i.e., the site of the cancer (and the overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or glycoside) that can be cleaved by an enzyme that is overexpressed in cancer cells. For example, the enzyme can be an esterase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme. In some cases, the enzyme can be a glycosidase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme.

[0283] In certain embodiments, the enzymatically cleavable moiety is an ester bond. For example, the first cleavable moiety (i.e., the cleavage moiety protected from premature cleavage by the second cleavable moiety) described above can comprise an ester. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by an esterase enzyme, thereby substantially reducing or preventing premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. In some cases, the portion of the linker adjacent to the first cleavable moiety is linked to or comprises a substituent, and the substituent comprises a second cleavable moiety. In some cases, the second cleavable moiety comprises a glycoside or a glycoside derivative.

[0284] In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycosyl) or glycoside derivative. In some cases, the glycoside or glycoside derivative can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain the glycoside or glycoside derivative. The glycoside or glycoside derivative can be any glycoside or glycoside derivative that is suitable for use in a cleavable linker and can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable moiety comprises an ester and the second cleavable moiety comprises a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. Optionally, the second cleavable moiety is a glucuronide. Optionally, the second cleavable moiety is a galactoside. Optionally, the second cleavable moiety is a glucoside. Optionally, the second cleavable moiety is a mannoside. Optionally, the second cleavable moiety is a fucoside. Optionally, the second cleavable moiety is O-GlcNAc. Optionally, the second cleavable moiety is O-GalNAc.

[0285] The glycoside or glycoside derivative can be covalently attached to the cleavable linker via a glycosidic bond. The glycosidic bond can be linked to the cleavable linker via various types of bonds, including, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside or glycoside derivative can be enzymatically cleaved from the cleavable linker (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). The glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme capable of effecting cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. One example of an enzyme that can be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker is a glycosidase, such as glucuronidase, galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes can also be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, e.g., a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate. In some cases, the enzyme is found at or near the target site where the enzyme that mediates cleavage of the first cleavable moiety is found.

[0286] Examples of conjugates according to the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0287] Examples of conjugates according to the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka] [ka]

[0288] Any of the chemical entities, linkers and conjugating moieties shown in the structures above can be adapted for use in the subject compounds and conjugates.

[0289] Additional disclosure regarding hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compounds and methods for producing the conjugates can be found in U.S. Pat. Nos. 9,310,374 and 9,493,413, the disclosures of each of which are incorporated herein by reference.

[0290] Compounds useful for generating conjugates The present disclosure provides compounds useful for producing the conjugates described herein. In certain embodiments, the compounds can be bound to two or more drugs or active agents and may include a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety useful for conjugating drugs or active agents to polypeptides (e.g., antibodies). For example, the conjugating moiety in the compound may be conjugated to a polypeptide (e.g., an antibody), thus indirectly linking the drug or active agent and the polypeptide (antibody) together.

[0291] In certain embodiments, the compound is a compound of formula (II): [ka] During the ceremony, Z 1 , Z 2 , Z3 , and Z 4 However, each independently, CR 4 , N, and CL B -W 2 and at least one Z 1 , Z 2 , Z 3 , and Z 4 But CL B -W 2 and R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R 2 and R 3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L A is the first linker, L B is the second linker, W 1 is the first drug, W 2 is the second drug, a compound.

[0292] With respect to compounds of formula (II), the substituent Z 1 , Z 2 , Z 3 , Z 4 , R 2 , R 3 , R 4 , L A , L B , W 1 , and W 2 is as described above for the conjugate of formula (I). Similarly, the first linker L of formula (II) A and a second linker L B Regarding T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , T 13 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 The substituents are as described above for the conjugates of formula (I).

[0293] For example, in some cases, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 is selected from the following: During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 But it doesn't exist, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 However, PABA and V 4 is -CO-, T 5 However, (C1-C 12 ) alkyl, and V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But it is 4AP, V 2 is -CO-, T 3 However, (C1-C 12 ) alkyl, and V 3 is -O-, T 4 However, (C1-C 12 ) alkyl, and V 4 is -CO-, T 5 But AA and V 5 But it doesn't exist, T 6 But PABC, V 6 does not exist, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 is an amino acid analogue, and V 2 But it doesn't exist, T 3 But AA and V 3 But it doesn't exist, T 4But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CONH-, T 3 is substituted (C1-C 12 ) alkyl, and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CO-, T 2 But AA and V 2 is -NH-, T 3 However, (PEG) n and V 3 is -CO-, T 4 But AA and V 4 But it doesn't exist, T 5 But PABC, V 5 But it doesn't exist, f is 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 However, (PEG) n and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 is PAP, and V 4 is -C(O)O-, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But AA and V 3 But it doesn't exist, T 4 But PABC, V 4 But it doesn't exist, e and f are each 0, or During the ceremony, T 1 However, (C1-C 12 ) alkyl, and V 1 is -CONH-, T 2 is substituted (C1-C 12 ) alkyl, and V 2 is -CO-, T 3 But PABC, V 3 But it doesn't exist, d, e, and f are each 0.

[0294] For example, in some cases, T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 , and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13is selected from the following: During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 However, PABA and V 11 is -CO-, T 12 However, (C1-C 12 ) alkyl, and V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But it is 4AP, V 9 is -CO-, T 10 However, (C1-C 12 ) alkyl, and V 10 is -O-, T 11 However, (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 is an amino acid analogue, and V 9 But it doesn't exist, T 10 But AA and V 10 But it doesn't exist, T 11 But PABC, V 11 But it doesn't exist, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CONH-, T 10 is substituted (C1-C 12 ) alkyl, and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 is -NH-, T 10 However, (PEG) n and V 10 is -CO-, T 11 But AA and V 11 But it doesn't exist, T 12 But PABC, V 12 But it doesn't exist, m is 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 However, (PEG) n and V 9 is -CO-, T 10 But AA and V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C12 ) alkyl, and V 8 is -CO-, T 9 But AA and V 9 But it doesn't exist, T 10 But PABC, V 10 But it doesn't exist, T 11 is PAP, and V 11 is -C(O)O-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 is -CONH-, T 9 is substituted (C1-C 12 ) alkyl, and V 9 is -CO-, T 10 But PABC, V 10 But it doesn't exist, k, l, and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 However, (PEG) n and V 11 is -CO-, l and m are each 0, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PAB, V 13 does not exist, or During the ceremony, T 7 But it doesn't exist, V 7 is -NHCO-, T 8 However, (C1-C 12 ) alkyl, and V 8 But it doesn't exist, T 9 is heteroaryl, and V 9 But it doesn't exist, T 10 However, (C1-C 12 ) alkyl, and V 10 is -CONH-, T 11 is substituted (C1-C 12 ) alkyl, and V 11 is -CO-, T 12 But AA and V 12 But it doesn't exist, T 13 But PABC, V 13 But it doesn't exist.

[0295] Compounds of formula (II) can be used in the conjugation reactions described herein, wherein two or more drugs or active agents attached to a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety are conjugated to a polypeptide (e.g., an antibody) to form an antibody-drug conjugate.

[0296] Examples of compounds according to the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0297] Examples of compounds according to the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka]

[0298] Any of the chemical entities, linkers and conjugating moieties shown in the structures above can be adapted for use in the subject compounds and conjugates.

[0299] Polypeptides and Antibodies As shown above, the subject conjugates may include a substituent W 3Examples of such polypeptides include polypeptides (e.g., antibodies). The amino acid sequence of the polypeptide (antibody) may be modified to include 2-formylglycine (fGly) residues. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and / or their standard one-letter abbreviations, for example, alanine or Ala or A, cysteine ​​or Cys or C, aspartic acid or Asp or D, glutamic acid or Glu or E, phenylalanine or Phe or F, glycine or Gly or G, histidine or His or H, isoleucine or Ile or I, lysine or Lys or K, leucine or Leu or L, methionine or Met or M, asparagine or Asn or N, proline or Pro or P, glutamine or Gln or Q, arginine or Arg or R, serine or Ser or S, threonine or Thr or T, valine or Val or V, tryptophan or Trp or W, and tyrosine or Tyr or Y.

[0300] In certain embodiments, the amino acid sequence of a polypeptide or antibody is modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) to a 2-formylglycine (fGly) residue by the action of formylglycine generating enzyme (FGE), either in vivo (e.g., during translation of the aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting the aldehyde-tagged protein with FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

[0301] sulfatase motif The minimum sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, and typically no more than 6 amino acid residues in length. The sulfatase motif provided in an Ig polypeptide is at least 5 or 6 amino acid residues in length, and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, defining a sulfatase motif that is less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acid residues in length.

[0302] In certain embodiments, a polypeptide of interest comprises one or more amino acid residues, e.g., 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues, inserted, deleted, or substituted (replaced) relative to the native amino acid sequence to provide a sulfatase motif sequence in the polypeptide. In certain embodiments, the polypeptide comprises modifications (insertion, addition, deletion, and / or substitution / replacement) of fewer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. When the native amino acid sequence for a polypeptide (e.g., an antibody) contains one or more residues of a desired sulfatase motif, the total number of residue modifications can be reduced, for example, by site-specific modification (insertion, addition, deletion, substitution / replacement) of amino acid residues adjacent to the native amino acid residues to provide the desired sulfatase motif sequence. In certain embodiments, the extent of modification of the native amino acid sequence of the target antibody is minimized so as to minimize the number of amino acid residues inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of modification of the amino acid sequence of the target antibody can minimize the effects that such modifications may have on the function and / or structure of the antibody.

[0303] Aldehyde tags of particular interest are those containing at least one minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), although it should be readily understood that longer aldehyde tags are contemplated and encompassed by the present disclosure and can be used in the compositions and methods of the present disclosure. Thus, an aldehyde tag can contain a minimal sulfatase motif of five or six residues, or can be longer and contain a minimal sulfatase motif that can be flanked on the N- and / or C-terminal sides of the motif by additional amino acid residues. For example, aldehyde tags of five or six amino acid residues, as well as longer amino acid sequences of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues, are contemplated.

[0304] The aldehyde tag can be located at or near the C-terminus of the Ig heavy chain, for example, the aldehyde tag can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a native, wild-type Ig heavy chain. The aldehyde tag can be located in the CH1 domain of the Ig heavy chain. The aldehyde tag can be located in the CH2 domain of the Ig heavy chain. The aldehyde tag can be located in the CH3 domain of the Ig heavy chain. The aldehyde tag can be located in an Ig light chain constant region, for example, a kappa light chain constant region or a lambda light chain constant region.

[0305] In certain embodiments, the sulfatase motif used may be described by the formula: X 1 Z 10 X 2 Z 20 X 3 Z 30 (I') During the ceremony, Z 10 is a cysteine ​​or serine (also represented by (C / S)), Z 20 is either a proline or alanine residue (also represented by (P / A)), Z 30is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 is present or absent, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, S, or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 may independently be any amino acid, but typically is an aliphatic amino acid, a polar uncharged amino acid, or a sulfur-containing amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V, or G.

[0306] The amino acid sequence of the heavy and / or light chain of the antibody is represented by the formula X 1 Z 10 X 2 Z 20 X 3 Z 30 may be modified to provide a sequence of at least five amino acids of the formula: Z 10 is cysteine ​​or serine, Z 20 is a proline or alanine residue, Z 30 is an aliphatic amino acid or a basic amino acid, X 1 is present or absent, and if present, is any amino acid, provided that when the heterologous sulfatase motif is at the N-terminus of the polypeptide, X 1 exists, X 2 and X 3are each independently any amino acid.

[0307] The sulfatase motif is generally selected so that it can be converted by a selected FGE, e.g., an FGE present in the host cell in which the aldehyde-tagged polypeptide is expressed, or an FGE contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0308] For example, when the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including human FGE), the sulfatase motif has the following formula: X 1 CX 2 PX 3 Z 30 (I”) During the ceremony, X 1 may or may not be present, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S, or V, except that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 may independently be any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G, or C, for example, S, T, A, V, or G; Z 30 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I).

[0309] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: / / ), MCTPSR (SEQ ID NO: / / ), VCTPSR (SEQ ID NO: / / ), LCSPSR (SEQ ID NO: / / ), LCAPSR (SEQ ID NO: / / ), LCVPSR (SEQ ID NO: / / ), LCGPSR (SEQ ID NO: / / ), ICTPAR (SEQ ID NO: / / ), LCTPSK (SEQ ID NO: / / ), MCTPSK (SEQ ID NO: / / ), VCTPSK (SEQ ID NO: / / ), LCSPSK (SEQ ID NO: / / ), LCAPSK (SEQ ID NO: / / ), LCVPSK (SEQ ID NO: / / ), LCGPSK (SEQ ID NO: / / ), LCTPSA (SEQ ID NO: / / ), ICTPAA (SEQ ID NO: / / ), MCTPSA (SEQ ID NO: / / ), VCTPSA (SEQ ID NO: / / ), LCSPSA (SEQ ID NO: / / ), LCAPSA (SEQ ID NO: / / ), LCVPSA ​​(SEQ ID NO: / / ), and LCGPSA (SEQ ID NO: / / ).

[0310] fGly-containing sequence When FGE acts on the heavy and / or light chains of an antibody, the serine or cysteine ​​in the sulfatase motif is modified to fGly. Thus, the fGly-containing sulfatase motif has the following formula: X 1 (fGly)X 2 Z 20 X 3 Z 30 (I''') During the ceremony, fGly is a formylglycine residue, Z 20 is either a proline or alanine residue (also represented by (P / A)), Z 30 is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1may or may not be present, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 may independently be any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G, or C, for example, S, T, A, V, or G.

[0311] As described above, to generate a conjugate, a polypeptide containing an fGly residue can be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety of a linker linked to the drug or active agent (e.g., a hydrazinyl-indolyl conjugating moiety or a hydrazinyl-pyrrolo-pyridinyl conjugating moiety, as described above), to generate an fGly'-containing sulfatase motif. As used herein, the term fGly' refers to the amino acid residue of the sulfatase motif that is coupled to the drug or active agent via a linker (e.g., a branched linker) as described herein. Thus, an fGly'-containing sulfatase motif can be of the formula: X 1 (fGly')X 2 Z 20 X 3 Z 30 (II) During the ceremony, fGly' is an amino acid residue that is coupled to a drug or active agent via a linker (e.g., a branched linker) described herein; Z 20 is either a proline or alanine residue (also represented by (P / A)), Z 30is a basic amino acid (e.g., arginine (R), which may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may or may not be present, and if present, may be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S, or T, e.g., L, M, or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists, X 2 and X 3 may independently be any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G, or C, for example, S, T, A, V, or G.

[0312] Modification site As noted above, the amino acid sequence of the antibody is modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) to a fGly residue by the action of FGE, either in vivo (e.g., upon translation of the aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting the aldehyde-tagged protein with FGE in a cell-free system). The antibodies used to generate the conjugates of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as "target Ig polypeptides" or "target antibodies."

[0313] The site in the antibody at which the sulfatase motif is introduced can be any convenient site. As noted above, in some cases, the extent of modification of the native amino acid sequence of the target polypeptide is minimized, so as to minimize the number of amino acid residues inserted (e.g., to the N- or C-terminus), deleted, substituted (replaced), and / or added. By minimizing the extent of modification of the amino acid sequence of the target antibody, the effect that such modifications may have on the function and / or structure of the antibody can be minimized.

[0314] The antibody heavy chain constant region can comprise an Ig constant region of any heavy chain isotype, including a non-naturally occurring Ig heavy chain constant region (including a consensus Ig heavy chain constant region). The Ig constant region amino acid sequence can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent to a solvent-accessible loop region of the Ig constant region. The Ig constant region amino acid sequence can be modified by the insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide the amino acid sequence of a sulfatase motif as described above.

[0315] In some cases, the aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least the CH1 domain; at least the CH1 and CH2 domains; the CH1, CH2, and CH3 domains; or the CH1, CH2, CH3, and CH4 domains). The aldehyde-tagged Ig heavy chain constant region can comprise a heavy chain constant region sequence of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain, or any allotypic variant thereof, such as a human heavy chain constant region sequence or a mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence modified by FGE to include at least one sulfatase motif that can generate an fGly-modified Ig polypeptide. Ig heavy chain allotypic variants are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

[0316] In some cases, the aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region comprises a kappa or lambda light chain light chain constant region sequence, such as a human kappa or lambda light chain constant region, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, modified to include at least one sulfatase motif that can be modified by FGE to generate an fGly-modified antibody. Exemplary constant regions include human gamma 1 and gamma 3 regions. Excluding the sulfatase motif, the constant region may have a wild-type amino acid sequence or may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90%, or at least 95% identical) to the wild-type amino acid sequence.

[0317] In some embodiments, the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. As noted above, the isolated aldehyde-tagged antibody may comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described above, wherein the sulfatase motif is within or adjacent to a surface-accessible loop region of the antibody heavy chain constant region.

[0318] A sulfatase motif can be provided within or adjacent to one or more of these amino acid sequences at such modification sites in an Ig heavy chain. For example, an Ig heavy chain polypeptide amino acid sequence can be modified at one or more of these amino acid sequences (e.g., where the modification comprises an insertion, deletion, and / or substitution of one or more amino acid residues) to provide a sulfatase motif adjacent to the N-terminus and / or adjacent to the C-terminus of these modification sites. Alternatively, or in addition, an Ig heavy chain polypeptide amino acid sequence can be modified at one or more of these amino acid sequences (e.g., where the modification comprises an insertion, deletion, and / or substitution of one or more amino acid residues) to provide a sulfatase motif between any two residues at an Ig heavy chain modification site. In some embodiments, an Ig heavy chain polypeptide amino acid sequence may be modified to contain two motifs, which may be adjacent to each other or separated by one, two, three, four or more amino acids (e.g., about 1 to about 25, about 25 to about 50, or about 50 to about 100 or more amino acids). Alternatively, or in addition, if the native amino acid sequence provides one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues at the modification site of the Ig heavy chain polypeptide amino acid sequence may be modified to provide a sulfatase motif at the modification site (e.g., if the modification includes insertion, deletion, and / or substitution of one or more amino acid residues).

[0319] The antibodies used in the antibody-drug conjugates of the present disclosure may have any of a variety of antigen binding specificities, including, but not limited to, antigens present on cancer cells, antigens present on autoimmune cells, antigens present on pathogenic microorganisms, antigens present on virus-infected cells (e.g., human immunodeficiency virus-infected cells), antigens present on diseased cells, etc. For example, the antibody conjugate may bind to an antigen, where the antigen is present on the surface of a cell. The antibody conjugates of the present disclosure may bind to an antigen with suitable binding affinity, for example, 5×10 -6 M~10 -7 M, 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, 5 x 10 -8 M~10 -9 M or 10 -9 It can bind to an antigen with a binding affinity greater than M.

[0320] As a non-limiting example, a subject antibody conjugate may bind to an antigen present on a cancer cell (e.g., a tumor-specific antigen, an antigen overexpressed on a cancer cell, etc.), and the conjugated moiety can be a drug, e.g., a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, a subject antibody conjugate may be specific for an antigen on a cancer cell, and the conjugated moiety is a drug, e.g., a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0321] As a further non-limiting example, a subject antibody conjugate may bind to an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus, where the antigen is expressed on a cell type infected by the virus, etc.), and the conjugated moiety can be a drug, e.g., a viral fusion inhibitor. For example, a subject antibody conjugate may bind to an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, e.g., a viral fusion inhibitor.

[0322] Drugs for conjugation to polypeptides As shown above, the conjugates or compounds of the present disclosure may comprise a substituent W 1 and W 2 The conjugate may include a drug or active agent. Any of several drugs are suitable for use, or can be modified to be suitable for use, as a reactive partner for conjugation to an antibody. Examples of drugs include small molecule drugs and peptide drugs.

[0323] As used herein, "small molecule drug" refers to a compound, e.g., an organic compound, that exhibits a desired pharmaceutical activity and that generally has a molecular weight of about 800 Da or less, or 2000 Da or less, but can include molecules up to 5 kDa and can be as large as 10 kDa. Small inorganic molecules refer to molecules that contain no carbon atoms, and small organic molecules refer to compounds that contain at least one carbon atom.

[0324] For example, the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as camptothecin, or an analog or derivative thereof, or a pharmaceutically active camptothecin moiety and / or portion thereof. The topoisomerase inhibitor (e.g., camptothecin, or an analog or derivative thereof) conjugated to the polypeptide can be any of a variety of topoisomerase inhibitors, including, but not limited to, camptothecin and its analogs and derivatives as described herein. Examples of drugs that may be used in the conjugates and compounds described herein include, but are not limited to, topoisomerase inhibitors, such as camptothecin or camptothecin derivatives, such as SN-38, belotecan, exatecan, 9-aminocamptothecin (9-AC), topotecan, des-Me-topotecan, and derivatives thereof. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT / US2022 / 012325, the disclosure of which is incorporated herein by reference.

[0325] In other embodiments, the drug or active agent may be maytansine. "Maytansine," "maytansine moiety," "maytansine active agent moiety," and "maytansinoid" refer to maytansine, as well as analogues and derivatives thereof, and pharmaceutically active maytansine moieties and / or portions thereof. The maytansine conjugated to the polypeptide may be any of a variety of maytansinoid moieties, including, but not limited to, maytansine and its analogues and derivatives (e.g., deacylmaytansine) as described herein.

[0326] In other examples, the drug or active agent may be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and / or portion thereof. The auristatin conjugated to the polypeptide may be any of a variety of auristatin moieties, including, but not limited to, auristatins as described herein and their analogs and derivatives. Examples of drugs that may be found for use in the conjugates and compounds described herein include, but are not limited to, auristatins or auristatin derivatives, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and derivatives thereof.

[0327] In other cases, the drug or active agent may be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and / or moiety thereof. The duocarmycin conjugated to the polypeptide may be any of a variety of duocarmycin moieties, including, but not limited to, the duocarmycins and analogs and derivatives thereof described herein. Examples of drugs that may be used in the conjugates and compounds described herein include, but are not limited to, duocarmycins or duocarmycin derivatives such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, and derivatives thereof. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.

[0328] In certain embodiments, the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulant, a Toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.

[0329] For example, a cytotoxin can include any compound that results in cell death (eg, necrosis or apoptosis) or a decrease in cell viability.

[0330] Kinase inhibitors include, but are not limited to, adavosertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitinib, tucatinib, vandetanib, and vemurafenib.

[0331] For example, selective estrogen receptor modulators include, but are not limited to, endoxifen, tamoxifen, afimoxifen, toremifene, and the like.

[0332] Immunostimulants include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony-stimulating factors, interferons, interleukins, etc. TLR agonists include, but are not limited to, imiquimod, resiquimod, etc.

[0333] Oligonucleotide drugs (dugs) include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, voranesorsen, inotersen, tofersen, tominersen, and the like.

[0334] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like.

[0335] Cytokines include albinterferon alfa-2B, aldesleukin, ALT-801, anakinra, ancestim, avotermin, valgrastim, bempegaldesleukin, binetrakin, syntredekin besudotox, CTCE-0214, darbepoetin alfa, denileukin diftitox, dulanermin, eddekin alfa, emfilamine, epoetin delta, erythropoietin, human interleukin-2, interferon alfa, interferon alfa-2c, interferon alfa-n1, interferon alfa-n3, interferon alfacon-1, interferon beta-1a, interferon beta-1b, interferon gamma-1b, and interferon kappa, interleukin-1 alpha, interleukin 10, interleukin 7, lenograstim, relidistim, lipegfilgrastim, lorcafusp alfa, Maxy-G34, methoxypolyethylene glycol epoetin beta, molgramostim, muprestim, nagressipene, oprelvekin, pegfilgrastim, pegilodecakin, peginterferon alfa-2a, peginterferon alfa-2b, peginterferon beta-1a, peginterferon lambda-1a, recombinant CD40 ligand, regramostim, romiplostim, sargramostim, thrombopoietin, tucotuzumab celmoleukin, viral macrophage inflammatory protein.

[0336] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.

[0337] As used herein, "peptide drug" refers to an amino acid containing polymeric compound and is meant to encompass natural and non-natural peptides, oligopeptides, cyclic peptides, and proteins, as well as peptidomimetics. Peptide drugs can be obtained by chemical synthesis or can be produced from genetically encoded sources (e.g., recombinant sources). Peptide drugs can range in molecular weight from 200 Da to 10 kDa or more. Suitable peptides include, but are not limited to, cytotoxic peptides, angiogenic peptides, antiangiogenic peptides, peptides that activate B cells, peptides that activate T cells, antiviral peptides, peptides that inhibit viral fusion, peptides that increase the production of one or more lymphocyte populations, antimicrobial peptides, growth factors, growth hormone-releasing factors, vasoactive peptides, anti-inflammatory peptides, peptides that regulate glucose metabolism, antithrombotic peptides, antinociceptive peptides, vasodilator peptides, platelet aggregation inhibitors, analgesics, and the like.

[0338] Additional examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to, tubulysin M, calicheamicin, STAT3 inhibitors, alpha-amanitin, aurora kinase inhibitors, belotecan, and anthracyclines.

[0339] Other examples of drugs include small molecule drugs such as cancer chemotherapeutic agents. For example, if the polypeptide is an antibody (or fragment thereof) with specificity for tumor cells, the antibody may be generated to contain modified amino acids as described herein and then conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, including cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, or steroid hormones. Peptide compounds may also be used.

[0340] Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives, and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, e.g., WO 96 / 33212, WO 96 / 14856, and US 6,323,315. For example, dolastatin 10 or auristatin PE may be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, e.g., EP 1391213, and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), duocarmycins and their active analogs and derivatives (including, e.g., synthetic analogs, KW-2189, and CB1-TM1), and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0341] Drugs that act to reduce cell proliferation are known in the art and are widely used. Such drugs include alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0342] Antimetabolites include, but are not limited to, folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0343] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines, daunorubicin salts, and the like. Examples of the anti-inflammatory drug include, but are not limited to, phenoxyzolidinone cyclopeptides such as cutinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindole diones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like.

[0344] Other antiproliferative cytotoxic agents are navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxaphene, cyclophosphamide, ifosfamide, and droloxifene.

[0345] Microtubule-interacting agents with antiproliferative activity are also suitable for use, including, but not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0346] Hormone modulating agents and steroids (including synthetic analogs) suitable for use include corticosteroids, such as prednisone, dexamethasone, and the like; estrogens and pregestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; and adrenocortical suppressants, such as aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosinate, and the like. Examples of estrogen-receptor-binding steroids include, but are not limited to, terone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation, and therefore compounds that bind to the estrogen receptor are used to inhibit this activity. Corticosteroids can inhibit T-cell proliferation.

[0347] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epiphyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporin, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), and the like.

[0348] Taxanes are suitable for use. "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, Taxol™, Taxotere™, the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see WO 94 / 07882, WO See also WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076, U.S. Patent Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP 590,267), or can be obtained from a variety of commercial sources including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0349] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., Taxotere™ docetaxel, as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0350] The term "taxane" also encompasses various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113; the piperazino and other derivatives described in WO99 / 14209; the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; and the taxol derivatives described in U.S. Patent No. 5,415,869. This also includes prodrugs of paclitaxel, including, but not limited to, those described in WO98 / 58927, WO98 / 13059, and U.S. Patent No. 5,824,701.

[0351] Suitable biological response modifiers for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.

[0352] Examples of drugs include small molecule drugs such as cancer chemotherapeutic agents. For example, if the polypeptide is an antibody (or fragment thereof) with specificity for tumor cells, the antibody may be generated to contain modified amino acids as described herein and then conjugated to a cancer chemotherapeutic agent such as a microtubule-interacting agent. In certain embodiments, the drug is a microtubule-interacting agent with antiproliferative activity, such as a maytansinoid.

[0353] Embodiments of the present disclosure include conjugates in which an antibody is conjugated to two or more drug moieties, for example, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 drug moieties. The drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugate has an average drug-to-antibody ratio (DAR) (molar ratio) ranging from 0.1 to 20, or 0.5 to 20, or 1 to 20, e.g., 1 to 19, or 1 to 18, or 1 to 17, or 1 to 16, or 1 to 15, or 1 to 14, or 1 to 13, or 1 to 12, or 1 to 11, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In certain embodiments, the conjugate has an average DAR of 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugate has an average DAR of 1 to 10. In certain embodiments, the conjugate has an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugate has an average DAR of 5 to 10 (e.g., 8). Average refers to the arithmetic mean.

[0354] In certain embodiments, the two drugs or active agents attached to the branched linker are the same drug or active agent. For example, the first branch of the branched linker can be attached to a drug or active agent, and the second branch of the branched linker can be attached to the same drug or active agent as the first branch. In other embodiments, the two drugs or active agents attached to the branched linker are different drugs or active agents. For example, the first branch of the branched linker can be attached to a first drug or a first active agent, and the second branch of the branched linker can be attached to a second drug or a second active agent that is different from the first drug or the first active agent attached to the first branch.

[0355] In some embodiments, when two different drugs or active agents are attached to a branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. For example, in some cases, the use of two different drugs or active agents attached to a branched linker may provide a low therapeutically effective concentration at which both payloads act, thereby increasing the overall efficacy of the ADC.

[0356] In some embodiments, when two different drugs or active agents are attached to a branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit compared to the drugs or active agents used separately. For example, the drug or active agent may provide increased efficacy in drug delivery of the ADC (e.g., some payloads, such as iRGD peptides, may increase extravasation into tissues and enhance tumor infiltration).

[0357] In some embodiments, when two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may result in a reduction in tumor drug resistance by targeting multiple pathways. Examples of payload combinations include, but are not limited to, cytotoxic drugs, immunomodulatory molecules for activating or inhibiting immune cell populations, cytokines, hormones, radioisotopes, and the like.

[0358] In some embodiments, when two different drugs or active agents are attached to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and an auristatin described herein (e.g., MMAE). In some embodiments, when two different drugs or active agents are attached to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and an iRGD peptide described herein. In some embodiments, when two different drugs or active agents are attached to a branched linker, the two different drugs or active agents are an auristatin described herein (e.g., MMAE) and an iRGD peptide described herein. In some embodiments, when two different drugs or active agents are attached to a branched linker, the two different drugs or active agents are an auristatin described herein (e.g., MMAE) and a kinase inhibitor described herein (e.g., sorafenib, lapatinib, gefitinib, etc.). In some embodiments, when two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and a kinase inhibitor described herein (e.g., sorafenib, lapatinib, gefitinib, etc.). In some embodiments, when two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin described herein (e.g., MMAE) and a selective estrogen receptor modulator described herein (e.g., endoxifen). In some embodiments, when two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and a selective estrogen receptor modulator described herein (e.g., endoxifen).

[0359] Drugs to be conjugated to polypeptides can be modified to incorporate a reactive partner for reaction with the polypeptide. When the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be located in the N-terminal region, N-terminus, C-terminal region, C-terminus, or at a position internal to the peptide. For example, one method involves synthesizing a peptide drug bearing an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide can be reacted with a compound containing a carboxylic acid group and an oxy-N-Boc group. As an example, the amino group of the peptide is reacted with 3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on compounds containing a carboxylic acid group and an oxy-N-protecting group can include different numbers of carbons in the alkylene linker and the substituents on the alkylene linker. The reaction between the amino group of the peptide and a compound containing a carboxylic acid group and an oxy-N-protecting group occurs through standard peptide ligation chemistry.Examples of peptide coupling reagents that can be used include DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluoroformamide), midinium hexafluorophosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate), TSTU(O-( N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyridin-1-yl)methyl) PyBrOP (Bromotris(pyrrolidino)phosphonium hexafluorophosphate), BrOP (Bromotris(dimethylamino)phosphonium tetrafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (Bromotris(pyrrolidino)phosphonium hexafluorophosphate). As non-limiting examples, HOBt and DIC can be used as peptide coupling reagents.

[0360] Deprotection to expose the aminooxy functionality is carried out on peptides containing N-protecting groups. Deprotection of the N-oxysuccinimide group occurs, for example, according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al. Specific deprotection conditions include hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of Boc protecting groups can be carried out using TFA. Other deprotection reagents include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. Products and intermediates can be purified by conventional means, such as HPLC purification.

[0361] Those skilled in the art will understand that factors such as pH and steric hindrance (i.e., accessibility of amino acid residues for reaction with the intended reactive partner) are important, and determining modification reaction conditions to provide optimal conjugation conditions is well within the skill of the art and routine in the art. When conjugation is performed with polypeptides present in or on living cells, conditions are selected to be physiologically compatible. For example, the pH may be temporarily lowered for a time sufficient to allow the reaction to occur, but not within a period tolerated by the cells (e.g., about 30 minutes to 1 hour). Physiological conditions for modifying polypeptides on the cell surface may be similar to those used in ketone-azide reactions during modification of cells bearing cell-surface azides (see, e.g., U.S. Pat. No. 6,570,040).

[0362] Small molecule compounds that contain, or have been modified to contain, an α-nucleophilic group that serves as a reactive partner with the compounds or conjugates disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure. General methods are known in the art for chemical synthesis schemes and conditions useful for synthesizing compounds of interest (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001, or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0363] Formulation The conjugates of the present disclosure can be formulated in a variety of different ways. Generally, when the conjugate is a gradient-drug conjugate, the conjugate is formulated in a manner that is compatible with the drug, antibody, the condition being treated, and the route of administration being used.

[0364] In some embodiments, a pharmaceutical composition is provided comprising any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.

[0365] The conjugates (e.g., antibody-drug conjugates) may be provided in any suitable form, for example, in the form of a pharmaceutically acceptable salt, and may be formulated for any suitable route of administration, for example, oral, topical, or parenteral administration. When the conjugates are provided as injectable liquids (such as in those embodiments in which they are administered intravenously or directly to a tissue), the conjugates may be provided as ready-to-use dosage forms or as reconstitutable, storage-stable powders or liquids comprised of pharmaceutically acceptable carriers and excipients.

[0366] Methods for formulating the conjugates can be adapted from those readily available. For example, the conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition can optionally include other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to a mammal, e.g., a human.

[0367] Treatment method The antibody-drug conjugates of the present disclosure are useful for treating conditions or diseases in subjects amenable to treatment by administration of the parent drug (ie, the drug prior to conjugation to the antibody).

[0368] In some embodiments, a method is provided that includes administering to a subject an effective amount (e.g., a therapeutically effective amount) of any of the conjugates of the present disclosure.

[0369] In certain aspects, a method of delivering a drug to a target site in a subject is provided, the method comprising administering to the subject a pharmaceutical composition comprising any of the conjugates of the present disclosure, wherein the administration is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject. For example, as described above, the antibody-drug conjugates of the present disclosure are cleavable linkers, such as enzymatically cleavable linkers comprising a first enzymatically cleavable moiety and a second enzymatically cleavable moiety. In some cases, the cleavable linker can be cleaved under appropriate conditions to separate or release the drug from the antibody at the desired target site of action for the drug. For example, a second cleavable linker that protects the first cleavable linker from cleavage may be cleaved to allow cleavage of the first cleavable moiety, thereby cleaving the cleavable linker into two or more moieties and thus releasing the drug from the antibody-drug conjugate at the desired site of action.

[0370] In certain embodiments, the first cleavable moiety can be an enzymatically cleavable moiety. In some cases, the enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is administered to the subject being treated (i.e., exogenous to the subject being treated). For example, the first enzyme can be administered before, simultaneously with, or after administration of the antibody-drug conjugate described herein.

[0371] In certain embodiments, the second cleavable moiety can be an enzymatically cleavable moiety. In some cases, the enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is administered to the subject being treated (i.e., exogenous to the subject being treated). For example, the second enzyme can be administered before, simultaneously with, or after administration of the antibody-drug conjugate described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

[0372] In other examples, the first enzyme that promotes cleavage of the first cleavable moiety is an enzyme present in the subject being treated (i.e., endogenous to the subject being treated). For example, the first enzyme can be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can be specifically targeted to the desired site of action (e.g., can specifically bind to an antigen present at the desired site of action), and the desired site of action also includes the presence of the first enzyme. In some cases, the first enzyme is present in an overabundance at the desired site of action compared to other regions of the body of the subject being treated. For example, the first enzyme can be overexpressed at the desired site of action compared to other regions of the body of the subject being treated. In some cases, the first enzyme is present in an overabundance at the desired site of action due to the localization of the first enzyme in a particular region or location. For example, the first enzyme can be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the first enzyme is present in an overabundance in lysosomes compared to other regions of the body of the subject. In some embodiments, the lysosome containing the first enzyme is found at the desired site of action for the drug of the antibody-drug conjugate, e.g., the site of a cancer or tumor being treated with the drug. In certain embodiments, the first enzyme is an esterase.

[0373] In certain embodiments, the second enzyme that promotes cleavage of the second cleavable moiety is an enzyme present in the subject being treated (i.e., endogenous to the subject being treated). For example, the second enzyme can be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can be specifically targeted to the desired site of action (e.g., can specifically bind to an antigen present at the desired site of action), and the desired site of action also includes the presence of the second enzyme. In some cases, the second enzyme is present in an overabundance at the desired site of action compared to other regions of the body of the subject being treated. For example, the second enzyme can be overexpressed at the desired site of action compared to other regions of the body of the subject being treated. In some cases, the second enzyme is present in an overabundance at the desired site of action due to the localization of the second enzyme in a particular region or location. For example, the second enzyme can be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the second enzyme is present in an overabundance in lysosomes compared to other regions of the body of the subject. In some embodiments, the lysosome containing the second enzyme is found at ...

Claims

1. A conjugate of formula (I): 【Chemistry 1】 During the ceremony, Z １ , Z ２ , Z ３ , and Z ４ However, each independently, CR ４ , N, and C-L Ｂ -W ２ and at least one Z １ , Z ２ , Z ３ , and Z ４ But C-L Ｂ -W ２ and R １ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R ２ and R ３ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R ２ and R ３ are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R ４ are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L Ａ is the first linker, L Ｂ is a second linker, W １ is the first drug, W ２ is the second drug, W ３ is a polypeptide.

2. Z １ But, CR ４ That is, Z 1 is N; or The conjugate of claim 1, wherein Z 3 is C-L B -W 2 .

3. L Ａ but, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ - Including, During the ceremony, a, b, c, d, e, and f are each independently 0 or 1; T １ , T ２ , T ３ , T ４ , T ５ , and T ６ each independently represents a covalent bond, (C １ -C １２ ) alkyl, substituted (C １ -C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer from 1 to 20; each n is an integer from 1 to 30; each p is an integer from 1 to 20; and each x is an integer from 1 to 12; V １ , V ２ , V ３ , V ４ , V ５ , and V ６ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and optionally T 1 may be selected from (C 1 -C 12 )alkyl and substituted (C 1 -C 12 )alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 may each independently be selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) x —, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, hydrazine, and an ester; V 1 , V 2 , V 3 , V 4 , V 5 and V 6 may each independently be selected from the group consisting of a covalent bond, —CO—, —NR 15 —, —NR 15 (CH 2 ) q —, —NR 15 (C 6 H 4 )—, —CONR 15 —, —NR 15 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 15 —, —NR 15 SO 2 —, and —P(O)OH—; During the ceremony, (PEG) n is and n is an integer from 1 to 30; EDA has the structure: where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP), and each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring; optionally, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside; MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP may each be substituted with a glycoside; The conjugate of claim 1 or 2, wherein the glycoside may be selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

4. During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is AA, and V ２ But it doesn't exist. T ３ But PABC, V ３ But it doesn't exist. d, e, and f are each 0; or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is an amino acid analogue, and V ２ is -NH-, T ３ But (PEG) ｎ And V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABA, V ４ is -CO-, T ５ However, (C １ -C １２ ) alkyl, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is 4AP, and V ２ is -CO-, T ３ However, (C １ -C １２ ) alkyl, V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is 4AP, and V ２ is -CO-, T ３ However, (C １ -C １２ ) alkyl, V ３ is -O-, T ４ However, (C １ -C １２ ) alkyl, V ４ is -CO-, T ５ is AA, and V ５ But it doesn't exist. T ６ But PABC, V ６ does not exist, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is an amino acid analogue, and V ２ But it doesn't exist. T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CONH-, T ３ is replaced by substitution (C １ -C １２ ) alkyl, V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is AA, and V ２ is -NH-, T ３ But (PEG) ｎ And V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ is PAP, and V ４ is -C(O)O-, e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ But PABC, V ３ But it doesn't exist. The conjugate of claim 3 , wherein d, e, and f are each 0.

5. L Ｂ but, -(T ７ -V ７ ) ｇ -(T ８ -V ８ ) ｈ -(T ９ -V ９ ) ｉ -(T １０ -V １０ ) ｊ -(T １１ -V １１ ) ｋ -(T １２ -V １２ ) ｌ -(T １３ -V １３ ) ｍ - Including, During the ceremony, g, h, i, j, k, l, and m are each independently 0 or 1; T ７ , T ８ , T ９ , T １０ , T １１ , T １２ , and T １３ each independently represents a covalent bond, (C １ -C １２ ) alkyl, substituted (C １ -C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer from 1 to 20; each n is an integer from 1 to 30; each p is an integer from 1 to 20; and each x is an integer from 1 to 12; V ７ , V ８ , V ９ , V １０ , V １１ , V １２ , and V １３ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and optionally T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 may each be substituted with a glycoside; MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP may each be substituted with a glycoside; The conjugate of any one of claims 1 to 4, wherein the glycoside may be selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

6. T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ But it doesn't exist. T １０ But PABC, V １０ But it doesn't exist. k, l, and m are each 0; or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is an amino acid analogue, and V ９ is -NH-, T １０ But (PEG) ｎ And V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ is replaced by substitution (C １ -C １２ ) alkyl, V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABA, V １１ is -CO-, T １２ However, (C １ -C １２ ) alkyl, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is 4AP, and V ９ is -CO-, T １０ However, (C １ -C １２ ) alkyl, V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is 4AP, and V ９ is -CO-, T １０ However, (C １ -C １２ ) alkyl, V １０ is -O-, T １１ However, (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ But PABC, V １３ does not exist, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is an amino acid analogue, and V ９ But it doesn't exist. T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CONH-, T １０ is replaced by substitution (C １ -C １２ ) alkyl, V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ is -NH-, T １０ But (PEG) ｎ And V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ is PAP, and V １１ is -C(O)O-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ But it doesn't exist. T １０ But PABC, V １０ But it doesn't exist. T １１ is PAP, and V １１ is -C(O)O-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ is replaced by substitution (C １ -C １２ ) alkyl, V ９ is -CO-, T １０ But PABC, V １０ But it doesn't exist. k, l, and m are each 0; or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ But (PEG) ｎ And V １１ is -CO-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ is replaced by substitution (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ is PAB, and V １３ does not exist, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ is replaced by substitution (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ But PABC, V １３ The conjugate of claim 5 , wherein is not present.

7. The conjugate of claim 1 , wherein the conjugate is selected from the following: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 【Chemistry 5-5】 【Chemistry 5-6】 【Chemistry 5-7】 【Chemistry 5-8】 【Chemistry 5-9】 【Chemistry 5-10】 【Chemistry 5-11】 【Chemistry 5-12】 【Chemistry 5-13】 【Chemistry 5-14】 【Chemistry 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 【Chemistry 6-4】 【Chemistry 6-5】 【Chemistry 6-6】

8. A compound of formula (II) 【Chemistry 7】 During the ceremony, Z １ , Z ２ , Z ３ , and Z ４ However, each independently, CR ４ , N, and C-L Ｂ -W ２ and at least one Z １ , Z ２ , Z ３ , and Z ４ But C-L Ｂ -W ２ and R ２ and R ３ are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or R ２ and R ３ are optionally cyclically linked to form a 5- or 6-membered heterocyclyl; Each R ４ are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L Ａ is the first linker, L Ｂ is a second linker, W １ is the first drug, W ２ is a second drug, the compound.

9. Z １ But, CR ４ That is, Z 1 is N; or The compound of claim 8, wherein Z 3 is C-L B -W 2 .

10. L Ａ but, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ - Including, During the ceremony, a, b, c, d, e, and f are each independently 0 or 1; T １ , T ２ , T ３ , T ４ , T ５ , and T ６ each independently represents a covalent bond, (C １ -C １２ ) alkyl, substituted (C １ -C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer from 1 to 20; each n is an integer from 1 to 30; each p is an integer from 1 to 20; and each x is an integer from 1 to 12; V １ , V ２ , V ３ , V ４ , V ５ , and V ６ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and optionally T 1 may be selected from (C 1 -C 12 )alkyl and substituted (C 1 -C 12 )alkyl; T 2 , T 3 , T 4 , T 5 , and T 6 may each independently be selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) x —, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, hydrazine, and an ester; V 1 , V 2 , V 3 , V 4 , V 5 and V 6 may each independently be selected from the group consisting of a covalent bond, —CO—, —NR 15 —, —NR 15 (CH 2 ) q —, —NR 15 (C 6 H 4 )—, —CONR 15 —, —NR 15 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 15 —, —NR 15 SO 2 —, and —P(O)OH—; During the ceremony, (PEG) n is and n is an integer from 1 to 30; EDA has the structure: where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP), and each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring; optionally, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside; MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP may each be substituted with a glycoside; The compound according to claim 8 or 9, wherein the glycoside may be selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

11. T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is AA, and V ２ But it doesn't exist. T ３ But PABC, V ３ But it doesn't exist. d, e, and f are each 0; or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is an amino acid analogue, and V ２ is -NH-, T ３ But (PEG) ｎ And V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABA, V ４ is -CO-, T ５ However, (C １ -C １２ ) alkyl, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is 4AP, and V ２ is -CO-, T ３ However, (C １ -C １２ ) alkyl, V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is 4AP, and V ２ is -CO-, T ３ However, (C １ -C １２ ) alkyl, V ３ is -O-, T ４ However, (C １ -C １２ ) alkyl, V ４ is -CO-, T ５ is AA, and V ５ But it doesn't exist. T ６ But PABC, V ６ does not exist, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is an amino acid analogue, and V ２ But it doesn't exist. T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CONH-, T ３ is replaced by substitution (C １ -C １２ ) alkyl, V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CO-, T ２ is AA, and V ２ is -NH-, T ３ But (PEG) ｎ And V ３ is -CO-, T ４ is AA, and V ４ But it doesn't exist. T ５ But PABC, V ５ But it doesn't exist. f is 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ But (PEG) ｎ And V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ is PAP, and V ４ is -C(O)O-, e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ is AA, and V ３ But it doesn't exist. T ４ But PABC, V ４ But it doesn't exist. e and f are each 0, or During the ceremony, T １ However, (C １ -C １２ ) alkyl, V １ is -CONH-, T ２ is replaced by substitution (C １ -C １２ ) alkyl, V ２ is -CO-, T ３ But PABC, V ３ But it doesn't exist.

11. The compound of claim 10, wherein d, e, and f are each 0.

12. L Ｂ but, -(T ７ -V ７ ) ｇ -(T ８ -V ８ ) ｈ -(T ９ -V ９ ) ｉ -(T １０ -V １０ ) ｊ -(T １１ -V １１ ) ｋ -(T １２ -V １２ ) ｌ -(T １３ -V １３ ) ｍ - Including, During the ceremony, g, h, i, j, k, and l are each independently 0 or 1; T ７ , T ８ , T ９ , T １０ , T １１ , T １２ , and T １３ each independently represents a covalent bond, (C １ -C １２ ) alkyl, substituted (C １ -C １２ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; (EDA) ｗ , (PEG) ｎ , (AA) ｐ , -(CR １３ O.H. ｘ -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazines, disulfides, and esters; EDA is an ethylenediamine moiety; PEG is polyethylene glycol; AA is an amino acid residue or amino acid analog; each w is an integer from 1 to 20; each n is an integer from 1 to 30; each p is an integer from 1 to 20; and each x is an integer from 1 to 12; V ７ , V ８ , V ９ , V １０ , V １１ , V １２ , and V １３ each independently represents a covalent bond, —CO—, or —NR １５ --, --NR １５ (CH ２ ) ｑ --, --NR １５ (C ６ H ４ ) -, -CONR １５ --, --NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ N.R. １５ --, --NR １５ SO ２ -, -P(O)OH-, and -P(O)OH-, where each q is an integer from 1 to 6; Each R １３ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; Each R １５ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and optionally T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 may each be substituted with a glycoside; MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP may each be substituted with a glycoside; The compound according to any one of claims 8 to 11, wherein the glycoside may be selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

13. T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ But it doesn't exist. T １０ But PABC, V １０ But it doesn't exist. k, l, and m are each 0; or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is an amino acid analogue, and V ９ is -NH-, T １０ But (PEG) ｎ And V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ is replaced by substitution (C １ -C １２ ) alkyl, V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ But PABA, V １１ is -CO-, T １２ However, (C １ -C １２ ) alkyl, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is 4AP, and V ９ is -CO-, T １０ However, (C １ -C １２ ) alkyl, V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is 4AP, and V ９ is -CO-, T １０ However, (C １ -C １２ ) alkyl, V １０ is -O-, T １１ However, (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ But PABC, V １３ does not exist, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is an amino acid analogue, and V ９ But it doesn't exist. T １０ is AA, and V １０ But it doesn't exist. T １１ But PABC, V １１ But it doesn't exist. l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CONH-, T １０ is replaced by substitution (C １ -C １２ ) alkyl, V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ is -NH-, T １０ But (PEG) ｎ And V １０ is -CO-, T １１ is AA, and V １１ But it doesn't exist. T １２ But PABC, V １２ But it doesn't exist. m is 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ But (PEG) ｎ And V ９ is -CO-, T １０ is AA, and V １０ But it doesn't exist. T １１ is PAP, and V １１ is -C(O)O-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CO-, T ９ is AA, and V ９ But it doesn't exist. T １０ But PABC, V １０ But it doesn't exist. T １１ is PAP, and V １１ is -C(O)O-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ is -CONH-, T ９ is replaced by substitution (C １ -C １２ ) alkyl, V ９ is -CO-, T １０ But PABC, V １０ But it doesn't exist. k, l, and m are each 0; or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ But (PEG) ｎ And V １１ is -CO-, l and m are each 0, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ is replaced by substitution (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ is PAB, and V １３ does not exist, or During the ceremony, T ７ But it doesn't exist, V ７ is -NHCO-, T ８ However, (C １ -C １２ ) alkyl, V ８ But it doesn't exist. T ９ is heteroaryl; V ９ But it doesn't exist. T １０ However, (C １ -C １２ ) alkyl, V １０ is -CONH-, T １１ is replaced by substitution (C １ -C １２ ) alkyl, V １１ is -CO-, T １２ is AA, and V １２ But it doesn't exist. T １３ But PABC, V １３ The compound of claim 12, wherein is not present.

14. 9. The compound of claim 8, wherein the compound is selected from the following: 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 【Chemistry 11-5】 【Chemistry 11-6】 【Chemistry 11-7】 【Chemistry 11-8】 【Chemistry 11-9】 【Chemistry 11-10】 【Chemistry 11-11】 【Chemistry 11-12】 【Chemistry 11-13】 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】 【Chemistry 12-6】

15. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 7 and a pharma- ceutically acceptable excipient.

16. A conjugate according to any one of claims 1 to 7 for use in a method for treating cancer.

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