Linkers, drug linkers and conjugates thereof and methods of use thereof

Abstract

Polar units, linker intermediates, linkers, drug-linkers and conjugates thereof are provided.SOLUTION: Linkers are provided that have hydrophilic properties that maintain the inherent properties of the antibody conjugated to the linker and the drug. In particular, the linker helps to maintain the hydrophilicity of the antibody at higher drug loadings and / or when conjugated to hydrophobic drugs and other agents. Also provided are conjugates comprising the drug-linkers and linkers, and methods of using such conjugates for the treatment of cancer and other diseases.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Electronic sequence list reference The contents of the electronic sequence listing (760270_40201WO_SEQUENCE_LISTING.xml; size: 40657 bytes; created June 30, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0002] The use of monoclonal antibodies (mAbs) for targeted delivery of cytotoxic agents to disease-related cells, such as cancer cells and other cells, in the form of antibody-drug conjugates (or ADCs) is of great interest. The design of antibody-drug conjugates, typically by attaching a cytotoxic agent, immunomodulator, or other agent (collectively "drug") to an antibody via a linker, involves consideration of various factors. These factors include the identity and location of the chemical group for drug attachment, the mechanism of drug release, the structural elements (if any) that provide for drug release, and structural modifications of the released free drug, if any. If the drug is released in the extracellular environment, the released form of the drug must be able to reach its target. If the drug is released after antibody internalization, the structural elements and mechanism of drug release must be consistent with the intracellular trafficking of the conjugate.

[0003] Another important factor in the design of antibody-drug conjugates is the amount of drug that can be delivered per targeting agent (i.e., the number of drugs attached to each targeting agent (e.g., antibody), referred to as drug load or drug loading). Historically, there was an assumption that higher drug loading was superior to lower drug loading (e.g., 8 loading vs. 4 loading). The rationale was that a more highly loaded conjugate would deliver more drug (e.g., cytotoxic agent) to the target cell. This rationale was supported by the observation that conjugates with higher drug loading were more active against cell lines in vitro. However, certain subsequent studies revealed that this assumption was not confirmed in animal models. Conjugates with drug loadings of 4 or 8 of a specific auristatin were observed to have similar activity in mouse models. See, e.g., Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004). Hamblett et al. further reported that more highly loaded ADCs were cleared more rapidly from the circulation in animal models. This faster clearance suggested PK liability of the highly loaded species compared to the less loaded species. See Hamblett et al. In addition, the more highly loaded conjugates had a lower maximum tolerated dose (MTD) in mice and, consequently, a narrower reported therapeutic index. Ibid. In contrast, ADCs with a drug loading of 2 at an engineered site on a monoclonal antibody have been reported to have the same or better PK and therapeutic index compared to certain 4-loaded ADCs. See, e.g., Junutula et al., Clinical Cancer Res. 16:4769 (2010). Therefore, the recent trend is to develop ADCs with lower drug loading.

[0004] Thus, there is a need for antibody-drug conjugate formats (and other conjugate formats more generally) that allow for higher drug loading while maintaining other properties of lower-loading conjugates, such as favorable PK properties. Surprisingly, the present invention addresses these needs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hamblett et al., Clinical Cancer Res.10:7063-70(2004) [Non-patent document 2] Junutula et al.,Clinical Cancer Res.16:4769(2010) Summary of the Invention [Problem to be solved by the invention]

[0006] Provided herein are linkers with hydrophilic properties that maintain the inherent properties of antibody conjugated with linkers and drugs.In particular, linkers help maintain the hydrophilicity of antibodies at higher drug loadings and / or when conjugated with hydrophobic drugs and other agents.Also provided are drug-linkers and conjugates comprising linkers, and methods for using such conjugates for the treatment of cancer and other diseases. [Means for solving the problem]

[0007] In some embodiments, the following formula (V): [ka] (wherein AA is an amino acid unit having 1 to 12 amino acid subunits; s is 0 or 1; L2 is a linker subunit having 1 to 4 binding sites for Drug units; Each wavy line (~) indicates a binding site for a Stretcher unit, and a double wavy line ( [ka] ) indicates the attachment site for the Drug unit) or a salt thereof, Linker intermediates are provided in which at least one polar unit is present within an amino acid unit, a linker subunit, or both, and the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and combinations thereof.

[0008] In some embodiments, the following formula (I): [ka] where L1 is a Stretcher unit having a binding site for a Targeting unit; AA is an amino acid unit with 1 to 12 subunits; s is 0 or 1; L2 is a linker subunit having 1 to 4 binding sites for Drug units; The wavy line (~) indicates the binding site for the targeting unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof, Linkers are provided in which at least one polar unit is present within an amino acid unit, a linker subunit, or both, and the polar unit is selected from a saccharide unit, a PEG unit, a carboxyl unit, and combinations thereof.

[0009] In some embodiments, the following formula (I): [ka] where L1 is a Stretcher unit having a binding site for a Targeting unit; AA is an amino acid unit with 1 to 12 subunits; s is 0 or 1; L2 is a linker subunit having 1 to 4 binding sites for Drug units; The wavy line (~) indicates the binding site for the targeting unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof, Linkers are provided in which at least one polar unit is present within an amino acid unit, a linker subunit, a stretcher unit, or a combination thereof, and the polar unit is selected from a saccharide unit, a PEG unit, a carboxyl unit, and a combination thereof.

[0010] sugar units In some embodiments, the sugar unit has the following formula: L3-**N(CH2-(CH(XR)) k -X1(X2))2 (X) wherein each X is independently selected from NH or O; each R is independently selected from hydrogen, acetyl, monosaccharides, disaccharides, and polysaccharides; each X1 is independently selected from CH2 and C(O); each X2 is independently selected from H, OH, and OR; k is 1 to 10; L3 has the following general formula (XI): L3a | *-NH-(CH2) p -CH-(CH2) o -C(O)-# (XI) (Wherein, L3a is C1-C 10 selected from alkylene and polyethylene glycol having 1 to 24 ethylene glycol subunits; p and o are independently 0 to 2; Each * and each # indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2 or stretcher unit (L1); L3a is covalently bonded to the N atom marked with a** in formula (X)) or a salt thereof) or a salt thereof.

[0011] In some embodiments, the linker intermediate or linker has a formula selected from the following: [ka] or [ka] wherein each R is independently selected from hydrogen, monosaccharides, disaccharides, and polysaccharides; p and o are independently 0 to 2; m is 1 to 8; n is 0 to 4; Each * and each # indicates a binding site for another subunit of an amino acid unit (AA), linker subunit L2 or stretcher unit (L1), or a sugar unit having a salt thereof.

[0012] PEG units In some embodiments, the linker intermediate or linker has a formula selected from the following: (a) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 21 and R 22 are each independently an optional C1-C3 alkylene; R24 and R 25 are respectively H; polyhydroxyl group; substituted polyhydroxyl group; -C(O)-polyhydroxyl group; substituted -C(O)-polyhydroxyl group; optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 are independently selected from the group consisting of sugar units of formula (XII) or (XIII); or -NR 24 R 25 are joined together from C3-C8 heterocycles; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26) or a salt thereof. or (b) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 21 and R 22 are each independently an optional C1-C3 alkylene; R 24 and R 25 one of which is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); R 24 and R 25 the other optionally is polyethylene glycol having 1 to 24 ethylene glycol subunits; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26;) or a salt thereof; or (c) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXI) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 26 and R 27 are optional, and C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- and -C(O)-C-C 12 independently selected from alkylene-NH-; R 24 and R 25one of which is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); R 24 and R 25 the other is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); and optionally polyethylene glycol having 1 to 24 ethylene glycol subunits; or —NR 24 R 25 are joined together from C3-C8 heterocycles; Each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—Ci-C6 alkenylene-, —NH—Ci-C6 alkenylene-, —Ci-C6 alkenylene-NH—, —Ci-C6 alkenylene-C(O)—, —NH(CO)NH—, and triazole; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26; n21 is 1 to 4; n27 is 1 to 4;) or a salt thereof.

[0013] In some embodiments, the R of the PEG unit 24 and R 25 and R are not both H. In some embodiments, the R of the PEG unit 24 and R 25 are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 and (b) are not H.

[0014] In some embodiments, a linker intermediate or linker is provided in which the polyhydroxyl group is a linear monosaccharide optionally selected from a C6 or C5 sugar, a sugar acid, or an amino sugar. the C6 or C5 sugar is selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idostulose, aldose, and ketose; the sugar acid is selected from gluconic acid, aldonic acid, uronic acid and uronic acid; or Linker intermediates or linkers are provided in which the amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

[0015] In some embodiments, a linker intermediate or linker, or a salt thereof, is provided, wherein the PEG unit is selected from the following: [ka] (In the formula, R 39 is selected from H, a linear monosaccharide, and optionally, a polyethylene glycol having 1 to 24 ethylene glycol subunits; the wavy line on the left indicates the site of attachment of an amino acid unit to the subunit or part of a linker subunit).

[0016] In some embodiments, the R of the PEG unit24 and R 25 In one embodiment, a linker intermediate or linker is provided, one of which is a linear monosaccharide and the other is a cyclic monosaccharide.

[0017] In some embodiments, a linker intermediate or linker, or a salt thereof, is provided, wherein the PEG unit is selected from the following: [ka] (In the formula, R 41 is a cyclic monosaccharide; the wavy line on the left indicates the site of attachment of an amino acid unit to a subunit or part of a linker subunit).

[0018] In some embodiments, the R of the PEG unit 24 and R 25 are independently selected from cyclic monosaccharides, disaccharides, and polysaccharides. In some embodiments, linker intermediates or linkers are provided wherein the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (In the formula, each R 45 is selected from H and a monosaccharide, a disaccharide, or a polysaccharide; R 46 is selected from cyclic monosaccharides, disaccharides, or polysaccharides; the wavy line on the right indicates the attachment site of an amino acid unit to a subunit or part of a linker subunit).

[0019] In some embodiments, the R of the PEG unit 24 and R 25 is independently selected from a linear monosaccharide and a substituted linear monosaccharide, and the substituted linear monosaccharide is replaced with a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, a linker intermediate or linker is provided in which the PEG unit is selected from the following, or a salt thereof: [ka] (In the formula, R 47 is a linear monosaccharide; each R 49 is selected from monosaccharides, disaccharides and polysaccharides; the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or part of the linker subunit).

[0020] In some embodiments, the R of the PEG unit 24 and R 25 are independently selected from a straight chain monosaccharide and a substituted monosaccharide, and the substituted straight chain monosaccharide is substituted with one or more substituents selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and optionally further substituted with a monosaccharide, disaccharide, or polysaccharide. In some embodiments, a linker intermediate or linker is provided in which the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (In the formula, each R 42 are independently selected from linear monosaccharides and substituted linear monosaccharides; each R 43 are independently selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, and amide; the wavy line on the left indicates the site of attachment of an amino acid unit to a subunit or part of a linker subunit).

[0021] In some embodiments, the R of the PEG unit 24 and R 25 is a -C(O)-polyhydroxyl group or a substituted -C(O)-polyhydroxyl group, and R 24 and R 25wherein the other is H, -C(O)-polyhydroxyl group, substituted -C(O)-polyhydroxyl group, polyhydroxyl group, or substituted polyhydroxyl group, and the substituted -C(O)-polyhydroxyl group and polyhydroxyl group are substituted with a monosaccharide, disaccharide, polysaccharide, alkyl, -O-alkyl, aryl, carboxyl, ester, or amide. In some embodiments, a linker intermediate or linker is provided wherein the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (where the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit or part of a linker subunit).

[0022] In some embodiments, the R of the PEG unit 24 and R 25 is independently selected from H, substituted -C1-C8 alkyl, substituted -C1-C4 alkyl, or substituted -C1-C3 alkyl, with the proviso that R 24 and R 25 and -C1-C8 alkyl, -C1-C4 alkyl, and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl, provided that both are not H. In some embodiments, linker intermediates or linkers are provided in which the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (In the formula, R 48is selected from H, OH, CHOH, COOH, or —C1-C6 alkyl substituted with hydroxyl or carboxyl; the wavy line on the left indicates the site of attachment to a subunit of an amino acid unit or part of a linker subunit).

[0023] In some embodiments, the R of the PEG unit 24 and R 25 is selected from H, substituted —C(O)—C-C alkyl, substituted —C(O)—C-C alkyl, and substituted —C(O)—C-C alkyl; and R 24 and R 25 and the other is selected from substituted -C(O)-C1-C8 alkyl, substituted -C(O)-C1-C4 alkyl, substituted -C(O)-C1-C3 alkyl, substituted -C1-C8 alkyl, substituted -C1-C4 alkyl, and substituted -C1-C3 alkyl, wherein the substituted -C(O)-C1-C8 alkyl, substituted -C(O)-C1-C4 alkyl, substituted -C(O)-C1-C3 alkyl, substituted -C1-C8 alkyl, -C1-C4 alkyl, and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl. In some embodiments, a linker intermediate or linker, or a salt thereof, is provided wherein the PEG unit is selected from: [ka] or [ka] (where the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit or part of a linker subunit).

[0024] In some embodiments, the R of the PEG unit 24 and R 25 is selected from H and optionally substituted aryl, with the proviso that R 24 and R 25and n are not H, and the optional substituents are as defined herein, for example, in some embodiments, the optional substituent is halo, such as F, Cl, or Br. In some embodiments, linker intermediates or linkers are provided in which the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (where the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit or part of a linker subunit).

[0025] In some embodiments, R 24 and R 25 taken together form an optionally substituted C3-C8 heterocycle or heteroaryl, in some embodiments, the C3-C8 heterocycle or heteroaryl is unsubstituted. [ka] or a salt thereof.

[0026] In some embodiments, the R of the PEG unit 24 and R 25 is independently selected from H and a chelating agent, and the chelating agent is connected to —NR by an alkylene, arylene, carbocyclo, heteroarylene, or heterocarbocyclo; 24 R 25 optionally bonded to the nitrogen of R 24 and R 25and N,N'-dialkyl-substituted piperazines are provided. In some embodiments, linker intermediates or linkers are provided wherein the chelator is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), benzyl-DTPA, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), benzyl-DOTA, 1,4,7-triazacyclododecane-N,N',N''-triacetic acid (NOTA), benzyl-NOTA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), and N,N'-dialkyl-substituted piperazines. In some embodiments, linker intermediates or linkers are provided wherein the PEG unit is selected from the following, or a salt thereof: [ka] or [ka] (where the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit or part of a linker subunit).

[0027] In some embodiments, linker intermediates or linkers are provided in which each monosaccharide of a sugar unit or PEG unit is independently selected from the following: a C5 or C6 sugar selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, gulose, idostulose, aldose, ketose, glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; a sugar acid selected from gluconic acid, aldonic acid, uronic acid, and urosonic acid; or The amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine.

[0028] In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or protected forms thereof.

[0029] In some embodiments, R 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or protected forms thereof.

[0030] In some embodiments, the PEG unit is a formula selected from: (a) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -R 30 (XXX) (In the formula, R 20 is a functional group for attachment to a subunit of the amino acid unit (if present) and / or part of the linker subunit L2; R 21 and R 22 are each optional and, when present, are independently a C1-C3 alkylene group; R 30 is an optionally substituted C3-C 10selected from carbocycles; thioureas; optionally substituted thioureas; ureas; optionally substituted ureas; sulfamides; alkylsulfamides; acylsulfamides, optionally substituted alkylsulfamides; optionally substituted acylsulfamides; sulfonamides; optionally substituted sulfonamides; guanidines (including alkyl and aryl guanidines); phosphoramides; or optionally substituted phosphoramides; or R 30 represents azido, alkynyl, substituted alkynyl, -NH-C(O)-alkynyl, -NH-C(O)-alkynyl-R 65 cyclooctyne; selected from -NH-cyclooctyne, -NH-C(O)-cyclooctyne or -NH-(cyclooctyne)2; R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26;) or a salt thereof; (b) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NH-C(O)-R 31 (XXXI) (In the formula, R 20 is a functional group for attachment to a subunit of an amino acid unit (if present) or part of the linker subunit L2; R 21 and R 22 are each independently an optional C1-C3 alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 having; R 35is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26;) or a salt thereof; (c) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -C(O)NH-R 31 (XXXII) (In the formula, R 20 is a functional group for attachment to a subunit of an amino acid unit (if present) or part of the linker subunit L2; R 21 and R 22 are each optional and independently a C1-C3 alkylene group; R 31 is a branched polyethylene glycol chain, each branch independently having 1 to 26 ethylene glycol subunits, and each branch has at its terminus R 35 having; R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, and optionally substituted heteroaryl; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26;) or a salt thereof, and (d) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -N-(R 33 -R 31 )2 (XXXIII) (In the formula, R 20 is a functional group for attachment to a subunit of an amino acid unit (if present) or part of the linker subunit L2; R 21 and R 22 are each optional and are a C1-C3 alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 having; R 33 is C1-C3 alkylene, C1-C3 alkylene-C(O), -C(O)-C1-C3 alkylene, or -C(O)-C1-C3 alkylene-C(O); R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicates the binding site for; n20 is 1 to 26; or a salt thereof.

[0031] In some embodiments, a linker intermediate or linker is provided in which the PEG unit has a formula selected from the following, or a salt thereof: ~R20 -R 21 -[O-CH2-CH2] n20 -R 22 -NH-C(O)-R 31 (XXXI), ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -C(O)NH-R 31 (XXXII), or ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -N-(R 33 -R 31 )2 (XXXIII); (In the formula, R 20 is a functional group for attachment to a subunit of an amino acid unit (if present) or part of the linker subunit L2; R 21 and R 22 are each optional and are a C1-C3 alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 R 33 is C1-C3 alkylene, -C1-C3 alkylene-C(O), -C(O)-C1-C3 alkylene or -C(O)-C1-C3 alkylene-C(O); R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, or optionally substituted heteroaryl; the wavy line (~) represents R 20and n20 is 1-26). In some embodiments, a linker intermediate or linker is provided in which the PEG unit is selected from the following: [ka] (In the formula, R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; the wavy line on the left indicates the site of attachment to a subunit of an amino acid unit or part of a linker subunit).

[0032] In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or protected forms thereof.

[0033] In some embodiments, R 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or protected forms thereof.

[0034] In some embodiments, the formula is selected from: ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 )n42 (XL) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; Each R 43 independently, absent or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, -C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene-C1-C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0035] In some embodiments, the formula is selected from: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; R 43 does not exist or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0036] In some embodiments, the formula is selected from: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C3 alkylene; R 43 is absent or C1-C6 alkylene, -NH-C1-C 12 Alkylene, -C1-C6 alkylene-NH-, -C(O)-C1-C6 alkylene, -C1-C6 alkylene-C(O)-, -NH-C1-C6 alkylene-C(O)-, -C(O)-C1-C6 alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C6 alkylene, -C(O)-NH-C1-C 12Alkylene, -heteroarylene, heteroaryl-C1-C6 alkylene, heteroaryl-C1-C6 alkylene-C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the groups is H or C1-C6 alkylene, and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1 to 16; n41 is 1 to 4; n42 is 1 to 4), or a salt thereof.

[0037] In some embodiments, R 40 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or protected forms thereof.

[0038] In some embodiments, R 40 but one of the following structures: [ka] or [ka] (Wherein, R=H or C1-6 alkyl; and n=0~12 (*) indicates the R for a subunit of an amino acid unit or a part of the linker subunit L2. 40 indicates the binding site of ( [ka] ) is the R 40 or a stereoisomer thereof.

[0039] In some embodiments, R 40 but one of the following structures: [ka] or [ka] (In the formula, n=0~12 (*) indicates the R for a subunit of an amino acid unit or a part of the linker subunit L2. 40 indicates the binding site of ( [ka] ) is the R 40 or a stereoisomer thereof.

[0040] In some embodiments, R 43 -(NR 44 R 45 ) n41 But R 43 When present, one of the following structures: [ka] or [ka] (In the formula, R=H, C1-6 Alkyl, polyhydroxyl, or substituted polyhydroxyl ( [ka] ) is the R 43 or a stereoisomer thereof.

[0041] In some embodiments, R 43 -(NR 44 R 45 ) n41 But R 43 When present, one of the following structures: [ka] or [ka] (In the formula, ( [ka] ) is the R 43 or a stereoisomer thereof.

[0042] In some embodiments, —NR 44 R 45 but one of the following structures: [ka] or [ka] (In the formula, ( [ka] ) is the -NR 44 R 45or a stereoisomer thereof.

[0043] In some embodiments, before the PEG unit is attached to an amino acid unit or portion of the linker subunit L2, a linker intermediate or linker is provided having one of the following structures: [ka] [ka] [ka] [ka] or [ka] (wherein R is H or alkyl and n is 1 to 12).

[0044] In some embodiments, the formula is selected from: ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 46 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLIII) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; Each R 43independently, absent or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, -C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene-C1-C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; R 46 is selected from amino, amino-alkyl-amino, or —NH—C(O)—NH—S(O)2—NH—; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0045] In some embodiments, before the PEG unit is attached to an amino acid unit or portion of the linker subunit L2, a linker intermediate or linker is provided having one of the following structures: [ka] (wherein R is H or alkyl and n is 1 to 12).

[0046] In some embodiments, the formula is selected from: [ka] or [ka] wherein each Y independently represents R 76 or [ka] and Each R 76 are independently H, acetyl, -P(=O)(OH)2, or -(CH2) v -OS(=O)2(OH); Each R a and R b are independently H or R a and R b together with the carbon to which they are attached to form an oxo group; each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; A linker intermediate or linker is provided that includes a PEG unit having an amino acid unit (AA), a linker subunit L2, or a Stretcher unit (L1) subunit (where each * indicates a binding site for the subunit), or a salt thereof.

[0047] In some embodiments, the formula is selected from: [ka] or [ka] (In the formula, each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH); each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; A linker intermediate or linker is provided that includes a PEG unit having an amino acid unit (AA), a linker subunit L2, or a Stretcher unit (L1) subunit (where each * indicates a binding site for the subunit), or a salt thereof.

[0048] In some embodiments, a linker intermediate or linker is provided that includes a PEG unit having a formula selected from the following, or a salt thereof: [ka] or [ka] (In the formula, each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; Each * indicates a binding site for a subunit of an amino acid unit (AA), linker subunit L2 or stretcher unit (L1).

[0049] In some embodiments, Y is R 76 A linker intermediate or linker is provided, which is

[0050] In some embodiments, Y is [ka] A linker intermediate or linker is provided, which is

[0051] In some embodiments, each R a and R b are independently H.

[0052] In some embodiments, R a and R b are provided which, together with the carbon to which they are attached, form an oxo group.

[0053] In some embodiments, a linker intermediate or linker is provided in which q is 10-20.

[0054] In some embodiments, a linker intermediate or linker is provided in which q is 12.

[0055] In some embodiments, a linker intermediate or linker, or a salt thereof, is provided, wherein the PEG unit is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] wherein each Z is joined by a * and is individually selected from: [ka] , and [ka] each [ka] indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2 or stretcher unit (L1).

[0056] Carboxyl Unit In some embodiments, linker intermediates or linkers are provided in which the carboxyl unit has the following formula, or a salt thereof: R 70 | L 70 | ~NH-(CH2) p1 -CH-(CH2) o1 -C(O)~ (XXXX) (In the formula, (a) L70 is selected from C-C alkylene, C-C alkylene-C(O)-, -C(O)-C-C alkylene- and -C(O)-C-C alkylene-C(O)-; R 70 is ~NR 71 (R 72 -R 73 ) where R 71 is H, C1-C 12 Alkyl, substituted C1-C 12 alkyl or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 72 is absent or selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl; R 73 is a carboxyl or polycarboxyl, wherein the polycarboxyl contains 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, and the carboxyl groups are interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2, or stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or (b) L 70 is selected from C-C alkylene, C-C alkylene-C(O)-, -C(O)-C-C alkylene- and -C(O)-C-C alkylene-C(O)-; R 70 is ~NR 71 (R 75 -(R 73 )2), wherein R71 is H, C1-C 12 Alkyl, substituted C1-C 12 alkyl or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 75 is a branched optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl, or optionally substituted heteroaryl, and each R 73 is independently carboxyl or polycarboxyl, wherein the polycarboxyl contains 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, and the carboxyl groups are interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2, or stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or (c) L 70 is selected from C-C alkylene, C-C alkylene-C(O)-, -C(O)-C-C alkylene- and -C(O)-C-C alkylene-C(O)-; R 70 is approximately N(R 74 -R 73 )(R 72 -R 73 ) where R 72 and R 74are each independently selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl, or optionally substituted heteroaryl; and each R 73 is independently carboxyl or polycarboxyl and contains 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkyl, heteroalkylene, amino and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2, or stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2).

[0057] In some embodiments, a linker intermediate or linker is provided that includes at least one sugar unit. In some embodiments, a linker intermediate or linker is provided that includes at least one PEG unit. In some embodiments, a linker intermediate or linker is provided that includes at least one carboxyl unit. In some embodiments, a linker intermediate or linker is provided that includes at least two polar units, each polar unit selected from a sugar unit, a PEG unit, and a carboxyl unit. In some embodiments, a linker intermediate or linker is provided that includes at least one sugar unit and a PEG unit or a carboxyl unit. In some embodiments, a linker intermediate or linker is provided that includes at least one carboxyl unit and a PEG unit.

[0058] In some embodiments, linker intermediates or linkers are provided in which an amino acid unit (AA) is present (s=1). In some embodiments, linker intermediates or linkers are provided in which the amino acid unit comprises at least one polar unit.

[0059] In some embodiments, linker intermediates or linkers are provided in which L2 or AA-L2 has one of the following structures: [ka] or [ka] (where the wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol).

[0060] In some embodiments, linker intermediates or linkers are provided in which ~AA-L2~ has a formula selected from the following: [ka] , [ka] ,or [ka] (wherein the square brackets indicate amino acid units, each aa is an optional subunit of AA, L2 is a linker subunit, and each wavy line (~) indicates a binding site for a Stretcher unit; aa1(PEG) is a PEG unit attached to an amino acid subunit of AA, SU is a sugar unit attached to a subunit of AA or L2, and CU is a carboxyl unit attached to a subunit of AA or L2; and the double wavy line ( [ka] ) indicates a binding site for a Drug unit, and aa and aa1 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

[0061] In some embodiments, linker intermediates or linkers are provided in which ~AA-L2~ has a formula selected from the following: [ka] [ka] ,or [ka] (wherein the square brackets indicate amino acid units, each aa is an amino acid subunit of AA, L2 is a linker subunit attached to the side chain of aa, the wavy line (~) indicates the attachment site for the Stretcher unit; aa1(PEG) is a PEG unit attached to aa, SU is a sugar unit attached to aa, CU is a carboxyl unit attached to aa, and the double wavy line ( [ka] ) indicates the attachment site for the Drug unit; aa and aa1 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

[0062] In some embodiments, a linker intermediate or linker is provided in which the amino acid unit comprises at least two polar units.

[0063] In some embodiments, linker intermediates or linkers are provided in which ~AA-L2~ has a formula selected from the following: [ka] , [ka] ,or [ka] (wherein the square brackets indicate amino acid units, aa is an optional subunit of AA, L2 is a linker subunit, the wavy line (~) indicates a binding site for a Stretcher unit; each of aa1(PEG) and aa2(PEG) is a PEG unit bound to aa or another PEG unit; each SU is a sugar unit bound to aa or another sugar unit, each CU is a carboxyl unit bound to aa or another carboxyl unit, and the double wavy line ( [ka] ) indicates the attachment site for the Drug unit; aa, aa1 and aa2 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

[0064] In some embodiments, linker intermediates or linkers are provided in which ~AA-L2~ has a formula selected from the following: [ka] [ka] ,or [ka] (wherein the square brackets represent amino acid units, aa is an amino acid subunit of AA, L2 is a linker subunit attached to the side chain of aa, each wavy line (~) represents a binding site for a Stretcher unit; each of aa1(PEG) and aa2(PEG) is a PEG unit attached to aa, each SU is a sugar unit attached to aa; each CU is a carboxyl unit attached to aa; and the double wavy line ( [ka] ) indicates a binding site for a Drug unit; each of aa, aa1 and aa2 is independently selected from alpha, beta and gamma amino acids and derivatives thereof).

[0065] In some embodiments, a linker intermediate or linker is provided in which the linker subunit L2 is a cleavable linker unit. In some embodiments, a linker intermediate or linker is provided in which the linker subunit L2 comprises a peptide cleavable by an intracellular protease. In some embodiments, a linker intermediate or linker is provided in which the cleavable peptide comprises a valine-citrulline peptide, a valine-alanine peptide, a valine-lysine peptide, a phenylalanine-lysine peptide, or a glycine-glycine-phenylalanine-glycine peptide.

[0066] In some embodiments, a linker intermediate or linker is provided in which the linker subunit L2 comprises at least one polar unit. In some embodiments, a linker intermediate or linker is provided in which the polar unit is a sugar unit (SU). In some embodiments, a linker intermediate or linker is provided in which the cleavable peptide comprises a SU-valine-citrulline peptide, a SU-valine-lysine peptide, a SU-valine-alanine peptide, a SU-phenylalanine-lysine peptide, or a SU-glycine-glycine-phenylalanine-glycine peptide.

[0067] In some embodiments, a linker intermediate or linker is provided in which the polar unit is a carboxyl unit (CU). In some embodiments, the cleavable peptide comprises a CU-valine-citrulline peptide, a CU-valine-lysine peptide, a valine-(CU-lysine) peptide, a CU-valine-alanine peptide, a CU-phenylalanine-lysine peptide, a phenylalanine-(CU-lysine) peptide, or a CU-glycine-glycine-phenylalanine-glycine peptide, wherein the CU-lysine is a carboxyl unit comprising a lysine residue.

[0068] In some embodiments, a linker intermediate or linker is provided in which the polar unit is a PEG unit (PEG). In some embodiments, a linker intermediate or linker is provided in which the cleavable peptide comprises a Lys(PEG)-valine-citrulline peptide, a valine-Cit(PEG) peptide, a Lys(PEG)-valine-lysine peptide, a valine-lysine(PEG) peptide, a Lys(PEG)-valine-alanine peptide, a Lys(PEG)-phenylalanine-lysine peptide, a phenylalanine-Lys(PEG) peptide, or a Lys(PEG)-glycine-glycine-phenylalanine-glycine peptide, wherein Lys(PEG) and Cit(PEG) comprise PEG units attached to lysine or citrulline residues, respectively.

[0069] In some embodiments, a linker intermediate or linker is provided in which a cleavable peptide is attached to a para-aminobenzyl alcohol self-immolative group (PABA).

[0070] In some embodiments, linker intermediates or linkers are provided in which ~AA-L2~ has one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] wherein each Z is joined by a * and is individually selected from: [ka] , and [ka] The wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol (i.e., the H of the benzyl alcohol is replaced by a bond with the Drug unit).

[0071] In some embodiments, a linker intermediate or linker is provided in which L2 is attached to a side chain of an AA subunit. [ka] However, a linker intermediate or linker having one of the following structures is provided: [ka] or [ka] where the wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is either attached to the terminal acid group or the benzyl alcohol (i.e., an H of the acid or benzyl alcohol is replaced by a bond to the Drug unit), or is attached to the wavy line ( [ka] ) indicates the binding site for the Drug unit).

[0072] In some embodiments, linker intermediates or linkers are provided in which an amino acid unit is linked to a linker subunit L2 by a non-peptidic linking group. In some embodiments, the non-peptidic linking group is C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 A linker intermediate or linker selected from alkynylene or polyethylene glycol is provided.

[0073] In some embodiments, a linker intermediate or linker is provided that further comprises a Stretcher unit to or from the linker. In some embodiments, the Stretcher unit is selected from: [ka] [ka] ,or [ka] (In the formula, R 17 is -C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C1-C8 alkylene-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -(CH2-O-CH2) b-C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C1-C8 alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b-C1-C8 alkylene-NH- (wherein b is 1 to 26), -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S-, or -(C3-C8 heterocyclo)-C1-C 10 alkylene-S-), or The stretcher unit is maleimide (C1-C 10 Alkylene-C(O)-, maleimide (CH2OCH2) p2 (C1-C 10 Alkyne) C(O)-, Maleimide (C1-C 10 Alkyne) (CH2OCH2) p2 Linkers are provided that include C(O)- (wherein p2 is 1-26) or open forms thereof.

[0074] In some embodiments, a linker is provided in which the Stretcher unit is selected from: [ka] and [ka] (wherein, wavy line [ka] indicates the attachment site of the Stretcher unit to the Amino Acid unit, and the attachment site to the Targeting unit is on the maleimide, primary amine or alkyne functional group).

[0075] In some embodiments, a linker is provided having one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] wherein each Z is joined by a * and is individually selected from: [ka] , and [ka] The Drug Unit is optionally attached to the terminal acid group or benzyl alcohol, or is represented by a wavy line ( [ka] ) indicates the binding site for the Drug unit).

[0076] In some embodiments, linkers are provided that further comprise at least one drug unit attached to the linker subunit L2 to form the drug linker. In some embodiments, drug linkers are provided wherein the drug unit is selected from a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand. In some embodiments, drug linkers are provided wherein the drug unit is a cytotoxic agent. In some embodiments, drug linkers are provided wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, and a calicheamicin. In some embodiments, drug linkers are provided wherein the cytotoxic agent is an auristatin. In some embodiments, drug linkers are provided wherein the cytotoxic agent is MMAE or MMAF. In some embodiments, drug linkers are provided wherein the cytotoxic agent is a camptothecin. In some embodiments, drug linkers are provided wherein the cytotoxic agent is exatecan or SN-38. In some embodiments, drug linkers are provided wherein the cytotoxic agent is exatecan. In some embodiments, Drug-Linkers are provided wherein the cytotoxic agent is calicheamicin. In some embodiments, Drug-Linkers are provided wherein the cytotoxic agent is a maytansinoid. In some embodiments, Drug-Linkers are provided wherein the maytansinoid is maytansine, maytansinol, or ansamatocin-2.

[0077] In some embodiments, Drug-Linkers are provided wherein the cytotoxic agent is calicheamicin. In some embodiments, Drug-Linkers are provided wherein the cytotoxic agent is a maytansinoid. In some embodiments, Drug-Linkers are provided wherein the maytansinoid is maytansine, maytansinol, or ansamatocin-2.

[0078] In some embodiments, a Drug-Linker is provided in which the Drug unit is an immunomodulatory agent. In some embodiments, a Drug-Linker is provided in which the immunomodulatory agent is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, a Drug-Linker is provided in which the immunomodulatory agent is a TLR7 agonist. In some embodiments, a Drug-Linker is provided in which the TLR7 agonist is an imidazoquinoline, imidazoquinoline amine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroaromatic thiol azide-2,2-dioxide, benzonaphthyridine, guanosine analog, adenosine analog, thymidine homopolymer, ssRNA, CpG-A, PolyG10, or PolyG3. In some embodiments, a Drug-Linker is provided in which the immunomodulatory agent is a TLR8 agonist. In some embodiments, a drug-linker is provided in which the TLR8 agonist is selected from imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, or ssRNA. In some embodiments, a drug-linker is provided in which the immunomodulatory agent is a STING agonist. In some embodiments, a drug-linker is provided in which the immunomodulatory agent is a RIG-I agonist. In some embodiments, a drug-linker is provided in which the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0079] In some embodiments, a Drug-Linker is provided in which the Drug unit is a chelating ligand. In some embodiments, the chelating ligand is selected from platinum (Pt), ruthenium (Ru), rhodium (Rh), gold (Au), silver (Ag), copper (Cu), molybdenum (Mo), titanium (Ti), or iridium (Ir); or a radioactive isotope such as yttrium-88, yttrium-90, technetium-99, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114, indium-115, lutetium-177, strontium-89, salarium-153, and lead-212.

[0080] In some embodiments, a Drug-Linker having the following structure is provided: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0081] In some embodiments, a conjugate is provided that includes a targeting unit attached to any of the drug-linkers described herein. In some embodiments, a conjugate is provided wherein the targeting unit is selected from an antibody or an antigen-binding portion thereof. In some embodiments, a conjugate is provided wherein the targeting unit is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody. In some embodiments, a conjugate is provided wherein the targeting unit is a diabody, DART, anticalin, affibody, avimer, DARPin, or adnectin. In some embodiments, a conjugate is provided wherein the targeting unit is monospecific. In some embodiments, a conjugate is provided wherein the targeting unit is bivalent. In some embodiments, a conjugate is provided wherein the targeting unit is bispecific. In some embodiments, a conjugate is provided wherein the average drug loading (p load ) is about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

[0082] In some embodiments, a conjugate selected from the following is provided: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0083] In some embodiments, the above conjugates are provided, wherein the targeting unit binds to a target molecule such as CD19, CD20, CD30, CD33, CD70, LIV-1, or EGFRv3.

[0084] In some embodiments, the above conjugate is provided, wherein the targeting unit is selected from scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv.

[0085] In some embodiments, the targeting unit is selected from the group consisting of cancer-associated antigens, such as CD19, CD20, CD30, CD33, CD38, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin (MLSN), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 1p19q, ABL1, AKT1, ALK, APC, AR, ATM, BRAF, BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, JAK2, KDR(VEGFR2), KRAS, MGMT, MGMT-Me, MLH1, MPL, NOTCH1, NRAS, PDGFRA, Pgp, PIK3CA, PR, PTEN, RET, RRM 1, SMO, SPARC, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1 , STK1, MLH1, MSH2, MSH6, PMS2, ROS1, ERCC1, 5T4(TPBG), B7-H3, CCR7, CD105, CD22, CD46, CD47, CD56, CD70, CD71, CD79b, CDH6 , CLDN6, CLDN18.2, CLEC12A, DLL3, DR5, ERBB3 (HER3), EPCAM, FOLR1, IGF1R, IL2RA (CD25), IL3RA, ITGB6, LIV-1, LRRC15, mesothelin (MSLN), NaPi2b (SLC34A2), nectin-4, PTK7, ROR1, SEZ6, SLC44A4, SLITRK6, tissue factor (TF), TROP2, or B7-H4.

[0086] In some embodiments, the targeting unit is selected from the group consisting of rituximab (Rituxan®), trastuzumab (Herceptin®), pertuzumab (Perjeta®), bevacizumab (Avastin®), ranibizumab (Lucentis®), cetuximab (Erbitux®), alemtuzumab (Campath®), panitumumab (Vectibix®), and ribozyme (Ribozyme®).

[0033] Provided are conjugates as described above that are selected from the group consisting of ibritumomab, ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), ipilimumab, zalutumumab, dalotuzumab, figitumumab, ramucirumab, galiumtuzumab, farletuzumab, ocrelizumab, ofatumumab (Arzerra®), tositumumab, ibritumomab, CD20 antibody 2F2 (HuMax-CD20), 7D8, IgM2C6, IgG1 2C6, 11B8, B1, 2H7, LT20, 1FS or AT80, daclizumab (Zenapax®), or an anti-LHRH receptor antibody, e.g., clones A9E4, F1G4, AT2G7, GNRH03 or GNRHR2.

[0087] In some embodiments, the above conjugate is provided, wherein the targeting unit is antibody F131 and the drug-linker is LD038. In certain embodiments, the targeting unit is antibody F131 (VH SEQ ID NO: 26 and VL SEQ ID NO: 27).

[0088] In some embodiments, the above conjugate is provided, wherein the targeting unit is an antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 arranged in a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR and LCDR3 arranged in a light chain variable region framework region, and wherein the VH and VL The CDRs have amino acid sequences selected from the set of amino acid sequences set forth in the group consisting of: (a) SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; and (b) SEQ ID NO:36, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In certain embodiments, the VH and VL regions have amino acid sequences selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:26 and SEQ ID NO:27, respectively; and the heavy and light chain framework regions are optionally modified with substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions. In certain embodiments, the antibody is F131 and the drug-linker is LD038.

[0089] In some embodiments, a pharmaceutical composition is provided comprising any of the conjugates described herein and a pharmaceutically acceptable carrier.

[0090] In some embodiments, provided is a method of treating a subject in need thereof comprising administering to the subject any of the conjugates described herein or any of the pharmaceutical compositions described herein, wherein the subject has cancer or an autoimmune disease, and the conjugate binds to a target antigen associated with the cancer or autoimmune disease.

[0091] These and other aspects of the present invention can be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief explanation of the drawings]

[0092] [Figure 1A] FIG. 1 shows the in vitro cytotoxicity of anti-huFOLR-1 conjugates against OV90 cells. [Figure 1B] FIG. 1 shows the in vitro cytotoxicity of anti-huFOLR-1 conjugates against OVCAR-3 cells. [Figure 1C]FIG. 1 shows the in vitro cytotoxicity of anti-huFOLR-1 conjugates against NCI-H292 cells. [Figure 2] The in vivo activity of the PA038 conjugate of human anti-huFOLR1 antibody was tested. Mice bearing established OV90 xenografts of approximately 117 mm were treated with 5 mg / kg of conjugate or PBS intravenously four times over two weeks, starting on day 8 after tumor cell inoculation. The mean tumor volume (mm) versus time (days) after cell inoculation is plotted. (N=6, mean ± SEM) [Figure 3] The in vivo activity of the PA038 conjugate of human anti-huFOLR1 antibody was tested. Mice bearing established NCI-H292 xenografts of approximately 123 mm were treated with 5 mg / kg of conjugate or PBS intravenously four times over two weeks, starting 11 days after cell inoculation. The mean tumor volume (mm) versus time (days) after cell inoculation is plotted. (N=6, mean ± SEM) [Figure 4] Figure 1 shows the in vivo activity of the PA038 conjugate of human anti-huFOLR1 antibody. Mice bearing established OV90 xenografts of approximately 110 mm were treated with 5 mg / kg of conjugate or PBS intravenously four times over two weeks, starting 13 days after cell inoculation. The mean tumor volume (mm) versus time (days) after cell inoculation is plotted. (N=6, mean ± SEM) [Figure 5] FIG. 1 shows the PK profiles of anti-huFOLR-1 conjugate F131-LD038 and naked Ab F131 evaluated at 3 mg / kg (N=3; mean±SD). [Figure 6] FIG. 1 shows a comparison of anti-FOLR1 antibody binding to Hela cells. [Figure 7] FIG. 1 shows a comparison of the binding ability of anti-FOLR1 antibodies to RPTEC / TERT1 cells. [Figure 8] FIG. 1 shows dose-dependent binding of anti-FOLR1 antibodies to Hela cells. [Figure 9] FIG. 1 shows dose-dependent binding of anti-FOLR1 antibodies to RPTEC / TERT1 cells. [Figure 10] FIG. 1 shows the internalization of anti-FOLR1 antibodies into Hela cells. [Figure 11] FIG. 1 shows the internalization of anti-FOLR1 antibodies into RPTEC / TERT1 cells. [Figure 12A] FIG. 1 shows F131 internalization in tumor cell lines. [Figure 12B] FIG. 1 shows F131-LD038 internalization in tumor cell lines. [Figure 13A] FIG. 1 shows in vitro cell cytotoxicity against KB. [Figure 13B] FIG. 1 shows in vitro cell cytotoxicity against OVCAR3. [Figure 13C] FIG. 1 shows in vitro cell cytotoxicity against JEG-3. [Figure 14A] FIG. 1 shows the in vivo efficacy of F131 and F131-LD038 in CDX against OVCAR-3. [Figure 14B] FIG. 1 shows the in vivo efficacy of F131 and F131-LD038 in CDX against KB. [Figure 14C] FIG. 1 shows the in vivo efficacy of F131 and F131-LD038 in CDX against HCC827. [Figure 14D] FIG. 1 shows the in vivo efficacy of F131 and F131-LD038 in CDX on H441. [Figure 14E] FIG. 1 shows the in vivo efficacy of F131 and F131-LD038 in CDX against OV90. [Figure 15A] FIG. 1 shows the in vivo efficacy of F131-038 and other conjugates in CDX against KB. [Figure 15B] FIG. 1 shows the in vivo efficacy of F131 conjugates in CDX against KB. [Figure 16A] FIG. 1 shows PK studies of F131 and conjugates in a rat model. [Figure 16B]FIG. 1 shows PK studies of F131 and conjugates in a rat model. [Figure 16C] FIG. 1 shows PK studies of F131 and conjugates in a rat model. [Figure 17A] FIG. 1 shows the tolerability of F131-deluktecan and F131-LD038 in a pilot cynomolgus monkey toxicity study. [Figure 17B] FIG. 1 shows the tolerability of F131-deluktecan and F131-LD038 in a pilot cynomolgus monkey toxicity study. [Figure 18] FIG. 1 shows F131-deluktecan and F131-LD038 PK in a pilot cynomolgus monkey toxicity study. DETAILED DESCRIPTION OF THE INVENTION

[0093] definition For convenience, certain terms in the specification, examples, and claims are defined here. Unless otherwise specified or implied from context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. 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.

[0094] As used herein, unless otherwise indicated, the terms "a" and "an" shall be construed to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include plurals and plural terms shall include the singular.

[0095] Unless the context requires otherwise, throughout the specification and claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense rather than an exclusive or exhaustive sense, i.e., meaning "including but not limited to".

[0096] The terms "reduce," "reduce," "reduced," "reduce," "reduce," and "inhibit" are all used generally herein to mean a decrease by a statistically significant amount relative to a reference.

[0097] The terms "increased," "increase," or "enhance," or "activate" are all used herein to generally mean an increase of a statically significant amount relative to a baseline.

[0098] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein to refer to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0099] As used herein, "epitope" refers to amino acids conventionally bound by an immunoglobulin VH / VL pair, e.g., antibodies, their antigen-binding portions, and other binding agents described herein. Other binding agents include non-antibody scaffolds. Epitopes can be formed on polypeptides from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more usually at least five, about nine, or about eight to ten amino acids in a unique spatial conformation. An epitope defines the minimal binding site of an antibody, its antigen-binding portion, and other binding agent and thus represents the target of specificity of the antibody, its antigen-binding portion, or other immunoglobulin-based binding agent. In the case of a single-domain antibody, an epitope represents the structural unit bound by the variable domain alone.

[0100] As used herein, "specifically binds" means that a binding agent (e.g., an antibody or antigen-binding portion thereof) described herein specifically binds to a target molecule. -5 M (10000nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 As used herein, "specifically binds" also refers to the ability of a molecule described herein (e.g., an antibody or antigen-binding portion thereof, or a non-antibody scaffold) to bind to a target with a KD of 10 or less. -5 M (10000nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12Specific binding refers to the ability to bind to a target with a KD of M or less. Specific binding can be influenced, for example, by the affinity and avidity of the antibody, antigen-binding portion, or other binding agent, as well as the concentration of the target polypeptide. One skilled in the art can determine appropriate conditions under which the antibodies, antigen-binding portions, and other binding agents described herein selectively bind to a target molecule using any suitable method, such as titrating the antibody or binding agent in a suitable cell-binding assay. A binding agent that specifically binds to a target molecule is not displaced by a dissimilar competitor. In certain embodiments, an antibody or antigen-binding portion thereof, or other binding agent, is said to specifically bind to a target molecule if it preferentially recognizes its target molecule in a complex mixture of proteins and / or macromolecules. Specific binding can be influenced, for example, by the affinity and avidity of the antibody, antigen-binding portion, or non-antibody scaffold, as well as the concentration of the target polypeptide. One skilled in the art can determine appropriate conditions under which the antibodies, antigen-binding portions, and non-antibody scaffolds described herein selectively bind to a target molecule using any suitable method, such as titrating the antibody or non-antibody scaffold in a suitable cell-binding assay. A molecule that specifically binds to a target molecule is not displaced by a dissimilar competitor. In certain embodiments, an antibody or antigen-binding portion thereof, or a non-antibody scaffold is said to specifically bind to a target molecule if it preferentially recognizes its target molecule in a complex mixture of proteins and / or macromolecules.

[0101] Unless otherwise indicated, the term "alkyl," by itself or as part of another term, refers to an alkyl group having the indicated number of carbon atoms (e.g., "-C1-C5 alkyl," "-C1-C8 alkyl," or "-C1-C 10"Alkyl" refers to a substituted or unsubstituted, straight or branched, saturated hydrocarbon having alkyl groups having 1 to 5, 1 to 8, or 1 to 10 carbon atoms, respectively. Examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH 3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (--CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1- Butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(C H3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3).

[0102] Unless otherwise indicated, "alkenyl," by itself or as part of another term, refers to an alkyl group having at least one site of unsaturation (i.e., carbon-carbon, sp 2"C" refers to a C2-C8 substituted or unsubstituted straight or branched chain hydrocarbon having a double bond. Examples include, but are not limited to, ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0103] Unless otherwise stated, "alkynyl," by itself or as part of another term, refers to a substituted or unsubstituted, straight or branched chain C2-C8 hydrocarbon having at least one site of unsaturation (i.e., a carbon-carbon, sp triple bond). Examples include, but are not limited to, acetylene and propargyl.

[0104] Unless otherwise indicated, "alkylene" refers to a saturated, branched, or straight-chain hydrocarbon radical of 1 to 8 carbon atoms having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0105] Unless otherwise indicated, "alkenylene" refers to an unsaturated, branched, or straight-chain hydrocarbon radical of 2 to 8 carbon atoms, which has two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0106] Unless otherwise indicated, "alkynylene" refers to an unsaturated, branched or straight-chain or cyclic hydrocarbon radical of 2 to 8 carbon atoms having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to, acetylene, propargyl, and 4-pentynyl.

[0107] Unless otherwise indicated, the term "heteroalkyl," by itself or in combination with another term, refers to a substituted or unsubstituted, stable, straight- or branched-chain hydrocarbon, or combinations thereof, saturated and containing from 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The O, N, and S heteroatoms may be placed at any interior position of the heteroalkyl group (i.e., as part of the backbone) or at the position at which the alkyl group is attached to the remainder of the molecule. The Si heteroatom may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples of heteroalkyl groups include the following: -CHCHOCH, -CHCHNHCH, -CHCHN(CH)CH, -CHSCHCH, CHCHS(O)CH, -CHCHS(O)CH, and -Si(CH)-. For example, up to two heteroatoms may be consecutive, such as -CH2NHOCH3 and CH2OSi(CH3)3. In some embodiments, a C1-C4 heteroalkyl has 1-4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl has 1-3 carbon atoms and 1 or 2 heteroatoms.

[0108] Unless otherwise indicated, the terms "heteroalkenyl" and "heteroalkynyl," alone or in combination with another term, refer to a substituted or unsubstituted, stable, straight- or branched-chain alkenyl or alkynyl having 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The O, N, and S heteroatoms may be placed at any interior position of the heteroalkenyl or heteroalkynyl group (i.e., as part of the backbone) or at the position at which the alkyl group is attached to the remainder of the molecule. The Si heteroatom may be placed at any position of the heteroalkenyl or heteroalkynyl group, including the position at which the alkyl group is attached to the remainder of the molecule.

[0109] Unless otherwise stated, the term "heteroalkylene," by itself or as part of another substituent, refers to a substituted or unsubstituted divalent group derived from heteroalkyl, as exemplified by -CH2CH2SCH2CH2- and -CH2SCH2CH2NHCH2- (as discussed above). In some embodiments, a C1-C4 heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0110] Unless otherwise stated, the terms "heteroalkenylene" and "heteroalkynylene," by themselves or as part of another substituent, refer to a substituted or unsubstituted divalent group derived from heteroalkenyl or heteroalkynyl (as discussed above). In some embodiments, a C2-C4 heteroalkenylene or heteroalkynylene has 1 to 4 carbon atoms. For heteroalkenylene and heteroalkynylene groups, heteroatoms can also occupy either or both of the chain termini. Additionally, for alkylene and heteroalkenylene and heteroalkynylene linking groups, no orientation of the linking group is implied.

[0111] Unless otherwise indicated, "C3-C8 carbocycle," by itself or as part of another term, refers to a substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocycle derived by removing one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C8 carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0112] Unless otherwise indicated, "C3-C8 carbocyclo" by itself or as part of another term refers to a substituted or unsubstituted C3-C8 carbocyclic group as defined above in which one or more of the carbocyclic group's hydrogen atoms have been replaced with a bond (i.e., it is divalent).

[0113] Unless otherwise indicated, "C3-C 10"Carbocycle," by itself or as part of another term, refers to a substituted or unsubstituted 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered monovalent substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic, bicyclic, or tricyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C 10 Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. 10 Carbocycles can further include fused cyclooctyne carbocycles, such as the fused cyclooctyne compounds disclosed in WO 2011 / 136645 (the disclosure of which is incorporated herein by reference), including BCN (bicyclo[6.1.0]nonyne) and DBCO (dibenzocyclooctyne).

[0114] Unless otherwise indicated, "C3-C8 heterocycle," by itself or as part of another term, refers to a substituted or unsubstituted, monovalent, substituted or unsubstituted, aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, derived by removing one hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in the heterocycle can be oxidized. A ring containing a heteroatom can be aromatic or non-aromatic. Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl. Unless otherwise specified, the term "heterocarbocycle" is synonymous with the term "heterocycle" or "heterocyclo" as used herein.

[0115] Unless otherwise indicated, "C3-C8 heterocyclo" by itself or as part of another term refers to a substituted or unsubstituted C3-C8 heterocyclic group as defined above in which one of the heterocyclic group's hydrogen atoms has been replaced with a bond (i.e., it is divalent).

[0116] Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6 to 20 carbon atoms (preferably 6 to 14 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar." Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is a phenyl group.

[0117] Unless otherwise indicated, an "arylene," by itself or as part of another term, is an unsubstituted or substituted aryl group, as defined above, in which one of the aryl group's hydrogen atoms has been replaced with a bond (i.e., it is divalent), and which may be in the ortho, meta, or para orientation.

[0118] Unless otherwise indicated, "heteroaryl" refers to a ring system in which one or more ring atoms is a heteroatom, such as nitrogen, oxygen, and sulfur. The heterocyclic radical contains 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heterocyclic ring can be a monocyclic ring (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) having 3 to 7 ring members or a bicyclic ring (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) having 7 to 10 ring members, such as a bicyclo[4,5], [5,5], [5,6], or [6,6] system.

[0119] Unless otherwise indicated, a "heteroarylene," by itself or as part of another term, is an unsubstituted or substituted heteroaryl group, as defined above, in which one of the heteroaryl group's hydrogen atoms has been replaced with a bond (i.e., it is divalent).

[0120] Unless otherwise indicated, "carboxyl" refers to COOH or COO - M + refers to M+ is a cation.

[0121] Unless otherwise indicated, "oxo" refers to (C=O).

[0122] Unless otherwise indicated, "substituted alkyl" and "substituted aryl" refer to alkyl and aryl, respectively, in which one or more hydrogen atoms have each been independently replaced with a substituent. Exemplary substituents include -X, -R 10 , -O - , -OR 10 , -SR 10 , -S - , -NR 10 2, -NR 10 3, =NR 10 , -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR 10 C(=O)R 10 , -C(=O)R 10 , -C(=O)NR 10 2, -SO3 - , -SO3H, -S(=O)2R 10 , -OS(=O)2OR 10 , -S(=O)2NR 10 , -S(=O)R 10 ,-OP(=O)(OR 10 )2, -P(=O)(OR 10 )2, -PO - 3, -PO3H2, -AsO2H2, -C(=O)R 10 , -C(=O)X, -C(=S)R 10 , -CO2R 10 , -CO2 - , -C(=S)OR 10 , C(=O)SR 10 , C(=S)SR 10 , C(=O)NR 10 2. C(=S)NR 10 2 or C(=NR 10 )NR 10 2, where each X is independently a halogen: -F, -Cl, -Br, or -I; 10 are independently -H, -C1-C20 Alkyl, -C6-C 20 Aryl, -C3-C 14 Heterocycle, protecting group or prodrug moiety. Typical substituents also include (=O). The above alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocycle and heterocyclo groups may also be similarly substituted.

[0123] Unless otherwise specified, a "polyhydroxyl group" refers to an alkyl, alkylene, carbocyclic, or carbocyclo group containing two or more or three or more hydroxyl groups substituted for hydrogen on carbon atoms of a carbon chain. In some embodiments, a polyhydroxyl group contains at least three hydroxyl groups. In some embodiments, a polyhydroxyl group contains carbon atoms containing only one hydroxyl group per carbon atom. A polyhydroxyl group may contain one or more carbon atoms that are not substituted with hydroxyl. A polyhydroxyl group may have each carbon atom substituted with a hydroxyl group. Examples of polyhydroxyl groups include linear (acyclic) or cyclic forms of monosaccharides such as C6 or C5 sugars such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, glucose, idose, talose, aldose, and ketose, sugar acids such as gluconic acid, aldonic acid, uronic acid, and amino sugars such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine. In some embodiments, polyhydroxyl groups include linear or cyclic forms of disaccharides and polysaccharides.

[0124] Unless otherwise indicated by context, "optionally substituted" refers to an alkyl, alkenyl, alkynyl, alkylaryl, arylalkylheterocycle, aryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, or other substituent, moiety, or group as defined or disclosed herein, in which a hydrogen atom of the substituent, moiety, or group is optionally replaced with a different moiety or group, or an alicyclic carbon chain comprising one of the substituents, moieties, or groups is interrupted by replacing a carbon atom of the chain with a different moiety or group. In some embodiments, an alkene functional group replaces two consecutive sp3 carbon atoms of an alkyl substituent, so long as the radical carbon of the alkyl moiety is not replaced, and thus the optionally substituted alkyl is an unsaturated alkyl substituent.

[0125] The optional substituents replacing a hydrogen on any one of the foregoing substituents, moieties, or groups are independently selected from the group consisting of aryl, heteroaryl, hydroxyl, alkoxy, aryloxy, cyano, halogen, nitro, fluoroalkoxy, and amino (including mono-, di-, and tri-substituted amino groups), and protected derivatives thereof, or -X, -OR', -SR', -NH2, -N(R')(R''), -N(R'')3, =NR, -CX3, -CN, -NO2, -NR'C(=O)H, -NR'C(=O)R, -NR'C(=O)R'', -C(=O)R', -C(=O)NH2, -C(=O)N(R')R'', -S(=O)2R'', -S(=O)2N H2, -S(=O)2N(R')R'', -S(=O)2NH2, -S(=O)2N(R')R'', -S(=O)2OR', -S(=O)R'', -OP(=O) (OR')(OR''), -OP(OH)3, -P(=O)(OR')(OR''), -PO3H2, -C(=O)R', -C(=S)R'', -CO2R', -C( and salts thereof, wherein each X is independently selected from the group consisting of halogens: -F, -CI, -Br, and -I; and each R" is selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl (C5-C 24 or two of R" together with the heteroatom to which they are attached define a heterocyclyl; R' is hydrogen or R" and R" is C-C 20 Alkyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl (C5-C 24 and protecting groups.

[0126] Typically, optional substituents include -X, -OH, -OR'', -SH, -SR'', -NH2, -NH(R''), -NR'(R'')2, -N(R'')3, =NH, =NR'', -CX3, -CN, -N O2, -NR'C(=O)H, NR'C(=O)R'', -CO2H, -C(=O)H, -C(=O)R'', -C(=O)NH2, -C(=O)NR'R''--S(=O)2R'', -S(=O)2NH2, and salts thereof, each X is independently selected from the group consisting of -S(=O)2N(R')R'', -S(=O)2NH2, -S(=O)2N(R')(R''), -S(=O)2OR', -S(=O)R'', -C(=S)R'', -C(=S)NH2, -C(=S)N(R')R'', -C(=NR')N(R'')2 and salts thereof, wherein each X is independently selected from the group consisting of -F and -Cl, and R'' is typically C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclyl (C5-C 10 R' is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C6 ... 10 Aryl, C3-C 10 Heterocyclyl (C5-C 10 is a protecting group independently selected from R″ (including heteroaryl) and R″. More typically, the substituents are selected from the group consisting of -X, -R", -OH, -OR", -NH, -NH(R"), -N(R"), -N(R"), -CX, -NO, -NHC(=O)H, -NHC(=O)R", -C(=O)NH, -C(=O)NHR", -C(=O)N(R"), -COH, -COR", -C(=O)H, -C(=O)R", -C(=O)NH, -C(=O)NH(R'"), -C(=O)N(R"), -C(=')NH, -C(=NR')NH(R"), -C(=NR')N(R"), protecting groups and salts thereof, wherein each X is -F and R" is C-C alkyl, C-C 10 Aryl, C5-C 10 is independently selected from the group consisting of heteroaryl and a protecting group; R' is selected from the group consisting of hydrogen, C1-C6 alkyl, and a protecting group independently selected from R''.

[0127] The phrase "pharmaceutically acceptable salt," as used herein, refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a linker, a drug linker, or a conjugate). The compound typically contains at least one amino group and, therefore, can form an acid addition salt with this amino group. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, linoleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate, succinate, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0128] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment.

[0129] As used herein, the term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0130] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean + / - 1%.

[0131] The term "statistically significant" or "significant" refers to statistical significance, generally meaning a difference of 2 standard deviations (2SD) above or below a reference value.

[0132] Other terms are defined herein within the context of the description of various aspects of the invention.

[0133] Provided herein are linkers comprising polar units, such as saccharide units, PEG units, and / or carboxyl units. Also provided are targeting unit-linkers, drug linkers, and conjugates thereof, comprising drug units, such as cytotoxic agents or immunomodulatory agents, as further described herein.

[0134] In some embodiments, the linker has the general formula (I) comprising a stretcher unit (L1) attached directly or via an optional amino acid unit (AA) to a linker subunit (L2), as shown in formula (I) below: [ka] (where s is 0 or 1, and the wavy line ( [ka] ) indicates a binding site for a targeting unit (L) or a drug unit (D) or a salt thereof. The linker includes at least one polar unit within the amino acid unit, the linker subunit L2, or both. Each polar unit can be a sugar unit, a PEG unit, or a carboxyl unit. The linker can include at least one sugar unit, at least one PEG unit, at least one carboxyl unit, or a combination thereof. The linker subunit L2 can have 1 to 4 binding sites for a drug unit. In some embodiments, the linker subunit L2 has one binding site for a drug unit. In some embodiments, the linker subunit L2 has two binding sites for a drug unit.

[0135] Also provided are linker conjugates comprising a targeting unit (L) attached to at least one linker, each linker being attached to at least one drug unit (D), as shown in formula (II) below: L-[[L1-AA s -L2]-D t ] pload (II) wherein L1, AA, and L2 comprise a linker and are as described above for formula (I), s is 0 or 1, t is 1 to 4, and p load is 1 to 20). The linker includes at least one polar unit in the amino acid unit, the linker subunit L2, or both. Each polar unit can be a saccharide unit, a PEG unit, or a carboxyl unit. The linker can include at least one saccharide unit, at least one PEG unit, at least one carboxyl unit, or a combination thereof. The linker subunit L2 can have 1 to 4 attachment sites for the Drug unit. In some embodiments, the linker subunit L2 has one attachment site for the Drug unit. In some embodiments, the linker subunit L2 has two attachment sites for the Drug unit.

[0136] The following formula (III) ~[L1-AAs -L2]-D t (III) Also provided are drug-linkers represented by the formula: (wherein L1, AA, L2, and D comprise a linker and are as described above with respect to Formula (II), s is 0 or 1, t is 1 to 4, and the wavy line represents a binding site for a targeting unit), or a salt thereof. The linker comprises at least one polar unit within the amino acid unit, the linker subunit L2, or both. Each polar unit can be a sugar unit, a PEG unit, or a carboxyl unit. The linker can comprise at least one sugar unit, at least one PEG unit, at least one carboxyl unit, or a combination thereof. The linker subunit L2 can have 1 to 4 binding sites for a drug unit. In some embodiments, the linker subunit L2 has one binding site for a drug unit. In some embodiments, the linker subunit L2 has two binding sites for a drug unit.

[0137] Formula (IV): [ka] (wherein L1, AA, and L2 comprise a linker, L, L1, AA, and L2 are as described above for formula (I), s is 0 or 1, d is 1 to 20, and the double wavy line ( [ka] Further provided are targeting unit-linker intermediates represented by the formula (I) or a salt thereof, wherein L2 denotes a binding site for a Drug unit. The linker comprises at least one polar unit within the amino acid unit, the linker subunit L2, or both. Each polar unit can be a sugar unit, a PEG unit, or a carboxyl unit. The linker can comprise at least one sugar unit, at least one PEG unit, at least one carboxyl unit, or a combination thereof. The linker subunit L2 can have 1 to 4 binding sites for a Drug unit. In some embodiments, the linker subunit L2 has one binding site for a Drug unit. In some embodiments, the linker subunit L2 has two binding sites for a Drug unit.

[0138] Polar Units The polar units (PU) provided herein include saccharide units, PEG units, and carboxyl units, as further described herein.

[0139] Sugar Unit (SU) In some embodiments, the sugar unit (SU) has the general formula (X): L3-**N(CH2-(CH(XR)) k -X1(X2))2 (X) wherein each X is independently selected from NH or O; each R is independently selected from hydrogen, acetyl, monosaccharides, disaccharides, and polysaccharides; each X is independently selected from CH and C(O); each X is independently selected from H, OH, and OR; and k is 1 to 10, or a salt thereof. In some embodiments, each (CH—(CH(XR)) k-X1(X2)) is a monosaccharide. In some embodiments, the monosaccharide is a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, glucose, idustalose, aldose, ketose, a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid, or an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine. Suitable disaccharides include sucrose, lactose, and maltose. Suitable polysaccharides include maltotriose, raffinose, kestose, starch, cellulose, and glycogen. The stereochemistry at the anomeric C-1 position can be either alpha or beta.

[0140] L3 has the following general formula (XI): L3a | *-NH-(CH2) p -CH-(CH2) o -C(O)-# (XI) (Wherein, L3a is C1-C 10 and L3a is covalently bonded to the N atom marked with a** in formula (X). Each * and each # indicates a binding site for another subunit of an amino acid unit (AA) or linker subunit (L2), a Stretcher unit (L1), or other component of a linker, as described herein.

[0141] In some embodiments, the sugar unit has the following formula (XII): [ka] (wherein R, p, and o are as defined above, n is 0 to 4, and each m is independently 1 to 4).

[0142] In some embodiments, the sugar unit has the following formula (XIII): [ka] (wherein n is 0 to 4, and each m is independently 1 to 4).

[0143] PEG units In some embodiments, the linker comprises a PEG unit. The PEG unit may be attached to a subunit of an amino acid unit or a portion of the linker subunit L2. The subunit of an amino acid unit may be, for example, an alpha, beta, or gamma amino acid or a derivative thereof. In some embodiments, the PEG unit may be attached to a stretcher unit.

[0144] In some embodiments, the PEG unit has the general formula: -(CHCHO) n20 -R 24 wherein R 24 is H or C1-C6 alkyl and n20 is 1 to 26. In some, n20 is 12 and R 24 is methyl.

[0145] In some embodiments, the PEG unit has the following general formula (XX): ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 21 and R 22 are each independently an optional C1-C3 alkylene; R 24 and R 25is as shown below; the wavy line (~) indicates the binding site; n20 is 1 to 26) or a salt thereof. In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0146] In some embodiments, the PEG unit has the following general formula (XX): ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 21 and R 22 are each independently an optional C1-C3 alkylene; R 24 and R 25 is as shown below; the wavy line (~) indicates the binding site; n20 is 1 to 26) or a salt thereof. In some embodiments, R 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0147] In some embodiments, the PEG unit has the following general formula (XXI): ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXI) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 26 and R 27 are optional C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- or -C(O)-C-C 12 alkylene-NH-; R 24 and R 25 are as shown below; each R 29 is optional and independently selected from -C(O)-, -NH-, -C(O)-C-C alkenylene-, -NH-C-C alkenylene-, -C-C alkenylene-NH-, and -C-C alkenylene-C(O)-; a wavy line (~) indicates the site of attachment; n20 is 1 to 26; n21 is 1 to 4; and n27 is 1 to 3) or a salt thereof. In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0148] In some embodiments, the PEG unit has the following general formula (XXI): ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXI) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 26 and R 27 are optional C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- or -C(O)-C-C 12 alkylene-NH-; R 24 and R 25 are as shown below; each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—Ci-C6 alkenylene-, —NH—Ci-C6 alkenylene-, —Ci-C6 alkenylene-NH—, —Ci-C6 alkenylene-C(O)—, —NH(CO)NH—, and triazole; a wavy line (~) indicates the site of attachment; n20 is 1 to 26; n21 is 1 to 4; and n27 is 1 to 3) or a salt thereof. In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0149] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are, respectively, H and a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 are sugar units of formula (XII) or (XIII), or —NR 24 R 25 are formed from C3-C8 heterocycles.

[0150] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 one of which is H and a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C-C 10 Carbocyclic ring; optionally substituted C1-C3 alkylene C3-C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted -C1-C8 alkyl; Substituted -C(O)-C1-C8 alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII), and R 24 and R 25 The other is optionally a polyethylene glycol having 1 to 24 ethylene glycol subunits.

[0151] In some embodiments of the PEG unit of formula (XX) or (XXI), R 24 and R 25and R are not H. In some embodiments of PEG units of formula (XX) or (XXI), R 24 and R 25 One of them is H.

[0152] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 provided that both are not H. The polyhydroxyl group can be linear or branched. In some embodiments, the polyhydroxyl group contains at least three hydroxyl groups. In some embodiments, the polyhydroxyl group is a linear monosaccharide. As used herein, a linear monosaccharide refers to the open-ring form of a monosaccharide. In some embodiments, a linear monosaccharide is a linear form of a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, gulose, idostulose, aldose, and ketose. In some embodiments, the linear monosaccharide can further comprise a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid. In some embodiments, the linear monosaccharide can further comprise an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

[0153] Examples of PEG units with linear monosaccharides include: [ka] [ka] (In the formula, R 39is selected from H, a linear monosaccharide, and polyethylene glycol). In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is optionally deprotected to form a bond between the left-most carboxyl or hydroxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or a portion of a linker subunit L2.

[0154] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 and (XXI) are not H. In some embodiments of PEG units of formula (XX) and (XXI), R 24 and R 25 One of R is selected from a polyhydroxyl group and the other is polyethylene glycol. In some embodiments, each polyhydroxyl group contains at least three hydroxyl groups. The polyhydroxyl group can be linear, branched, or cyclic. In some embodiments, R 24 and R 25 One of R is a linear monosaccharide and the other is a cyclic monosaccharide. 24 and R 25One of the monosaccharides is a cyclic monosaccharide, and the other is a linear or cyclic monosaccharide. In some embodiments, the linear monosaccharide is a linear (acyclic) form of a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, allulose, glucose, idostulose, aldose, and ketose. In some embodiments, the linear monosaccharide can further comprise a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid. In some embodiments, the linear monosaccharide can further comprise an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine. In some embodiments, the cyclic monosaccharide is a cyclic form of a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, glucose, idose, talose, aldose, and ketose. In some embodiments, the cyclic monosaccharide can further comprise a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid. In some embodiments, the cyclic monosaccharide can further comprise an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine. The stereochemistry at the anomeric C-1 position can be either alpha or beta.

[0155] Examples of PEG units include: [ka] (In the formula, R 41 is a linear monosaccharide, a cyclic monosaccharide, or polyethylene glycol). In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is optionally deprotected, and a bond is formed between the left-most carboxyl or hydroxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or a portion of a linker subunit L2.

[0156] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 and R are not H. In some embodiments, R 24 and R 25 Each of R is a cyclic monosaccharide, disaccharide, or polysaccharide. In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 one of R is selected from a cyclic monosaccharide, disaccharide, or polysaccharide; 24 and R 25 The other is polyethylene glycol. In some embodiments, the cyclic monosaccharide is a cyclic form of a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, glucose, idose, talose, aldose, and ketose. In some embodiments, the cyclic monosaccharide can further comprise a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid. In some embodiments, the cyclic monosaccharide can further comprise an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine. The stereochemistry at the anomeric C-1 position can be either alpha or beta.

[0157] In some embodiments, disaccharides include those containing any of the above monosaccharides. The term disaccharide may include linear, cyclic, and linear cyclic forms of disaccharides. Exemplary disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, and cellobiose. In some embodiments, polysaccharides include those containing any of the above monosaccharides. The term polysaccharide may include linear, cyclic, and linear cyclic forms of polysaccharides. Exemplary polysaccharides include, but are not limited to, maltotriose, raffinose, kestose, starch, cellulose, and glycogen.

[0158] In an exemplary embodiment, the PEG unit having a cyclic monosaccharide, disaccharide, or polysaccharide comprises: [ka]

[0159] In each of these examples, each R 45 is selected from H or a monosaccharide, a disaccharide, or a polysaccharide containing any of these amino sugars; R 46- is selected from H, or a monosaccharide, disaccharide, or polysaccharide containing any of these amino sugars, and polyethylene glycol. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of the linker subunit L2, it is deprotected as necessary, and a bond is formed between the carboxyl group at the right end (first four examples) or left end (last example) of the PEG unit and a reactive group on the subunit of an amino acid unit or a portion of the linker subunit L2.

[0160] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are each independently selected from a polyhydroxyl group that is a linear monosaccharide or a substituted linear monosaccharide. In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25one of R is selected from a polyhydroxyl group that is a linear monosaccharide or a substituted linear monosaccharide; 24 and R 25 The other is polyethylene glycol. In some embodiments, the linear monosaccharide is a linear form of a C6 or C5 sugar, such as glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idostulose, aldose, and ketose. In some embodiments, the linear monosaccharide can further comprise a sugar acid, such as gluconic acid, aldonic acid, uronic acid, or ulosonic acid. In some embodiments, the linear monosaccharide can further comprise an amino sugar, such as glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

[0161] In some embodiments, the substituted linear monosaccharide may be replaced with a monosaccharide, disaccharide, or polysaccharide, in either case, linear or cyclic. In some embodiments, disaccharides include those containing any of the above monosaccharides. The term disaccharide may include linear, cyclic, and linear cyclic forms of disaccharides. Exemplary disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, and cellobiose. In some embodiments, polysaccharides include those containing any of the above monosaccharides. The term polysaccharide may include linear, cyclic, and linear cyclic forms of polysaccharides. Exemplary polysaccharides include, but are not limited to, maltotriose, raffinose, kestose, starch, cellulose, and glycogen.

[0162] Examples of PEG units containing linear monosaccharides optionally substituted with saccharides include: [ka] (In the formula, R 47 is selected from H, a linear monosaccharide, and a polyethylene glycol, and each R 49is selected from monosaccharides, disaccharides, and polysaccharides. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is optionally deprotected to form a bond between the left-most carboxyl or hydroxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or a portion of a linker subunit L2.

[0163] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are each independently selected from a polyhydroxyl group that is a linear monosaccharide or a substituted linear monosaccharide, where the substituted linear monosaccharide is substituted with one or more substituents such as alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide. In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 is selected from a polyhydroxyl group that is a linear monosaccharide or a substituted linear monosaccharide, the substituted linear monosaccharide being substituted with one or more substituents such as alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and R 24 and R 25 The other is polyethylene glycol. Such substituted polyhydroxyl groups may optionally be further substituted with a monosaccharide, disaccharide, or polysaccharide.

[0164] In an exemplary embodiment, the PEG unit having a polyhydroxyl group comprising a linear or substituted linear monosaccharide comprises: [ka]

[0165] In each of these examples, each R 42 is independently selected from H, a monosaccharide, disaccharide, or polysaccharide as described herein, or polyethylene glycol; and each R 43is selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or a portion of a linker subunit L2.

[0166] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 at least one of R is a -C(O)-polyhydroxyl group or a substituted -C(O)-polyhydroxyl group; 24 and R 25 The other is a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group, a polyhydroxyl group, or a substituted polyhydroxyl group. In some embodiments, the substituted -C(O)-polyhydroxyl group and the polyhydroxyl group can be substituted with a monosaccharide, disaccharide, or polysaccharide (in each case, either linear or cyclic); an alkyl; an O-alkyl; an aryl; a carboxyl; an ester; or an amide. In some embodiments, disaccharides include those containing any of the above monosaccharides. The term disaccharide can include linear, cyclic, and linear cyclic forms of disaccharides. Exemplary disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, and cellobiose. In some embodiments, polysaccharides include those containing any of the above monosaccharides. The term polysaccharide can include linear, cyclic, and linear cyclic forms of polysaccharides. Exemplary polysaccharides include, but are not limited to, maltotriose, raffinose, kestose, starch, cellulose, and glycogen.

[0167] In an exemplary embodiment, the PEG unit having a -C(O)-polyhydroxyl group or a substituted -C(O)-polyhydroxyl group comprises: [ka]

[0168] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0169] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are independently selected from H and substituted -C1-C8 alkyl, with the proviso that R 24 and R 25 and R are not H. In some embodiments, R 24 and R 25 are independently selected from H and substituted -C1-C4 alkyl, with the proviso that R 24 and R 25 and R are not H. In some embodiments, R 24 and R 25 are independently selected from H and substituted -C1-C3 alkyl, with the proviso that R 24 and R 25 are not both H. The alkyl portion of the substituted -C1-C8, -C1-C4 and -C1-C3 alkyl can be linear or branched.

[0170] The substituted -C1-C8, -C1-C4, or -C1-C3 alkyl may be substituted with hydroxyl or carboxyl. In some embodiments, each carbon atom of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl is substituted with hydroxyl or carboxyl. In some embodiments, each carbon atom of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl is substituted with carboxyl. In some embodiments, one or two carbon atoms of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl are substituted with hydroxyl or carboxyl. In some embodiments, one or two carbon atoms of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl are substituted with carboxyl. In some embodiments, the terminal carbon atom of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl is substituted with carboxyl. In some embodiments, the terminal carbon atom of the substituted -C1-C8, -C1-C4, or -C1-C3 alkyl is substituted with hydroxyl.

[0171] Exemplary embodiments of PEG units having a substituted -C1-C8, -C1-C4, or -C1-C3 alkyl are as follows: [ka] [ka] (In the formula, R 48 can be H, OH, CHOH, COOH, or -C1-C6 alkyl substituted with hydroxyl and / or carboxyl). In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and the reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0172] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25is selected from H and substituted —C(O)—C-C alkyl; 24 and R 25 and the other is selected from substituted —C(O)—C-C alkyl, —C(O)—C-C alkyl, and —C(O)—C-C alkyl, and substituted —C-C alkyl, —C-C alkyl, and —C-C alkyl (as described above). 24 and R 25 is independently selected from H and substituted —C(O)—C-C alkyl; 24 and R 25 and the other is selected from substituted —C(O)—C-C alkyl, —C(O)—C-C alkyl, and —C(O)—C-C alkyl, and substituted —C-C alkyl, —C-C alkyl, and —C-C alkyl (as described above). 24 and R 25 is selected from H and substituted —C(O)—C1-C3 alkyl, and R 24 and R 25 is selected from substituted -C(O)-C-C alkyl, -C(O)-C-C alkyl, and -C(O)-C-C alkyl, and substituted -C-C alkyl, -C-C alkyl, and -C-C alkyl (as described above). The alkyl of the substituted -C(O)-C-C alkyl, -C(O)-C-C alkyl, and -C(O)-C-C alkyl can be linear or branched. The alkyl moiety of the substituted -C-C, -C-C, and -C-C alkyl can be linear or branched.

[0173] The substituted -C(O)-C1-C8 alkyl, -C(O)-C1-C4 alkyl, and -C(O)-C1-C3 alkyl can be substituted with hydroxyl or carboxyl. In some embodiments, each carbon atom of the substituted -C(O)-C1-C8 alkyl, -C(O)-C1-C4 alkyl, and -C(O)-C1-C4 alkyl is substituted with hydroxyl or carboxyl. In some embodiments, one or two carbon atoms of the substituted -C(O)-C1-C8 alkyl, -C(O)-C1-C4 alkyl, and -C(O)-C1-C3 alkyl are substituted with hydroxyl or carboxyl. In some embodiments, one or two carbon atoms of the substituted -C(O)-C1-C8 alkyl, -C(O)-C1-C4 alkyl, and -C(O)-C1-C3 alkyl are substituted with carboxyl. In some embodiments, the terminal carbon atom of the substituted -C(O)-C-C alkyl, -C(O)-C-C alkyl, and -C(O)-C-C alkyl is substituted with carboxyl. In some embodiments, the terminal carbon atom of the substituted -C(O)-C-C alkyl, -C(O)-C-C alkyl, and -C(O)-C-C alkyl is substituted with hydroxyl.

[0174] Exemplary embodiments of such PEG units include: [ka] [ka]

[0175] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0176] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25is selected from H and optionally substituted aryl, with the proviso that R 24 and R 25 and R are not H. In some embodiments, substituted aryl includes aryl substituted with halogen (such as chloro, fluoro, and bromo).

[0177] In an exemplary embodiment, the substituted aryl-containing PEG unit comprises: [ka]

[0178] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0179] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are taken together to form an optionally substituted C-C heterocycle or heteroaryl. In some embodiments, the optional substituents include a heterocycle or aryl substituted with a halogen (such as chloro, fluoro, and bromo).

[0180] In an exemplary embodiment, the PEG unit comprising an optionally substituted C3-C8 heterocycle comprises: [ka]

[0181] In this exemplary embodiment, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0182] In some embodiments of the PEG units of formula (XX) and (XXI), R 24 and R 25 are independently selected from H and chelators, with the proviso that R 24 and R 25 and N,N'-dialkyl-substituted piperazines. In some embodiments, the chelator is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), benzyl-DTPA, 1,4,7,10 tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), benzyl-DOTA, 1,4,7 triazacyclononane-N,N',N''-triacetic acid (NOTA), benzyl-NOTA, 1,4,8,11 tetraazacyclotetradecane-1,4,8,11 tetraacetic acid (TETA), and N,N'-dialkyl-substituted piperazines. In some embodiments, the chelator is selected from -NR 24 R 25 In some embodiments, the chelator is attached via an alkylene, arylene, carbocyclo, heteroarylene, or heterocarbocyclo (in each case either substituted or unsubstituted).

[0183] In some exemplary embodiments, the PEG unit comprising the chelator comprises: [ka]

[0184] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0185] In some embodiments of the PEG units of formula (XX), (XXI), (XXX), (XXXI), (XXXII), and (XXXIII), the chelator may be an R 24 , R 25 and / or R 30 The chelator can be attached to any of the R groups described herein. In some embodiments, the chelator is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), benzyl-DTPA, 1,4,7,10-tetraazacyclododecane-,N,N',N'',N'''-tetraacetic acid (DOTA), benzyl-DOTA, 1,4,7 triazacyclononane-N,N',N''-triacetic acid (NOTA), benzyl-NOTA, 1,4,8,11 tetraazacyclotetradecane-1,4,8,11 tetraacetic acid (TETA), and N,N'-dialkyl-substituted piperazines ... 24 , R 25 or R 30 In some embodiments, the chelator is attached via an alkylene, arylene, carbocycle, heteroaryl, or heterocarbosyl (in each case, either substituted or unsubstituted).

[0186] In some embodiments, the PEG unit has the following general formula (XXX): ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -R 30 (XXX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 21 and R 22 are each an optional C1-C3 alkylene group; R 30 is an optionally substituted C3-C 10In some embodiments, R is selected from a carbocycle; a thiourea; an optionally substituted thiourea; a urea; an optionally substituted urea; a sulfamide; an alkylsulfamide; an acylsulfamide, an optionally substituted alkylsulfamide; an optionally substituted acylsulfamide; a sulfonamide; an optionally substituted sulfonamide; a guanidine (including alkyl and aryl guanidines); a phosphoramide; or an optionally substituted phosphoramide; a wavy line (~) indicates the site of attachment; and n20 is 1 to 26. 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0187] In some embodiments, the PEG unit has the following general formula (XXX): ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -R 30 (XXX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 21 and R 22 are each an optional C1-C3 alkylene group; R 30 is an optionally substituted C3-C 10In some embodiments, R is selected from a carbocycle; a thiourea; an optionally substituted thiourea; a urea; an optionally substituted urea; a sulfamide; an alkylsulfamide; an acylsulfamide, an optionally substituted alkylsulfamide; an optionally substituted acylsulfamide; a sulfonamide; an optionally substituted sulfonamide; a guanidine (including alkyl and aryl guanidines); a phosphoramide; or an optionally substituted phosphoramide; a wavy line (~) indicates the site of attachment; and n20 is 1 to 26. 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0188] In some embodiments of the PEG unit of formula (XXX), R 30 is an optionally substituted C3-C 10 In some embodiments, an optionally substituted C-C 10 The carbocycle is a fused cyclooctyne compound as disclosed in WO 2011 / 136645, the disclosure of which is incorporated herein by reference. An exemplary PEG unit having a fused cyclooctyne is shown below: [ka]

[0189] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is optionally deprotected to form a bond between the left-most carboxyl or amino group of the PEG unit and a reactive group on the subunit of an amino acid unit or a portion of a linker subunit L2.

[0190] As will be understood by those skilled in the art, the above compounds and other compounds disclosed in WO 2011 / 136645 can be used as intermediates for click chemistry to attach additional compounds. In some embodiments, the additional compound is a drug unit. In some embodiments, the additional compound is a linker subunit L2 as described herein.

[0191] In some embodiments of the PEG unit of formula (XXX), R 30 is a thiourea; a substituted thiourea, a urea, or a substituted urea. The thiourea and urea groups can be substituted, for example, with an optionally substituted alkyl, an optionally substituted carbocycle, or an optionally substituted aryl.

[0192] Exemplary PEG units comprising thiourea; substituted thiourea; urea; or substituted urea include the following: [ka]

[0193] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0194] In some embodiments of the PEG unit of formula (XXX), R 30 is sulfamide, alkylsulfamide, acylsulfamide, optionally substituted alkylsulfamide, optionally substituted acylsulfamide, sulfonamide, or optionally substituted sulfonamide. The optionally substituted alkylsulfamide, optionally substituted acylsulfamide, and optionally substituted sulfonamide may be substituted with additional groups, such as linkers, groups for attaching drugs or other compounds, to enhance solubility or in other embodiments.

[0195] Exemplary PEG units comprising a sulfamide; an alkylsulfamide; an acylsulfamide, an optionally substituted alkylsulfamide; an optionally substituted acylsulfamide; a sulfonamide; or an optionally substituted sulfonamide include the following: [ka]

[0196] In these examples, R 50 can be, for example, an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of a subunit of an amino acid unit or a portion of a linker subunit L2.

[0197] In some embodiments of the PEG unit of formula (XXX), R 30 is guanidine or an optionally substituted guanidine. The optionally substituted guanidine may be substituted with an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl.

[0198] Exemplary PEG units containing guanidine or optionally substituted guanidine include: [ka]

[0199] In these examples, R 55can be, for example, an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is optionally deprotected to form a bond between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or a portion of a linker subunit L2.

[0200] In some embodiments of the PEG unit of formula (XXX), R 30 is a phosphoramide or an optionally substituted phosphoramide. The optionally substituted phosphoramide may be substituted with an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl.

[0201] Exemplary PEG units that contain a phosphoramide or an optionally substituted phosphoramide include the following: [ka] In these examples, R 60 R can be, for example, an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl. 61 can be, for example, an optionally substituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocarbocycle, or heteroaryl. In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or a portion of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of a subunit of an amino acid unit or a portion of a linker subunit L2.

[0202] In some embodiments of PEG units of formula (XXX), the PEG unit comprises a functional group for attaching additional moieties. In some embodiments of PEG units of formula (XXX), R 30represents azido, alkynyl, substituted alkynyl, -NH-C(O)-alkynyl, -NH-C(O)-alkynyl-R 65 cyclooctyne; selected from -NH-cyclooctyne, -NH-C(O)-cyclooctyne or -NH-(cyclooctyne)2; R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, or optionally substituted heteroaryl. In some embodiments, such PEG units can be used as intermediates for click chemistry to attach additional compounds. In some embodiments, the additional compound is a drug unit. In some embodiments, the additional compound is a linker subunit L2 as described herein. In some embodiments, the additional compound is another linker or a drug linker.

[0203] Exemplary PEG units containing an azide, alkynyl, or cyclooctyne group include the following: [ka] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0204] In some embodiments, the PEG unit has the following formula: ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NH-C(O)-R 31 (XXXI) ~R 20 -R 21 -[O-CH2-CH2] n20 -R22 -C(O)NH-R 31 (XXXII) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -N-(R 33 -R 31 )2 (XXXIII) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NC(O)-R 31 (XXXIV) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -C(O)NH-R 31 (XXXV) or ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXXVI) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 21 and R 22 are each an optional C1-C3 alkylene group; R 26 and R 27 are optional C1-C 12 Alkylene, -NH-C1-C 12Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- or -C(O)-C-C 12 alkylene-NH-; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 R 33 is C-C alkylene, C-C alkylene-C(O), -C(O)-C-C alkylene, or -C(O)-C-C alkylene-C(O); each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—C-C alkenylene-, —NH—C-C alkenylene-, —C-C alkenylene-NH—, and —C-C alkenylene-C(O)—; R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, or optionally substituted heteroaryl; the wavy line (~) indicates the point of attachment; n20 is 1 to 26; n21 is 1 to 4; and n27 is 1 to 4) or a salt thereof. In some embodiments, R 20 is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0205] In some embodiments, the PEG unit has the following formula: ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NH-C(O)-R 31 (XXXI) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -C(O)NH-R 31 (XXXII) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -N-(R 33 -R 31 )2 (XXXIII) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NC(O)-R 31 (XXXIV) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -C(O)NH-R 31 (XXXV) or ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXXVI) (In the formula, R 20is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 21 and R 22 are each an optional C1-C3 alkylene group; R 26 and R 27 are optional C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- or -C(O)-C-C 12 alkylene-NH-; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having an R 35 R 33 is C-C alkylene, C-C alkylene-C(O), -C(O)-C-C alkylene, or -C(O)-C-C alkylene-C(O); each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—C-C alkenylene-, —NH—C-C alkenylene-, —C-C alkenylene-NH—, —C-C alkenylene-C(O)—, —NH(CO)NH—, and triazole; R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, or optionally substituted heteroaryl; the wavy line (~) indicates the point of attachment; n20 is 1 to 26; n21 is 1 to 4; and n27 is 1 to 4), or a salt thereof. In some embodiments, R 20is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or protected forms thereof. Suitable protecting groups include carboxylic acid, amine, and sulfonyl protecting groups typically used in the art.

[0206] As will be understood by those skilled in the art, such PEG units can be used to attach additional compounds. In some embodiments, the additional compound is a drug unit. In some embodiments, the additional compound is a linker subunit L2 as described herein. In some embodiments, the additional compound is a linker or drug linker.

[0207] Exemplary PEG units comprising branched polyethylene glycol chains include: [ka] [ka] In these exemplary embodiments, when the PEG unit is attached to a subunit of an amino acid unit or part of a linker subunit L2, it is deprotected and a bond is formed between the left-most carboxyl group of the PEG unit and a reactive group of the subunit of an amino acid unit or part of a linker subunit L2.

[0208] In some embodiments, the formula is selected from: ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XL) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; Each R 43 independently, absent or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, -C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene-C1-C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene, and the other is C-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0209] In some embodiments, the formula is selected from: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; R 43 does not exist or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0210] In some embodiments, the formula is selected from: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C3 alkylene; R 43 is absent or C1-C6 alkylene, -NH-C1-C 12 Alkylene, -C1-C6 alkylene-NH-, -C(O)-C1-C6 alkylene, -C1-C6 alkylene-C(O)-, -NH-C1-C6 alkylene-C(O)-, -C(O)-C1-C6 alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C6 alkylene, -C(O)-NH-C1-C 12Alkylene, -heteroarylene, heteroaryl-C1-C6 alkylene, heteroaryl-C1-C6 alkylene-C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the groups is H or C1-C6 alkylene, and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicates the binding site for; n40 is 1 to 16; n41 is 1 to 4; n42 is 1 to 4), or a salt thereof.

[0211] In some embodiments, R 40 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or protected forms thereof.

[0212] In some embodiments, R 40 but one of the following structures: [ka] or [ka] (Wherein, R=H or C1-6 alkyl; and n=0~12 (*) indicates the R for a subunit of an amino acid unit or a part of the linker subunit L2. 40 indicates the binding site of ( [ka] ) is the R 40 or a stereoisomer thereof.

[0213] In some embodiments, R 40 is one of the following structures: [ka] or [ka] (In the formula, n=0~12 (*) indicates the R for a subunit of an amino acid unit or a part of the linker subunit L2. 40 indicates the binding site of ( [ka] ) is the R 40 or a stereoisomer thereof.

[0214] In some embodiments, R 43 -(NR 44 R 45 ) n41 But R 43 When present, one of the following structures: [ka] or [ka] (In the formula, R=H, C1-6 alkyl, polyhydroxyl or substituted polyhydroxyl; ( [ka] ) is the R 43 or a stereoisomer thereof.

[0215] In some embodiments, R 43 -(NR 44 R 45 ) n41 But R 43 When present, one of the following structures: [ka] or [ka] (In the formula, ( [ka] ) is the R 43 or a stereoisomer thereof.

[0216] In some embodiments, —NR 44 R 45 but one of the following structures: [ka] or [ka] (In the formula, ( [ka] ) is the -NR 44 R 45or a stereoisomer thereof.

[0217] In some embodiments, before the PEG unit is attached to an amino acid unit or portion of the linker subunit L2, a linker intermediate or linker is provided having one of the following structures: [ka] [ka] [ka] [ka] or [ka] (wherein R is H or alkyl and n is 1 to 12).

[0218] In some embodiments, the formula is selected from: ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 46 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLIII) (In the formula, R 40 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 41 and R 42 are absent or each independently C1-C6 alkylene; Each R 43independently, absent or C1-C 12 Alkylene, -NH-C1-C 12 Alkylene, -C1-C 12 Alkylene-NH-, -C(O)-C-C 12 Alkylene, -C1-C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene-C(O)-, -C(O)-C-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1-C 12 Alkylene, -C(O)-NH-C1-C 12 Alkylene, -heteroarylene, heteroaryl-C1-C 12 Alkylene-C1-C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C1-C 12 alkylene and the other is C1-C 12 is alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; R 46 is selected from amino, amino-alkyl-amino, or —NH—C(O)—NH—S(O)2—NH—; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6), or a salt thereof.

[0219] In some embodiments, before the PEG unit is attached to an amino acid unit or portion of the linker subunit L2, a linker intermediate or linker is provided having one of the following structures: [ka] (wherein R is H or alkyl and n is 1 to 12).

[0220] In some embodiments, the formula is selected from: [ka] or [ka] (wherein each Y is independently R 76 or [ka] and Each R 76 are independently H, acetyl, -P(=O)(OH)2, or -(CH2) v -OS(=O)2(OH); Each R a and R b are independently H or R a and R b together with the carbon to which they are attached to form an oxo group; each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; and A linker intermediate or linker is provided that includes a PEG unit having an amino acid unit (AA), a linker subunit L2, or a Stretcher unit (L1) subunit (where each * indicates a binding site for the subunit), or a salt thereof.

[0221] In some embodiments, the formula is selected from: [ka] or [ka] (In the formula, each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH); each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; A linker intermediate or linker is provided that includes a PEG unit having an amino acid unit (AA), a linker subunit L2, or a Stretcher unit (L1) subunit (where each * indicates a binding site for the subunit), or a salt thereof.

[0222] In some embodiments, the formula is selected from: [ka] or [ka] (wherein each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; A linker intermediate or linker is provided that includes a PEG unit having an amino acid unit (AA), a linker subunit L2, or a Stretcher unit (L1) subunit (where each * indicates a binding site for the subunit), or a salt thereof.

[0223] In some embodiments, Y is R 76 A linker intermediate or linker is provided, which is

[0224] In some embodiments, Y is [ka] A linker intermediate or linker is provided, which is

[0225] In some embodiments, each R a and R b are independently H.

[0226] In some embodiments, R a and R b are provided which, together with the carbon to which they are attached, form an oxo group.

[0227] In some embodiments, a linker intermediate or linker is provided in which q is 10-20.

[0228] In some embodiments, a linker intermediate or linker is provided in which q is 12.

[0229] In some embodiments, a linker intermediate or linker is provided in which the PEG unit is selected from the following, or a salt thereof: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] wherein each Z is joined by a * and is individually selected from: [ka] , and [ka] each [ka] indicates the subunit of the amino acid unit (AA), part of the linker subunit L2 or the binding site to the stretcher unit (L1).

[0230] Carboxyl Unit In some embodiments, the linker comprises a carboxyl unit. The carboxyl unit may be a subunit of an amino acid unit or may be attached to a portion of the linker subunit L2. In some embodiments, the carboxyl unit has the following general formula (XXXX): R 70 | L 70 | ~NH-(CH2) p1 -CH-(CH2) o1 -C(O)~ (XXXX) (In the formula, L 70is selected from C-C alkylene, C-C alkylene-C(O)-, -C(O)-C-C alkylene- and -C(O)-C-C alkylene-C(O)-; R 70 is ~NR 71 (R 72 R 73 ) and R 71 is H, C1-C 12 Alkyl, substituted C1-C 12 alkyl, or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 72 is absent or selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted arylene, or optionally substituted heteroarylene; R 73 is carboxyl or polycarboxyl; each of p1 and o1 is independently selected from 0 to 2) or a salt thereof. As used herein, the term "polycarboxyl" refers to a group containing 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, where the carboxyl groups are interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amido. As used herein, polycarboxyl includes carboxylate forms.

[0231] In some embodiments, R 70 is ~NR 71 (R 75 -(R 73 )2), wherein R 71 is H, C1-C 12 Alkyl, substituted C1-C 12 alkyl or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 75is a branched, optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted arylene, or optionally substituted heteroarylene; and each R 73 is carboxyl or polycarboxyl, and each of p1 and o1 is independently selected from 0 to 2.

[0232] In some embodiments, R 70 is ~N(R 74 -R 73 )(R 72 -R 73 ) where R 72 and R 74 are each independently selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted arylene, or optionally substituted heteroarylene; and each R 73 is independently carboxyl or polycarboxyl, and each of p1 and o1 is independently selected from 0 to 2.

[0233] In some of the above embodiments, R 73 can be selected from: [ka] and ~COOH; (In the formula, the wavy line represents R 72 , R 74 or R 75 (showing the bond to

[0234] Linker subunit L2 The linker comprises at least one linker subunit L2, each of which has a binding site for at least one Drug unit (D), as further described herein. In some embodiments, a Drug unit (D) binds to each binding site for a Drug unit on the linker subunit L2. In various embodiments, the linker subunit L2 can be a cleavable or non-cleavable linker subunit. The linker subunit L2 also has a binding site for an Amino Acid unit (AA) or a Stretcher unit (L1).

[0235] In some embodiments, the linker subunit L2 comprises a polar unit such as a sugar unit, a PEG unit, or a carboxyl unit. In some embodiments, the linker subunit L2 does not comprise a polar unit, and the amino acid unit comprises a polar unit. In some embodiments, both the linker subunit L2 and the amino acid unit (if present) comprise a polar unit.

[0236] In some embodiments, the linker subunit L2 is a cleavable linker subunit. As used herein, the term "cleavable" refers to a metabolic process or reaction within a cell or extracellular environment in which the covalent bond between the drug unit (e.g., a cytotoxic agent) and the linker subunit L2, or a portion thereof, is cleaved, resulting in a free drug unit or other metabolic product of the linker subunit L2 drug unit dissociated from the remainder of the linker subunit L2.

[0237] In some embodiments, the linker subunit L2 is a protease-cleavable linker subunit, an acid-cleavable linker subunit, a disulfide linker subunit, a disulfide-containing linker subunit, or a disulfide-containing linker subunit having a dimethyl group adjacent to the disulfide bond (e.g., an SPDB linker) (see, e.g., Jain et al., Pharm. Res. 32:3526-3540 (2015); Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020), a cleavable self-stabilizing linker (see, e.g., WO 2018 / 031690 and WO 2015 / 095755 and Jain et al. al., Pharm. Res. 32:3526-3540 (2015)), and / or a cleavable hydrophilic linker (see, e.g., WO 2015 / 123679). In some embodiments, the linker subunit L2 comprises a photolabile linker subunit. In some embodiments, the linker subunit L2 has a non-cleavable linker unit (see, e.g., WO 2007 / 008603).

[0238] In some embodiments, the linker subunit L2 is a cleavable linker that is cleavable under intracellular conditions such that cleavage of or within the linker subunit L2 releases the drug unit from the linker subunit L2 or the remainder of the linker subunit L2 in the intracellular environment. For example, in some embodiments, the linker subunit L2 is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). As used herein, the terms "cleavable under intracellular conditions," "cleaved intracellularly," and "intracellular cleavage" refer to a metabolic process or reaction within a cell in which the covalent bond between the drug unit (e.g., a cytotoxic agent) and the linker subunit L2, or a portion thereof, is cleaved, resulting in a free drug unit or other metabolic product of the linker subunit L2 drug unit dissociated from the remainder of the linker subunit L2 within the cell. Thus, the cleaved portion of the conjugate is an intracellular metabolic product.

[0239] In some embodiments, the linkage between the linker subunit L2 and the Drug unit can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to release the Drug unit (D). Linker subunit L2 can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease (see, e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 20150297748, U.S. Patent Application Publication No. 2008 / 0166363, U.S. Patent Application Publication No. 20120328564, and U.S. Patent Application Publication No. 20200347075). Intracellular cleavage agents may include cathepsins B, C, and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The peptidyl linker may be cleavable by enzymes present in target antigen-expressing cells. For example, a peptidyl linker subunit that can be cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissues, may be used (e.g., having a Phe-Leu, Val-Ala, Val-Cit, or Gly-Phe-Leu-Gly peptide).

[0240] Typically, the peptidyl linker is at least one amino acid long or at least two amino acids long. In certain embodiments, the peptidyl linker is a dipeptide, tripeptide, tetrapeptide, or pentapeptide. In certain embodiments, the peptidyl linker subunit can contain only natural amino acids. In some embodiments, for example, the peptidyl linker subunit can have a Phe-Leu, Val-Ala, Val-Cit, or Gly-Phe-Leu-Gly peptide. Other such cleavable linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by an intracellular protease comprises a Val-Cit peptide or a Phe-Lys peptide (see, e.g., U.S. Pat. No. 6,214,345) or a Gly-Gly-Phe-Gly linker (see, e.g., U.S. Patent Application Publication No. 2015 / 0297748). One advantage of using intracellular proteolytic release of the Drug unit is that the activity of the Drug unit is typically attenuated when conjugated, and the serum stability of the conjugate is typically high. See also U.S. Patent No. 9,345,785.

[0241] In some embodiments, a peptidyl linker subunit can comprise only unnatural amino acids. In some embodiments, a peptidyl linker subunit can comprise a natural amino acid linked to an unnatural amino acid. In some embodiments, a peptidyl linker subunit can comprise a natural amino acid linked to a D-isomer of the natural amino acid. In some embodiments, at least one amino acid of a peptidyl linker subunit is an L-amino acid. In some embodiments, at least one amino acid is a D-amino acid.

[0242] In some embodiments, the peptidyl linker subunits contain one or more of the following glycine and / or L-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and polar units (including PEG units attached to glycine or L-amino acids). In some embodiments, the peptidyl linker subunits contain one or more of the following glycine and / or D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and polar units (including PEG units attached to glycine or D-amino acids). In some embodiments, the peptidyl linker subunits contain one or more of the following: glycine and / or a mixture of L- and D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and a polar unit (including a PEG unit attached to a glycine or amino acid).

[0243] In some embodiments, the peptidyl linker subunit contains one or more of the following glycine and / or naturally occurring L-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and at least one polar unit, such as a sugar unit, or a carboxyl unit or PEG unit attached to the glycine or L-amino acid. In some embodiments, the peptidyl linker subunits contain one or more of the following glycine and / or D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and at least one polar unit, such as a sugar unit, or a carboxyl unit or PEG unit attached to the glycine or D-amino acid.

[0244] In some embodiments, the amino acids of the peptidyl linker subunit have the formula shown below within the brackets: [ka] (In the formula, R 190 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH-CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-ρpyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, [ka] )

[0245] In some embodiments, the peptidyl linker subunit comprises one or more of the following L-(natural) amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine; and at least one polar unit, e.g., a sugar unit, or a carboxyl unit or PEG unit attached to the glycine or natural amino acid.

[0246] In some embodiments, the peptidyl linker subunit does not contain a cysteine. In some embodiments, the peptidyl linker does not contain a proline.

[0247] In some embodiments, the peptidyl linker subunit comprises one or more of the following D-isomers of these naturally occurring amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine; and at least one polar unit, e.g., a sugar unit, or a carboxyl unit or PEG unit attached to the glycine or D-amino acid.

[0248] In some embodiments, peptidyl linker subunit comprises the following amino acid: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, ornithine, penicillamine, β-alanine, aminoalkanoic acid, aminoalkanoic acid, aminoalkane diacid, aminobenzoic acid, amino-heterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, and derivatives thereof; and at least one polar unit, for example, a sugar unit, or one or more of a carboxyl unit or PEG unit bonded to amino acid.Illustrative examples of such amino acid derivatives are shown below in the section describing amino acid subunit.

[0249] In some embodiments, the peptidyl linker subunit contains a sugar unit as part of the cleavable peptide. For example, the sugar unit contains lysine or citrulline as part of the cleavable peptide. In some embodiments, the peptidyl linker subunit contains a carboxyl unit as part of the cleavable peptide. For example, the carboxyl unit contains lysine or citrulline as part of the cleavable peptide.

[0250] In some embodiments, the cleavable linker subunit is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value.Typically, the pH-sensitive linker subunit is hydrolyzable under acidic conditions.For example, acid-labile linker subunits (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) that can be hydrolyzed in lysosomes can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linker subunits are relatively stable under neutral pH conditions, e.g., in blood, but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker unit is a thioether linker, such as a thioether attached to the drug unit via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0251] In some embodiments, the linker subunit L2 is cleavable under reducing conditions (e.g., a disulfide linker subunit). For example, a variety of disulfide linkers are known, including those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.S. Press, 1987. See also U.S. Pat. No. 4,880,935.

[0252] In some embodiments, the linker subunit L2 is a malonic acid linker (Johnson et al. al., 1995, Anticancer Res. 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). In some embodiments, the linker subunit L2 is not cleavable, such as a maleimidocaproyl linker, and the drug unit is released by metabolic degradation of the drug-linker. (See, e.g., U.S. Patent Application Publication No. 2005 / 0238649.)

[0253] In some embodiments, the linker subunit L2 is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of the linker subunit L2 means that when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5% or less, about 3% or less, or about 1% or less of the linker subunit L2 in a sample of the conjugate is cleaved. Whether the linker subunit L2 is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the conjugate ("conjugate sample") and (b) an equal molar amount of unconjugated targeting unit or drug unit ("control sample") independently with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated targeting unit or drug unit present in the conjugate sample with that present in the control sample, as measured, for example, by high performance liquid chromatography.

[0254] In some embodiments, the linker or linker subunit L2 promotes cellular internalization. In some embodiments, the linker or linker subunit L2 promotes cellular internalization when conjugated to a drug unit, such as a cytotoxic agent (i.e., in the context of the linker-drug unit portion of a conjugate described herein). In yet other embodiments, the linker or linker subunit L2 promotes cellular internalization when conjugated to both a drug unit and a targeting unit (i.e., in the context of a conjugate described herein).

[0255] Various linker subunits L2 that can be used with the compositions and methods of the present invention are described, for example, in WO2004010957. In some embodiments, the linker subunit L2 comprises a protease-cleavable linker comprising a thiol-reactive spacer and a dipeptide (e.g., maleimidyl caproyl valine alanine). In some embodiments, the linker subunit L2 comprises a protease-cleavable linker comprising a thiol-reactive maleimidocaproyl spacer, an amino acid or peptide, and a self-immolative group. In some embodiments, the linker subunit L2 comprises a protease-cleavable linker comprising a thiol-reactive maleimidocaproyl spacer, a valine-citrulline dipeptide, and a p-aminobenzyloxycarbonyl self-immolative group.

[0256] In some embodiments, the linker subunit L2 comprises an acid-cleavable linker such as a hydrazine linker or a quaternary ammonium linker (see, e.g., WO 2017 / 096311 and WO 2016 / 040684).

[0257] In some embodiments, the linker subunit L2 comprises a self-stabilizing moiety comprising a maleimide group as described in WO 2013 / 173337.

[0258] In some embodiments, the linker subunit L2 comprises a hydrophilic linker, such as, for example, the hydrophilic peptides of WO 2015 / 123679 and the sugar alcohol polymer-based linkers disclosed in WO 2013 / 012961 and WO 2019 / 213046.

[0259] In other embodiments, the linker subunit L2 can be generated using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxyl (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Chelating agents for the conjugation of radionucleotides are described, for example, in WO 94 / 11026.

[0260] In some embodiments, linker subunit L2 can be prepared using cross-linkers including, but not limited to, commercially available (e.g., Pierce Biotechnology, Inc. (Rockford, IL, USA)) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0261] Amino acid (AA) units The linker optionally comprises an amino acid unit (AA). When present in the linker, the amino acid unit connects the stretcher unit (L1) to the linker subunit L2. When s in AA is 0, the amino acid unit is absent (e.g., any of Formulas I-IV). In some embodiments, the amino acid unit comprises 0 to 12 subunits. Each subunit of the amino acid unit is selected from natural or unnatural alpha, beta, or gamma amino acids or polar units, such as sugar units (SU), or carboxyl units or PEG units attached to the subunits of the amino acid unit.

[0262] In some embodiments, the amino acid unit is an amino acid or a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide, and one or more of the subunits is optionally modified to form a polar unit, such as a sugar unit, a PEG unit, or a carboxyl unit.

[0263] In some embodiments, the subunits of the amino acid unit are selected from glycine and / or L-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and polar units (including PEG units attached to glycine or L-amino acids). In some embodiments, the subunits of the amino acid unit are selected from glycine and / or D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and polar units. In some embodiments, the subunits of the amino acid unit are selected from glycine and / or a mixture of L- and D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and polar units (including PEG units attached to glycine or D-amino acids).

[0264] In some embodiments, the subunits of the amino acid unit are selected from glycine and / or natural L-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and at least one polar unit, such as a sugar unit, or a carboxyl unit or PEG unit, attached to glycine or an L-amino acid. In some embodiments, the subunits of the amino acid unit are selected from glycine and / or D-amino acids, such as arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, and at least one polar unit, such as a sugar unit, or a carboxyl unit or PEG unit, attached to glycine or an D-amino acid.

[0265] In some embodiments, the subunits of the amino acid unit independently have the formula shown below in square brackets: [ka] (In the formula, R 190 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH-CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-ρpyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, [ka] )

[0266] In some embodiments, each subunit of the amino acid unit is independently selected from the group consisting of the following L-(naturally occurring) amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine; and at least one polar unit, e.g., a sugar unit, or a carboxyl unit or PEG unit attached to a natural amino acid.

[0267] In some embodiments, the subunit of the amino acid unit is not cysteine. In some embodiments, the subunit of the amino acid unit is not proline.

[0268] In some embodiments, each subunit of the amino acid unit is independently selected from the group consisting of the following D-isomers of these naturally occurring amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, and valine; and at least one polar unit, e.g., a sugar unit, or a carboxyl unit or PEG unit attached to glycine or an L-amino acid.

[0269] In some embodiments, each subunit of the amino acid unit is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, ornithine, penicillamine, β-alanine, aminoalkanoic acid, aminoalkanoic acid, aminoalkanedioic acid, aminobenzoic acid, amino-heterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, and derivatives thereof; and at least one polar unit, for example, a sugar unit, or a carboxyl unit or PEG unit attached to one of the subunits.

[0270] Illustrative examples of alanine and its derivatives include alanine (Ala), N-alkyl-alanine, dehydro-alanine, 4-thiazolylalanine, 2-pyridylalanine, 3-pyridylalanine, 4-pyridylalanine, β-(1-naphthyl)-alanine, β-(2-naphthyl)-alanine, α-aminobutyric acid, β-chloro-alanine, β-cyano-alanine, β-cyclopentyl-alanine, β-cyclohexyl-alanine, β-iodo-alanine, β-cyclopentenyl- These include, but are not limited to, alanine, β-tBu-alanine, β-cyclopropyl-alanine, β-diphenyl-alanine, β-fluoro-alanine, β-piperazine-alanine with or without a protected piperazine ring, β-(2-quinolyl)-alanine, β-(1,2,4-triazole-1-yi)-alanine, β-ureido-alanine, H-β-(3-benzothienyl)-Ala-OH, and H-β-(2-thienyl)~Ala-OH.

[0271] Illustrative examples of arginine and its derivatives include, but are not limited to, arginine (Arg), N-alkyl-arginine, H-Arg(Me)-OH, H-Arg(NH)-OH, H-Arg(NO)-OH, H-Arg(Ac)-OH, H-Arg(Me)-OH (asymmetric), H-Arg(Me)-OH (symmetric), 2-amino-4-(2'-hydroxyguanidino)-butyric acid (N-ω-hydroxy-nor-arginine), and homoarginine.

[0272] Illustrative examples of aspartic acid and its derivatives include, but are not limited to, aspartic acid (Asp), N-alkyl-aspartic acid, and H-Asp(OtBu)-OH.

[0273] Illustrative examples of asparagine and its derivatives include, but are not limited to, asparagine (Asn), N-alkyl-asparagine, and isoasparagine (H-Asp-NH2).

[0274] Illustrative examples of cysteine ​​(Cys) derivatives (not containing a free SH group) include, but are not limited to, H-Cys(Acm)-OH, H-Cys(Trt)-OH, H-Cys(tBu)-OH, H-Cys(Bzl)-OH, H-Cys(Et)-OH, H-Cys(SOH)-OH, H-Cys(aminoethyl)-OH, H-Cys(carbamoyl)-OH, H-Cys(phenyl)-OH, H-Cys(Boc)-OH, and H-Cys(hydroxyethyl)-OH.

[0275] Illustrative examples of histidine and its derivatives include, but are not limited to, histidine (His), N-alkyl-histidine, H-His(Boc)-OH, H-His(Bzl)-OH, H-HBs(I-Me)-OH, H-His(l-Tos)-OH, H-2,5-diiodo-His-OH, and H-His(3-Me)-OH.

[0276] Illustrative examples of glycine and its derivatives include glycine (GIy), N-alkyl-glycines, H-propargylglycine ( [ka] CH); α These include, but are not limited to, aminoglycine (protected or unprotected), β-cyclopropyl-glycine, cyclopentyl-glycine, cyclohexyl-glycine, α-allylglycine, t-butyl-glycine, neopentylglycine and phenylglycine.

[0277] Illustrative examples of glutamic acid and its derivatives include, but are not limited to, glutamic acid (GIu), N-alkyl-glutamic acid, H-GIu(OtBu)-OH, H-γ-hydroxy-Glu-OH, H-γ-methylene-Glu-OH, H-γ-carboxy-Glu(OtBu)2-OH, and pyroglutamic acid.

[0278] Illustrative examples of glutamine and its derivatives include, but are not limited to, glutamine (GIn), N-alkyl-glutamine, isoglutamine (H-GIu-NH2), H-GIn(Trt)-OH, and H-Gln(isopropyl)-OH.

[0279] Illustrative examples of phenylalanine and its derivatives include, but are not limited to, phenylalanine (Phe), N-alkyl-phenylalanine, Hp-amino-Phe-OH, Hp-amino-Phe(Z)-OH, Hp-bromo-Phe-OH, Hp-benzyl-Phe-OH, Hp-tBu-Phe-OH, Hp-carboxy-Phe(OtBu)-OH, Hp-carboxy-Phe-OH, Hp-cyano-Phe-OH, Hp-fluoro-Phe-OH, H-3,4-dichloro-Phe-OH, Hp-iodo-Phe-OH, Hp-nitro-Phe-OH, Hp-methyl-Phe-OH, H-pentafluoro-Phe-OH, Hm-fluoro-Phe-OH, H-α-Me-Phe-OH, H-4-phenyl-Phe-OH, homoalanine, chloro-phenylalanine, and β-homophenylalanine.

[0280] Illustrative examples of lysine and its derivatives include lysine (Lys), N-alkyl-lysine, H-Lys(Boc)-OH, H-Lys(Ac)-OH, H-Lys(formyl)-OH, H-Lys(Me)-OH, H-Lys(nicotinoyl)-OH, H-Lys(Me)-OH, H-trans-4,5-dehydro-Lys-OH, H-Lys(Aloc)-OH, HH-δ-hydroxy-Lys-OH, H-δ- Illustrative examples of leucine and its derivatives include, but are not limited to, leucine (Leu), N-alkyl-leucine, 4,5-dehydroleucine-leucine, H-α-Me-Leu-OH, homoleucine, norleucine, and t-leucine, including, but not limited to, hydroxy-Lys(Boc)-OH, H-Lys(acetamidoyl)-OH, and H-Lys(isopropyl)-OH.

[0281] Illustrative examples of methionine and its derivatives include, but are not limited to, methionine (Met), H-Met(O)-OH, and H-Met(O)2-OH.

[0282] Illustrative examples of serine and its derivatives include, but are not limited to, serine (Ser), N-alkyl-serine, H-Ser(Ac)-OH, H-Ser(tBu)-OH, H-Ser(Bzl)-OH, H-Ser(ρ-chloro-Bzl)-OH, H-β-(3,4-dihydroxyphenyl)-Ser-OH, H-β-(2-thienyl)-Ser-OH, isoserine N-alkyl-isoserine, and 3-phenylisoserine.

[0283] Illustrative examples of tyrosine and its derivatives include, but are not limited to, tyrosine (Tyr), N-alkyl-tyrosine, H-3,5-dinitro-Tyr-OH, H-3-amino-Tyr-OH, H-3,5-dibromo-Tyr-OH, H-3,5-diiodo-Tyr-OH, H-Tyr(Me)-OH, H-Tyr(tBu)-OH, H-Tyr(Boc)-OH, H-Tyr(Bzl)-OH, H-Tyr(Et)-OH, H-3-iodo-Tyr-OH and H-3-nitro-Tyr-OH.

[0284] Illustrative examples of threonine and its derivatives include, but are not limited to, threonine (Thr), N-alkyl-threonine, allo-threonine, H-Thr(Ac)-OH, H-Thr(tBu)-OH, and H-Thr(Bzl)-OH.

[0285] Illustrative examples of isoleucine and its derivatives include, but are not limited to, isoleucine (He), N-alkyl-isoleucine, allo-isoleucine, and norleucine.

[0286] Illustrative examples of tryptophan and its derivatives include, but are not limited to, tryptophan (Tip), N-alkyl-tryptophan, H-5-Me-Trp-OH, H-5-hydroxy-Trρ-OH, H-4-Me-Trp-OH, H-α-Me-Trp-OH, H-Trp(Boc)-OH, H-Trp(formyl)-OH, and H-Trp(mesitylene-2-sulfonyl)-OH.

[0287] Illustrative examples of proline and its derivatives include, but are not limited to, proline (Pro), N-alkyl-proline, homoproline, thioproline, hydroxyproline (H-Hyp-OH), H-Hyp(tBu)-OH, H-Hyp(Bzl)-OH, H-3,4-dehydro-Pro-OH, 4-keto-proline, α-Me-Pro-OH, and H-4-fluoro-Pro-OH.

[0288] Illustrative examples of valine and its derivatives include, but are not limited to, valine (Val), N-alkyl-valine, H-α-Me-Val-OH, and norvaline.

[0289] Illustrative examples of ornithine and its derivatives include, but are not limited to, ornithine, N-alkyl-ornithine, H-Orn(Boc)-OH, H-Om(Z)-OH, H-α-difluoro-Me-Orn-OH (eflornithine), and H-Orn(Aloc)-OH.

[0290] Illustrative examples of penicillamine and its derivatives include, but are not limited to, penicillamine, H-penicillum(Acm)-OH (H-β,β-dimethylbis(Acm)-OH), and N-alkyl-penicillamine.

[0291] Illustrative examples of β-alanine and its derivatives include, but are not limited to, β-alanine, N-alkyl-β-alanine, and dehydro-alanine.

[0292] Illustrative examples of aminoalkanoic acids and derivatives thereof include, but are not limited to, N-alkylaminoalkanoic acids, aminobutyric acid, 4-(neopentyloxysulfonyl)-aminobutyric acid, ε-aminocaproic acid, α-aminoisobutyric acid, piperidylacetic acid, 3-amurnopropionic acid, 3-amino-3-(3-pyridyl)-propionic acid, and 5-aminopentanioic acid (aminovaleric acid).

[0293] Illustrative examples of aminoalkynoic acids and derivatives thereof include, but are not limited to, N-alkylaminoalkynoic acids, 6-amino-4-hexynoic acid, 6-(Boc-amino)-4-hexynoic acid.

[0294] Illustrative examples of aminoalkanedioic acids and derivatives thereof include, but are not limited to, N-alkylaminoalkanedioic acids, 2-aminohexanedioic acid, 2-aminoheptanedioic acid, 2-aminooctanedioic acid (H-Asu-OH).

[0295] Illustrative examples of aminobenzoic acids and derivatives thereof include, but are not limited to, N-alkylaminobenzoic acids, 2-aminobenzoic acids, 3-aminobenzoic acids, and 4-aminobenzoic acids.

[0296] Illustrative examples of amino-heterocycloalkanoic acids and derivatives thereof include, but are not limited to, N-alkylamino-heterocycloalkanoic acids, 4-amino-1-methyl-1H-imidazole-2-carboxylic acid, 4-amino-1-methyl-1H-pyrrole-2-carboxylic acid, 4-amino-piperidine-4-carboxylic acid (H-pipe-OH; 1-protected or unprotected), 3-amino-3-(3-pyridyl)-propionic acid.

[0297] Illustrative examples of heterocyclocarboxylic acids and derivatives thereof include, but are not limited to, azetidine-2-carboxylic acid, azetidine-3-carboxylic acid, piperidine-4-carboxylic acid, and thiazolidine-4-carboxylic acid.

[0298] Illustrative examples of citrulline and its derivatives include, but are not limited to, citrulline (cit), N-alkyl-citrulline, thiocitrulline, S-methyl-thiocitrulline, and homocitrulline.

[0299] Illustrative examples of statins and derivatives thereof include, but are not limited to, statins, N-alkyl-statins, cyclohexylstatins, and phenylstatins.

[0300] Illustrative examples of diaminoalkanoic acids (Dab) and derivatives thereof include, but are not limited to, N-alkyl-diamino-alkanoic acids, N,N-dialkylamino-alkanoic acids, α,γ-diaminobutyric acid (H-Dab-OH), H-Dab(Aloc)-OH, H-Dab(Boc)-OH, H-Dab(Z)-OH, α,β-diaminopropionic acid and side chain protected versions thereof.

[0301] In some embodiments, the amino acid unit may be terminated with a capping group, such as a straight or branched alkyl group, or a polyethylene chain (1-30 subunits) or PEG unit.

[0302] Exemplary embodiments of amino acid units include the following, where SU is a saccharide unit, PEG is a PEG unit, and CU is a carboxyl unit:

[0303] In some embodiments, the amino acid unit comprises SU.

[0304] In some embodiments, the amino acid unit comprises SU-Lys-SU.

[0305] In some embodiments, the amino acid unit comprises SU-Lys-SU-tert-butyl.

[0306] In some embodiments, the amino acid unit comprises SU-Lys.

[0307] In some embodiments, the amino acid unit comprises Lys-SU.

[0308] In some embodiments, the amino acid unit comprises Lys-SU-Lys(PEG).

[0309] In some embodiments, the amino acid unit comprises SU-Lys(PEG)-SU.

[0310] In some embodiments, the amino acid unit comprises SU-Glu-SU.

[0311] In some embodiments, the amino acid unit comprises Lys(PEG).

[0312] In some embodiments, the amino acid unit comprises Lys(PEG)-Lys(PEG).

[0313] In some embodiments, the amino acid unit comprises CU.

[0314] In some embodiments, the amino acid unit comprises CU-CU.

[0315] In some embodiments, an amino acid unit is present and is linked to the peptide of linker subunit L2 via a peptide bond. In some embodiments, such an amino acid unit-linker subunit L2 comprises SU-Val-Cit~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises SU-Val-Ala~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises SU-Val-Lys~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises SU-Gly-Gly-Phe-Gly~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit.

[0316] In some embodiments, such an amino acid unit-linker subunit L2 comprises Val-Lys(PEG)~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises Val-Cit(PEG)~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises Lys(PEG)-Val-Cit~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises Lys(PEG)-Gly-Gly-Phe-Gly~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit.

[0317] In some embodiments, such an amino acid unit-linker subunit L2 comprises CU-Val-Cit~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises CU-Val-Lys~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises CU-Val-Ala~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit. In some embodiments, such an amino acid unit-linker subunit L2 comprises Val-CU~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit, where CU comprises a lysine residue. In some embodiments, such an amino acid unit-linker subunit L2 comprises CU-Gly-Gly-Phe-Gly~, where the wavy line indicates the bond to the remainder of the linker subunit L2 or to a Drug unit.

[0318] In some embodiments, the amino acid unit is present and is attached to the linker subunit L2 by a non-peptide bond. In some embodiments, the amino acid unit is C1-C 10Alkylene, C2-C 10 Alkenylene, C2-C 10 It is connected to the linker subunit L2 by a peptidic linking group such as alkynylene or polyethylene glycol.

[0319] In some embodiments, linker intermediates or linkers are provided in which L2 or AA-L2 has one of the following structures: [ka] or [ka] (where the wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol).

[0320] Stretcher unit (L1) The Stretcher unit (L1) can link a targeting unit to an amino acid unit (AA) or a linker subunit L2. The Stretcher unit has a functional group that can form a bond with a functional group of a targeting unit. In some embodiments of the linker, the Stretcher unit is attached to an amino acid unit that is attached to a linker subunit L2 (i.e., when s of AA is 1, see e.g., Formulas (I)-(IV)). In some embodiments, the Stretcher unit is attached to a linker subunit L2 (i.e., when s of AA is 0, see e.g., Formulas (I)-(IV)). In some embodiments, the Stretcher unit is attached to an amino acid unit-linker subunit L2 after the amino acid unit-linker subunit L2 bond is formed. In some embodiments, the Stretcher unit is attached to an amino acid unit-linker subunit L2-drug unit after the amino acid unit-linker subunit L2-drug unit bond is formed. In some embodiments, the Stretcher unit is attached to the linker-subunit L2-Drug unit after the linker-subunit L2-Drug unit has been formed.

[0321] The functional group of the Stretcher unit for attachment to the targeting unit can include, for example, a maleimide, a haloacetamide, a sulfhydryl group, an NHS ester, an aldehyde, a ketone, a carbonyl, a hydrazide, a hydroxylamine, an amine, an amino, a hydrazine, a thiosemicarbazone, a hydrazine carboxyl, or an aryl hydrazide.

[0322] Functional groups that may be present on a targeting unit, either naturally or through chemical manipulation, include, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, anomeric hydroxyl groups of carbohydrates, and carboxyl groups. In one embodiment, the functional groups of a targeting unit are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of intramolecular disulfide bonds of a targeting unit. Alternatively, sulfhydryl groups can be generated by reaction of the amino groups of lysine moieties of a targeting unit with 2-iminothiolane (Traut's reagent) or another sulfhydryl-generating reagent.

[0323] In some embodiments, the Stretcher unit forms a bond with the sulfur atom of the targeting unit via the maleimide group of the Stretcher unit. The sulfur atom can be, for example, derived from a sulfhydryl group of the targeting unit (e.g., a thiol group of an interchain disulfide bond). Representative Stretcher units of this embodiment are shown in Formulas 100 and 101 below, where L is a targeting unit and the wavy line indicates the binding site for an amino acid unit or linker subunit L2: [ka]

[0324] In some embodiments, a linker is provided in which the Stretcher unit is selected from: [ka] and [ka] (wherein, wavy line [ka] indicates the binding site of the Stretcher unit to the amino acid unit).

[0325] In Equation 100 and Equation 101, R 17 is -C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C1-C8 alkylene-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b-C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C1-C8 alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or -(C3-C8 heterocyclo)-C1-C 10 Alkylene-S-. 17 Any of the substituents can be substituted or unsubstituted (also referred to as unsubstituted). In some embodiments, R 17 The substituent is unsubstituted. In some embodiments, R 17 The substituents are optionally substituted. In some embodiments, for example, -(CH) x NH2, -(CH2) x NHR a and -(CH2) x NR a R such as 2 17 group (see, for example, WO 2013 / 173337), where x is an integer from 1 to 4, and each R a are independently selected from the group consisting of C-C alkyl and C-C haloalkyl, or two R a The groups, taken together with the nitrogen to which they are attached, form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0326] In some embodiments of Formula 100, R 17 is -C1-C6 alkylene-C=O)-. In some embodiments, R 17 is -C1 alkylene-C(=O)-.

[0327] In some embodiments of Formula 100, R 17 is -(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C(=O), -(wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O), - (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-C(=O), - (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C1-C8 alkylene-(C(=O)), -NH-(CH2-O-CH2) b -C(=O), -(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O)), -NH-(CH2-O-CH2) b -C1-C8 alkylene-C(=O), -(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O)), -(CH2-O-CH2) b -NH- (wherein b is 1 to 26), or -C1-C8 alkylene-NH-(C(=O)), -(CH2-O-CH2) b-C1-C8 alkylene-NH- (wherein b is 1 to 26).

[0328] In other embodiments, the Stretcher unit is linked to the targeting unit via a disulfide bond between a sulfur atom of the Stretcher unit and a sulfur atom of the targeting unit. A representative Stretcher unit of this embodiment is shown in Formula 102 below, where L is a targeting unit, the wavy line indicates the attachment site for an amino acid unit or linker subunit, and L and R 17 is as described above for Equation 100 and Equation 101. [ka]

[0329] In yet another embodiment, the reactive group of the Stretcher unit contains a reactive site that can form a bond with a primary or secondary amino group of the targeting unit. Examples of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative Stretcher units of this embodiment are shown in Formulas 103, 104, and 105, where L is a targeting unit, the wavy line indicates the attachment site for an amino acid unit or linker subunit, and L and R 17 is as above for equations 100 and 101: [ka]

[0330] In yet another embodiment, the reactive group of the Stretcher unit contains a reactive site that is reactive to a modified carbohydrate (-CHO) group that may be present on the targeting unit. For example, the carbohydrate can be mildly oxidized using a reagent such as sodium periodate, and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher unit containing a functional group such as hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxyl, or arylhydrazide (such as those described by Kaneko, T. et al. (1991) Bioconjugate Chem. 2:133-41). Representative Stretcher units of this embodiment are shown in Formulas 106, 107, and 108 below, where L is a targeting unit, the wavy line indicates the attachment site for an amino acid unit or linker subunit, and L and R 17 is as above for Equation 100 and Equation 101: [ka]

[0331] In some embodiments, it may be desirable to extend the length of the Stretcher unit. Thus, the Stretcher unit may include additional components. A representative Stretcher unit of this embodiment is shown in Formula 109 below, where L is a targeting unit, the wavy line indicates a binding site for an amino acid unit or linker subunit, and L and R 17 is as above for equations 100 and 101: [ka]

[0332] In some aspects of this embodiment, R 17 is -C1-C5 alkylene-C(=O)-. R 13 is -C1-C6 alkylene-, -(CH2-O-CH2) b-(wherein b is 1 to 26), -C3-C8 carbocyclo-, -arylene-, -C1-C 10 Heteroalkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 heterocyclo)- or -(C3-C8 heterocyclo)-C1-C 10 In a preferred embodiment, R 13 is -(CH2-O-CH2) b - and b is 1 to 26.

[0333] Target Unit In some embodiments, the linker is attached to the targeting unit to form a targeting unit-linker. In some embodiments, the linker is attached to the targeting unit via a stretcher unit (L1) and to the drug unit via a linker subunit L2 to form a conjugate. In some embodiments, the linker is attached to the targeting unit via a stretcher unit (L1) and to the drug unit via a linker subunit L2 to form a conjugate. The targeting unit can be an antibody, an antigen-binding portion thereof, or a non-antibody targeting unit. A non-antibody targeting unit can also be referred to as a non-antibody scaffold.

[0334] In some embodiments, the targeting unit specifically binds to a target molecule. As used herein, "specifically binds" refers to a targeting unit (e.g., an antibody or portion thereof) described herein specifically binds to a target molecule. -5 M (10000nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12Specific binding refers to the ability to bind to a target with a KD of M or less. Specific binding can be affected, for example, by the affinity and avidity of the targeting unit and the concentration of the target polypeptide. Those skilled in the art can determine the appropriate conditions under which the antibodies, antibody-binding moieties, and non-antibody scaffolds described herein selectively bind to a target using any suitable method, for example, titration of the binding agent in a suitable cell binding assay. A targeting unit that specifically binds to its target is not displaced by a dissimilar competitor. In certain embodiments, a targeting unit is said to specifically bind to a target if it preferentially recognizes its target in a complex mixture of proteins and / or macromolecules.

[0335] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds to a target antigen. The term generally refers to antibodies composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (having heavy and light chain constant regions).

[0336] Each heavy chain is typically composed of a variable region (abbreviated as VH region) and a constant region. The heavy chain constant region may include three domains, CH1, CH2, and CH3, and optionally a fourth domain, CH4. Each light chain is composed of a variable region (abbreviated as VL region) and a constant region. The light chain constant region is a CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), and may be interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region includes three CDRs and four FRs arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.

[0337] As used herein, the "antigen-binding portion" of an antibody refers to the portion of an antibody that has the VH and / or VL sequence or the CDR of the antibody and specifically binds to a target antigen. Examples of antigen-binding portions include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, single domain antibodies (also referred to as VHH, VNAR, sdAb or nanobody) or diabodies (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). As used herein, the terms Fab, F(ab')2, and Fv refer to the following: (i) Fab is a monovalent fragment composed of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 is a bivalent fragment comprising two Fab fragments linked together in the hinge region via disulfide bridges; and (iii) Fv is composed of the VL and VH domains. The two domains of the Fv fragment, i.e., VL and VH, are encoded by separate coding regions, but they may be separated by a synthetic linker, such as the poly G4S amino acid sequence ("(G4S)" disclosed as SEQ ID NO: 1). n(where n=1-5)), allowing them to be prepared as a single protein chain in which the VL and VH regions combine to form monovalent molecules (known as single-chain Fvs or scFvs). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as "diabodies," are also included herein. Diabodies are bivalent, bispecific antibodies in which the VH and VL regions are expressed on a single polypeptide chain, but the VH and VL regions are allowed to pair with complementary regions (VL and VH, respectively) on different chains to form two antigen-binding sites, using a linker connecting the VH and VL regions that is too short to allow the two regions to be combined on the same chain (see, e.g., Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0338] A single domain antibody is an antigen-binding portion of an antibody that contains a single monomeric variable antibody region. A single domain antibody can be derived from the variable region of an antibody heavy chain from a camelid (e.g., a nanobody or VHH portion). Furthermore, the term single domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR portion derived from a shark (see, for example, Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0339] Techniques for producing single domain antibodies (e.g., DABs or VHHs) are known in the art, as disclosed, for example, in Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017). Single domain antibodies can be obtained, for example, from camels, alpacas, or llamas by standard immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007.) VHHs can have strong antigen-binding ability and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camel heavy chain-only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0340] In some embodiments, the targeting unit is an antibody, or its antigen-binding portion is a bispecific or multispecific binding agent. Bispecific and multispecific antibodies include the following: scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv. In some embodiments, the IgG-scFv is IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. For example, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0341] In some embodiments, the targeting unit is a cancer-associated antigen, e.g., CD19, CD20, CD30, CD33, CD38, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin (MLSN), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2 , GD2, 1p19q, ABL1, AKT1, ALK, APC, AR, ATM, BRAF, BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, FGFR1, FGFR2, FLT3, GNA11, GNA Q, GNAS, HRAS, IDH1, IDH2, JAK2, KDR(VEGFR2), KRAS, MGMT, MGMT-Me, MLH1, MPL, NOTCH1, NRAS, PDGFRA, Pgp, PIK3CA, PR, PTE N, RET, RRM1, SMO, SPARC, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SM AD4, SMARCB1, STK1, MLH1, MSH2, MSH6, PMS2, ROS1, ERCC1, 5T4(TPBG), B7-H3, CCR7, CD105, CD22, CD46, CD47, CD56, CD70, CD 71, CD79b, CDH6, CLDN6, CLDN18.2, CLEC12A, DLL3, DR5, ERBB3 (HER3), EPCAM, FOLR1, IGF1R, IL2RA (CD25), IL3RA, ITGB6, LIV-1, LRRC15, mesothelin (MSLN), NaPi2b (SLC34A2), Nectin-4, PTK7, ROR1, SEZ6, SLC44A4, SLITRK6, tissue factor (TF), TROP2, or B7-H4. According to the present invention, the terms "cancer-associated antigen," "tumor antigen," "tumor-expressed antigen," "cancer antigen," "cancer-associated antigen," and "cancer-expressed antigen" are equivalent and are used interchangeably herein.

[0342] In some embodiments, the targeting unit specifically binds to a target such as CD19, CD20, CD30, CD33, CD70, LIV-1, or EGFRv3.

[0343] In some embodiments, the targeting unit is an antibody (or fragment thereof) that binds to a target having a sequence disclosed in Leuschner et al., U.S. Patent Application Publication No. 2022 / 0048951 and / or Lerchen et al., U.S. Patent Application Publication No. 2022 / 0016258. Non-limiting examples of monoclonal antibodies include rituximab (Rituxan®), trastuzumab (Herceptin®), pertuzumab (Perjeta®), bevacizumab (Avastin®), ranibizumab (Lucentis®), cetuximab (Erbitux®), alemtuzumab (Campath®), pancreatic cancer (PMC), erythromycin (Protein® ... Tumumab (Vectibix®), ibritumomab (Zevalin®), tositumomab (Bexxar®), ipilimumab, zalutumumab, dalotuzumab, figitumumab, ramucirumab, galiximab, farletuzumab, oclezumab, ofatumumab (Arzerra®), CD20 antibody 2F2 (HuMax-CD20), 7D8, IgM2C6, IgG1 2C6, 11B8, B1, 2H7, LT20, 1FS or AT80 (see Teeling et al., J. Immunol. 177:362-371 (2006)), daclizumab (Zenapax®), and anti-LHRH receptor antibodies such as clones A9E4, F1G4, AT2G7, GNRH03, GNRHR2, among others, which can be used in combination with the conjugates according to the invention.

[0344] In some embodiments, FOLR1 antibodies, their antigen-binding portions, and other binding agents, as well as conjugates of such antibodies, antigen-binding portions, and other binding agents, are provided. Methods of using FOLR1 antibodies, antigen-binding portions, and other binding agents and their conjugates for the treatment of cancer and other diseases are also provided. The inventions disclosed herein are based in part on FOLR1 antibodies, their antigen-binding portions, and other binding agents, as well as their conjugates that specifically bind to FOLR1 and exhibit improved properties. FOLR1 is an important and advantageous therapeutic target for the treatment of certain cancers. FOLR1 antibodies, their antigen-binding portions, other binding agents, and their conjugates provide compositions and methods based on the use of such antibodies, antigen-binding portions, and related binding agents, as well as their conjugates, in the treatment of FOLR1+ cancers and other diseases.

[0345] In some embodiments, the targeting unit is a non-antibody scaffold. In some embodiments, the targeting unit is a non-antibody protein scaffold. Such non-antibody scaffolds include, for example, affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., adnectins, centilins, pronectins, and Tn3), fynomers, Kunitz domains, and O-bodies. (See, for example, Vazquez-Lombardi et al., Drug Discovery Today 20(10):1271(2015) and references cited therein.) Such non-antibody protein scaffolds include, for example, affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., adnectins, centilins, pronectins, and Tn3), fynomers, Kunitz domains, and O-bodies. (See, e.g., Vazquez-Lombardi et al., Drug Discovery Today 20(10):1271 (2015) and references cited therein.) Non-antibody scaffolds can be considered to fall into two structural categories: domain-sized constructs (range 6-20 kDa) and constrained peptides (range 2-4 kDa). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centrins), finomers, Kunitz domains, pronectins, and O-bodies. Peptide-sized non-antibody scaffolds include, for example, avimers, bicyclic peptides, and cysteine ​​knots. Non-antibody protein scaffolds can be considered to fall into two structural categories: domain-sized constructs (range 6-20 kDa) and constrained peptides (range 2-4 kDa). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centirins), fynomers, Kunitz domains, pronectins, and O-bodies.Non-antibody scaffolds of peptide size include, for example, avimers, bicyclic peptides and cysteine ​​knots.These non-antibody scaffolds and the underlying proteins or peptides that they are based on or that they are derived from are, for example, reviewed by Simeon and Chen, Protein Cell 9(1):3-14(2018); Vazquez-Lombardi et al., Drug Discovery Today 20:1271-1283(2015) and Binz et al., Nature Biotechnol.23:1257-1268(2005), the contents of each of which are incorporated herein by reference in their entirety.

[0346] The advantages of using non-antibody scaffolds include increased affinity, target neutralization, and stability.Various non-antibody scaffolds can also overcome some of the limitations of antibody scaffolds, such as tissue penetration, smaller size, and thermal stability.Some non-antibody scaffolds can also be constructed more easily, for example, when a bispecific construct is desired, without being hindered by the concern of potential light chain association.Methods for constructing constructs on non-antibody scaffolds are known to those skilled in the art.

[0347] Thus, in some embodiments, the targeting unit can comprise a non-antibody scaffold.Thus, in some embodiments, the targeting unit can comprise a non-antibody scaffold protein. Those skilled in the art will appreciate that targeting units may, in some embodiments, be selected from adnectin scaffolds or portions derived from, for example, the human tenth fibronectin type III domain (10Fn3); anti-callin scaffolds derived from human lipocalins (such as, for example, those described in WO 2015 / 104406); avimer scaffolds or protein fragments derived from the A domain of low density related protein (LRP) and / or very low density lipoprotein receptor (VLDLR); fynomer scaffolds or portions of the SH3 domain of FYN tyrosine kinase; Kunitz domain scaffolds or portions of Kunitz-type protease inhibitors, such as human trypsin inhibitor, aprotinin (bovine pancreatic trypsin inhibitor), Alzheimer's amyloid precursor protein, and tissue factor pathway inhibitor; knottin scaffolds (cysteine ​​knot miniproteins), for example, those based on trypsin inhibitor from E. elaterium; S. aureus protein A (S. aureus protein B); It will be understood that the scaffold may include the affibody scaffold or all or part of the Z domain of A); a β-hairpin mimic scaffold; a designed ankyrin repeat protein (DARPin) scaffold or an artificial protein scaffold based on ankyrin repeat (AR) protein; or any scaffold derived from or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin. For example, the binding site of human transferrin to the human transferrin receptor can be diversified to create a diverse library of transferrin mutants, some of which have acquired affinity for different antigens. For example, see Ali et al. (1999) J.Biol.Chem.274:24066-24073. The part of human transferrin that is not involved in binding to the receptor remains unchanged and functions as a scaffold like the framework region of an antibody, presenting mutant binding sites.Similar to antibody libraries, the library is then screened against the target antigen of interest according to the methods described herein to identify variants with optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol. 23(10):514-522.

[0348] FOLR1 targeting unit In some embodiments, the targeting agent is an anti-FOLR1 antibody or an antigen-binding portion thereof that specifically binds to FOLR1. In some embodiments, conjugates of such antibodies and their antigen-binding portions are provided. Conjugates comprising targeting agents that specifically bind to FOLR1 are useful in methods for treating cancer and other diseases. Such conjugates of FOLR1 antibodies and their antigen-binding portions, when coupled to linker drugs described herein, exhibit improved properties compared to other FOLR1 conjugates. FOLR1 is an important and advantageous therapeutic target for the treatment of certain cancers. The FOLR1 conjugates provide compositions and methods based on the use of such conjugates in the treatment of FOLR1+ cancers and other diseases.

[0349] In some embodiments, the FOLR1 targeting agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs have amino acid sequences selected from the set of amino acid sequences set forth in the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; and SEQ ID NO:36, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In some embodiments, the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. In some embodiments, the framework regions are human framework regions.

[0350] In some embodiments, the VH and VL regions have amino acid sequences selected from the pairs of amino acid sequences set forth in the group consisting of SEQ ID NO:6 and SEQ ID NO:7, respectively; SEQ ID NO:8 and SEQ ID NO:9, respectively; SEQ ID NO:10 and SEQ ID NO:11, respectively; SEQ ID NO:12 and SEQ ID NO:13, respectively; SEQ ID NO:14 and SEQ ID NO:15, respectively; SEQ ID NO:16 and SEQ ID NO:17; SEQ ID NO:18 and SEQ ID NO:19, respectively; SEQ ID NO:20 and SEQ ID NO:21, respectively; SEQ ID NO:22 and SEQ ID NO:23, respectively; SEQ ID NO:24 and SEQ ID NO:25, respectively; SEQ ID NO:26 and SEQ ID NO:27, respectively; and SEQ ID NO:28 and SEQ ID NO:29, respectively, wherein the heavy chain framework regions and light chain framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids within the framework regions.

[0351] In some embodiments, the VH region and VL region have amino acid sequences selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:6 and SEQ ID NO:7, respectively; SEQ ID NO:8 and SEQ ID NO:9, respectively; SEQ ID NO:10 and SEQ ID NO:11, respectively; SEQ ID NO:12 and SEQ ID NO:13, respectively; SEQ ID NO:14 and SEQ ID NO:15, respectively; SEQ ID NO:16 and SEQ ID NO:17; SEQ ID NO:18 and SEQ ID NO:19, respectively; SEQ ID NO:20 and SEQ ID NO:21, respectively; SEQ ID NO:22 and SEQ ID NO:23, respectively; SEQ ID NO:24 and SEQ ID NO:25, respectively; SEQ ID NO:26 and SEQ ID NO:27, respectively; and SEQ ID NO:28 and SEQ ID NO:29, respectively.

[0352] In some embodiments, the VH region and the VL region have an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:8 and SEQ ID NO:9, respectively; SEQ ID NO:12 and SEQ ID NO:13, respectively; SEQ ID NO:14 and SEQ ID NO:15, respectively; SEQ ID NO:16 and SEQ ID NO:17, respectively; SEQ ID NO:20 and SEQ ID NO:21, respectively; SEQ ID NO:22 and SEQ ID NO:23, respectively; SEQ ID NO:24 and SEQ ID NO:25, respectively; and SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0353] In some embodiments, the VH and VL regions have amino acid sequences selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:8 and SEQ ID NO:9, respectively; SEQ ID NO:12 and SEQ ID NO:13, respectively; and SEQ ID NO:26 and SEQ ID NO:27, respectively. In some embodiments, the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:8 and SEQ ID NO:9, respectively. In some embodiments, the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:12 and SEQ ID NO:13, respectively. In some embodiments, the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0354] In some embodiments, the heavy chain variable region further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is of the IgG isotype. In some embodiments, the heavy chain constant region is an IgG1 constant region. In some embodiments, the IgG1 constant region has the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the heavy chain constant region is an IgG4 constant region. In some embodiments, the heavy chain constant region further comprises at least an amino acid modification that reduces binding affinity to human Fc gamma RIII. In some embodiments, the light chain variable region further comprises a light chain constant region. In some embodiments, the light chain constant region is of the kappa isotype. In some embodiments, the light chain constant region has the amino acid sequence set forth in SEQ ID NO: 42.

[0355] In some embodiments, the FOLR1 conjugate is monospecific. In some embodiments, the FOLR1 conjugate is bivalent. In some embodiments, the FOLR1 conjugate is bispecific.

[0356] In some embodiments, a FOLR1 conjugate comprises a targeting unit that is an antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in the heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in the light chain variable region framework region, and the VH and VL CDRs have an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: (a) SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, and (b) SEQ ID NO:36, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In certain embodiments, the VH and VL regions have an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:26 and SEQ ID NO:27, respectively; and the heavy chain framework region and the light chain framework region are optionally modified with substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions. In certain embodiments, the targeting unit is antibody F131 (VH SEQ ID NO: 26 and VL SEQ ID NO: 27). In certain embodiments, the antibody is F131 and the drug-linker is LD038.

[0357] constant region In some embodiments, a targeting unit, such as an antibody or antigen-binding portion thereof, or other targeting unit, has an antibody constant region. In some embodiments, the constant region is a fully human constant region. In some embodiments, the constant region is a humanized constant region. In some embodiments, the constant region is a non-human constant region. An immunoglobulin constant region refers to a heavy or light chain constant region. The amino acid sequences of human heavy and light chain constant regions are known in the art. The constant region can be of any suitable type selected from the immunoglobulin classes IgA, IgD, IgE, IgG, and IgM. Some immunoglobulin classes can be further divided into isotypes, such as IgG1, IgG2, IgG3, IgG4, or IgA1 and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. The light chain can be one of either kappa (or κ) or lambda (or λ).

[0358] In some embodiments, the constant region can have an IgG isotype. In some embodiments, the constant region can have an IgG1 isotype. In some embodiments, the constant region can have an IgG2 isotype. In some embodiments, the constant region can have an IgG3 isotype. In some embodiments, the constant region can have an IgG4 isotype. In some embodiments, the constant region can have a hybrid isotype comprising constant regions from more than one isotype. In some embodiments, the immunoglobulin constant region can be an IgG1 or IgG4 constant region. In some embodiments, the constant region is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the constant region is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO:3.

[0359] Furthermore, the targeting unit comprising antibody or its antigen binding portion or non-antibody scaffold can be part of a larger molecule formed by covalent or non-covalent binding of antibody or antigen binding portion with one or more other proteins or peptides.Related to this targeting unit is, for example, the use of streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995), Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidinyl peptides, such as hexahistidinyl tag (disclosed as SEQ ID NO: 4 "hexahistidinyl tag") to produce bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol.31:10471058).

[0360] Fc domain modifications to alter effector function In some embodiments, the Fc region or Fc domain of a targeting unit, such as an antibody or antigen-binding portion thereof, or a non-antibody scaffold, does not substantially bind to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, the Fc region or domain does not substantially bind to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, "does not substantially bind" refers to weak to no binding to one or more selected Fc gamma receptors. In some embodiments, "does not substantially bind" refers to at least a 1000-fold decrease in binding affinity (i.e., an increase in Kd) for an Fc gamma receptor. In some embodiments, the Fc domain or region is Fc null. As used herein, "Fc null" refers to an Fc region or Fc domain that does not bind to any of the Fc gamma receptors, from an Fc region or Fc domain that has weak binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold decrease in binding affinity (i.e., an increase in Kd) to the Fc gamma receptor.

[0361] In some embodiments, the Fc domain has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, or CDC activity compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, and CDC compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate or effect ADCC, ADCP, or CDC). As used herein, "substantially no effector function" refers to at least a 1000-fold reduction in effector function activity compared to a wild-type or reference Fc domain.

[0362] In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to a decrease in the ADCC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0363] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to a decrease in the CDC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0364] To confirm the reduction / depletion of ADCC and / or CDC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fc gamma receptor binding (and therefore likely lacks ADCC activity). The primary cells for mediating ADCC, NK cells, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.); and the CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0365] C1q binding assays can also be performed to confirm that antibodies or Fc domains or regions cannot bind to C1q and therefore lack or have reduced CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402.CDC assays can also be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996); Cragg, MS et al., Blood 101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743(2004)).

[0366] In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, reduced or no ADCP activity refers to a reduction in the ADCP activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0367] To confirm that antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity, ADCP binding assay can be carried out.See, for example, US Patent Application Publication No. 20190079077 and US Patent Application Publication No. 20190048078 and the references disclosed therein.

[0368] Targeting units such as antibodies or antigen-binding portions thereof or non-antibody scaffolds with reduced effector function activity include those with substitutions of one or more of the Fc region residues, such as 238, 265, 269, 270, 297, 327, and 329, according to the EU numbering of Kabat (see, for example, U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, according to the EU numbering of Kabat, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (see, for example, U.S. Pat. No. 7,332,581). Certain antibody variants with reduced binding to FcR are also known. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).) Targeting units, such as antibodies or antigen-binding portions thereof or non-antibody scaffolds, that have reduced binding to FcRs can be prepared to contain such amino acid modifications.

[0369] In some embodiments, the targeting unit, such as an antibody or antigen-binding portion thereof, or a non-antibody scaffold, comprises an Fc domain or region having one or more amino acid substitutions that reduce Fc gamma R binding, for example, substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In some embodiments, the substitutions are L234A and L235A (LALA) according to Kabat EU numbering. In some embodiments, the Fc domain comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region according to Kabat EU numbering. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region according to Kabat EU numbering. (See, e.g., WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A, and D265A in the Fc region derived from a human IgG1 Fc region according to EU numbering of Kabat (LALA-DA).

[0370] In some embodiments, modifications to the Fc region are made that result in altered (i.e., decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0371] Methods for producing antibodies and antigen-binding moieties and other targeting units In various embodiments, targeting units such as antibodies and their antigen-binding portions can be produced in human, mouse, or other animal-derived cell lines. Recombinant DNA expression can be used to produce antibodies and their antigen-binding portions. This allows for the production of a wide range of antibodies and antigen-binding portions (including fusion proteins) in the host species of choice. Production of antibodies and their antigen-binding portions in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives to cell-based production systems. A major advantage of transgenic animals is the potential high yield from renewable resources.

[0372] Nucleic acid molecules encoding the amino acid sequence of a targeting unit, such as an antibody or its antigen-binding portion, can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding the antibody or antigen-binding portion. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding antibodies or antigen-binding portions. The nucleic acid sequence encoding at least the antibody or antigen-binding portion thereof described herein, or a polypeptide thereof, can be recombined with vector DNA according to conventional techniques, such as ligation, restriction enzyme digestion to provide suitable ends, filling in cohesive ends as necessary, alkaline phosphatase treatment to avoid undesired ligation, and blunting or zigzag ends for ligation using an appropriate ligase or other techniques known in the art. Techniques for such manipulations are disclosed, for example, in Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and can be used to construct nucleic acid sequences and vectors encoding antibodies or antigen-binding portions thereof, or VH or VL polypeptides thereof.

[0373] As used herein, the terms "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" or "nucleotide" refer to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be either single-stranded or double-stranded. A single-stranded nucleic acid can be a single-stranded nucleic acid of denatured double-stranded DNA. In some embodiments, a nucleic acid can be cDNA, e.g., a nucleic acid lacking introns.

[0374] A nucleic acid molecule, such as DNA, is said to be "capable of being expressed" into a polypeptide when it contains a nucleotide sequence containing transcriptional and translational regulatory information, and such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence desired to be expressed (e.g., an antibody or antigen-binding portion thereof) are connected in a manner that allows for gene expression of recoverable amounts of the polypeptide or antigen-binding portion. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0375] Thus, expression of targeting units, such as antibodies or antigen-binding portions thereof, can occur in either prokaryotic or eukaryotic cells. Suitable hosts include yeast, insect, fungal, avian, and mammalian cells in vivo or in situ, or bacterial or eukaryotic hosts, including host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although other mammalian cells may also be used. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus produced can be processed in vivo or purified and processed in vitro, allowing for the synthesis of antibodies or antigen-binding portions thereof described herein with specific amino-terminal sequences. Furthermore, problems associated with retaining the methionine residue from the start codon in direct yeast (or bacterial) expression can be avoided. (See, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).) Recombinant antibodies, or antigen-binding portions thereof, can be produced using any of a range of yeast gene expression systems that incorporate promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0376] Production of antibodies or antigen-binding portions in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express the polypeptide by methods known to those of skill in the art. See Ausubel et al., 1987-1993.

[0377] In some embodiments, the introduced nucleic acid sequence (encoding the antibody or antigen-binding portion thereof, or the polypeptide) is incorporated into a plasmid or viral vector capable of autonomous replication in recipient host cells. Any of a wide variety of vectors can be used for this purpose and are known and available to those skilled in the art. See, for example, Ausubel et al., 1987-1993. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the copy number of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0378] Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replicating in E. coli. Other gene expression elements useful for expressing DNA encoding an antibody or antigen-binding portion thereof include (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 2003). Mol.Cell.Biol.280(1983)), Rous sarcoma virus LTR(Gorman et al.,79 PNAS 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayama et al., 1983), and (c) polyadenylation sites such as those in SV40 (Okayama et al., 1983). DNA genes encoding immunoglobulins can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin heavy chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin intervening sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element as described below by Liu et al. and Weidle et al., 51 Gene 21 (1987).

[0379] In the case of immunoglobulin encoding nucleotide sequences, the transcription promoter can be, for example, human cytomegalovirus and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin.

[0380] In some embodiments, for expression of a DNA coding region in rodent cells, the transcription promoter can be a viral LTR sequence, and the transcription promoter enhancer can be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the above expression elements to achieve protein expression in mammalian cells.

[0381] Each coding region or gene fusion is assembled or inserted into an expression vector. Then, the nucleotides encoding the antibody or antibody polypeptide or its antigen-binding portion are transfected alone, or the polynucleotides encoding the VH and VL chain coding regions are co-transfected into recipient cells capable of expressing the variable region or its antigen-binding portion. The transfected recipient cells are cultured under conditions that allow the expression of the incorporated coding regions, and the expressed antibody chains or intact antibodies or antigen-binding portions are recovered from the culture.

[0382] In some embodiments, nucleic acids containing coding regions encoding antibodies or antigen-binding portions thereof are assembled into separate expression vectors that are then used to co-transfect recipient host cells. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, one designed for selection in a bacterial system and the other designed for selection in a eukaryotic system, with each vector carrying a set of coding regions. This strategy initially results in a vector that directs the production of nucleotide sequences in a bacterial system and allows for amplification. The DNA vectors so produced and amplified in the bacterial host are then used to co-transfect eukaryotic cells, allowing for the selection of co-transfected cells carrying the desired transfected nucleic acids (e.g., containing the heavy and light chains of an antibody). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, fused nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0383] For transfection of the expression vector and production of the antibody or antigen-binding portion thereof, the recipient cell line can be a Chinese hamster ovary cell line (e.g., DG44) or a myeloma cell. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin genes and possess the machinery for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is recombinant Ig-producing myeloma cell SP2 / 0. SP2 / 0 cells produce only the immunoglobulin encoded by the transfected gene. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, and secreted immunoglobulins can be obtained from the ascites fluid.

[0384] Expression vectors encoding antibodies or antigen-binding portions thereof can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection and microprojectile bombardment, as known to those skilled in the art (see, e.g., Johnston et al., 240 Science 1538 (1988)).

[0385] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., prepolypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol (Montpellier, France, 1982).

[0386] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion, and stability of antibodies, as well as assembled antibodies and antigen-binding portions thereof. A variety of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium are available. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1 alpha promoter, such as that from Chinese hamster cells. Several approaches can be taken to evaluate the optimal expression plasmid for immunoglobulin expression in yeast. See, for example, II DNA Cloning 45 (Glover, ed., IRL Press, 1985) and U.S. Patent Application Publication No. 2006 / 0270045 A1.

[0387] Bacterial strains can also be utilized as hosts for the production of antibody molecules or antigen-binding portions thereof as described herein. E. coli K12 strains such as E. coli W3110, Bacillus species, Enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vector contains a replication site and specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of antibodies and their antigen-binding portions in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0388] Host mammalian cells can be grown in vitro or in vivo and provide post-translational modifications of immunoglobulin molecules, including removal of leader peptides, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or antigen-binding portions thereof.

[0389] In addition to the cells of lymphoid origin described above, mammalian cells that may be useful as hosts for producing antibody proteins include cells of fibroblast origin, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PERC6™ cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0390] In some embodiments, one or more antibodies or antigen-binding portions thereof can be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.

[0391] In some embodiments, the antibody or antigen-binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems are, for example, Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); and Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0392] Many vector systems are available for the expression of VH and VL chains in mammalian cells (see Glover, 1985). Various approaches can be followed to obtain intact antibodies. As discussed above, VH and VL chains, and optionally associated constant regions, can be coexpressed in the same cell to achieve intracellular association and linkage of the VH and VL chains into a complete tetrameric H2L2 antibody or its antigen-binding portion. Coexpression can occur by using either the same or different plasmids in the same host. Nucleic acids encoding the VH and VL chains or their antigen-binding portions can be placed on the same plasmid, which is then transfected into cells, thereby directly selecting for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the VL chain, and then the resulting cell line can be transfected with a VH chain plasmid containing a second selection marker. Cell lines producing antibodies or antigen-binding portions thereof via either route could be transfected with plasmids encoding additional copies of the peptide, VH, VL or VH+VL chains, along with additional selectable markers, to generate cell lines with enhanced properties, e.g., higher production of assembled antibodies or antigen-binding portions thereof or enhanced stability of the transfected cell line.

[0393] Furthermore, plants have emerged as a convenient, safe, and economical alternative expression system for recombinant antibody production based on large-scale culture of microbial or animal cells. Antibodies or antigen-binding portions thereof can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, restricted to intracellular plastids, or restricted to seeds (endosperm). See, for example, U.S. Patent Application Publication No. 2003 / 0167531; U.S. Patent No. 6,080,560; U.S. Patent No. 6,512,162; and International Publication No. 0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0394] In the case of intact antibodies, the variable regions of the antibody (VH and VL regions) are typically linked to at least a portion of an immunoglobulin constant region (Fc) or domain, typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, according to well-known procedures (WO 87 / 02671). An antibody can contain both a light chain constant region and a heavy chain constant region. The heavy chain constant region can include a CH1, hinge, CH2, CH3, and optionally a CH4 region. In some embodiments, the CH2 domain can be deleted or omitted.

[0395] Techniques described for the production of single chain antibodies (e.g., U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989), which are incorporated herein by reference in their entireties, can be adapted to produce single-chain antibodies that specifically bind to target antigens. Single-chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Techniques for assembly of functional Fv portions in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988), which are incorporated herein by reference in their entireties).

[0396] In some embodiments, the antigen-binding portion comprises one or more scFvs. An scFv can be a fusion protein of the variable regions of an antibody's heavy chain (VH) and light chain (VL), connected by a short linker peptide, e.g., 10 to about 25 amino acids. The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96. Methods for generating scFv molecules and designing suitable peptide linkers are described, for example, in U.S. Pat. Nos. 4,704,692; 4,946,778; and Raag. and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9:132-137 (1991). scFv-Fcs have been described by Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050, 2017.

[0397] In some embodiments, the antigen-binding portion is a single-domain antibody, an antibody portion consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domain of an antibody heavy chain from a camelid (e.g., a nanobody or VHH portion). Furthermore, single-domain antibodies can be autonomous human heavy chain variable domains (aVH) or VNAR portions derived from sharks (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0398] Techniques for producing single domain antibodies (DABs or VHHs) are known in the art, for example, as disclosed in Cossins et al. (2006, Prot Express Purif 51:253-259) and Li et al. (Immunol. Lett. 188:89-95, 2017). Single domain antibodies can be obtained, for example, from camels, alpacas, or llamas by standard immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007.) VHHs can have strong antigen-binding ability and can interact with epitopes that are inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camel heavy chain-only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0399] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168; Carter (2001), J Immunol Methods 248, 7-15). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004 A1); cross-linking two or more antibodies or antigen-binding portions thereof (e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody portions (e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (e.g., Gruber et al. al., J. Immunol., 152:5368 (1994)); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0400] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," can also be targeting units (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576A1).

[0401] In some embodiments, the targeting unit comprises different antigen binding sites fused to one or the other of the two subunits of the Fc domain; therefore, the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of bispecific molecules in recombinant production, it may be advantageous to introduce modifications to the Fc domain of the targeting unit that promote the association of the desired polypeptides.

[0402] Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domains with a charged amino acid residue, such that homodimer formation is electrostatically unfavored, but heterodimerization is electrostatically favored.

[0403] In some embodiments, the targeting unit is a "bispecific T cell engager" or BiTE (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain can include two single-chain Fv (scFv) portions, each having a variable heavy (VH) and variable light (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further includes a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different epitope, and these epitopes can be specific for different proteins such that both proteins are bound by the BiTE.

[0404] Because it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic expression system known in the art, such as a CHO cell line. However, specific purification techniques (see, e.g., EP 1691833) may be necessary to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activity other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted with a gradient of imidazole concentration. The eluate is further purified using anion exchange chromatography, and the polypeptide is eluted with a gradient of sodium chloride concentration. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species. In some embodiments, the targeting unit is a bispecific antibody composed of a single polypeptide chain comprising two single-chain FV moieties (scFVs) fused to each other by a peptide linker.

[0405] In some embodiments, the targeting unit is multispecific, such as an IgG-scFv. IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods for making them are described, for example, in Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0406] Igg-like dual variable domain antibodies (DVD-Ig) are described in Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol. 75:28-37, 2016, and WO 08 / 024188 and WO 07 / 024715. Triomabs are described in Chelius et al., MAbs 2(3):309-319, 2010. 2-in-1 IgGs are described in Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tandem antibodies or TandAbs are described in Kontermann et al., id. ScFv-HSA-scFv antibodies are also described in Kontermann et al. (id.).

[0407] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen-binding portions thereof can be recovered and purified by known techniques, such as immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See generally, Scopes, Protein Purification (Springer-Verlag, New York, 1982). Substantially pure antibodies or antigen-binding portions thereof of at least about 90% to 95% homogeneity are advantageous, with antibodies or antigen-binding portions thereof having 98% to 99% or greater homogeneity also being advantageous, particularly for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the intact antibodies or antigen-binding portions thereof can then be used therapeutically or in the development and implementation of assay procedures, immunofluorescence staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, New York, 1979 and 1981).

[0408] Drug Unit In some embodiments, the linker is attached to the drug unit, the targeting unit, and / or the targeting unit and the drug unit (the latter also referred to as a conjugate, ADC, or antibody-drug conjugate). In some embodiments, the linker is attached to at least one drug unit via the linker subunit L2. As used herein, in the context of a conjugate, the term "drug unit" or drug refers to a cytotoxic agent (such as a chemotherapeutic agent or drug), an immunomodulator, a nucleic acid (including siRNA), a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), a radioisotope, a PROTAC, and other compounds that are active against target cells when delivered to those cells.

[0409] cytotoxic agents In some embodiments, the Drug Unit is a cytotoxic agent. "Cytotoxic agent" refers to an agent that has a cytotoxic effect on a cell. "Cytotoxic effect" refers to the depletion, elimination, and / or death of a target cell. Cytotoxic agents include, for example, tubulin-disrupting agents, topoisomerase inhibitors, DNA minor groove binders, and DNA alkylating agents.

[0410] Tubulin-disrupting agents include, for example, auristatins, dolastatins, tubulysins, colchicine, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterins, and other tubulin-disrupting agents. Auristatins are derivatives of the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine), MMAF (N-methylvaline-valine-dolaisoleucine-dolaproine-phenylalanine), and AFP (see WO 2004 / 010957 and WO 2007 / 008603). Other auristatin-like compounds are disclosed, for example, in U.S. Patent Application Publication Nos. 2021 / 0008099, 2017 / 0121282, 2013 / 0309192, and 2013 / 0157960. Dolastatins include, for example, dolastatin 10 and dolastatin 15 (see, e.g., Pettit et al., J. Am. Chem. Soc., 1987, 109, 6883-6885; Pettit et al., Anti-Cancer Drug Des., 1998, 13, 243-277; and U.S. Patent Application Publication No. 2001 / 0018422). Additional dolastatin derivatives contemplated for use herein are disclosed in US Pat. No. 9,345,785, which is incorporated herein by reference.

[0411] Tubulysins include, but are not limited to, tubulysin D, tubulysin M, tubuphenylalanine, and tubutyrosine. International Publication Nos. WO 2017 / 096311 and WO 2016 / 040684 describe tubulysin analogs, including tubulysin M.

[0412] Colchicines include, but are not limited to, colchicine and CA-4.

[0413] Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VOS).

[0414] Taxanes include, but are not limited to, paclitaxel and docetaxel.

[0415] Cryptophycins include, but are not limited to, cryptophycin-1 and cryptophycin-52.

[0416] Maytansinoids include, but are not limited to, maytansine, maytansinol, maytansine analogs in DM1, DM3, and DM4, and ansamatocin-2. Exemplary maytansinoid drug moieties include those with modified aromatic rings, such as C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chloride), as well as those with modifications at other positions.

[0417] Maytansinoid drug moieties include C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (see U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (see U.S. Pat. No. 4,450,254) (prepared from Nocardia); C-15-hydroxy / acyloxy (see U.S. Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces); C-15-methoxy (see U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared from Trewia nudiflora); C-18-N-demethyl (see U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by Streptomyces demethylation of maytansinol); and 4,5-deoxy (see U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

[0418] Hemiasterins include, but are not limited to, hemiasterin and HTI-286.

[0419] Other tubulin disrupting agents include takalonolide A, takalonolide B, takalonolide AF, takalonolide AJ, takalonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0420] In some embodiments, the cytotoxic agent may be a topoisomerase inhibitor, such as camptothecin. Exemplary camptothecins include, for example, camptothecin, irinotecan (also known as CPT-11), belotecan, (7-(2-(N-isopropylamino)ethyl)camptothecin), topotecan, 10-hydroxy-CPT, SN-38, exatecan, and exatecan analog DXd (see U.S. Patent Application Publication No. 20150297748). Other camptothecins are disclosed in International Publication Nos. WO 1996 / 021666, WO 00 / 08033, U.S. Patent Application Publication No. 2016 / 0229862, and WO 2020 / 156189.

[0421] In some embodiments, the cytotoxic agent is a duocarmucisin, including the synthetic analogs KW-2189 and CBI-TMI.

[0422] immunomodulators In some embodiments, the Drug Unit is an immunomodulatory agent. The immunomodulatory agent can be, for example, a TLR7 and / or TLR8 agonist, a STING agonist, a RIG-I agonist, or other immunomodulatory agent.

[0423] In some embodiments, the Drug Unit is an immunomodulatory agent, such as a TLR7 and / or TLR8 agonist. In some embodiments, the TLR7 agonist is selected from imidazoquinolines, imidazoquinoline amines, thiazoquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, heteroaromatic azides-2,2-dioxides, benzonaphthyridines, guanosine analogs, adenosine analogs, thymidine homopolymers, ssRNA, CpG-A, PolyG10, and PolyG3. In some embodiments, the TLR7 agonist is selected from imidazoquinolines, imidazoquinoline amines, thiazoquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, heteroaromatic azides-2,2-dioxides, or benzonaphthyridines. In some embodiments, the TLR7 agonist is a non-naturally occurring compound. Examples of TLR7 modulators include GS-9620, GSK-2245035, imiquimod, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7795, and compounds disclosed in U.S. Patent Application Publication Nos. 20160168164, 20150299194, 20110098248, 20100143301, and 20090047249.

[0424] In some embodiments, the TLR8 agonist is selected from benzazepines, imidazoquinolines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, tetrahydropyridopyrimidines, or ssRNA. In some embodiments, the TLR8 agonist is selected from benzazepines, imidazoquinolines, thiazoloquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamines, pyrimidine-2,4-diamines, 2-aminoimidazoles, 1-alkyl-1H-benzimidazol-2-amines, and tetrahydropyridopyrimidines. In some embodiments, the TLR8 agonist is a non-naturally occurring compound. Examples of TLR8 agonists include motolimod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763, and VTX-1463.

[0425] In some embodiments, the TLR8 agonist can be any of the compounds described in WO 2018 / 170179, WO 2020 / 056198, and WO 2020056194.

[0426] Other TLR7 and TLR8 agonists are described in, for example, WO 2016142250, WO 2017046112, WO 2007024612, WO 2011022508, WO 2011022509, WO 2012045090, WO 2012097173, WO 2012097177, WO 2017079283, U.S. Patent Application Publication No. 20160008374, U.S. Patent Application Publication No. 20160008374, U.S. Patent Application Publication No. 20160008375, U.S. Patent Application Publication No. 20160008376, U.S. Patent Application Publication No. 20160008377, U.S. Patent Application Publication No. 20160008378, U.S. Patent Application Publication No. 20160008379 ... No. 160194350, U.S. Patent Application Publication No. 20160289229, U.S. Patent No. 6,043,238, U.S. Patent Application Publication No. 20180086755, International Publication No. 2017216054, International Publication No. 2017190669, International Publication No. 2017202704, International Publication No. 2017202703, International Publication No. 20170071944, U.S. Patent Application Publication No. 20140045849, U.S. Patent Application Publication No. 20140073642 Specification, International Publication No. 2014056953, International Publication No. 2014076221, International Publication No. 2014128189, U.S. Patent Application Publication No. 20140350031, International Publication No. 2014023813, U.S. Patent Application Publication No. 20080234251, U.S. Patent Application Publication No. 20080306050, U.S. Patent Application Publication No. 20100029585, U.S. Patent Application Publication No. 20110092485, U.S. Patent Application Publication No. 2011011 No. 8235, U.S. Patent Application Publication No. 20120082658, U.S. Patent Application Publication No. 20120219615, U.S. Patent Application Publication No. 20140066432, U.S. Patent Application Publication No. 20140088085, U.S. Patent Application Publication No. 20140275167 and U.S. Patent Application Publication No. 20130251673, International Publication No. 2018198091 and U.S. Patent Application Publication No. 20170131421.

[0427] In some embodiments, the immunomodulatory agent is a STING agonist.Examples of STING agonists include those disclosed in, for example, International Publication No. 2020059895, International Publication No. 2015077354, International Publication No. 2020227159, International Publication No. 2020075790, International Publication No. 2018200812 and International Publication No. 2020074004.

[0428] In some embodiments, the immunomodulatory agent is a RIG-I agonist. Examples of RIG-I agonists include KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0429] toxin In some embodiments, the Drug Unit is an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0430] radioactive isotope In some embodiments, the drug unit is a radioactive atom.Various radioisotopes are available for producing radioconjugates.Examples include yttrium-88, yttrium-90, technetium-99, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114, indium-115, lutetium-177, strontium-89, salarium-153 and lead-212.

[0431] PROTAC In some embodiments, the drug unit is a proteolytic targeting chimera (PROTAC). PROTACs are described, for example, in U.S. Patent Application Publication Nos. 20210015942, 20210015929, 20200392131, 20200216507, 20200199247, and 20190175612; the disclosures of which are incorporated herein by reference.

[0432] Ligand In some embodiments, the Drug Unit comprises a ligand that can be bound by a carboxyl unit such as platinum (Pt), ruthenium (Ru), rhodium (Rh), gold (Au), silver (Ag), copper (Cu), molybdenum (Mo), titanium (Ti), or iridium (Ir); a radioisotope such as yttrium-88, yttrium-90, technetium-99, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114, indium-115, lutetium-177, strontium-89, salarium-153, and lead-212.

[0433] Drug burden A conjugate can contain one or more drug units per targeting unit. The number of drug units per targeting unit is referred to as drug loading. The drug loading of a conjugate is the average number of drug units (drug molecules (e.g., cytotoxic agents)) per targeting unit (e.g., antibody or antigen-binding moiety or non-antibody scaffold or non-antibody protein) in the conjugate, p load For example, p load is about 4, the average drug loading, taking into account all of the targeting units (e.g., antibodies or antigen-binding moieties or non-antibody scaffolds or non-antibody proteins) present in the composition, is about 4. In some embodiments, p load In some embodiments, p is in the range of about 3 to about 5, about 3.6 to about 4.4, or about 3.8 to about 4.2. load can be about 3, about 4, or about 5. In some embodiments, p load In some embodiments, p is in the range of about 6 to about 8, more preferably about 7.5 to about 8.4. load can be about 6, about 7, or about 8. In some embodiments, p load is in the range of about 8 to about 16.

[0434] The average number of drug units per targeting unit (e.g., antibody or antigen-binding moiety or non-antibody scaffold) in a preparation can be characterized by conventional means such as UV, mass spectrometry, capillary electrophoresis (CE), and HPLC. load The quantitative distribution of the conjugate with respect to p can also be determined. load Separation, purification, and characterization of homogeneous conjugates of a particular value from conjugates with other drug loads can be achieved by means such as reverse-phase HPLC or hydrophobic interaction chromatography (HIC) HPLC.

[0435] Exemplary Linkers and Linker Unit-Drug Unit Combinations In some embodiments, the linker intermediate [ka] is of the general formula: [ka] where AA is an amino acid unit having 1 to 12 subunits selected from alpha, beta, and gamma amino acids and derivatives thereof, sugar units, carboxyl units, and amino acid subunits, optionally substituted with at least one PEG unit, provided that the amino acid unit comprises at least one sugar unit, PEG unit, or carboxyl unit; L1 is a Stretcher unit; the wavy line (~) indicates a binding site for a targeting unit, and the double wavy line ( [ka] ) indicates a binding site for the linker subunit L2) or a salt thereof. In some embodiments, the amino acid unit comprises at least one saccharide unit, PEG unit, carboxyl unit, or a combination thereof.

[0436] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] (wherein AA is an amino acid unit having 1 to 12 subunits selected from alpha, beta, and gamma amino acids and derivatives thereof, amino acid subunits optionally substituted with a sugar unit, a carboxyl unit, and at least one PEG unit; L2 is a linker subunit optionally substituted with at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof; the wavy line (~) indicates the attachment site of a Stretcher unit; the double wavy line ( [ka] ) indicates a binding site for the Drug unit, with the proviso that [ka] (containing at least one sugar unit, PEG unit, carboxyl unit, or a combination thereof) or a salt thereof.

[0437] In some embodiments, the drug linker intermediate AA-L2-D has the following general formula: ~AA-L2-D

[0192] wherein AA is an amino acid unit having 1 to 12 subunits selected from alpha, beta, and gamma amino acids and derivatives thereof, amino acid subunits optionally substituted with a sugar unit, a carboxyl unit, and at least one PEG unit; L2 is a linker subunit optionally substituted with at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof; D is a Drug unit; and the wavy line (~) indicates an attachment site for a Stretcher unit, with the proviso that -AA-L2- comprises at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof, or a salt thereof.

[0438] In some embodiments, the linker [ka] is of the general formula: [ka] where L1 is a stretcher unit; AA is an amino acid unit having 1 to 12 subunits selected from alpha, beta, and gamma amino acids and derivatives thereof, amino acid subunits optionally substituted with a sugar unit, a carboxyl unit, and at least one PEG unit; L2 is a linker subunit optionally substituted with at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof; the wavy line (~) indicates a binding site for a targeting unit, and the double wavy line ( [ka] ) indicates a binding site for a Drug unit, with the proviso that: [ka] comprises at least one saccharide unit, PEG unit, carboxyl unit, or a combination thereof) or a salt thereof. In some embodiments, L2 is attached to the side of the chain of AA subunits.

[0439] In some embodiments, the drug linker -L1-AA-L2-D has the following general formula: ~L1-AA-L2-D

[0194] (wherein L1 is a Stretcher unit; AA is an amino acid unit having 1 to 12 subunits selected from alpha-, beta-, and gamma-amino acids and derivatives thereof, amino acid subunits optionally substituted with a sugar unit, a carboxyl unit, and at least one PEG unit; L2 is a linker subunit optionally substituted with at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof; D is a Drug unit; and the wavy line (~) indicates an attachment site for a targeting unit, with the proviso that -AA-L2- comprises at least one sugar unit, a PEG unit, a carboxyl unit, or a combination thereof) or a salt thereof. In some embodiments, L2 is attached to the side of the AA subunit chain.

[0440] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] (where [SU] is an amino acid unit, each SU is a sugar unit, L1 is a stretcher unit, the wavy line (~) indicates the binding site for the targeting unit, and the double wavy line ( [ka] ) indicates a binding site for the linker subunit L2) or a salt thereof.

[0441] Exemplary embodiments of such linker intermediates include: [ka] wherein the carboxyl group on the right side of the sugar unit is the attachment site for the linker subunit L2) or a salt thereof.

[0442] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] (wherein [SU-aa-] is an amino acid unit, each SU is a sugar unit, aa is an optional subunit of AA selected from alpha, beta and gamma amino acids and derivatives thereof, L1 is a Stretcher unit, the wavy line (~) indicates the attachment site for the targeting unit, and the double wavy line ( [ka] ) indicates a binding site for the linker subunit L2) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, [SU-aa] is [SU-Lys-].

[0443] Exemplary embodiments of such linker intermediates include: [ka] wherein the carboxyl group to the right of the lysine is the attachment site for the linker subunit L2, or a salt thereof.

[0444] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] (wherein [SU-aa-SU] is an amino acid unit, each SU is a sugar unit, aa is an AA subunit selected from alpha, beta and gamma amino acids and derivatives thereof, L1 is a Stretcher unit, the wavy line (~) indicates a binding site for a targeting unit, and the double wavy line ( [ka] ) indicates a binding site for the linker subunit L2) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, [SU-aa-SU] is [SU-Lys-SU].

[0445] Exemplary embodiments of such linker intermediates include: [ka] wherein the protected carboxyl group on the right side of the sugar unit is the attachment site for the linker subunit L2, or a salt thereof.

[0446] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] ([SU-aa] is an amino acid unit, where each SU is a sugar unit, aa is an optional subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit, or combinations thereof, a wavy line (~) indicates a binding site for a Stretcher unit, and a double wavy line ( [ka] ) indicates a binding site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, [Su-aa] is [Su-Lys]. In some embodiments, linker subunit L2 is a cleavable linker subunit.

[0447] Exemplary embodiments of such linkers include the following: [ka] wherein the amino group on the left side of the Sugar unit is the attachment site for the Stretcher unit and the benzyl alcohol group on the right side is the attachment site for the Drug unit, or a salt thereof.

[0448] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] where [SU-aa-SU] are amino acid units, each SU is a sugar unit, aa is an AA subunit selected from alpha, beta, and gamma amino acids and derivatives thereof, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit, or combinations thereof, L2 is attached to a site on aa, the wavy line (~) indicates the attachment site for a Stretcher unit, and the double wavy line ( [ka] ) indicates a binding site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, [Su-aa-Su] is [Su-Lys-Su]. In some embodiments, linker subunit L2 is a cleavable linker subunit.

[0449] Exemplary embodiments of such linker intermediates include: [ka] wherein the amino group on the left side of the Sugar unit is the attachment site for the Stretcher unit, and the Drug unit is attached to the terminal acid group or the benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the Drug unit), or a salt thereof.

[0450] In some embodiments, the linker has the following general formula: [ka] (wherein [SU-aa-SU] is an amino acid unit, each SU is a sugar unit, aa is an optional subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, L1 is a stretcher unit, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit, or combinations thereof, L2 is attached to a site on aa or to SU, the wavy line (~) indicates the attachment site for the targeting unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is present and is lysine.

[0451] Exemplary embodiments of such linkers include the following: [ka] wherein the maleimide group to the left of the sugar unit is the attachment site for the targeting unit, and the drug unit is attached to the benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the drug unit), or a salt thereof.

[0452] In some embodiments, the linker has the following general formula: [ka] where [SU-aa-SU] are amino acid units, each SU is a sugar unit, aa is an optional subunit of AA selected from alpha, beta and gamma amino acids and derivatives thereof, L1 is a stretcher unit, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit or combinations thereof, L2 is attached to AA, the wavy line (~) indicates the attachment site for the targeting unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is present and is lysine.

[0453] Exemplary embodiments of such linkers include the following: [ka] wherein the maleimide group to the left of the sugar unit is the attachment site for the targeting unit, and the drug unit is attached to the benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the drug unit), or a salt thereof.

[0454] In some embodiments, the linker has the following general formula: [ka] where [SU-aa-SU] is an amino acid unit, each SU is a sugar unit, aa is an AA subunit selected from alpha, beta, and gamma amino acids and derivatives thereof, L1 is a stretcher unit, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit, or combinations thereof, and L2 is attached to a site on aa, the wavy line (~) indicates the attachment site for a targeting unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is present and is lysine.

[0455] Exemplary embodiments of such linkers include the following: [ka] [ka] wherein the maleimide or bromoacetamide group to the left of the saccharide unit is the attachment site for the targeting unit, and the drug unit is attached to the terminal acid group or benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the drug unit), or a salt thereof.

[0456] In some embodiments, the drug-linker intermediate AA-L2-D has the following general formula: ~[SU-aa]-L2-D

[0216] wherein [SU-aa] is an amino acid unit, each SU is a saccharide unit, aa is an optional subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, L2 is a linker subunit optionally substituted with at least one saccharide unit, PEG unit, carboxyl unit, or combinations thereof, D is a Drug unit, and the wavy line (~) indicates an attachment site for a Stretcher unit), or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, [Su-aa] is [Su-Lys]. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0457] Exemplary embodiments of such drug-linkers include the following: [ka] wherein the amino group on the left side of the sugar unit is the attachment site for the stretcher unit, or a salt thereof.

[0458] In some embodiments, the drug-linker intermediate AA-L2-D has the following general formula: ~[SU-aa-SU] | L2-D

[0218] wherein [SU-aa-SU] is an amino acid unit, each SU is a saccharide unit, aa is a subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, L2 is a linker subunit optionally substituted with at least one saccharide unit, PEG unit, carboxyl unit, or combinations thereof, L2 is attached to a site on aa, D is a Drug unit, and the wavy line (~) indicates an attachment site for a Stretcher unit), or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, [Su-aa-Su] is [Su-Lys-Su]. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0459] Exemplary embodiments of such drug-linker intermediates include the following: [ka] wherein the amino group on the left side of the sugar unit is the attachment site for the stretcher unit, or a salt thereof.

[0460] In some embodiments, the linker intermediate ~AA-L2~ has the following general formula: [ka] where [SU-aa(PEG)-SU] is an amino acid unit, each SU is a sugar unit, aa is an AA subunit selected from alpha, beta, and gamma amino acids and derivatives thereof, PEG is a PEG unit attached to aa, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit, or combination thereof, L2 is attached to AA, the wavy line (~) indicates the attachment site for a Stretcher unit, and the double wavy line ( [ka] ) indicates a binding site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, linker subunit L2 is a cleavable linker subunit.

[0461] Exemplary embodiments of such linker intermediates include: [ka] wherein the amino group to the left of the Sugar unit is the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the Drug unit), or a salt thereof.

[0462] In some embodiments, the drug-linker intermediate AA-L2-D has the following general formula: ~[SU-aa(PEG)-SU]-L2-D

[0222] wherein [SU-aa(PEG)-SU] is an amino acid unit, each SU is a saccharide unit, aa is a subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, PEG is a PEG unit attached to aa, L2 is a linker subunit optionally substituted with at least one saccharide unit, PEG unit, carboxyl unit, or combinations thereof, L2 is attached to AA, D is a Drug unit, and the wavy line (~) indicates an attachment site for a Stretcher unit), or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0463] Exemplary embodiments of such drug-linker intermediates include the following: [ka] wherein the amino group on the left side of the sugar unit is the attachment site for the stretcher unit, or a salt thereof.

[0464] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] where [aa(PEG)] is an amino acid unit, aa is a subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, PEG is a PEG unit attached to aa, L2 is a linker subunit optionally substituted with at least one saccharide unit, PEG unit, carboxyl unit, or combination thereof, L2 is attached to AA, the wavy line (~) indicates the attachment site for a Stretcher unit, and the double wavy line ( [ka] ) indicates a binding site for a Drug unit) or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, linker subunit L2 is a cleavable linker subunit.

[0465] Exemplary embodiments of such linker intermediates include: [ka] [ka] wherein the amino group on the left side of the molecule is the attachment site for the Stretcher unit, or a salt thereof.

[0466] In some embodiments, ~AA-L2~ is one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] wherein each Z is joined by a * and is individually selected from: [ka] , and [ka] A linker intermediate or linker is provided having a wavy line over the amino group indicating the attachment site for a Stretcher unit and a Drug unit attached to the benzyl alcohol (i.e., the H of the benzyl alcohol is replaced with a bond to the Drug unit), or a salt thereof.

[0467] In some embodiments, the drug-linker intermediate AA-L2-D has the following general formula: ~[aa(PEG)]-L2-D

[0226] wherein [aa(PEG)] is an amino acid unit, aa is a subunit of AA selected from alpha, beta, and gamma amino acids and derivatives thereof, PEG is a PEG unit attached to aa, L2 is a linker subunit optionally substituted with at least one saccharide unit, PEG unit, carboxyl unit, or combinations thereof, L2 is attached to AA, D is a Drug unit, and the wavy line (~) indicates an attachment site for a Stretcher unit, or a salt thereof. In some embodiments, aa is an amino acid selected from glycine, lysine, and glutamic acid. In some embodiments, aa is lysine. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0468] Exemplary embodiments of such drug-linker intermediates include the following: [ka] [ka] wherein the amino group on the left side of the molecule is the attachment site for the Stretcher unit, or a salt thereof.

[0469] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] where there is no amino acid unit, L2 is a linker subunit, PEG is a PEG unit attached to L2, the wavy line (~) indicates the attachment site for the Stretcher unit, and the double wavy line ( [ka] ) indicates a linking site for the Drug unit) or a salt thereof. In some embodiments, the PEG unit is attached to an amino acid selected from lysine, glutamate, and citrulline. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0470] Exemplary embodiments of such linker intermediates include: [ka] wherein the amino group on the left side of the molecule is the attachment site for the Stretcher unit, or a salt thereof.

[0471] In some embodiments, the drug-linker intermediate ~L2-D has the following general formula: ~L2~D | PEG

[0230] wherein the Amino Acid unit is absent, L2 is a Linker subunit, D is a Drug unit, PEG is a PEG unit attached to L2, and the wavy line (~) indicates an attachment site for a Stretcher unit or an Amino Acid unit, or a salt thereof. In some embodiments, the PEG unit is attached to an amino acid selected from lysine, glutamate, and citrulline. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0472] Exemplary embodiments of such drug-linker intermediates include the following: [ka] wherein the amino group on the left side of the molecule is the attachment site for the Stretcher unit, or a salt thereof.

[0473] In some embodiments, the linker intermediate [ka] is of the general formula: [ka] where [CU] is an amino acid unit, CU is a carboxyl unit, L2 is a linker subunit optionally substituted with at least one sugar unit, PEG unit, carboxyl unit or combination thereof, L2 is attached to AA, the wavy line (~) indicates the attachment site for the Stretcher unit, and the double wavy line ( [ka] ) indicates an attachment site for a Drug unit) or a salt thereof. In some embodiments, the linker subunit L2 is a cleavable linker subunit.

[0474] Exemplary embodiments of such linker intermediates include: [ka] wherein the amino group on the left side of the molecule is the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol (i.e., an H is removed from the benzyl alcohol to form a bond between the benzyl oxygen and the Drug unit), or a salt thereof.

[0475] In some embodiments, the drug-linker intermediate AA-L2-D has the following general formula: ~[CU]-L2-D

[0234] wher...

Claims

1. The following formula (V): 【Chemistry 1】 where AA is an amino acid unit having 1 to 12 amino acid subunits; s is 0 or 1; L2 is a linker subunit having 1 to 4 attachment sites for Drug units; Each wavy line (~) indicates a binding site for a Stretcher unit; Double wavy line ( 【Chemistry 2】 ) indicates the binding site for the Drug unit) or a salt thereof, wherein at least one polar unit is present within the amino acid unit, the linker subunit, or both, and the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and combinations thereof.

2. Formula (I): 【Transformation 3】 where L1 is a Stretcher unit that has a binding site for a targeting unit; AA is an amino acid unit having 1 to 12 subunits; s is 0 or 1; L2 is a linker subunit having 1 to 4 attachment sites for Drug units; The wavy line (~) indicates the binding site for the targeting unit; Double wavy line ( 【Chemistry 4】 ) indicates an attachment site for a Drug unit) or a salt thereof, wherein at least one polar unit is present within the Amino Acid unit, the Linker subunit, the Stretcher unit, or a combination thereof, and the polar unit is selected from a saccharide unit, a PEG unit, a carboxyl unit, and a combination thereof.

3. The sugar unit has the following formula: L3-**N(CH 2 -(CH(XR)) k -X 1 (X) 2 )) 2 （X） wherein each X is independently selected from NH or O; each R is independently selected from hydrogen, acetyl, monosaccharides, disaccharides, and polysaccharides; each X 1 is CH 2 and C(O); each X 2 is independently selected from H, OH, and OR; k is 1 to 10; L3 is represented by the following general formula (XI): L3a ｜ *-NH-(CH 2 ) p -CH-(CH 2 ) o -C(O)-# (XI) (Wherein, L3a is C 1 -C 10 selected from alkylene and polyethylene glycol having 1 to 24 ethylene glycol subunits; p and o are independently 0 to 2; Each * and each # indicates a binding site for another subunit of the amino acid unit (AA), linker subunit L2 or stretcher unit (L1); 3. The linker intermediate or linker of claim 1, wherein L3a is covalently bonded to the N atom marked with ** in formula (X)) or a salt thereof) or a salt thereof.

4. The sugar unit is selected from the following formula: 【Transformation 5】 or 【Transformation 6】 wherein each R is independently selected from hydrogen, monosaccharides, disaccharides, and polysaccharides; p and o are independently 0 to 2; m is 1 to 8; n is 0 to 4; 4. The linker intermediate or linker of claim 1, wherein each * and each # indicates a binding site for another subunit of the amino acid unit (AA), the linker subunit L2 or the stretcher unit (L1), or a salt thereof.

5. The PEG unit is selected from the following formula: （a） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of the amino acid unit or a portion of the linker subunit L2; R 21 and R 22 are each independently an optional C 1 -C 3 alkylene; R 24 and R 25 are respectively H; polyhydroxyl group; substituted polyhydroxyl group; -C(O)-polyhydroxyl group; substituted -C(O)-polyhydroxyl group; optionally substituted C 3 -C 10 Carbocyclic ring; optionally substituted C 1 -C 3 Alkylene C 3 -C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted-C 1 -C 8 Alkyl; Substituted -C(O)-C 1 -C 8 Alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 are sugar units of formula (XII) or (XIII); or —NR 24 R 25 C 3 -C 8 Composed of heterocyclic rings; The wavy line (~) is R 20 indicates the binding site for n20 is 1 to 26; or a salt thereof. or （b） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NR 24 R 25 (XX) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit or a part of the linker subunit L2; R 21 and R 22 are each independently an optional C 1 -C 3 alkylene; R 24 and R 25 one of which is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C 3 -C 10 Carbocyclic ring; optionally substituted C 1 -C 3 Alkylene C 3 -C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted-C 1 -C 8 Alkyl; Substituted -C(O)-C 1 -C 8 Alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); R 24 and R 25 the other optionally is polyethylene glycol having 1 to 24 ethylene glycol subunits; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; or a salt thereof. or （c） ~R 20 -[-R 26 -[R 29 -[O-CH 2 -CH 2 -] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXI) (In the formula, R 20 is a functional group for binding to a subunit of an amino acid unit and / or a portion of the linker subunit L2; R 26 and R 27 are optional, and C 1 -C 12 Alkylene, -NH-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene -NH-, -C(O)-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene-C(O)-, -NH-C 1 -C 12 Alkylene -C(O)- and -C(O)-C 1 -C 12 alkylene-NH-; R 24 and R 25 one of which is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C 3 -C 10 Carbocyclic ring; optionally substituted C 1 -C 3 Alkylene C 3 -C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted-C 1 -C 8 Alkyl; Substituted -C(O)-C 1 -C 8 Alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); R 24 and R 25 the other is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C 3 -C 10 Carbocyclic ring; optionally substituted C 1 -C 3 Alkylene C 3 -C 10 Carbocycle; Optionally substituted heteroaryl; Optionally substituted carbocycle; Substituted-C 1 -C 8 Alkyl; Substituted -C(O)-C 1 -C 8 Alkyl; Chelating agent; -C(O)-R 28 (In the formula, R 28 is a sugar unit of formula (XII) or (XIII); and optionally polyethylene glycol having 1 to 24 ethylene glycol subunits; or -NR 24 R 25 C 3 -C 8 Composed of heterocyclic rings; Each R 29 is optional, -C(O)-, -NH-, -C(O)-C 1 -C 6 Alkenylene-, -NH-C 1 -C 6 Alkenylene-, -C 1 -C 6 Alkenylene-NH-, -C 1 -C 6 independently selected from alkenylene-C(O)-, -NH(CO)NH-, and triazole; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; n21 is 1 to 4; n27 is 1 to 4; or a salt thereof.

6. R 24 and R 25 and n is 0 or 1. The linker intermediate or linker of claim 5, wherein both are not H.

7. R 24 and R 25 are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 7. The linker intermediate or linker of claim 5 or 6, provided that both of

8. 8. The linker intermediate or linker of any one of claims 5 to 7, wherein the polyhydroxyl group is optionally a straight chain monosaccharide selected from a C6 or C5 sugar, a sugar acid, or an amino sugar.

9. the C6 or C5 sugar is selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, and ketose; the sugar acid is selected from gluconic acid, aldonic acid, uronic acid, and uronic acid; or 9. The linker intermediate or linker of claim 8, wherein the amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

10. 10. The linker intermediate or linker of any one of claims 5 to 9, or a salt thereof, wherein the PEG unit is selected from the following: 【Transformation 7】 (In the formula, R 39 is selected from H, a linear monosaccharide, and optionally, polyethylene glycol having 1 to 24 ethylene glycol subunits; the wavy line on the left indicates the site of attachment of the amino acid unit to the subunit or to the portion of the linker subunit).

11. R 24 and R 25 7. The linker intermediate or linker according to claim 5 or 6, wherein one of said monosaccharides is a linear monosaccharide and the other is a cyclic monosaccharide.

12. 12. The linker intermediate or linker of claim 11, wherein the PEG unit is selected from the following: 【Transformation 8】 (In the formula, R 41 is a cyclic monosaccharide; the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or the portion of the linker subunit).

13. R 24 and R 25 7. The linker intermediate or linker of claim 5 or 6, wherein is independently selected from cyclic monosaccharides, disaccharides and polysaccharides.

14. 14. The linker intermediate or linker of claim 13, wherein the PEG unit is selected from the following: 【Chemistry 9】 or 【Chemistry 10】 (In the formula, each R 45 is selected from H and a monosaccharide, disaccharide, or polysaccharide; R 46 is selected from cyclic monosaccharides, disaccharides, or polysaccharides; the wavy line on the right indicates the attachment site of the amino acid unit to the subunit or the portion of the linker subunit).

15. R 24 and R 25 are independently selected from a linear monosaccharide and a substituted linear monosaccharide, wherein the substituted linear monosaccharide is substituted with a monosaccharide, a disaccharide, or a polysaccharide.

16. 16. The linker intermediate or linker of claim 15, wherein the PEG unit is selected from the following: 【Chemistry 11】 (In the formula, R 47 is a linear monosaccharide; each R 49 is selected from monosaccharides, disaccharides and polysaccharides; the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or to the portion of the linker subunit).

17. R 24 and R 25 are independently selected from linear monosaccharides and substituted monosaccharides, wherein said substituted linear monosaccharides are substituted with one or more substituents selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester or amide, and optionally further substituted with a monosaccharide, disaccharide or polysaccharide.

18. 18. The linker intermediate or linker of claim 17, wherein the PEG unit is selected from the following: 【Chemistry 12】 or 【Chemistry 13】 (In the formula, each R 42 are independently selected from linear monosaccharides and substituted linear monosaccharides; each R 43 are independently selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, and amide; the wavy line on the left indicates the site of attachment of the amino acid unit to the subunit or the portion of the linker subunit).

19. R 24 and R 25 is a -C(O)-polyhydroxyl group or a substituted -C(O)-polyhydroxyl group, and R 24 and R 25 and the other is H, -C(O)-polyhydroxyl, substituted -C(O)-polyhydroxyl, polyhydroxyl or substituted polyhydroxyl; and the substituted -C(O)-polyhydroxyl and polyhydroxyl groups are substituted with monosaccharide, disaccharide, polysaccharide, alkyl, -O-alkyl, aryl, carboxyl, ester or amide.

20. 20. The linker intermediate or linker of claim 19, wherein the PEG unit is selected from the following: 【Chemistry 14】 or 【Chemistry 15】 (where the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or the portion of the linker subunit).

21. R 24 and R 25 But H, substitution -C 1 -C 8 Alkyl, substituted -C 1 -C 4 Alkyl or substituted -C 1 -C 3 alkyl, with the proviso that R 24 and R 25 and C are not H; 1 -C 8 Alkyl, -C 1 -C 4 Alkyl and -C 1 -C 3 7. The linker intermediate or linker of claim 5 or 6, wherein the alkyl is substituted with hydroxyl and / or carboxyl.

22. 22. The linker intermediate or linker of claim 21 , wherein the PEG unit is selected from the following: 【Chemistry 16】 or 【Chemistry 17】 (In the formula, R 48 are H, OH, and CH 2 -C substituted with OH, COOH, or hydroxyl or carboxyl 1 -C 6 alkyl; the wavy line on the left indicates the site of attachment of the amino acid unit to the subunit or the portion of the linker subunit).

23. R 24 and R 25 One of the groups is H, substituted -C(O)-C 1 -C 8 Alkyl, substituted -C(O)-C 1 -C 4 Alkyl and substituted -C(O)-C 1 -C 3 alkyl, and R 24 and R 25 The other is a substituted -C(O)-C 1 -C 8 Alkyl, substituted -C(O)-C 1 -C 4 Alkyl, substituted -C(O)-C 1 -C 3 Alkyl, substituted -C 1 -C 8 Alkyl, substituted -C 1 -C 4 Alkyl and Substituted-C 1 -C 3 alkyl, substituted —C(O)—C 1 -C 8 Alkyl, substituted -C(O)-C 1 -C 4 Alkyl, substituted -C(O)-C 1 -C 3 Alkyl, substituted -C 1 -C 8 Alkyl, -C 1 -C 4 Alkyl and -C 1 -C 3 7. The linker intermediate or linker of claim 5 or 6, wherein the alkyl is substituted with hydroxyl and / or carboxyl.

24. 24. The linker intermediate or linker of claim 23, wherein the PEG unit is selected from the following: 【Chemistry 18-1】 【Chemistry 18-2】 or 【Chemistry 19】 (where the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or the portion of the linker subunit).

25. R 24 and R 25 is selected from H and optionally substituted aryl, with the proviso that R 24 and R 25 7. The linker intermediate or linker of claim 5 or 6, provided that both of

26. 26. The linker intermediate or linker of claim 25, wherein the PEG unit is selected from the following: 【Chemistry 20】 or 【Chemistry 21】 (where the wavy line on the left indicates the binding site of the subunit of the amino acid unit or the portion of the linker subunit).

27. R 24 and R 25 together with an optionally substituted C 3 -C 8 7. The linker intermediate or linker of claim 5 or 6, which forms a heterocycle or heteroaryl.

28. The PEG units are 【Chemistry 22】 ’ 28. The linker intermediate or linker of claim 27, which is:

29. R 24 and R 25 is independently selected from H and a chelator, and said chelator is optionally linked to —NR by alkylene, arylene, carbocyclo, heteroarylene, or heterocarbocyclo. 24 R 25 is bonded to the nitrogen of R 24 and R 25 7. The linker intermediate or linker of claim 5 or 6, provided that both of

30. 30. The linker intermediate or linker of claim 29, wherein the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), benzyl-DTPA, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), benzyl-DOTA, 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), benzyl-NOTA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), and N,N'-dialkyl substituted piperazines.

31. 31. The linker intermediate or linker of claim 30, wherein the PEG unit is selected from the following: 【Chemistry 23】 or 【Chemistry 24】 (where the wavy line on the left indicates the attachment site of the amino acid unit to the subunit or the portion of the linker subunit).

32. Each monosaccharide is C5 or C6 sugars selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altose, gulose, idostulose, aldose, ketose, glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; a sugar acid selected from gluconic acid, aldonic acid, uronic acid, and urosonic acid; or 20. The linker intermediate or linker of any one of claims 5 to 19, wherein the amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine.

33. R 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

34. The PEG unit is selected from the following formula: （a） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -R 30 (XXX) (In the formula, R 20 is a functional group for attachment to a subunit of said amino acid unit (if present) and / or part of the linker subunit L2; R 21 and R 22 are each optional and, if present, independently 1 -C 3 an alkylene group; R 30 is an optionally substituted C 3 -C 10 Carbocycle; Thiourea; optionally substituted thioureas; ureas; optionally substituted urea; sulfamide; alkylsulfamide; acylsulfamide, optionally substituted alkylsulfamide; optionally substituted acylsulfamide; sulfonamide; optionally substituted sulfonamide; guanidine (including alkyl and aryl guanidine); phosphoramide; or optionally substituted phosphoramide; or R 30 is azide, alkynyl, substituted alkynyl, -NH-C(O)-alkynyl, -NH-C(O)-alkynyl-R 65 Cyclooctyne; -NH-cyclooctyne, -NH-C(O)-cyclooctyne or -NH-(cyclooctyne) 2 Selected from: R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; or a salt thereof. （b） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NH-C(O)-R 31 (XXXI) (In the formula, R 20 is a functional group for attachment to a subunit of the amino acid unit (if present) or to a portion of the linker subunit L2; R 21 and R 22 are each independently an optional C 1 -C 3 an alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 having R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; or a salt thereof. （c） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -C(O)NH-R 31 (XXXII) (In the formula, R 20 is a functional group for attachment to a subunit of the amino acid unit (if present) or to a portion of the linker subunit L2; R 21 and R 22 are each optional and independently 1 -C 3 an alkylene group; R 31 is a branched polyethylene glycol chain, each branch independently having 1 to 26 ethylene glycol subunits, and each branch having at its terminus R 35 having R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, and optionally substituted heteroaryl; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; or a salt thereof, and （d） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -N-(R 33 -R 31 ) 2 (XXXIII) (In the formula, R 20 is a functional group for attachment to a subunit of the amino acid unit (if present) or to a portion of the linker subunit L2; R 21 and R 22 are optional, and C 1 -C 3 an alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 having R 33 is C 1 -C 3 Alkylene, C 1 -C 3 Alkylene-C(O), -C(O)-C 1 -C 3 Alkylene or -C(O)-C 1 -C 3 alkylene-C(O); R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; The wavy line (~) is R 20 indicating the binding site to n20 is 1 to 26; or a salt thereof.

35. 5. The linker intermediate or linker of any one of claims 1 to 4, wherein the PEG unit has a formula selected from the following: or a salt thereof: ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NH-C(O)-R 31 (XXXI), ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -C(O)NH-R 31 (XXXII), or ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -N-(R 33 -R 31 ) 2 (XXXIII); (In the formula, R 20 is a functional group for binding to a subunit of the amino acid unit (if present) or a portion of the linker subunit L2; R 21 and R 22 are optional, and C 1 -C 3 an alkylene group; R 31 is a branched polyethylene glycol chain, each branch having 1 to 26 ethylene glycol subunits, and each branch having at its end R 35 R 33 is C 1 -C 3 Alkylene, -C 1 -C 3 Alkylene-C(O), -C(O)-C 1 -C 3 Alkylene or -C(O)-C 1 -C 3 alkylene-C(O); R 35 is azide, alkynyl, alkynyl-R 65 , cyclooctyne or cyclooctyne-R 65 and R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle, or optionally substituted heteroaryl; the wavy line (~) indicates R 20 n20 is 1-26).

36. 36. The linker intermediate or linker of claim 35, wherein the PEG unit is selected from: 【Chemistry 25】 (In the formula, R 65 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle, optionally substituted aryl, optionally substituted heterocarbocycle or optionally substituted heteroaryl; the wavy line on the left indicates the site of attachment of the amino acid unit to the subunit or to the portion of the linker subunit).

37. R 20 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

38. A formula selected from the following: ~R 40 -(R 43 -R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XL) (In the formula, R 40 is a functional group for binding to a subunit of the amino acid unit or a portion of the linker subunit L2; R 41 and R 42 are absent or, independently, C 1 -C 6 alkylene; Each R 43 is independently absent or C 1 -C 12 Alkylene, -NH-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene -NH-, -C(O)-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene-C(O)-, -NH-C 1 -C 12 Alkylene -C(O)-, -C(O)-C 1 -C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C 1 -C 12 Alkylene, -C(O)-NH-C 1 -C 12 Alkylene, -heteroarylene, heteroaryl-C 1 -C 12 Alkylene-C 1 -C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C 1 -C 12 alkylene and the other is C 1 -C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicating the binding site to n40 is 1 to 26; n41 is 1 to 6; 5. The linker intermediate or linker of claims 1 to 4, comprising a PEG unit having n42, n43, n44, n45, n46, n47, n48, n49, n50, n51, n52, n53, n54, n55, n56, n57, n58, n59, n60, n61, n62, n63, n64, n65, n66, n67

39. A formula selected from the following: ~R 40 -(R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) (In the formula, R 40 is a functional group for binding to a subunit of the amino acid unit or a portion of the linker subunit L2; R 41 and R 42 are absent or, independently, C 1 -C 6 alkylene; R 43 does not exist or is C 1 -C 12 Alkylene, -NH-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene -NH-, -C(O)-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene-C(O)-, -NH-C 1 -C 12 Alkylene -C(O)-, -C(O)-C 1 -C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C 1 -C 12 Alkylene, C(O)-NH-C 1 -C 12 Alkylene, -heteroarylene, heteroaryl-C 1 -C 12 Alkylene, Heteroaryl-C 1 -C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C 1 -C 12 alkylene and the other is C 1 -C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicating the binding site to n40 is 1 to 26; n41 is 1 to 6; 5. The linker intermediate or linker of claims 1 to 4, comprising a PEG unit having n42, n43, n44, n45, n46, n47, n48, n49, n50, n51, n52, n53, n54, n55, n56, n57, n58, n59, n60, n61, n62, n63, n64, n65, n66, n67

40. A formula selected from the following: ~R 40 -(R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) (In the formula, R 40 is a functional group for binding to a subunit of the amino acid unit or a portion of the linker subunit L2; R 41 and R 42 are absent or, independently, C 1 -C 3 alkylene; R 43 does not exist or is C 1 -C 6 Alkylene, -NH-C 1 -C 12 Alkylene, -C 1 -C 6 Alkylene -NH-, -C(O)-C 1 -C 6 Alkylene, -C 1 -C 6 Alkylene-C(O)-, -NH-C 1 -C 6 Alkylene -C(O)-, -C(O)-C 1 -C 6 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C 1 -C 6 Alkylene, -C(O)-NH-C 1 -C 12 Alkylene, -heteroarylene, heteroaryl-C 1 -C 6 Alkylene, Heteroaryl-C 1 -C 6 Alkylene -C(O)- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C 1 -C 6 alkylene and the other is C 1 -C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; The wavy line (~) is R 40 indicating the binding site to n40 is 1 to 16; n41 is 1 to 4; 5. The linker intermediate or linker of claims 1 to 4, comprising a PEG unit having n42, n43, n44, n45, n46, n47, n48, n49, n50, n51, n52, n53, n54, n55, n56, n57, n58, n59, n60, n61, n62, n63, n64, n65, n66, n67

41. R 40 is selected from halo, aldehyde, carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

42. R 40 but one of the following structures: 【Chemistry 26】 or 【Chemistry 27】 (Wherein, R=H or C 1－6 alkyl; and n＝0～12 (*) indicates R for a subunit of the amino acid unit or a part of the linker subunit L2. 40 and ( 【Chemistry 28】 ) is the R 40 41. The linker intermediate or linker of any one of claims 38 to 40, having a linking site of:

43. R 40 but one of the following structures: 【Chemistry 29】 or 【Transformation 30】 (In the formula, n = 0 to 12 (*) indicates R for a subunit of the amino acid unit or a part of the linker subunit L2. 40 and ( 【Chemistry 31】 ) is the R 40 43. The linker intermediate or linker of claim 42, having a linking site of:

44. R 43 - (NR 44 R 45 ) n41 But NR 43 If present, one of the following structures: 【Chemistry 32】 or 【Transformation 33】 (In the formula, R=H, C 1－6 Alkyl, polyhydroxyl, or substituted polyhydroxyl （ 【Transformation 34】 ) is the R 43 41. The linker intermediate or linker of claims 38 to 40, having a linking site of:

45. R 43 - (NR 44 R 45 ) n41 But NR 43 If present, one of the following structures: 【Chemistry 35】 or 【Transformation 36】 (In the formula, ( 【Chemistry 37】 ) is the R 43 45. The linker intermediate or linker of claim 44, having a linking site of:

46. -NR 44 R 45 is one of the following structures: 【Transformation 38】 or 【Chemistry 39】 (In the formula, ( 【Chemistry 40】 ) is —NR ) to the remainder of the PEG unit. 44 R 45 41. The linker intermediate or linker of any one of claims 38 to 40, having a linking site of:

47. 47. The linker intermediate or linker of any one of claims 1 to 46, wherein the PEG unit, prior to attachment to the amino acid unit or portion of the linker subunit L2, has one of the following structures: 【Chemistry 41-1】 【Chemistry 41-2】 【Chemistry 41-3】 【Chemistry 41-4】 or 【Chemistry 42】 where R is H or alkyl and n is 1 to 12.

48. A formula selected from the following: ~R 40 -(R 43 -R 41 --[O-CH 2 -CH 2 ] n40 -R 46 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLIII) (In the formula, R 40 is a functional group for binding to a subunit of the amino acid unit or a portion of the linker subunit L2; R 41 and R 42 are absent or, independently, C 1 -C 6 alkylene; Each R 43 is independently absent or C 1 -C 12 Alkylene, -NH-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene -NH-, -C(O)-C 1 -C 12 Alkylene, -C 1 -C 12 Alkylene-C(O)-, -NH-C 1 -C 12 Alkylene -C(O)-, -C(O)-C 1 -C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C 1 -C 12 Alkylene, -C(O)-NH-C 1 -C 12 Alkylene, -heteroarylene, heteroaryl-C 1 -C 12 Alkylene-C 1 -C 12 Alkylene- or -C(O)NR 46 R 47 Selected from R 46 and R 47 One of the two is H or C 1 -C 12 alkylene and the other is C 1 -C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, wherein the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; R 46 is amino, amino-alkyl-amino, or -NH-C(O)-NH-S(O) 2 -NH-; The wavy line (~) is R 40 indicating the binding site to n40 is 1 to 26; n41 is 1 to 6; 5. The linker intermediate or linker of claims 1 to 4, comprising a PEG unit having n42, n43, n44, n45, n46, n47, n48, n49, n50, n51, n52, n53, n54, n55, n56, n57, n58, n59, n60, n61, n62, n63, n64, n65, n66, n67

49. 49. The linker intermediate or linker of claim 48, wherein the PEG unit, prior to attachment to the amino acid unit or portion of the linker subunit L2, has one of the following structures: 【Chemistry 43】 where R is H or alkyl and n is 1 to 12.

50. A formula selected from the following: 【Chemistry 44】 or 【Chemistry 45】 wherein each Y is independently R 76 or 【Chemistry 46】 and Each R 76 are independently H, acetyl, -P(=O)(OH) 2 , or -(CH 2 ) v -O-S (=O) 2 (OH); Each R a and R b are independently H or R a and R b together with the carbon to which they are attached form an oxo group; each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; 5. The linker intermediate or linker of claim 1, comprising a PEG unit having a * or salt thereof, wherein each * indicates a binding site for the amino acid unit (AA), the linker subunit L2, or a subunit of the stretcher unit (L1).

51. A formula selected from the following: 【Chemistry 47】 or 【Chemistry 48】 (In the formula, each R 76 are independently H, acetyl, -P(=O)(OH) 2 or - (CH 2 ) v S (=O) 2 (OH); each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; 51. The linker intermediate or linker of claim 50, comprising a PEG unit having a *, or a salt thereof, wherein each * indicates a binding site for the amino acid unit (AA), the linker subunit L2, or a subunit of the Stretcher unit (L1).

52. A formula selected from the following: 【Chemistry 49】 or [Transformation 50] wherein each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; 52. The linker intermediate or linker of claim 50 or 51, comprising a PEG unit having a *, or a salt thereof, wherein each * indicates a binding site for the amino acid unit (AA), the linker subunit L2, or a subunit of the Stretcher unit (L1).

53. Y is R 76 51. The linker intermediate or linker of claim 50, wherein:

54. Y is 【Chemistry 51】 51. The linker intermediate or linker of claim 50, wherein:

55. Each R a and R b is independently H.

56. R a and R b together with the carbon to which they are attached form an oxo group.

57. 53. The linker intermediate or linker of any one of claims 50 to 52, wherein q is 10 to 20.

58. 53. The linker intermediate or linker of any one of claims 50 to 52, wherein q is 12.

59. 53. The linker intermediate or linker of any one of claims 1 to 4, 38 to 40, or 50 to 52, or a salt thereof, wherein the PEG unit is selected from the following: 【Chemistry 52-1】 【Chemistry 52-2】 【Chemistry 52-3】 【Chemistry 52-4】 【Chemistry 52-5】 【Chemistry 52-6】 【Chemistry 52-7】 【Chemistry 52-8】 【Chemistry 52-9】 【Chemistry 52-10】 【Chemistry 52-11】 【Chemistry 52-12】 and 【Chemistry 53】 wherein each Z is joined by a * and is individually selected from: 【Chemistry 54】 and 【Transformation 55】 each 【Transformation 56】 indicates a binding site for the amino acid unit (AA), the linker subunit L2 or another subunit of the stretcher unit (L1).

60. 5. The linker intermediate or linker of any one of claims 1 to 4, wherein the carboxyl unit has the following formula or a salt thereof: R 70 ｜ L 70 ｜ ~NH-(CH 2 ) p1 -CH-(CH 2 ) o1 -C(O)~ (XXXX) (In the formula, （a） L 70 is C 1 -C 8 Alkylene, C 1 -C 8 Alkylene -C(O)-, -C(O)-C 1 -C 8 Alkylene- and -C(O)-C 1 -C 8 alkylene-C(O)-; R 70 , ~NR 71 (R 72 -R 73 ) where R 71 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 alkyl or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 72 is absent or optionally replaced by C 1 -C 3 selected from alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl; R 73 is a carboxyl or polycarboxyl, said polycarboxyl containing 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), the linker subunit L2, or the stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or （b） L 70 is C 1 -C 8 Alkylene, C 1 -C 8 Alkylene -C(O)-, -C(O)-C 1 -C 8 Alkylene- and -C(O)-C 1 -C 8 alkylene-C(O)-; R 70 , ~NR 71 (R 75 - (R 73 ) 2 ) where R 71 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 alkyl or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 75 is a branched, optionally substituted C 1 -C 3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl, and each R 73 independently comprise carboxyl or polycarboxyl, wherein the polycarboxyl comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, said carboxyl groups being interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), the linker subunit L2, or the stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or （c） L 70 is C 1 -C 8 Alkylene, C 1 -C 8 Alkylene -C(O)-, -C(O)-C 1 -C 8 Alkylene- and -C(O)-C 1 -C 8 alkylene-C(O)-; R 70 is ~N (R 74 -R 73 ) (R 72 -R 73 ) where R 72 and R 74 each independently represents an optionally substituted C 1 -C 3 selected from alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl; and each R 73 are independently carboxyl or polycarboxyl and contain 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, said carboxyl groups being interconnected by alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkyl, heteroalkylene, amino and / or amido; Each wavy line (~) indicates a binding site for another subunit of the amino acid unit (AA), the linker subunit L2, or the stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2).

61. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least one sugar unit.

62. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least one PEG unit.

63. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least one carboxyl unit.

64. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least two polar units, each polar unit selected from a sugar unit, a PEG unit, and a carboxyl unit.

65. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least one sugar unit and a PEG unit or a carboxyl unit.

66. 61. The linker intermediate or linker of any one of claims 1 to 60, comprising at least one carboxyl unit and a PEG unit.

67. 61. The linker intermediate or linker of any one of claims 1 to 60, wherein the amino acid unit (AA) is present (s=1).

68. 68. The linker intermediate or linker of any one of claims 1 to 67, wherein the amino acid unit comprises at least one polar unit.

69. 69. The linker intermediate or linker of any one of claims 1 to 68, wherein L2 or AA-L2 has one of the following structures: 【Chemistry 57】 or 【Transformation 58】 (where the wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol).

70. 68. The linker intermediate or linker of any one of claims 1 to 67, wherein ~AA-L2~ has a formula selected from: 【Chemistry 59】 、 【Transformation 60】 ,or 【Chemistry 61】 where the square brackets represent the amino acid units, each aa is an optional subunit of AA, L2 is the linker subunit, and each wavy line (~) represents a binding site for a Stretcher unit; 1 (PEG) is a PEG unit attached to an amino acid subunit of AA, SU is a sugar unit attached to a subunit of AA or L2, and CU is a carboxyl unit attached to a subunit of AA or L2; 【Transformation 62】 ) indicates the binding site for the drug unit, and aa and aa 1 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

71. 68. The linker intermediate or linker of any one of claims 1 to 67, wherein ~AA-L2~ has a formula selected from: 【Transformation 63】 ,or 【Chemistry 64】 (wherein the square brackets represent the amino acid units, each aa is an amino acid subunit of AA, L2 is the linker subunit attached to the side chain of aa, and the wavy line (~) represents the attachment site for the Stretcher unit; aa 1 (PEG) is a PEG unit attached to aa, SU is a sugar unit attached to aa, CU is a carboxyl unit attached to aa, and the double wavy line ( 【Transformation 65】 ) indicates the attachment site for the Drug unit; aa and aa 1 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

72. 69. The linker intermediate or linker of any one of claims 1 to 68, wherein the amino acid unit comprises at least two polar units.

73. 73. The linker intermediate or linker of claim 72, wherein ~AA-L2~ has a formula selected from: 【Chemical Formula 66】 、 【Transformation 67】 ,or 【Transformation 68】 where the square brackets represent the amino acid units, aa is an optional subunit of AA, L2 is the linker subunit, and the wavy line (~) represents the attachment site for the Stretcher unit; 1 (PEG) and aa 2 Each of the (PEG)s is a PEG unit bonded to an aa or other PEG unit; each SU is a saccharide unit bonded to an aa or other saccharide unit; each CU is a carboxyl unit bonded to an aa or other carboxyl unit, and is represented by a double wavy line ( 【Transformation 69】 ) indicates the attachment site for the Drug unit; aa, aa 1 and aa 2 are independently selected from alpha, beta and gamma amino acids and derivatives thereof).

74. 73. The linker intermediate or linker of claim 72, wherein ~AA-L2~ has a formula selected from: 【Transformation 70】 ,or 【Chemistry 71】 (wherein the square brackets represent the amino acid units, aa is an amino acid subunit of AA, L2 is a linker subunit attached to the side chain of aa, and each wavy line (~) represents a binding site for a Stretcher unit; aa 1 (PEG) and aa 2 Each of (PEG) is a PEG unit attached to aa, SU is a sugar unit attached to aa; each CU is a carboxyl unit attached to aa; 【Chemistry 72】 ) indicates the attachment site for the Drug unit; aa, aa 1 and aa 2 each of which is independently selected from alpha, beta and gamma amino acids and derivatives thereof).

75. 75. The linker intermediate or linker of any one of claims 1 to 74, wherein the linker subunit L2 is a cleavable linker unit.

76. 76. The linker intermediate or linker of claim 75, wherein the linker subunit L2 comprises a peptide that is cleavable by an intracellular protease.

77. 77. The linker intermediate or linker of claim 76, wherein the cleavable peptide comprises a valine-citrulline peptide, a valine-alanine peptide, a valine-lysine peptide, a phenylalanine-lysine peptide, or a glycine-glycine-phenylalanine-glycine peptide.

78. 78. The linker intermediate or linker of any one of claims 1 to 77, wherein the linker subunit L2 comprises at least one polar unit.

79. 79. The linker intermediate or linker of any one of claims 1 to 78, wherein the polar unit is a sugar unit (SU).

80. 80. The linker intermediate or linker of claim 79, wherein the cleavable peptide comprises an SU-valine-citrulline peptide, an SU-valine-lysine peptide, an SU-valine-alanine peptide, an SU-phenylalanine-lysine peptide, or an SU-glycine-glycine-phenylalanine-glycine peptide.

81. 79. The linker intermediate or linker of claim 78, wherein the polar unit is a carboxyl unit (CU).

82. 82. The linker intermediate or linker of claim 81, wherein the cleavable peptide comprises a CU-valine-citrulline peptide, a CU-valine-lysine peptide, a valine-(CU-lysine) peptide, a CU-valine-alanine peptide, a CU-phenylalanine-lysine peptide, a phenylalanine-(CU-lysine) peptide, or a CU-glycine-glycine-phenylalanine-glycine peptide, wherein CU-lysine is a carboxyl unit comprising a lysine residue.

83. 79. The linker intermediate or linker of claim 78, wherein the polar unit is a PEG unit (PEG).

84. 84. The linker intermediate or linker of claim 83, wherein the cleavable peptide comprises a Lys(PEG)-valine-citrulline peptide, a valine-Cit(PEG) peptide, a Lys(PEG)-valine-lysine peptide, a valine-lysine(PEG) peptide, a Lys(PEG)-valine-alanine peptide, a Lys(PEG)-phenylalanine-lysine peptide, a phenylalanine-Lys(PEG) peptide, or a Lys(PEG)-glycine-glycine-phenylalanine-glycine peptide, wherein Lys(PEG) and Cit(PEG) comprise PEG units attached to a lysine or citrulline residue, respectively.

85. 85. The linker intermediate or linker of any one of claims 75 to 84, wherein the cleavable peptide is attached to a para-aminobenzyl alcohol self-immolative group (PABA). 【Request Item 86】 【Chemistry 73】 86. The linker intermediate or linker of claim 85, wherein said linker intermediate or linker has one of the following structures: 【Chemistry 74-1】 【Chemistry 74-2】 【Chemistry 74-3】 (where the wavy line over the amino group indicates the attachment site for the Stretcher unit, and the Drug unit is attached to the benzyl alcohol).

87. 3. The linker intermediate or linker of claim 1 or 2, wherein ~AA-L2~ has one of the following structures: 【Chemistry 75-1】 【Chemistry 75-2】 【Chemistry 75-3】 【Chemistry 75-4】 【Chemistry 75-5】 【Chemistry 75-6】 【Chemistry 75-7】 【Chemistry 75-8】 【Chemistry 75-9】 【Chemistry 75-10】 【Chemistry 75-11】 【Chemistry 75-12】 【Chemistry 75-13】 【Chemistry 75-14】 【Chemistry 75-15】 【Chemistry 75-16】 【Chemistry 75-17】 【Chemistry 75-18】 【Chemistry 75-19】 【Chemistry 75-20】 or 【Transformation 76】 wherein each Z is joined by a * and is individually selected from: 【Chemical 77】 and 【Transformation 78】 The wavy line over the amino group indicates the attachment site for a Stretcher unit, and the Drug unit is attached to the benzyl alcohol (i.e., the H of the benzyl alcohol is replaced by a bond to the Drug unit).

88. 86. The linker intermediate or linker of any one of claims 75 to 85, wherein L2 is attached to a side chain of a subunit of AA. 【Request Item 89】 【Chemistry 79】 89. The linker intermediate or linker of claim 88, wherein said linker intermediate or linker has one of the following structures: 【Chemistry 80】 or 【Chemistry 81】 where the wavy line over the amino group indicates the attachment site for a Stretcher unit, and the Drug unit is attached to the terminal acid group, the benzyl alcohol, or the wavy line ( 【Chemistry 82】 ) indicates the binding site for the Drug unit).

90. 86. The linker intermediate or linker of any one of claims 1 to 85, wherein the amino acid unit is linked to the linker subunit L2 by a non-peptidic linking group.

91. The non-peptidic linking group is C 1 -C 10 Alkylene, C 2 -C 10 Alkenylene, C 2 -C 10 91. The linker intermediate or linker of claim 90, selected from alkynylene or polyethylene glycol.

92. 92. The linker intermediate or linker of any one of claims 1 to 91, further comprising a Stretcher unit.

93. The stretcher unit is selected from: 【Chemistry 83-1】 【Chemistry 83-2】 or 【Chemical 84】 (In the formula, R 17 is -C 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 3 -C 8 Carbocyclo-, -O-(C 1 -C 8 alkylene)-, -(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b - (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene- (wherein b is 1 to 26), -arylene-, -C 1 -C 10 Alkylene-arylene-, -arylene-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-, -C 3 -C 8 Heterocyclo-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-C(=O)-, C 1 -C 10 Heteroalkylene -C(=O)-, -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b -C(=O)- (wherein b is 1 to 26), -(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-C(═O)- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-C(═O)- (wherein b is 1 to 26), -C 3 -C 8 Carbocyclo-C(=O)-, -O-(C 1 -C 8 alkyl)-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-C(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 3 -C 8 Heterocyclo-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-C(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Heteroalkylene -NH-, -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b -NH- (wherein b is 1 to 26), -(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-NH- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-NH- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(C(=O))-NH-(CH 2 -O-CH 2 ) b -C(=O)- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-(C(=O))-NH-(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-C(═O)- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-NH-(C(=O))-(CH 2 -O-CH 2 ) b -NH- (wherein b is 1 to 26), -C 1 -C 8 Alkylene-NH-(C(=O))-(CH 2 -O-CH 2 ) b -C 1 -C 8 alkylene-NH- (wherein b is 1 to 26), -C 3 -C 8 Carbocyclo-NH-, -O-(C 1 -C 8 alkyl)-NH-, -arylene-NH-, -C 1 -C 10 Alkylene-arylene-NH-, -arylene-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 carbocyclo)-NH-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-NH-, -C 3 -C 8 Heterocyclo-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 heterocyclo)-NH-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-S-, C 1 -C 10 Heteroalkylene -S-, -C 3 -C 8 Carbocyclo-S-, -O-(C 1 -C 8 alkyl)-S-, -arylene-S-, -C 1 -C 10 Alkylene-arylene-S-, -arylene-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-S-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-S-, -C 3 -C 8 Heterocyclo-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 heterocyclo)-S-, or -(C 3 -C 8 Heterocyclo)-C 1 -C 10 alkylene-S-) or The Stretcher unit is a maleimide (C 1 -C 10 Alkylene-C(O)-, maleimide (CH 2 OCH 2 ) p2 (C 1 -C 10 Alkyne) C(O)-, Maleimide (C 1 -C 10 Alkyen) (CH 2 OCH 2 ) p2 93. The linker of claim 92, comprising C(O)-, wherein p2 is 1 to 26, or an open form thereof.

94. 93. The linker intermediate or linker of claim 92, wherein said Stretcher unit is selected from: 【Chemical 85】 and 【Chemical 86】 (Where, the wavy line 【Transformation 87】 indicates the attachment site for the Stretcher unit to an Amino Acid unit, and the attachment site for the Targeting unit is on a maleimide, primary amine, or alkyne functional group).

95. 94. The linker of claim 93, having one of the following structures: 【Chemistry 88-1】 【Chemistry 88-2】 【Chemistry 88-3】 【Chemistry 88-4】 wherein the Drug unit is attached to a terminal acid group, a benzyl alcohol, or is represented by a wavy line ( 【Chemistry 89】 ) indicates the binding site for the Drug unit).

96. 93. The linker of claim 92, having one of the following structures: 【Chemistry 90-1】 【Chemistry 90-2】 【Chemistry 90-3】 【Chemistry 90-4】 【Chemistry 90-5】 【Chemistry 90-6】 【Chemistry 90-7】 【Chemistry 90-8】 【Chemistry 90-9】 【Chemistry 90-10】 【Chemistry 90-11】 【Chemistry 90-12】 【Chemistry 90-13】 【Chemistry 90-14】 【Chemistry 90-15】 【Chemistry 90-16】 【Chemistry 90-17】 【Chemistry 90-18】 【Chemistry 90-19】 【Chemistry 90-20】 【Chemistry 90-21】 or 【Chemistry 91】 wherein each Z is joined by a * and is individually selected from: 【Chemistry 92】 and 【Chemistry 93】 The Drug unit is optionally attached to a terminal acid group, a benzyl alcohol, or is represented by a wavy line ( 【Chemical 94】 ) indicates the binding site for the Drug unit).

97. 97. The linker of any one of claims 1 to 96, further comprising at least one drug unit attached to the linker subunit L2 to form a drug-linker.

98. 98. The Drug-Linker of claim 97, wherein said Drug unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand.

99. 99. The Drug-Linker of claim 98, wherein the Drug unit is a cytotoxic agent.

100. 100. The drug-linker of claim 99, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, and a calicheamicin.

101. The drug-linker of claim 100, wherein the cytotoxic agent is an auristatin.

102. The drug-linker of claim 101, wherein the cytotoxic agent is MMAE or MMAF.

103. The drug-linker of claim 100, wherein the cytotoxic agent is camptothecin.

104. The drug-linker of claim 103, wherein the cytotoxic agent is exatecan or SN-38.

105. The drug-linker of claim 104, wherein the cytotoxic agent is exatecan.

106. 100. The drug-linker of claim 99, wherein the cytotoxic agent is calicheamicin.

107. 100. The drug-linker of claim 99, wherein the cytotoxic agent is a maytansinoid.

108. The drug-linker of claim 107, wherein the maytansinoid is maytansine, maytansinol, or ansamatocin-2.

109. 99. The Drug-Linker of claim 98, wherein the Drug unit is an immunomodulatory agent.

110. The drug-linker of claim 109, wherein the immunomodulatory agent is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist.

111. The drug-linker of claim 110, wherein the immunomodulatory agent is a TLR7 agonist.

112. The drug-linker of claim 111, wherein the TLR7 agonist is imidazoquinoline, imidazoquinoline amine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroarothiadiazide-2,2-dioxide, benzonaphthyridine, guanosine analogue, adenosine analogue, thymidine homopolymer, ssRNA, CpG-A, PolyG10 or PolyG3.

113. The drug-linker of claim 110, wherein the immunomodulatory agent is a TLR8 agonist.

114. The drug-linker of claim 113, wherein the TLR8 agonist is selected from imidazoquinoline, thiazoloquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine or ssRNA.

115. The drug-linker of claim 110, wherein the immunomodulatory agent is a STING agonist.

116. The drug-linker of claim 110, wherein the immunomodulatory agent is a RIG-I agonist.

117. The drug-linker of claim 116, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000.

118. 99. The Drug-Linker of claim 98, wherein the Drug unit is a chelating ligand.

119. 119. The drug-linker of claim 118, wherein the chelating ligand is selected from platinum (Pt), ruthenium (Ru), rhodium (Rh), gold (Au), silver (Ag), copper (Cu), molybdenum (Mo), titanium (Ti), or iridium (Ir); a radioactive isotope such as yttrium-88, yttrium-90, technetium-99, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114, indium-115, lutetium-177, strontium-89, salarium-153, and lead-212.

120. 98. The drug-linker of claim 97, having the following structure: 【Chemistry 95-1】 【Chemistry 95-2】 【Chemistry 95-3】 【Chemistry 95-4】 【Chemistry 95-5】 【Chemistry 95-6】 【Chemistry 95-7】 【Chemistry 95-8】 【Chemistry 95-9】 【Chemistry 95-10】 【Chemistry 95-11】 【Chemistry 95-12】 【Chemistry 95-13】 【Chemistry 95-14】 【Chemistry 95-15】 【Chemistry 95-16】 【Chemistry 95-17】 【Chemistry 95-18】 【Chemistry 95-19】 【Chemistry 95-20】 【Chemistry 95-21】 【Chemistry 95-22】 【Chemistry 95-23】 【Chemistry 95-24】 【Chemistry 95-25】 【Chemistry 95-26】 【Chemistry 95-27】 【Chemistry 95-28】 【Chemistry 95-29】 【Chemical 95-30】 【Chemistry 95-31】 【Chemistry 95-32】 【Chemistry 95-33】 【Chemistry 95-34】 【Chemistry 95-35】 【Chemistry 95-36】 wherein each Z is joined by a * and is individually selected from: 【Chemistry 96】 and 【Chemistry 97】 or 【Chem.98】 wherein each Z is joined by a * and is individually selected from the following: 【Chem.99】 and 【Chemistry 100】 ）。

121. 121. A conjugate comprising a targeting unit attached to a drug-linker according to any one of claims 97 to 120.

122. 122. The conjugate of claim 121, wherein the targeting unit is selected from an antibody or an antigen-binding portion thereof.

123. 123. The conjugate of claim 122, wherein said targeting unit is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody or multispecific antibody.

124. 122. The conjugate of claim 121, wherein said targeting unit is a diabody, DART, anticalin, affibody, avimer, DARPin or adnectin.

125. 125. The conjugate of any one of claims 121 to 124, wherein the targeting unit is monospecific.

126. 126. The conjugate of any one of claims 121 to 125, wherein the targeting unit is bivalent.

127. 125. The conjugate of any one of claims 121 to 124, wherein the targeting unit is bispecific.

128. The average drug loading of the conjugate (p load 128. The conjugate of any one of claims 121 to 127, wherein R, R, and R are from about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

129. 129. The conjugate of any one of claims 121 to 128, selected from: 【Chemistry 101-1】 【Chemistry 101-2】 【Chemistry 101-3】 【Chemistry 101-4】 【Chemistry 101-5】 【Chemistry 101-6】 【Chemistry 101-7】 【Chemistry 101-8】 【Chemistry 101-9】 【Chemistry 101-10】 【Chemistry 101-11】 【Chemistry 101-12】 【Chemistry 101-13】 【Chemistry 101-14】 【Chemistry 101-15】 【Chemistry 101-16】 【Chemistry 101-17】 【Chemistry 101-18】 【Chemistry 101-19】 【Chemistry 101-20】 【Chemistry 101-21】 【Chemistry 101-22】 【Chemistry 101-23】 【Chemistry 101-24】 【Chemistry 101-25】 【Chemistry 101-26】 【Chemistry 101-27】 【Chemistry 101-28】 【Chemistry 101-29】 【Chemistry 101-30】 【Chemistry 101-31】 【Chemistry 101-32】 【Chemistry 101-33】 【Chemistry 101-34】 【Chemistry 101-35】 【Chemistry 101-36】 wherein each Z is joined by a * and is individually selected from: 【Chemical Engineering 102】 and 【Chemistry 103】 or 【Chemical 104】 wherein each Z is joined by a * and is individually selected from the following: 【Chemistry 105】 and 【Chemistry 106】 Ab is the targeting unit and n is p load (It is).

130. 130. The conjugate of any one of claims 121, 128 or 129, wherein the targeting unit is bound to a target molecule.

131. The conjugate of claim 130, wherein the target molecule is CD19, CD20, CD30, CD33, CD70, LIV-1 or EGFRv3.

132. 130. The conjugate of any one of claims 121, 128 or 129, wherein said targeting unit is selected from scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb and scFv-HSA-scFv.

133. 130. The conjugate of any one of claims 121, 128, or 129, wherein said targeting unit is a cancer-associated antigen.

134. The targeting unit is selected from the group consisting of CD19, CD20, CD30, CD33, CD38, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin (MLSN), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 1p19q, ABL1, AKT1, AL K, APC, AR, ATM, BRAF, BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, JAK 2, KDR (VEGFR2), KRAS, MGMT, MGMT-Me, MLH1, MPL, NOTCH1, NRAS, PDGFRA, Pgp, PIK3CA, PR, PTEN, RET, RRM1, SMO, SPARC, TLE3, T OP2A, TOPO1, TP53, TS, TUBB3, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1, STK1, MLH1, MSH2, MS H6, PMS2, ROS1, ERCC1, 5T4 (TPBG), B7-H3, CCR7, CD105, CD22, CD46, CD47, CD56, CD70, CD71, CD79b, CDH6, CLDN6, CLDN18.2, CL 130. The conjugate of any one of claims 121, 128 or 129, which is EC12A, DLL3, DR5, ERBB3 (HER3), EPCAM, FOLR1, IGF1R, IL2RA (CD25), IL3RA, ITGB6, LIV-1, LRRC15, mesothelin (MSLN), NaPi2b (SLC34A2), nectin-4, PTK7, ROR1, SEZ6, SLC44A4, SLITRK6, tissue factor (TF), TROP2 or B7-H4.

135. The targeting unit may be selected from the group consisting of rituximab (Rituxan®), trastuzumab (Herceptin®), pertuzumab (Perjeta®), bevacizumab (Avastin®), ranibizumab (Lucentis®), cetuximab (Erbitux®), alemtuzumab (Campath®), panitumumab (Vectibix®), ), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), ipilimumab, zalutumumab, dalotuzumab, figitumumab, ramucirumab, galiumtuzumab, farletuzumab, ocrelizumab, ofatumumab (Arzerra®), tositumumab, ibritumomab, CD20 antibody 2F2 (HuMax-CD20), 7D8, IgM2C6, IgG1 130. The conjugate of any one of claims 121, 128 or 129, which is an antibody or fragment thereof comprising 2C6, 11B8, B1, 2H7, LT20, 1FS or AT80, daclizumab (Zenapax®), or an anti-LHRH receptor antibody comprising clone A9E4, F1G4, AT2G7, GNRH03 or GNRHR2.

136. 122. The conjugate of claim 121, wherein the targeting unit is antibody F131 and the drug-linker is LD038.

137. The targeting unit comprises: The antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 arranged in heavy chain variable region framework regions, and the VL region comprises LCDR1, LCDR, and LCDR3 arranged in light chain variable region framework regions, and the VH and VL CDRs are (a) SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; and (b) the conjugate of any one of claims 121, 128 or 129, having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of SEQ ID NO: 36, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

138. the VH region and the VL region having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:26 and SEQ ID NO:27, respectively; 138. The conjugate of claim 137, wherein the heavy chain framework regions and the light chain framework regions are optionally modified by substitution, deletion or insertion of 1 to 8 amino acids within the framework regions.

139. The conjugate of claim 137, wherein the antibody is F131 and the drug-linker is LD038.

140. A conjugate comprising a targeting unit attached to said drug-linker, wherein said targeting unit is antibody F131 and said drug-linker is LD038.

141. 141. A pharmaceutical composition comprising the conjugate of any one of claims 121 to 140 and a pharmaceutically acceptable carrier.

142. 142. A method of treating a subject in need thereof comprising administering to the subject the conjugate of any one of claims 121 to 140 or the pharmaceutical composition of claim 141, wherein the subject has cancer or an autoimmune disease, and wherein the conjugate binds to a target antigen associated with the cancer or autoimmune disease.

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